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Master’s dissertation submitted in partial fulfilment of the requirements for the joint degree of International Master of Science in Environmental Technology and Engineering an Erasmus+: Erasmus Mundus Master Course jointly organized by Ghent University, Belgium University of Chemistry and Technology, Prague, Czech Republic UNESCO-IHE Institute for Water Education, Delft, the Netherlands Academic year 2015 – 2016 Enhanced Extraction Method for the Isolation of Difficult-To-Culture Soil Bacteria Host University: University of Chemistry and Technology, Prague, Czech Republic Marco Antonio Lopez Marin Promotor: Assoc. Prof. Ondřej Uhlík, M.Sc., Ph.D. This thesis was elaborated and defended at the University of Chemistry and Technology, Prague, Czech Republic, within the framework of the European Erasmus Mundus Programme “Erasmus Mundus International Master of Science in Environmental Technology and Engineering " (Course N° 2011-0172) © 2016, Prague, Czech Republic, Marco Antonio Lopez Marin, Ghent University, all rights reserved. Declaration This thesis was written in the Department of Water Technology and Environmental Engineering of the University of Chemistry and Technology, Prague from February to August of 2016 I hereby declare that this thesis is my own work. Where other sources of information have been used, they have been acknowledged and referenced in the list of used literature and other sources. I have been informed that the rights and obligation implied by Act No. 121/2000 Coll. On Copyright, Rights Related to Copyright and on the Amendment of Certain Laws (Copyright Act) apply to my work. In particular I am aware of the fact that the University of Chemistry and Technology, Prague has a right to sign a license agreement for use of these work as schoolwork under § 60 paragraph 1of the Copyright Act. I have also been informed that in the case this work will be used by myself or that a license will be granted for its usage by another entity, the University of Chemistry and Technology, Prague is entitled to require from me a reasonable contribution to cover the cost incurred in the creation of the work, according to the circumstances up to the full amount. I agree to the publication of my work in accordance with the Act No. 111/1998 Coll. On Higher Education and the amendment of the related laws (Higher Education Act). In Prague, 17th of August of 2016 Abstract Only around 1% of all the estimated microorganisms that exist on Earth have been cultured in the laboratory (Vartoukian et al 2010). The vast majority of Earth’s microorganisms is still uncharacterized, and their roles in the environment may be not totally known (Kell et al 1998). It is thought that the majority of these yet-uncultured organisms can be dormant (Rittershaus et al 2013) and have thus escaped cultivation efforts throughout decades. An enhanced extraction method for the isolation of difficult-to-culture soil bacteria was developed using two growth factors of the bacterium Micrococcus luteus: the liquid cultures’ supernatants of this bacterium containing the extracellular protein “resuscitation promoting factor” (Rpf), and M. luteus cell walls. Compared to a control soil extraction, the enhanced extraction with M. luteus supernatant-Rpf (SRpf) increased the number of CFU formed on solid medium by ca. one order of magnitude. These CFUs were analyzed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) and their spectra were analyzed and clustered to determine the resulting diversity in terms of different operational taxonomical units (OTUs) present. The SRpf-enhanced extraction not only increased the CFU number, but also the diversity in terms of different cultured OTUs. The cell wall-enhanced extraction also increased the CFU number and the diversity, albeit not as efficiently as the former extraction approach. SRpf increases the culturability of soil bacteria. Because dormancy can be an universal phenomenon (Rittershaus et al 2013) these enhanced extraction methods, specially the SRpfenhanced extraction, have a great potential in improving the culture of difficult-to culture soil bacteria not only in soil, as it proved to be, but also in different environments. Yet-uncultured microorganisms can be cultured for the first time following this enhanced extraction method. i ii Acknowledgements I would like to express my sincere gratitude to my promoter, Dr. Ondřej Uhlík and my tutor, Ing. Michal Strejček, whose constant support and guidance have made this work possible. I also owe a deep sense of gratitude to Ing. Jáchym Šuman and Petra Junková, PhD, for their assistance with the laboratory work and for their invaluable counselling. I would also like to thank the Mexican National Council for Science and Technology (CONACYT) for the financial support received during the course of the master. Finally, thanks to my parents for their unconditional support and for giving me the opportunity to keep studying. This work is mainly the result of their endless support. iii iv Content Abstract ..................................................................................................................... i Acknowledgements ................................................................................................. iii List of abbreviations and acronyms ........................................................................ vii Chapter 1.0. Rationale ............................................................................................ 9 Chapter 2.0. Literature Review. ............................................................................ 11 2.1 It is not dead. It is only sleeping................................................................... 11 2.1.1 Viable but non culturable bacteria: a mysterious bacterial state ........... 11 2.1.2 The VBNC state as an adaptive response for survival .......................... 14 2.1.3 Resuscitation of bacteria from the VBNC state ..................................... 17 2.1.4 An alternative model: VBNC cells as a result of cellular deterioration ... 19 2.1.5 Implications of the VBNC state ............................................................. 20 2.2 Culturing the unculturable ............................................................................ 20 2.2.1 Culturing the unculturable: breaking down a misnomer ........................ 20 2.2.2 Media and cultivation generalities ......................................................... 21 2.2.3 In situ culturing and recreating the environment in vitro ........................ 22 2.2.4 Co-culturing........................................................................................... 23 2.2.5 Improving cultivation using growth-promoting factors ........................... 24 2.2.6 Cultivation based on genomic data ....................................................... 25 2.2.7 Miscellaneous novel culturing techniques ............................................. 26 2.3 Peptidoglycan and Rpf, a bacterial cytokine ................................................ 26 2.3.1 Resuscitation promoting factor (Rpf) ..................................................... 26 2.3.2 Rpf’s mode of action ............................................................................. 28 2.3.3 Peptidoglycan-mediated resuscitation .................................................. 28 Chapter 3.0. Objectives......................................................................................... 31 Chapter 4.0. Materials and Methods ..................................................................... 33 4.1 Materials ...................................................................................................... 33 4.1.1 Media and buffers ................................................................................. 33 4.1.2 Soil ........................................................................................................ 33 4.2 Methods ....................................................................................................... 34 4.2.1 Bacterial Cultures .................................................................................. 34 4.2.2 Supernatant retrieval ............................................................................. 34 4.2.3 Micrococcus luteus cell wall preparation ............................................... 34 4.2.4 Supernatant activity determination by absorbance ............................... 35 4.2.5 Supernatant activity determination by fluorometry ................................ 35 4.2.6 Supernatant purification ........................................................................ 36 v 4.2.7 Rpf identification ................................................................................... 36 4.2.8 Soil extractions and cultivation .............................................................. 37 4.2.9 Diversity screening ................................................................................ 38 4.2.10 LC-ESI-Q-TOF mass spectrometry analyses ...................................... 39 Chapter 5.0. Results ............................................................................................. 41 5.1 Micrococcus luteus growth curve................................................................. 41 5.2 Micrococcus luteus supernatant activity to lyse peptidoglycan .................... 41 5.3 Micrococcus luteus supernatant activity against fluorogenic substrate ........ 44 5.4 Rpf detection ............................................................................................... 48 5.5 Soil extractions and cultivation: quantitative analyses ................................. 48 5.6 Soil extractions and cultivation: qualitative analyses (microbial diversity) ... 50 Chapter 6.0. Discussion ........................................................................................ 53 6.1 Supernatant activity ..................................................................................... 53 6.2 Enhanced extraction experiments ............................................................... 54 Chapter 7.0. Conclusion........................................................................................ 57 Bibliography .......................................................................................................... 59 Appendix. OTU Table............................................................................................ 69 vi List of abbreviations and acronyms AHL (n-acyl homoserine lactone) cAMP (adenosine 3',5'-cyclic monophosphate) CFU (colony forming unit) DVC (direct viable counting) DTT (dithiotreitol) IAA (iodacetamide) LB broth (Luria-Bertani broth) LC-ESI-Q-TOF-MS (liquid chromatography-electrospray ionization-quadrupole-time of flight-mass spectrometry) LMM (lactate minimal medium) MALDI-TOF MS (matrix-assisted laser desorption/ionization-time of flight mass spectrometry) MPN (most probable number) MUF tri-NAG (4-methylumbelliferyl- -D-N,N’,N’’-triacetylchitotrioside) OUT (operational taxonomical unit) PBS (phosphate buffer saline) PCA (plate count agar) PUF (polyurethane foam) Rpf (resuscitation promoting factor) Rpf (resuscitation promoting factor gene) SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) SPS (stationary phase survival) SRpf (supernatant resuscitation promoting factor) UHPLC (ultra high pressure liquid chromatography) VBNC (viable but non-culturable) vii 8 Chapter 1.0. Rationale The planet we know today is largely the result of the microorganisms’ activity in the biosphere. Earth’s smallest and simplest organisms created the conditions for the development of more complex life forms; that is, all the present biodiversity of animals, plants, and other organisms. Despite the central role of microorganisms in several biogeochemical processes, and unlike plants and animals, whose importance to humanity has been acknowledged since ancient times, the microbial world remained hidden from the human consciousness until the seventeenth century. Antoine van Leeuwenhoek described microorganisms for the first time using his self-built lenses, the first microscope. Microbiology developed during the course of the succeeding centuries, always constrained by the limitations of our own senses. Instruments and techniques had to be developed to overcome these limitations and to start uncovering the immense diversity of microorganisms. Despite technological advances that allowed microbiology to establish the solid body of knowledge it has today, microbiologists realized that only a few organisms were rewarding their endeavors: total microscopic counts greatly exceed viable counts in Petri dishes. This inconsistency is known as the “great plate count anomaly”, a term coined in 1985 by Staley and Konopka (Staley and Konopka 1985). In recent years, the improvement of molecular techniques for microbial analyses has open new horizons, and has shed light upon some features of the uncultivable microbial representatives. This implicates that today it is not necessary to culture bacteria to research community structure and dynamics. Owing to the ever developing molecular technologies, it would seem that the relevance of cultivation in today’s microbiology could be questioned. In fact, only 1% of microorganisms can be cultured (Vartoukian et al 2010), but metagenomic data can only be fully resolved based on knowledge derived from cultivation. This means that cultivation is still necessary to ground the ever increasing data that is being obtained through molecular techniques. Culture approaches give context to environmental microbiology: a basis upon which to confirm the knowledge that metagenomics creates (Nichols 2007). Only through the isolation of pure cultures can bacteria be fully characterized (Vartoukian et al 2010), and solid knowledge on their actual behavior and function in the environment can be acquired (Pham and Kim 2012). Moreover, the yet-uncultured bacteria may also be a reservoir of bio-molecules, which could be useful for all kinds of industrial and health applications (Alain and Querellou 2009). 9 10 Chapter 2.0. Literature Review. 2.1 It is not dead. It is only sleeping. 2.1.1 Viable but non culturable bacteria: a mysterious bacterial state The physiological characterization of macroorganisms is easy to determine compared to that of microorganisms (Bogosian and Bourneuf 2001). The “great plate count anomaly” persuaded microbiologists to search for the existence of other states in which bacteria may exist in nature, apart from being alive and dead. For a long time, it was considered that the cells observed through a microscope but which at the same time were unable to grow in laboratory media were dead cells; however, evidence has shown that they are not necessarily dead, but instead they are waiting for environmental conditions to be favorable to revert to a state in which they can grow and replicate normally (Kaprelyants et al 1994). By combining the direct viable counting method (DVC) with direct plating on solid medium, Xu et al. (1982) observed that Escherichia coli and Vibrio cholerae, when exposed to stresses such as incubation in saltwater, maintained their numbers while measured with direct microscopic examinations, but plate counts and most probable number (MPN) results exhibited declines in the number of counts. The DVC method was proposed by Kogure et al. in 1979. In this method, the number of viable bacterial cells is determined not by the bacterium’s ability to divide, but rather by the elongation or growth of single cells once they are exposed to substrate (Kogure et al 1979). Xu et al. (2012) resolved that the non-dividing cells were not dead: although they did not divided, they remained substrate-responsive. They were not dead; they were “only sleeping”. This study was the turning point to sharpen the concepts of culturability and viability (Pinto et al 2015). The definition of viability of a microorganism which stated as: “An organism is viable if it is capable of multiplying to form two or more progeny [sic] in conditions that are optimal for the species and strain of microbe concerned” (Postgate 1969), was ultimately challenged. A cell is nowadays considered viable if it keeps its membrane integrity and potential, enzymatic activity, respiration, responsiveness to nutritional stimuli and mRNA production (Pinto et al 2015). Assigning a name to this phenomenon of “sleeping cells” has been a problematic task for microbiology. Several terms have been used to describe this bacterial dormant state, being 11 “viable but non-culturable” (VBNC) the most common name that appears in the literature. Cells are not dead (are viable because they exhibit some of the characteristics previously described), but they simply do not divide in cultures. This naming problem is mainly because no large scale studies exist to compare the effects of diverse environmental conditions in the induction of the VBNC state (Li et al 2014, Pinto et al 2015), and there has been no consensus among different researchers on how to call the physiological states they observe. Some of the terms commonly used in literature which can relate to “VBNC” include cryptobiotic, dormant, latent, moribund, pseudosenescent, and somniscent cells (Kell et al 1998); injured, and somnambulant cells (Colwell 2000). Other similar terms are “not immediately culturable” (Kell et al 1998); “not readily culturable” (Bovill and Mackey 1997); “transiently non culturable” (Mukamolova et al 2003); non culturable, active but non culturable (Sachidanandham and Yew-Hoong Gin 2008); and “quiescence” (Rittershaus et al 2013). All these terms are not necessarily synonyms. Not only different terms have been used to describe the same physiological state, but it is also possible that different states and conditions have been categorized under a single VBNC state (Kell et al 1998). For instance, Kell et al (1998) define “dormancy” as a state of metabolic inactivity: dormant cells are those which exhibit negligible metabolic activity, but can further transit into a growing state; whereas VBNC (they call them active but non culturable) cells are metabolically active but fail to grow to detectable levels. To determine which bacteria are metabolically more inactive than others would imply setting a threshold at which a bacterium can be considered to be in the VBNC state, what will indeed be problematic. Oliver (2005) defines the VBNC state as a state of dormancy (dormant state). The metabolic state and the cell’s ability to divide are not considered separately to name the state in which bacteria are. What is then the VBNC state? Kaprelyants et al (1993) proposed a simple definition: VBNC cells are not alive in the sense of colony formation, but are not dead because they can again be ‘alive’ if conditions become favorable again. The VBNC state is considered a general survival strategy (Mukamolova et al 2003), since it is observed across several and very diverse bacterial phyla (Pinto et al 2015). Resting stages can be mechanisms of microbial adaptation to environmental changes to assure the dissemination of the population when appropriate conditions return (Suzina et al 2006). The environmental phenomena which can trigger stress in bacteria include changes in temperature, nutrient unavailability, salinity, osmotic pressure, pH (Colwell 2000); and desiccation (Suzina et al 2006). 12 Other factors influencing the entry into the VBNC state include pollutants present, redox conditions of the environment, and organic matter availability (Trevors 2011). Some of these damaging environmental conditions may temporarily comprise the cell´s ability to divide (Kell et al 1998), and trigger some responses that seem to be universal (Rittershaus et al 2013). Owing to the possibility that different states have been described under one term, the consideration of Ayrapetyan et al (2015) is interesting: they proposed the existence of a “dormancy continuum”, in which the VBNC state is a deeper state of dormancy but part of a common, shared phenomenon. Two different physiological states are contained within this “dormancy continuum”: the VBNC state, and a state called “bacterial persistency” (Ayrapetyan et al 2015). Both persisters and VBNC cells are highly tolerant to antibiotics (Ayrapetyan et al 2015, Lewis 2010). Several environmental factors induce the formation of persisters, such as starvation, carbon-source changes, pH, oxidative damage, etc (Ayrapetyan et al 2015). These factors are also some of those responsible to induce the formation of VBNC cells (Mukamolova et al 2003). Persisters might also share with the VBNC cells the “shut down” mechanism of cellular protection that appears when DNA-damaging agents are present (Lewis 2010). Nevertheless, while persisters can appear in exponentially growing populations (Lewis 2010), VBNC cells are the consequence of a “low metabolic cost existence” (Mukamolova et al 2003), which definitely is not the case during exponential growth. VBNC cells require a longer resuscitation-promoting treatment than persisters, which can be readily cultivable e.g. by removing the antibiotics (Ayrapetyan et al 2015). It must be also noted that “starved” or “injured” cells have been described as a different condition from the VBNC (Morita 1993, Mukamolova et al 2003). Injured cells have the ability to grow on non-selective medium but will not grow on a selective medium (Kell et al 1998) or in the presence of selective agents (Mukamolova et al 2003). Injured cells are formed by means of cell degradation and are dying instead of adapting to adverse environmental conditions (Pinto et al 2015). In contrast to injured cells, one of the characteristics of the cells in the VBNC state is their cell integrity (Mukamolova et al 2003). In laboratory conditions, entry into the stationary phase means that bacteria must also start to adapt to an increasingly harsh environment, since many factors such as oxidative stress, pH, and nutrient availability are changing (Mukamolova et al 2003). Even though the stationary phase observed during laboratory culture is not the same as the low-nutrient conditions observed in the environment (Mukamolova et al 2003), during this phase nutrients can become limiting, so it is possible to conceive a stationary culture in the laboratory as an 13 occasion for the formation of VBNC cells. It must be also noted that the term “dormancy” was coined for describing a physiological state of bacteria in nature (Stevenson 1977) before the term VBNC was coined to describe an issue of non-culturability in the laboratory (Xu et al 1982). The fact notwithstanding, throughout this work the terms VBNC and dormant will be considered as synonyms: VBNC cells are non culturable because they are physiologically dormant. The view that the VBNC state is a survival strategy (Mukamolova et al 2003) is not the only explanation for the origin of VBNC cells. Three models have been proposed to explain the origins of the VBNC phenomenon (Nyström 2003): (1) it is the culmination of an adaptive, genetically programmed pathway generating dormant, survival forms; (2) it appears due to cellular deterioration; and (3) it is the activation of genetic modules preceding cell death. The two first models will be further analyzed in the present work. 2.1.2 The VBNC state as an adaptive response for survival The view that the VBNC state is a general, genetically driven mechanism to generate dormant and surviving cells is supported by a wide number of observations. Bacteria in the VBNC state exhibit several types of morphological changes. Cells under these conditions can predominate in cyst-like forms, have thickened cell walls, fine-grained cytoplasms, round and reduced shape, condensed nucleoids, etc (Suzina et al 2006). Of particular relevance is the cell wall state, since it is the structure which protects the cell from the environment. Enterococcus faecalis cells in the VBNC state were twice as resistant as measured for mechanical disruption with glass beads in a homogenizer and their peptidoglycan appeared to be more crosslinked (Signoretto et al 2000). Other Enterococcus species produce O-acetylated peptidoglycan (at the C-6 hydroxyl group of muramoyl residues) when they enter the VBNC state, a modification that inhibits the activity of hen egg white lysozyme (Pfeffer et al 2006) and regulates the sensitivity to hydrolytic enzymes (Pinto et al 2015). The cell wall of Vibrio cholerae changes during its transition from the exponential growth phase to the stationary phase, that is, when nutrients become limiting (Lam et al 2009). The content of D-amino acids, specially D-methionine and D-leucine increases, which may influence the strength of the cell wall (Lam et al 2009). Staphylococcus aureus cell wall also thickens when the cells enter the stationary phase (Zhou and Cegelski 2012). Membranes of cells exposed to conditions that are thought to trigger the VBNC state also show a modified 14 structure, like changes in the lipid composition (Denich et al 2003). When exposed to starvation, visible radiation or seawater, E. coli changes the protein composition of its outer membrane (Muela et al 2008). Bacteria better adapted to survive starvation conditions are small sized and have lower metabolic activity (Colwell 2000). A size reduction when entering the stationary phase can mean a reduction of the cell´s energy requirements (Mukamolova et al 2003). This reduction can be as big as 15- to 300-fold (Colwell 2000). For Micrococcus luteus cells, a decrease in the mean cell diameter (from 1.3 to 0.4 µm) was observed after the cells entered the stationary phase (Kaprelyants et al 1993). Small bacteria, called ultramicrobacteria, predominate in the environment (Bakken and Olsen 1987). Their smaller size can give them an advantage on resource scavenging due to the increased area per unit volume (Morita 1988). Bacteria respond to challenging conditions changing their morphology to a coccoid form (Asakura et al 2007). Several bacteria have been observed to adopt a coccoid form once entering adverse conditions, among them Campylobacter jejuni (Bovill and Mackey 1997), Vibrio cholerae (Lam et al 2009), and Helicobacter pylori (Nilsson et al 2002). In chemostat experiments at low dilution rates and in old bacterial batch cultures, bacteria exhibit also a reduction in size (Kaprelyants et al 1993). For instance, Campylobacter jejuni grown under different temperature and aereation conditions in sterile stream water microcosms has been shown to form a continuum of morphological forms when entering the nonculturable phase (Rollins and Colwell 1986). These shapes ranged from spiral to coccoid, and its predominance depended upon their growth stage (Rollins and Colwell 1986). When entering the VBNC state, bacteria still exhibit some detectable metabolic activity (Oliver 2005). Gene expression occurs in Escherichia coli and Vibrio cholerae after being exposed to low nutrient concentrations (Asakura et al 2007, Rozen et al 2002). In Escherichia coli, enzymes essential for several catabolic pathways are upregulated (Rozen et al 2002). The gene VCA0700 coding for chitodextrinase in Vibrio cholerae, important for the chitin utilization by the bacterium, is also upregulated in dormancy (Asakura et al 2007). This is relevant for the metabolism since the bacterium can rely on chitin as its sole carbon source (Asakura et al 2007). VBNC cells preserve their membrane potential and ATP synthesis despite their dormancy (Rittershaus et al 2013). ATP level in dormant Mycobacterium tuberculosis cells under hypoxic conditions is five times lower, but they keep their membranes energized to drive ATP production at the same level as replicating cells (Rao et al 2008). 15 Protein synthesis accelerates during the VBNC state (Rittershaus et al 2013). It was found in Vibrio parahaemolyticus that, when incubated at 4 ºC in mineral medium, proteins with functions associated with transcription, translation, ATP synthase, gluconeogenesis, antioxidation and transmembrane solute uptake were enhanced (Lai et al 2009). In bacterial cultures, protein production during the stationary phase can be achieved upon the degraded proteins produced during the exponential phase (Shaikh et al 2010). The protein profiles of Enterococcus faecalis cells in the VBNC state differ from its starved cell counterparts (Heim et al 2002). While in VBNC cells, proteins homologous to the elongation factor Ts (EF-Ts) , fructose bisphosphate aldolase, and a catabolite regulator protein (CAA09491) were upregulated, in starved cells they were down-regulated (Heim et al 2002). EF-Ts plays a role in the regulation of protein synthesis, and fructose bisphosphate aldolase is an enzyme needed during growth in nitrogen limiting conditions (Heim et al 2002). The mannose-specific PTS system component IIAB, a protein expressed under stress conditions, was present in starved cells but not in VBNC cells (Heim et al 2002). This study supports the fact that the VBNC state might not be only a product of cellular deterioration (Nyström 2003) because protein expression patterns of both conditions differ and some up-regulated proteins, i.e. fructose biphosphate aldolase, play an important role in responses against hostile conditions. The regulation of mRNA stability and the preservation of DNA integrity are important responses against stress in bacteria (Takayama and Kjelleberg 2000). An increase in mRNA stability and half-life has been observed for Staphylococcus aureus (Anderson et al 2006) and Mycobacterium tuberculosis (Rustad et al 2012) when these bacteria are exposed to temperature shocks and hypoxic conditions, respectively. Stable mRNA transcripts were also observed in the starved marine Vibrio sp. strain S14, which could be translated after a glucose upshift (Marouga and Kjelleberg 1996). For a cell to remain viable, it needs to keep its DNA protected from potential lesions. Both spores and dormant cells alter their chromosomes to form more stable structures (Rittershaus et al 2013). When E. coli cells starve for carbon, they induce a DNA binding protein that renders the genome resistant to DNAses and hydrogen peroxide (Almiron et al 1992). This protein, Dps, prevents DNA damage by free radicals, yet does not interfere with normal DNA metabolism, and can also be involved in nucleoid condensation (Martinez and Kolter 1997). When Vibrio vulnificus enters the VBNC state, i.e. when cells are exposed to a cold shock, they express several genes responsible for combating numerous environmental stresses, such as rpoS and vvhA, the latter having a possible role in osmoprotection (Smith and Oliver 2006). A condensed nucleoid can be an indication that the cell is in the VBNC state. 16 Ghost cells, bacteria without visible nucleoids, regain nucleoid visibility (and activity) once they are placed under growth permissive conditions (Cole 1999). Choi et al (1996) described that the proportion of starved marine bacteria with visible nucleoids, when resupplied with nutrients, increased from 27% to 100%. The entry into the VBNC state also implies storage of carbon reserves, in forms such as glycogen, triglycerides, wax esters, polyhydroxyalcanoates, and poly-β-hydroxybutyric acid, which are quickly mobilized for use once the conditions are restored (Rittershaus et al 2013). Acetyl-CoA that enters the Krebs cycle during active growth is redirected into lipid synthesis pathways and stored in the mentioned chemical forms; this both reduces metabolic rate and increases energy reserves for the cell to survive during dormancy (Rittershaus et al 2013). 2.1.3 Resuscitation of bacteria from the VBNC state At some point during their dormant existence, bacteria must ‘wake up’ to start dividing again or ultimately die, because the VBNC state is time-limited (Mukamolova et al 2003). Dormant bacteria can resume division once the appropriate conditions for their growth reappear, a process called resuscitation (Mukamolova et al 2003). VBNC cells become culturable again (Pinto et al 2015). Determining whether a cell resuscitated or not can be a difficult task, since VBNC cells may coexist with multiple phenotypic states, and interfering signals from other physiological types within the same population may impede a correct result interpretation (Sachidanandham and Yew-Hoong Gin 2008). Bacterial populations must likely contain a mixture of VBNC/actively growing cells at the same developmental stage (Mukamolova et al 2003). If the state in which bacteria are found cannot be correctly determined, it is not possible to determine whether a regrowth was due to resuscitation, or only because of the growth of active cells (Kaprelyants et al 1994), or repair processes of injured cells (Kell et al 1998). Kaprelyants et al (1994) determined the resuscitation of M. luteus cells starved for 3 months, after which they were transferred to fresh lactate minimal medium containing 0.5 µg ml-1 penicillin G. They concluded that the posterior growth observed in the cell population was caused only because of the resuscitation of dormant cells, since the antibiotic targeted only dividing cells and left VBNC cells intact (Kaprelyants et al 1994). Campylobacter jejuni 17 cells subjected to dormancy by rendering their atmosphere microaerophilic, resuscitated when they returned to aerobic conditions (Bovill and Mackey 1997). Wai et al (1996) researched the resuscitation of Vibrio cholerae, after the application of a heat shock from 15 ºC (temperature which triggered the VBNC formation) to 45 ºC for 1 minute. Cells that were not culturable in liquid or solid media became culturable only after this heat shock (Wai et al 1996). Bacteria can resuscitate when they are cultured together with other microorganisms, either other bacteria or eukaryotes. Senoh et al (2010) demonstrated that resuscitation of V. cholerae was possible when cultured with eukaryotic cells. Kaprelyants et al (1994) used the autoclaved supernatant from centrifuged suspensions of starved cultures that may have contained compounds excreted by some microbial species, which are necessary for others to grow. When the cells were counted through the MPN method, it was observed that the use of the autoclaved supernatant yielded better resuscitation results than the use of fresh liquid growth medium (Kaprelyants et al 1994). Resuscitation is also possible in E. coli supplementing dormant cells with amino acids, especially polar amino acids (Pinto et al 2011). The role that other microorganisms have in the resuscitation of dormant bacteria (or culturing of VBNC bacteria in a technique called co-culture) and the use of growth factors for the improvement of bacterial cultivation techniques will be further discussed in the following section (2.2). Epstein (2009) proposed a different resuscitation approach, based on the fact that cells can resuscitate stochastically instead of responding to environmental signals. If the conditions found by a resuscitating bacterium are growth supportive, it will survive and start dividing; otherwise the cell will simply die (Epstein 2009). Other cells will resuscitate later in the same way to assess the environmental conditions; these cells are called ’scout cells‘ (Epstein 2009). The sensing of extracellular signals can be impaired by dormancy (Sturm and Dworkin 2015). Nevertheless, the scout theory and the resuscitation mediated by environmental cues are not mutually exclusive, since scout cells which resuscitate when the conditions are growth supportive can at the same time produce growth inducing signaling compounds that would accumulate in the extracellular media, helping other bacteria in the population to resuscitate (Epstein 2009, Pinto et al 2015). 18 2.1.4 An alternative model: VBNC cells as a result of cellular deterioration Despite the fact that the VBNC state has recurrently appeared in the literature for more than 30 years, its existence as a defined physiological phenomenon generating dormant, resistant cells has been challenged by several researchers. VBNC cells, as previously discussed, exhibit some changes that differentiate them from actively growing bacteria, but these changes do not necessarily imply that bacteria entered the VBNC state (Pinto et al 2015). One of the main arguments against many of the studies that research on VBNC bacteria, has been that they fail to adequately determine whether alleged resuscitation was indeed due to resuscitation, or to the growth of some culturable cells initially present in the population (Kell et al 1998). Bacterial unculturability can also be a condition preceding cellular death (Sachidanandham and Yew-Hoong Gin 2008). Clear degenerative changes can be observed in cells that enter the stationary phase in laboratory cultures (Mukamolova et al 2003, Nyström 2003) . In a viability study of Vibrio vulnificus, CFU on medium supplemented with catalase and sodium pyruvate to diminish H2O2 in the culture showed 1,000-fold higher counts than on unsupplemented media (Bogosian et al 2000), supporting the idea that non-culturability is really due to the damaging action of free radicals (Mukamolova et al 2003). Cells subjected to nutrient starvation have an increased need for oxidation management (Nyström 2001), so they express proteins aimed to protect themselves against oxidation damages. Upon starvation, production of cytoplasmic superoxide dismutases, enzymes involved in protecting the cell against oxidation, increase (Dukan and Nyström 1999). Depriving the starved cells from oxygen makes them survive for longer periods (Dukan and Nyström 1999). This close relationship between nonculturability and oxidation damages, especially protein carbonylation, favors the stochastic deterioration theory (Nyström 2003). Specific determinants to support the theory of VBNC phenomenon as adaptive, genetically programmed pathway generating dormant cells are yet to be uncovered (Kell et al 1998, Pinto et al 2015). Until now, not enough light has been shed upon the molecular mechanisms behind the entry and exit from the VBNC state (Pinto et al 2015). If the genetic machinery behind the VBNC state is neither characterized nor identified, the phenomenon will remain one of microbiology’s biggest questions (Nyström 2003). But if there are several common features of VBNC cells such as morphology, biochemistry and physiology alterations, there must definitely be some basic genetic mechanisms orchestrating all these changes (Mukamolova et al 2003). 19 2.1.5 Implications of the VBNC state The VBNC state may have many implications in the environment and human health, so unveiling the molecular basis of its perplexing nature is one of the pending milestones for today’s microbiology. It is estimated that 60% of the Earth’s microbial biomass exist in a dormant state (Lewis 2007, Rittershaus et al 2013). In oceanic ecosystems, the proportion of dormant cells, which are inactive and can activate when conditions become suitable, ranges from 20 to 90% (Cole 1999). Slow replication rates must be the norm in the environment: organisms usually live in habitats which are not suitable for rapid growth, since the basic nutrients for growth are not always abundant (Rittershaus et al 2013). If these dormant cells maintain metabolic activity, as they have been shown to do, they might have a role in environmentally significant processes such as nutrient cycling (Kell et al 1998). Jones and Lennon (2010) proposed that bacteria, when dormant, become members of a ‘seedbank’ – a reservoir of inactive bacteria which can contribute to microbial diversity and dynamics in future generations (or when pertinent environmental conditions for each of them arise). The dynamics of this reservoir can be season-dependent (Mukamolova et al 2003). VBNC cells must also play an important role for medicine. Infections could be able to enter a dormant state, thus bacteria can become latent and resistant to antibiotics, since the antibiotic’s action usually requires the organisms to be actively reproducing (Kell et al 1998). When an environmental sample is inoculated on medium, the inability to observe colonies of bacteria that can be otherwise observed under the microscope can be due to two reasons: the suitable in vitro culture conditions have not been discovered, as is the case of the majority of microorganisms, or the cells are temporarily present in the VBNC state and could become culturable at a later time point (Kell et al 1998). Bacteria which have not been cultured in vitro might be staying (or entering) the VBNC state once inoculated in laboratory medium (Lewis 2007, Watve et al 2000). 2.2 Culturing the unculturable 2.2.1 Culturing the unculturable: breaking down a misnomer Absolutely unculturable microorganisms are not likely to exist: since all microbes can grow in nature, unculturability is only the microbiologists’ failure to mimic conditions 20 necessary for growth of many organisms (Stewart 2012, Watve et al 2000). The environment in which bacteria live in nature is very different from the conditions we create for them in the laboratory. In nature, the conditions are more likely oligotrophic (Kaprelyants et al 1993). Bacteria live under what Koch (1971) called a “feast and famine existence”. Bacterial growth observed under laboratory conditions, namely the lag phase, exponential phase, and stationary phase, does not necessarily exist in nature, where bacteria are constantly experiencing environmental changes (Pinto et al 2015). Oligotrophs are likely genetically adapted to low nutrient environments, while copiotrophs are physiologically adapted to nutrient fluctuations (Mukamolova et al 2003). Oligotrophs and copiotrophs can also be related to the strategies which categorize higher organisms: R strategists for fast growing, copiotrophic bacteria, and K strategists for oligotrophs, slow growers (Watve et al 2000). In natural environments, the selective pressure favors cells with a low metabolic cost existence (Mukamolova et al 2003). Laboratory conditions must mimic as close as possible natural environments of bacteria (Mukamolova et al 2003). However, when isolating novel bacterial representatives, highly specialized conditions or media can be disadvantageous, since as yet uncultured bacteria are entities whose needs may not be totally characterized (Durbin 1961). This is why most of the novel media for culturing difficult-to-culture bacteria are not designed but are rather taken directly from nature, e.g. creating media based solely on water taken directly from the environment and with a low inoculum of organisms (low concentrations) reflecting that of the environment (Mukamolova et al 2003). Pham and Kim (2012) mention some common mistakes when attempting to culture soil bacteria: (1) addition of inhibitors, (2) high concentration of nutrients, and (3) no consideration for density dependent cell/signaling mechanisms. In the environment, bacterial populations are very heterogeneous, with some individuals actively growing, some other injured, and some dormant. In vitro, opportunities for these bacteria to grow should be equalized. The overgrowth of oligotrophs by copiotrophs should be avoided (Giovannoni and Stingl 2007). Several novel culturing strategies will be described in the following sections. 2.2.2 Media and cultivation generalities Indeed, one of the successful modifications that led to the culture of previously unculturable bacteria is the use of dilute nutrient media, since slow growers can be inhibited when inoculated in substrate rich conventional media (Vartoukian et al 2010). High nutrient 21 media might work well for human-residing bacteria and pathogens, since the interior of a human being must be very high in carbon resources and nutrients (Dewi Puspita et al 2012) and has a stable temperature. If most of natural environments in which microorganisms live are oligotrophic, then laboratory media aimed to their recovery must also have a low nutrient concentration. Inhibitors found in media such as oxidative agents (e.g. H2O2) can cause oxidative stress (Dewi Puspita et al 2012), and could hinder the growth of injured cells. Other relevant variables when culturing microorganisms are cultivation time and inoculum size. When incubation time is increased, some rarely isolated groups appear in the culture medium (Davis et al 2005). Song et al (2009) attempted the isolation of SAR11 cells, abundant members of bacterioplankton in the ocean. The cells did not appear under laboratory conditions until 8 weeks of culturing. Since oligotrophs are slow growers, they likely require higher cultivation times. At the same time, if the scout cell awakening theory proposed by Epstein (2009) is the case for resuscitation of VBNC cells, a longer cultivation time (and also larger inocula) would allow a higher formation rate of scouts and thus increase the chance of observing the growth of colonies (Epstein 2013). Choosing the right inoculum size is relevant when culturing difficult-to-culture bacteria, since some bacteria appear to be culturable only when they are inoculated above a certain cell density (Stewart 2012). Also, some of the signaling mechanisms among bacteria are density dependent, so the lack of consideration for initial density may have a direct relation with the VBNC phenomenon. If bacteria do not detect the environmental signals necessary for their resuscitation, they will not start dividing in vitro. Dilution of the inoculum can be beneficial, because in this way the inoculum would contain the most abundant cells rather than the most nutrient tolerant, fast grower organisms (Button et al 1993), which may exist in lower numbers but adapt and grow quickly. Low inoculum sizes can also prevent bacteria from being affected by antimicrobials produced by other community members (Dewi Puspita et al 2012). 2.2.3 In situ culturing and recreating the environment in vitro If laboratory conditions are unable to mimic the natural environment, then culturing in situ can be an option (Alain and Querellou 2009). Yasumoto-Hirose et al (2006) brought their cultivation chambers, polyurethane foam (PUF) supplemented with agar medium, directly to the ocean to culture marine bacteria. They targeted different bacteria by supplying the PUF 22 with different culture media, and in the laboratory, the PUFs where homogenized with sterile seawater, diluted and bacteria were inoculated into agar medium (Yasumoto-Hirose et al 2006). For bacteria which grow in biofilms, colonization carriers can be used (Alain and Querellou 2009). Titanium rings pierced with numerous apertures were used as colonization carriers for bacteria in deep-sea vent worms, which were further isolated through a series of enrichment cultures in media enriched with electron donors and acceptors found in situ such as iron, nitrate and sulphate (Alain et al 2004). Natural environments can also be constructed in the laboratory (Stewart 2012), or the environment where bacteria live can be mimicked using directly natural matrices as media, such as water or soil (Vartoukian et al 2010). Ferrari et al (2008) developed a protocol for cultivating previously uncultured soil bacteria employing, what they call, a soil substrate membrane system. The medium of this system consists of natural non-sterilized wet soil and a polycarbonate membrane where colonies grow. Some methane oxidizing bacteria were also cultured for the first time on a polycarbonate membrane, using non sterile soil slurry as a medium while incubated in a methane-air atmosphere (Svenning et al 2003). Bacteria inoculated in the polycarbonate membranes profit from the nutrients and chemicals from their own environment (the natural medium used), since these substances can pass through the membrane but bacteria cannot. The basic reasoning behind diffusion chambers is to provide bacteria with their natural environment while in culture by artificially creating this environment, such as in an aquarium (Bollmann et al 2007, Kaeberlein et al 2002). After bacterial colonies appear on the membrane, they can be selected and propagated with successive reinoculations (Stewart 2012). Sterile natural lake water has been used as a culture medium for low nucleic acid bacteria (Stingl et al 2008, Wang et al 2009). 2.2.4 Co-culturing Because microbes do not live isolated in their environment and establish relationships with other microorganisms in the community, co-culture approaches have been established (Pham and Kim 2012). It must be remembered that resuscitation of some bacteria was possible in the presence of other living cells, either with growing cells of the same species or with different cells (Kaprelyants and Kell 1993, Senoh et al 2010). So, in a petri dish, cells might be releasing signaling molecules which indicate dormant cells that they are in a suitable environment for their growth (Lewis 2007). 23 The isolation of novel microorganisms from a given environment can be done by coculture with cultured specimens of that environment, so called helper cells (D'Onofrio et al 2010). Kaeberlein et al (2002) were able to culture previously uncultured marine bacteria in nutrient medium only in the presence of other marine microorganisms. Pagnier et al (2008) isolated novel bacteria in co-culture with amoebae, since they live in symbiosis with the eukaryote. Marine bacteria isolated via the diffusion chamber might be difficult to be further cultured in a pure culture, but they can grow in co-culture with culturable marine bacteria on an agar medium (Nichols et al 2008). Taking advantage of symbiotic relationships with higher organisms such as plants and insects might help to bring some of the most challenging as-yet-uncultured bacteria to culture (Stewart 2012). The genomes of symbiotic bacteria can be reduced due to close host association and can lack critical genes essential for microbial reproduction (Kikuchi 2009, Stewart 2012). Bacteria which live in symbiosis with insects have been mainly cultured using insect cell lines (Kikuchi 2009). Associations of bacteria in communities require them to have an effective cell-to-cell communication system, which is called ‘quorum sensing’ (Bassler 1999). Quorum sensing occurs when bacteria are at high population densities (Bassler 1999) and this system involves several substances for cell-cell communication. These substances include growth factors, which can also be incorporated in the cultivation strategies and thus increase the efficiency for culturing environmental bacteria (Dewi Puspita et al 2012). 2.2.5 Improving cultivation using growth-promoting factors Media can be also supplied with metabolites that microbes need in their natural environment for growth. A lot of different substances can be mentioned that have helped to culture as-yet-uncultured microorganisms in vitro: different carbon sources, electron donors and acceptors, inorganic ions, aminoacids (Pham and Kim 2012, Vartoukian et al 2010), and the number of these substances might be as vast as the number of as-yet-uncultured microorganisms. Two exemplatory growth promoting factors are the adenosine 3',5'-cyclic monophosphate (cAMP) and n-acyl homoserine lactone (AHL). A possible link between cAMP and the VBNC state has been proposed, since cAMP can be a signal for the regulation of starvation survival proteins (Schultz et al 1988). In Mycobacterium tuberculosis, gene Rv3676 codes for a transcriptional factor belonging to the 24 cAMP receptor family, which is a direct regulator of the gene rpfA, one of the M. tuberculosis genes that encodes for a resuscitation promoting factor (Rickman et al 2005). Cyclic AMP is widely present dissolved in aquatic ecosystems; it is released from other organisms, including multicellular eukaryotes (Ammerman and Azam 1981). AHL is a quorum sensing molecule; its extracellular concentration increases when the cell density increases (Dewi Puspita et al 2012). When attempting to recover Acidobacteria and Verrucomicrobia uncultured representatives, Stevenson et al (2004) determined that the supplementation of medium with catalase or AHL resulted in one of the best culture modifications among several for a greater occurrence of Acidobacteria in culture. Similarly to cAMP, AHL could also be related to the VBNC state. Gorschkov et al (2010) suggested that, when starved, cell sensitivity toward AHL increases, which allows the quorum-dependent stress response to start at lower population densities. Other molecules that could act as growth factors are resuscitation promoting factor (Rpf) of Micrococcus luteus and peptidoglycan/cell wall fragments. Their role in the VBNC phenomenon and their potential as signaling compounds will be discussed in section 2.3. 2.2.6 Cultivation based on genomic data When designing new culturing techniques, there is no need for trial and error approaches: finding out which growth factors do bacteria need can be found within their genomes (Alain and Querellou 2009). For instance, genome analysis of three members of the SAR11 alphaproteobacterial clade showed a deficiency in sulphate reduction genes (Tripp et al 2008). In nature, members of this clade take reduced sulphur compounds exclusively from other organisms; therefore supplementing reduced sulphur species to their growth medium could bring them to culture (Tripp et al 2008). Bomar et al (2011) cultured for the first time a leech symbiont based on the information provided by sequencing the transcriptome of the leech’s gut. The information suggested that the metabolism of the bacterium relied on the conversion of fermented sialylated-mucin glycans to acetate, so addition of these glycans to the designed medium brought the bacterium into culture. 25 2.2.7 Miscellaneous novel culturing techniques Other approaches for optimizing culturing techniques involve looking after individual cells. Zengler et al (2002) developed a technique for encapsulating cells individually in gel microdroplets, sorting the droplets through flow cytometry, and inoculating each cell in the wells of microtiter plates. A similar approach using agar spheres to encapsulate bacteria directly from a simulated environment was developed by Ben-Dov et al (2009). Bacteria inside the spheres can be cultured in their own natural medium, from which they can be isolated and enriched in agar plates (Ben-Dov et al 2009). The use of microbial culture chips is another high-throughput culture technology that can be used to segregate and grow microorganisms (Ingham et al 2007). As an analogy, these devices resemble microscopic egg carton boxes, with a porous aluminium oxide base so that they allow the exchange of nutrients between the medium and the sorted cells. An advantage of microbial culture chips is their high throughput screening capacity (Ingham et al 2007). Individual cells can be physically manipulated using optical tweezers in the form of infrared lasers to isolate them and transfer them to a growing medium (Vartoukian et al 2010). 2.3 Peptidoglycan and Rpf, a bacterial cytokine 2.3.1 Resuscitation promoting factor (Rpf) Bacterial growth factors, or bacterial cytokines, are compounds whose name is derived from substances present in higher organisms that control cell growth and division, called growth factors (Kell and Young 2000). In eukaryotic cells, cytokines activate receptor tyrosine kinases, which communicate with the nucleus through phosphorylation cascades (Kell and Young 2000). It was observed that, when supplementing dormant Micrococcus luteus cells with sterile supernatant from their actively growing counterparts, dormant cells were resuscitated and the viable count as estimated by MPN raised several orders of magnitude (Mukamolova et al 1998). M. luteus is a gram positive bacterium with a high G+C content that can be found in diverse environments such as soil, water and human skin (Mukamolova et al 2002). Mukamolova et al (1998) isolated a protein from the Micrococcus luteus culture supernatant: a small protein called resuscitation promoting factor, Rpf, essential for M. luteus growth. 26 The lytic activity of Rpf was hypothesized by Cohen-Gonsaud et al (2004), since the enzyme revealed a high structural similarity with lysozyme. Mukamolova et al (2006) confirmed the muralytic activity of Rpf. When expressed in E. coli and secreted into its periplasm, Rpf lysed the bacterium; it also lysed fluorescamine labelled cell walls of M. luteus, the artificial substrate 4-methylumbelliferyl β-D-N,N′,N′′-triacetylchitotrioside (MUF tri-NAG) (Mukamolova et al 2006), and crude preparations of M. luteus cell wall (Telkov et al 2006). It cleaves the -glycosidic bond found between N-acetyl-muramic acid and N- acetylglucosamine in the peptidoglycan (Mukamolova et al 2006), while releasing its fragments. Rpf is a member of a broad protein group found throughout high G+C gram positive bacteria (Mukamolova et al 2003). Firmicutes possess a protein family related to the actinobacterial Rpf, which also improves their culturability, the SPS (stationary phase survival) protein family (Ravagnani et al 2005). Rpf is encoded for by the M. luteus gene rpf (Koltunov et al 2010). The protein is formed by a 70-residue lysozyme like domain and a LysM domain found in cell-wall-associated proteins, which is thought to facilitate binding to peptidoglycan (Koltunov et al 2010). A linker region which connects both domains has a variable size in different strains; this determines whether Rpf remains bound to the surface of the cell, or is released to the medium (Koltunov et al 2010). Rpf production reaches maximum before the late exponential phase of M. luteus growth, and rapidly declines before entering the stationary phase (Mukamolova et al 2002). Optimum activity of Rpf is at pH 6 (Telkov et al 2006). Rpf proteins control culturability of one of the largest taxonomic units within the bacterial domain, Actinobacteria (Mukamolova et al 2006, Ventura et al 2007). This phylum is of extreme relevance because it includes some of the main antibiotic-producing organisms for the pharmaceutical industry (Ventura et al 2007). Rpf stimulates also the growth of other high G+C gram positive organisms like Mycobacterium spp. Rpf genes are not exclusive to Micrococcus luteus: Mycobacterium tuberculosis has 5 analogous proteins with similar properties to those of Micrococcus Rpf (Mukamolova et al 2002), and these genes can be widely distributed among high G+C gram positive bacteria (Kaprelyants et al 1999). Much research has been done to understand the role of each of the 5 rpf genes of M. tuberculosis (Kana and Mizrahi 2010), but their structure and specific roles in Mycobacterium spp. dormancy will not be further discussed in this work. 27 2.3.2 Rpf’s mode of action Although the resuscitating mechanism of Rpf in dormant bacteria is not clear, several models have been proposed (Pinto et al 2015): (1) Rpf acts as a cytokine, binding to specific receptors in dormant cells and triggering resuscitation; (2) Rpf hydrolyzes bacterial cell wall of dormant cells, remodeling it and thus starting resuscitation; (3) Rpf hydrolyzes the cell wall, resulting in peptidoglycan fragments release, which bind to specific cell receptors that trigger resuscitation; and (4) Rpf mediated hydrolysis of the cell wall releases muropeptides, which then bind to specific receptors and trigger resuscitation. Rpf is active at picomolar concentrations, so its mode of action can be more similar to a cytokine (1) rather than by restructuring the cell wall (2) (Mukamolova et al 2003). Nevertheless, a receptor for the Rpf has not been identified yet (Pinto et al 2015, Telkov et al 2006). As previously discussed, one of the characteristics of VBNC cells is their thickened cell wall, and cells might need a specialized enzyme for cleaving that reinforced structure (Keep et al 2006, Pinto et al 2015). When bacteria divide, they must cleave peptidoglycan to separate the new daughter cell attached at the septum (Hett et al 2007). The lytic activity of Rpf against peptidoglycan can aid the cell during the divisions after resuscitation occurs (Hett et al 2007). Telkov et al (2006) determined by directed mutagenesis that an alteration in the catalytic site of Rpf, namely the replacement of the glutamate residue at position 54 with glutamine, alanine and lysine, and cysteine residues 53 and 114 with threonine and lysine, respectively, reduced its activity and at the same time minimized its resuscitation potential. This finding strongly favors the hypothesis that activity of Rpf is directly related to its resuscitation potential (Telkov et al 2006). 2.3.3 Peptidoglycan-mediated resuscitation The third Rpf’s mode of action described previously (3), implies catalytic activity of Rpf and the possibility that the peptidoglycan fragments resulting from hydrolysis are detected by receptors in other cells (Pinto et al 2015), possibly promoting growth and, in the case of dormant bacteria, triggering resuscitation (Dworkin and Shah 2010). Peptidoglycan is constantly released in the environment when bacteria replicate (Dworkin and Shah 2010), and this polymer comprises a large fraction of the organic material found in aquatic environments (Dewi Puspita et al 2012). Peptidoglycan has the potential of activating bacterial spores and 28 non-spore forming dormant bacteria (Dewi Puspita et al 2012). Nikitushkin et al (2013) observed that peptidoglycan fragments obtained either by lysis with Rpfs or by sonication had stimulatory effects on the resuscitation of dormant Mycobacterium smegmatis and Mycobacterium tuberculosis. 29 30 Chapter 3.0. Objectives The present work proposes a method to increase the yield of the extraction and cultivation approaches for soil bacteria, employing growth promoting factors. These include purified supernatants from the liquid culture of the bacterium Micrococcus luteus, which contains the Rpf protein, and cell wall fragments from the same bacterium. The main goal is to ‘resuscitate’, or promote the culturability of VBNC bacteria from soil during the soil extraction step employing these growth factors, and then culture these bacteria on low nutrient solid media. The results will be analyzed in terms of (1) differences in counts of total colony forming units (CFU) between the extraction with versus without purified supernatant and cell wall fragments; and (2) the difference in the microbial diversity observed in both approaches. The hypothesis is that difficult-to-culture soil bacteria will be isolated with this approach, with some being cultured for the first time. Prior to the extraction experiments, the lytic activity of the M. luteus supernatant will be proved. The presence of Rpf in the purified supernatant of M. luteus will also be addressed. 31 32 Chapter 4.0. Materials and Methods 4.1 Materials 4.1.1 Media and buffers 1. Lactate minimal medium (LMM), prepared according to Kaprelyants and Kell (1992): 4.0 g l-1 NH4Cl; 1.4 g l-1 KH2PO4; 0.005 g l-1 biotin*; 0.02 g l-1 L-methionin*; 0.04 g l1 thiamin*; 1.0 g l-1 inosin*; 0.07 g l-1 MgSO4*; 0.000024 g l-1 CuSO4*; 0.0005 g l-1 MnCl2*; 0.001 g l-1 FeSO4*; 0.000025 g l-1 Na2MoO4*; 0.0005 g l-1 ZnSO4*; 10 g l-1 lithium lactate. The pH was adjusted to 7.5 and the medium was autoclaved. 2. PCA: Tryptone glucose yeast agar (17.50 g l-1) was mixed with 7 g l-1 agar bacteriological (agar no. 1) and the medium was autoclaved. 3. Half-strength LB broth: 12.50 g l-1 LB, autoclaved. 4. Sodium pyruvate (0.5%) mineral medium: 10.955 g l-1 Na2HPO4·12H2O; 2.7 g l-1 KH2PO4; 1.0 g l-1 (NH4)2SO4; 0.03 g l-1 Ca(NO3)2*; 0.2 g l-1 MgSO4*; 0.01 g l-1 FeSO4*; 5.0 g l-1 sodium pyruvate . The pH was adjusted to 7.5, and the medium was autoclaved. 5. Modified R2 broth: 0.50 g l-1 casein acid hydrolysate; 0.50 g l-1 yeast extract; 0.50 g l-1 special peptone; 0.50 g l-1 starch; 0.03 g l-1 K2HPO4; 0.024 g l-1 MgSO4; 0.30 g l-1 sodium pyruvate. 15 g l-1 agar bacteriological (agar no.1) were used for the modified R2 agar. The pH was adjusted to 7 and the medium was autoclaved 6. Phosphate buffer saline (PBS)*: 8.0 g l-1 NaCl; 0.2 g l-1 KCl; 1.42 g l-1 Na2HPO4; 0.24 g l-1 KH2PO4. 7. Physiological solution: 0.85% NaCl, autoclaved. Marked (*) components were filter-sterilized (0.22 m) and added to media after autoclaving. 4.1.2 Soil The soil used for the extraction experiments was retrieved from Prague-Suchdol, Czech Republic. It is a chernozem soil with a clay-loamy texture (Casova et al 2009). 33 4.2 Methods 4.2.1 Bacterial Cultures Micrococcus luteus NCTC2665 (“Fleming strain”) was grown in 200 ml LMM at 28°C and 120 rpm. Its growth curve was determined during 250 hours by absorbance at 600 nm using an IMPLEN Nanophotometer® spectrophotometer, model P300. The purity of the M. luteus liquid cultures was assessed by plating on PCA at 28°C. Colonies formed were analyzed by MALDI-TOF MS (4.2.9). Escherichia coli was grown overnight in 150 ml half-strength LB broth at 200 rpm and 28°C. The culture was autoclaved to use it in the supernatant activity determination experiments (4.2.4). 4.2.2 Supernatant retrieval M. luteus cultures in LMM at different growth stages were centrifuged at 5000 × g for 10 minutes, after which the pellet was collected to prepare cell walls (4.2.3). The supernatant was sterilized using a 0.22 m filter and further used for its activity determination. As activity control in all the experiments, a supernatant aliquot boiled for 10 minutes was used. 4.2.3 Micrococcus luteus cell wall preparation M. luteus cell wall for the supernatant activity determination was prepared according to the procedure described by Mukamolova et al (2006): collected M. luteus cells (exponential phase) after centrifugation (10 min, 5000 × g, described in 4.2.2) were washed with deionized water, and then resuspended in 40 ml SDS (5% w/v). This was boiled for 20 min and centrifuged again. The pellet was resuspended in 20 ml SDS (4% w/v), and boiled for another 20 min. In order to remove the SDS, the pellet was washed six times with hot water (60°C). The remaining solids were washed with 20 ml acetone, centrifuged, air-dried, and stored at 20°C until they were used. For the determination of Rpf lytic activity towards the cell walls, 1.47 g of prepared cell wall were suspended in 40 ml LMM, and diluted 8 times (in LMM) to get a final cell wall concentration below 5 mg ml-1. 34 4.2.4 Supernatant activity determination by absorbance Both the supernatant and the boiled aliquot (control) were mixed in the wells of a microtiter plate (0,25 ml total volume in each well) with the diluted cell wall preparation (target 1) and the autoclaved E. coli suspension (target 2), in the following ratios (supernatant or boiled supernatant: target 1 or 2): 1:20, 1:15, 1:10, 1:5, 1:2, 3:1, 4:1, 5:1. These ratios were prepared in triplicates. For the ratio 3:1 onward, the only target used were the cell walls. Absorbance was also measured (in triplicates) for the E. coli suspension, the cell wall suspension (<5 mg ml-1 in LMM), as well as the supernatants (fresh and boiled), and the lactate medium. To measure the lytic activity towards the bacterial cell wall, a procedure similar to that reported by Telkov et al (2006) was performed. The change in absorbance (600 nm) after the mixtures was followed for 2 hours in 2 minutes intervals in an EON microplate spectrophotometer (Biotek®) at 28°C and 180 rpm. 4.2.5 Supernatant activity determination by fluorometry Supernatant activity was also determined using the fluorogenic substrate 4methylumbelliferyl- -D-N,N’,N’’-triacetylchitotrioside (MUF tri-NAG), as described by Mukamolova et al (2006). Fluorescence was measured with a Fluoroskan Ascent FL microplate fluorometer and luminometer with one dispenser (ThermoLabsystems), with an excitation wavelength of 355 nm. In each well, 250 l of supernatant were placed together with MUF tri-NAG in a concentration of 80 M. As a negative control for the supernatant activity, boiled supernatant (10 min) was used. As positive control, lysozyme from chicken egg white (1x105 units mg-1) was used (2.50 mg of lysozyme were used per well, dissolved in LMM). The fluorescence of the supernatant, the boiled supernatant and the lactate medium (background fluorescence) were also measured. Measurements were done in triplicates for 4 hours, at 28°C and 480 rpm. Supernatant activity was measured at different times during the M. luteus culture growth to determine the point where supernatant lytic activity was at its maximum: 18, 41, 64, 71, 88, 95, and 256 hours after inoculation in LMM (200 ml). 35 4.2.6 Supernatant purification After collecting the supernatant from the Micrococcus luteus culture (4.2.2) its high molecular weight content was concentrated through ultrafiltration using Millpore™ centrifugal filter units (10 kDa) and CENTRIPLUS® disposable centrifugal concentrators (3 kDa). For the filtration in the 10 kDa filters, 12 ml of sterile M. luteus supernatant were filtered at 4000 × g for 10 minutes. In the case of the 3 kDa filters, 13 ml of supernatant were filtered for 2,5 hours at 3000 × g to remove the excess lactate. This retentate was then washed with 5 ml LMM without lithium lactate and further concentrated for 2 more hours. The activity of the retentate and the filtrate in both filtrations was measured using the fluorogenic substrate MUF tri-NAG as previously described (4.2.5). Boiled retentate and lysozyme were used as negative and positive controls, respectively. The washed retentate resulting from the filtration with 3 kDa filters was used for the enhanced extraction from soil (4.2.8). The background fluorescence of the retentates and the filtrates was measured as previously described (4.2.5). 4.2.7 Rpf identification To identify the Rpf protein from the M. luteus culture, the supernatant was purified as previously described (4.2.6 in the 3 kDa filters), except that the retentate was washed with 5 ml distilled water to reduce its salt content. This purified supernatant (containing the high molecular weight molecules) was lyophilized for 24 hours. 2 mg of the lyophilized, purified supernatant were dissolved in 25 l final sample buffer (Laemmli 1970). SDS-PAGE gel electrophoresis was done according to Laemmli (1970) (modified). The protein fractions were excised from the gel and cut in small pieces (approximately 1 mm2). A piece of empty gel was also excised and used as a blank. The gel pieces were placed in Eppendorf tubes. The pieces were then washed for 5 minutes with water and with 0.1 M NH4HCO3/acetonitrile 1:1 solution three times for 5 minutes. The solution was discarded and pieces were washed with acetonitrile three times for 5 minutes until they became white. The acetonitrile was discarded and the pieces were incubated for 45 min at 56 ºC in 10 mM dithiotreitol (DTT) solution in 0,1 M NH4HCO3. DTT solution was discarded, and freshly prepared 55 mM solution iodacetamide (IAA) IN 0.1 M NH4HCO3 was added to the gel pieces for 30 minutes. After the IAA treatment, gel pieces were washed again with 0.1 36 M NH4HCO3/acetonitrile 1:1 solution three times for 5 minutes and then with acetonitrile also three times for five minutes, until they became white. Trypsin solution (12.5 ng l-1 in 50 mM NH4HCO3) was added to the gel pieces and incubated in ice for 30 minutes. The trypsin solution that did not get into the gel was discarded, and the gel pieces were covered with 30 l of 50 mM NH4HCO3 solution and incubated overnight at 37 ºC. The next day, the gel pieces were spinned and sonicated to extract the peptides. The aliquots extracted from the gels were purified using ZipTip pipette tips. The peptides were analyzed by LC-ESI-Q-TOF mass spectrometry (4.2.10). 4.2.8 Soil extractions and cultivation For the soil extraction experiments, extraction buffer was mixed with soil in a 1:10 ratio (1.0 g of soil, 9.0 ml of extraction buffer) in falcon tubes. Two independent experiments consisting of 3 different extractions were performed. Each extraction was done in triplicate (biological replicates). In the first experiment, the 3 extractions were (1) extraction with PBS amended with purified supernatant, (2) extraction with PBS amended with M. luteus cell walls (obtained from 4.2.3), and (3) extraction with PBS alone (control). 1. Extraction with purified M. luteus supernatant: 1.67 ml of the supernatant after purification (4.2.6) was added to 3 falcon tubes (three biological replicates, 1 g of soil each) together with 7.33 ml of extraction buffer (PBS). The concentration of the high molecular weight fraction of this extraction buffer was slightly lower than the concentration in the original M. luteus culture supernatant due to the filtration process. 2. Extraction with M. luteus cell wall: prepared M. luteus cell wall (4.2.3) was dissolved in PBS (cell wall concentration: 100 g ml-1). 1.67 ml of LMM without lithium lactate and 7.33 ml of PBS with dissolved cell wall were added to the falcon tube (1 g of soil). 3. Control extraction: 1.67 ml LMM without lithium lactate, and 7.33 ml PBS. LMM without lithium lactate was included in the cell wall-amended extraction (2) and in the control (3) to discard any effect of the salts in the bacteria resuscitation. 37 In a second extraction experiment, the effect of the boiled supernatant was tested. After purification of the supernatant (4.2.6), half of the supernatant volume was boiled for 10 min. The same ratio of soil to extraction buffer was used: 1. Extraction with purified M. luteus supernatant: extraction with 2.91 ml of purified supernatant, 6.09 ml of PBS. 2. Extraction with boiled, purified M. luteus supernatant: 2.91 ml boiled, purified supernatant, and 6.09 ml of PBS. 3. Control extraction: 9 ml PBS. In all the cases, the extraction proceeded at 120 rpm and 28°C. Three extraction times were evaluated: 4, 24 and 48 hours in the first experiment, 2, 24 and 48 hours in the second. The dilutions used for the total viable counts were 103, 104, and 105 in physiological solution. There were no technical replicates for each dilution. Total viable counts of the 3 biological replicates of each extraction (for both experiments three times) were done between 24-48 hours of cultivation on modified R2 agar medium. 4.2.9 Diversity screening The diversity screening was performed only for the first extraction experiment described in the previous section (extraction purified supernatant, cell wall and control). All the colonies which grew at the highest dilution plates (that is the 105 dilution, 3 plates after 4 hours, 3 plates after 24 hours, and 3 plates after 48 hours, 9 plates in total) for each treatment were recollected and inoculated in 96-well microtiter plates in modified R2 broth. For the purified supernatant treatment, only colonies in 8 out of the 9 plates were collected, since recollection of single colonies in the last plate was not possible. In total, 763 colonies from the 105 dilution plates were collected in microtiter plates, which were cultivated at 28 °C and 120 rpm. All the bacteria were further re-inoculated on modified R2 agar. The analysis of the 763 isolates was performed by Autoflex speed MALDI-TOF mass spectrometer (Bruker Daltonik GmbH, Germany). Bacterial samples were prepared from the fresh R2 agar cultures using direct transfer procedure and mixed with HCCA matrix, both according to the manufacturer's instructions (Bruker Daltonik GmbH, Germany). Spectra were measured automatically by the Real Time Classification software (Bruker Daltonik GmbH, Germany). To identify the microorganisms the spectra were processed and analyzed in Biotyper software 38 version 2.0 (Bruker Daltonik GmbH, Germany) by the MALDI Biotyper standard preprocessing method and MALDI Biotyper standard identification method. Furthermore, to determine richness of the isolated microorganisms in different treatments, cluster analysis of the raw spectra was carried out. Spectral processing and clustering were done in R project following an in-house R script with the use of MALDIquant and MALDIforeign packages (Gibb and Strimmer 2012). In short, the procedure was: (1) m/z values were trimmed to the range of 3000 – 15000 Da; (2) intensities were square root transformed and (3) smoothed by Savitzky-Golay method; (4) baseline correction was done by SNIP algorithm; and (4) finally data were normalized by the Total Ion Current method. Peaks in all spectra were detected and transformed into a sample-feature matrix. The Pearson product-moment correlation coefficient was then computed for all pairs of samples. The distance matrix was constructed by subtracting absolute values of correlation coefficients from 1. Hierarchal clustering with average linkage was conducted and clusters or Operation Taxonomic Units (OTU) were created by applying cut-off distance of 0.2. If two replicates of the same sample resulted in different clusters, the OTUs were merged together. 4.2.10 LC-ESI-Q-TOF mass spectrometry analyses Mass spectrometry measurements were carried out using UHPLC Dionex Ultimate3000 RSLC nano (Dionex, Germany) connected with mass spectrometer ESI-Q-TOF Maxis Impact (Bruker, Germany). Samples were dissolved in a 10 μl mixture of water : acetonitrile : formic acid (97:3:0.1%), and 3 μl were loaded on a trap column Acclaim PepMap 100 C18 (100 μm x 2 cm, particle size 5 μm, Dionex, Germany) with mobile phase A (0.1% formic acid in water) and flow rate of 5 μL/min for 5 min. The peptides were eluted from the trap column to the analytical column Acclaim PepMap RSLC C18 (75 μm x 150 mm, particle size 2 μm) by mobile phase B (0.1% formic acid in acetonitrile) using the following gradient: 0-5 min 3 % B, 5-35 min 5-35 % B, 37 min 90 % B, 37-50 90 %B, 51 min 3 % B, 51-60 min 3 % B. The flow rate during gradient separation was set to 0.3 μL/min. Peptides were eluted directly to the ESI source – Captive spray (Bruker Daltonics, Germany). Measurements were carried out in positive ion mode with precursor ion selection in the range of 400–1400 m/z; up to ten precursor ions were selected for fragmentation from each MS spectrum. Peak lists were extracted from raw data by Data Analysis version 4.1 (Bruker Daltonics, Germany). Proteins were identified using Mascot version 2.4.1 (Matrix Science, UK). 39 40 Chapter 5.0. Results 5.1 Micrococcus luteus growth curve The growth of M. luteus in LMM medium was monitored for 250 hours. Growth was measured by the increase in absorbance at 600 nm. Figure 1. M. luteus growth curve. The beginning of the stationary phase corresponds to an absorbance value of 11.40 (95.5 hours). The longer the M. luteus colonies stayed in PCA plates (before cultivation in LMM), the longer was the lag phase observed in the liquid culture. 5.2 Micrococcus luteus supernatant activity to lyse peptidoglycan The ability of the M. luteus culture supernatant to lyse peptidoglycan was determined by the absorbance (at 600 nm) change of a cell wall suspension in LMM in the presence of the bacterial supernatant (4.2.4). The supernatant was collected at the beginning of the stationary phase (figure 1). A decrease in the measured absorbance is caused by the lysis of cell wall fragments in the suspension (Telkov et al 2006). As a negative control, boiled supernatant was used. 41 A) B) Figure 2. Absorbance change for the ratios A) 1:20 and B) 1:5 (supernatant : cell wall suspension). Absorbances were measured in triplicates. Means (points) and standard deviations (bars) are shown. All the different ratios (4.2.4), exhibited an increase in absorbance over time (figure 2). This can be due to the sedimentation of the cell walls (section 6.1). Nevertheless, the higher the proportion of supernatant in the mixtures was, the lower were the absorbance values for the unboiled supernatant compared to the boiled supernatant (plot B, figure 2). The absorbance of the sole boiled and unboiled supernatants did not change in time. For both supernatants the mean absorbance value for the 61 measurements in time and for each triplicate was 0.094 (SD=0.001) for the unboiled supernatant and 0.095 (SD= 0.010) for the boiled one. Significant differences in the mean between the absorbances of the boiled and the unboiled supernatant/target mixtures were tested for each ratio and both targets, either M. luteus cell wall suspension or autoclaved E. coli suspension (for absorbance values after 120 minutes). Results are displayed in table 1. Significant differences between the mixtures with boiled and unboiled supernatant started to be observable for increased supernatant contents. Causes for non-significance and drawbacks from this experiment will be further analyzed in the Discussion chapter 6.1. Table 1. Results of the t-tests for independent samples, testing the difference in absorbance (600 nm) for the targets mixed either with boiled or unboiled supernatant after 120 minutes of reaction (figure 2). Target Ratio (Supernatant:Target) 1:20 1:15 Autoclaved E. coli suspension 1:10 1:5 1:2 1:20 1:15 1:10 Cell wall suspension 1:5 1:2 3:1 4:1 5:1 t-test for independent samples t-value = 0.9705 p-value=0.386756 t-value = -0.7372 p-value=0.501899 t-value = -1.9385 p-value=0.12459 t-value = -2.8235 p-value=0.04765 t-value = -3.0632 p-value=0.037542 t-value = -0.6256 p-value=0.565507 t-value = -0.1928 p-value=0.85647 t-value = -1.0067 p-value=0.37103 t-value = -3.4213 p-value=0.026748 t-value = -1.7801 p-value=0.149658 t-value = -13.795 p-value=0.00016 t-value = -1.7693 p-value=0.15156 t-value = -4.6663 p-value=0.009544 43 Mean differences for the targets with boiled/unboiled supernatant at p<0.05 No significant difference observed No significant difference observed No significant difference observed Unboiled supernatant has a significantly lower absorbance Unboiled supernatant has a significantly lower absorbance No significant difference observed No significant difference observed No significant difference observed Unboiled supernatant has a significantly lower absorbance No significant difference observed Unboiled supernatant has a significantly lower absorbance No significant difference observed Unboiled supernatant has a significantly lower absorbance 5.3 Micrococcus luteus supernatant activity against fluorogenic substrate The lytic activity of M. luteus supernatant was also determined by its ability to lyse fluorogenic glycanase substrate MUF tri-NAG (figure 3). In peptidoglycan, Rpf cleaves the glycosidic bond between N-acetylglucosamine and N-acetyl-muramic acid (Mukamolova et al 2006). In MUF tri-NAG, Rpf cleaves the -glycosidic bond between triacetylchitotriose and 4-methylumbelliferone (figure 3), which emits fluorescence when excited (Chen 1968). The excitation wavelength used in this experiment was 355 nm. Supernatant lytic activity was determined by an increase in fluorescence in time due to the increase of coumarin concentration in the medium (4-methylumbelliferone emission wavelength = 460nm). Figure 3. Molecule of 4-methylumbelliferyl- -D-N,N’,N’’-triacetylchitotrioside (MUF tri-NAG). In this state, the fluorescence of the molecule is quenched. Rpf cleaves the molecule, releasing the coumarin portion (4methylumbelliferone, top-right), which emits fluorescence when excited (maximum absorbance wavelength in 0.01 M NaOH is 360 nm (Chen 1968)). For the first activity determination by fluorescence, the supernatant was retrieved when M. luteus culture absorbance was 11.38 (early stationary phase, figure 1). The result of this experiment is depicted in figure 4. During the first 50 minutes of the experiment, background fluorescence (i.e., fluorescence of the supernatant without MUF tri-NAG substrate) dropped exponentially (data not shown). Since the background fluorescence was substracted from the recorded fluorescence (where the MUF tri-NAG lysis reaction took place), the effect of the exponential drop in the relative fluorescence can be seen in the first 50 minutes of the experiment (figure 4). The background fluorescence was in some cases higher than the recorded fluorescence, explaining the existance of negative relative fluorescence values. The background fluorescence stabilized after 50 minutes, following a linear trend. Figure 4. Fluorescence increases for MUF tri-NAG solution in the prescence of supernatant (●); boiled supernatant (●); chicken white egg lysozyme in LMM (positive control) (●). Each measurement was done in triplicates at 28 ºC. Points are the mean values and bars are standard deviations. Background fluorescence (from the supernatants and the lysozyme in LMM without fluorogenic substrate) was substracted from the recorded fluorescence. M. luteus supernatant is likely a very complex mixture, so it is difficult to ascertain the cause of this exponential drop in fluorescence, which masks the lytic activity in the first 50 minutes of the experiment. It must be noted that only few minutes after the supernatant retrieval (4.2.2), a color change in the sterile supernatant could be observed. The relative fluorescence trend after 50 minutes was analyzed with a simple linear regression to determine if there was a functional dependancy of the increase of relative fluorescence in time (H0: =0, no linear dependence of relative fluorescence on time; HA: 1≠1, linear dependence of relative fluorescence on time) for each curve (figure 4). The linear regression (dependence of relative fluorescence on the elapsed time) is highly significant at = 5% for the supernatant (Y = -0.3457 (±0.2468) + 0.01735 (±0.0014) X; r2 = 0.8836; F1,19 = 144.2; p < 2.56 × 10-10) and the chicken egg white lysozyme (Y = 4.1511 (±0.451) + 0.1353 (±0.0026) X; r2 = 0.993; F1,19 = 2627; p < 2.2 × 10-16); for the boiled supernatant, it is not significant (Y = 3.6830 (±1.0467) - 0.00244 (±0.00613) X; r2 = 0.0083; F1,19 = 0.1591; p= 0.6944). In order to determine the bacterial growth stage at which the supernatant had maximum lytic activity, M. luteus supernatant was retrieved at different times during its 45 growth: 18, 41, 64, 71, 88, 95, and 256 hours after inoculation in LMM (200 ml, figure 5). Since a linear model could be approximated to the supernatant fluorescence change over time after 50-minute reaction (r2 = 0.8836), the growth stage with maximum lytic activity was that with the highest increase of relative fluorescence per hour (highest slope). In this experiment, the fluorescence changes for the supernatant at different growth stages were measured from 50 minutes (initial time) to 240 minutes. Figure 5. Relative fluorescence per hour (●) at different growth stages (●) for the supernatant. Means and standard deviations are shown. Supernatant activity against MUF tri-NAG was higher during the exponential growth, and diminished starting the stationary phase. At late stages of the stationary phase (256 hours, absorbance = 11.375), the supernatant had no remarkable activity. The supernatant activity after purification (4.2.6) was also determined by fluorimetry. After ultrafiltration using both the 10 and 3 kDa filters, the lytic activity of the retentates (high molecular weight fractions) and of the filtrates was determined. The results of both filtrations are shown in figure 6. It can be observed in figure 6 A) that the boiling impaired the increase in fluorescence (supernatant lytic activity). Both the filtrate (<10 kDa) and the retentate (>10 kDa) fractions exhibited an increase in relative fluorescence in time. In the case of the 3 kDa filtration (figure 6 B), the retentate had activity, but the filtrate’s activity was reduced as compared with the activity of the filtrate obtained by the filtration with 10 kDa (figures 6 and 7). It must be noted that a fluorescence decay is reported for the coumarin 4-methylumbeliferone (figure 3) (Chen 1968). This fluorescence decay can be observed in figure 7 B). In this figure, production of 4methylumbelliferone is stopped in the 3 kDa filtrate (figure 7 B), while in the 10 kDa filtrate (figure 7 A) the fluorescence keeps increasing for the measured reaction time. 46 A) B) Figure 6. Fluorescence increase for retentate (●);boiled retentate (●);and filtrate (●). A) Filtration with 10 kDa filter; B) filtration with 3 kDa filter. Measurements done in triplicates (means and standard deviations shown). The determination of the lytic capacities of the retentate and filtrate is a necessary step for the extraction experiments (4.2.8). The presence of lithium lactate or the salts contained in the LMM (still likely present in the supernatant) can contribute to an increase in the number of CFUs observed on solid media, masking the effect of the growth factors secreted by M. luteus (like Rpf). Since the filtration with the 10 kDa filter still exhibited activity in its filtrate fraction, the 3 kDa filter was chosen for the supernatant purification to: (1) get rid of the excess lithium lactate and other low-molecular-weight compounds that could favor bacterial growth (and maybe resuscitation); and (2) to concentrate the growth factors in the retentate. B) 5 Relative fluorescnce unit Relative fluorescence unit A) 4 3 2 1 0 0 100 200 Time (minutes) 300 6 4 2 0 -2 -4 0 100 200 Time (minutes) 300 Figure 7. Fluorescence increases for filtrates (♦) after 50 minutes of reaction. A) Filtration with 10 kDa filter; B) filtration with 3 kDa filter. 47 5.4 Rpf detection The Rpf identification procedure (4.2.7) was carried out when M. luteus was in exponential phase (absorbance 8). Telkov et al (2006) reported that the size of secreted Rpf is 19 kDa. In the SDS-PAGE gel, two bands were observed around this size (figure 8, white arrow). The bands were excised from the gel (4.2.7) and its protein content was analyzed by LC-ESI-Q-TOF-MS (4.2.10). By the time of the publication of this work, the determination of the identity of the bands was still ongoing. Figure 8. SDS-PAGE gel of the lyophilized M. luteus supernatant. 5.5 Soil extractions and cultivation: quantitative analyses The first extraction experiment consisted of the extraction of soil bacteria from soil using three different buffers: 1) PBS and purified M. luteus supernatant (4.2.6), the “SRpfenhanced extraction”; 2) PBS and M. luteus cell wall suspension (4.2.3, 100 g ml-1), the “cell wall-enhanced extraction”; and 3) PBS alone, as a control. LMM without lithium lactate was added to both the cell wall-enhanced extraction and the control extraction in the same volume as the volume of purified supernatant added to the supernatant-enhanced extraction; in this way, any effect of the LMM salts in the experiment was excluded. The extractions proceeded for 4, 24 and 48 hours at 120 rpm shaking and 28 °C. Plating on modified R2 agar at different dilutions (103, 104, and 105) was performed. CFUs were counted in the 103 and 104 dilutions. Figure 10 shows the CFUs observed on R2 modified agar for the different extractions, extraction times, and dilutions. In the 105 dilution plates it can be seen that the increase in 48 CFUs due to the presence of SRpf was ca. one order of magnitude (figure 10 B). This increase can also be seen after 48 hours of extraction (figure 10 C). After 4 hours of extraction (figure 10 A), no relevant difference was observed between the control and the two enhanced extractions. In the case of the cell wall-enhanced extraction after 24 and 48 hours (figure 10 B and C, respectively), the CFU number increased in the dilution 104, but sharply decreased in the 105 dilution (even slightly lower than the PBS extraction). In figure 10 B) in the 104 dilution, only one of the 3 plates was counted (single point of the cell wall-enhanced extraction), because the other two were already overgrown. The second extraction experiment, where the effect of the boiled supernatant was tested, yielded a similar increase in the number of observed CFUs for the SRpf-enhanced compared to the control extraction. In this experiment, the extraction times were 2, 24 and 28 hours. The extraction attempt with the boiled supernatant suggests that boiling reduces the efficiency of the extraction. For the 105 dilution and after 24 hours of extraction, the SRpfenhanced extraction plates were already overgrown (+200 CFU), whereas for the boiled supernatant extraction the CFU number was 131 (±64.58). After 48 hours of extraction and for the same dilution, the supernatant-enhanced extraction yielded on average 163.67 CFU (±24.94), while the boiled supernatant extraction yielded on average 138 CFU (±37.27). In order to prove the differences to be statistically significant, the experiment will have to be repeated with more dilutions. Figure 9. Modified R2 agar plates for the SRpf-enhanced extraction (top three) and the PBS (control) extraction. The plates correspond to the 105 dilution and 24 hours extraction. 49 A) B) - C) Figure 10. CFUs observed on modified R2 agar after 4 (A), 24 (B) and 48 (C) hours of extraction for the PBS extraction (Ctrl), cell wall-enhanced extraction (Pg) and SRpf-enhanced extraction (Sup). Horizontal line at CFU = 200 expresses overgrowth. 5.6 Soil extractions and cultivation: qualitative analyses (microbial diversity) The CFUs from the 105 dilution from all the extraction times were inoculated for storage at 28 °C in microtiter plates containing R2 modified broth. For the control extraction and the cell wall-enhanced extraction, colonies were selected from all the 9 plates for the 105 dilution (three for each extraction time, three for each extraction, control or cell wallenhanced). For the supernatant-enhanced extraction only colonies from 8 plates were isolated 50 due to overgrowth in one plate. In total, 763 isolates (161 colonies from the cell wallenhanced extraction, 127 from the control, and 475 from the SRpf-enhanced extraction) were retrieved from the 26 plates. The isolates were then reinoculated on R2 modified agar to screen for their diversity using Autoflex speed MALDI-TOF mass spectrometry (4.2.9, each isolate analyzed in duplicate). From the 763 isolates, after the spectral processing and clustering (4.2.9), 241 operational taxonomical units (OTUs) were determined. The supernatant-enhanced extraction not only did increase the total number of CFUs observed, but also the richness in terms of different OTUs. The number of OTUs isolated with the different extraction approaches is shown in figure 11 (for more comprehensive results refer to the Appendix, OTU table). A total of 119 OTUs from the 241 identified OTUs across the three extractions were observed only in the SRpf-enhanced extraction; that is almost half of all the observed OTUs (49.38%). Diversity indices (Simpson’s index, Shannon’s entropy, total number of OTUs, and Chao1 estimator) (Chao 1984, Hill 1973) were obtained for each of the extraction methods (table 2). Figure 11. OTUs observed for the 3 different extractions: cell wall- and supernatant-enhanced extraction, and PBS extraction (control). Table 2. Diversity indexes for the different extraction approaches SRpf-enhanced Cell wall-enhanced Control 0.9655 0.9710 0.9511 Shannon’s entropy 4.2048 4.0208 3.6158 Total OTU number 161 88 64 Chao1 Estimator 255 161 103 Simpson’s index of diversity 51 All the treatments shared some common OTUs which clustered to taxa with the highest number of CFUs observed. In the SRpf-enhanced extraction, the CFU number of these common OTUs increased. The bacterial community observed in the SRpf-enhanced extraction was highly uneven, with some of the SRpf-exclusive OTUs having only one CFU, thus being “rare”. The richness increase observed for the SRpf-enhanced extraction can also be observed OTU Richness in figure 12. Figure 12. Rarefaction curves for the cell wall-enhanced (Pg), SRpf-enhanced (Sup) and PBS (ctrl) extractions. 52 Chapter 6.0. Discussion 6.1 Supernatant activity As reported by Telkov et al (2006) and Mukamolova et al (2006), M. luteus supernatant has lytic activity towards its cell wall suspension and the substrate MUF tri-NAG. The lytic activity against the cell wall suspension and the autoclaved E. coli suspension is more apparent when the supernatant fraction increases (table 1). Absorbance (600 nm) is significantly lower for the supernatant/cell wall or E. coli suspension mixtures with higher supernatant content, as compared against the mixtures with boiled supernatant (table 1). Boiling does not change supernatant’s absorbance, but it seems to impair its lytic properties (figure 2, table 1). The enzyme in the supernatant responsible for its lytic activity, most likely Rpf (Mukamolova et al 2006, Telkov et al 2006), is denaturated by the supernatant boiling. The increase in absorbance observed in figure 2 is a trend that is present in all the ratios; it is relatively faster in the beginning, and then it tends to stabilize at the end of the measurements. This increase can be the result of the cell wall sedimentation. The cell wall suspension sedimentedin the wells of the microtiter plate several hours after the beginning of the experiments (4.2.4). The fact that the cell wall preparation (4.2.3) resulted in a suspension rather than a solution decreases the reliability of the experiment and increases variability in the absorbance (figure 2). This increased variability can be the cause of non-significance between supernatant and boiled supernatant mixtures observed even at some highly diluted cell wall concentrations (table 1). Telkov et al (2006) determined the ability of Rpf to lyse M. luteus cell walls. They observed a decrease in absorbance at 600 nm, but the absorbance of their control also decreased. They also saw lysis zones in polyacrylamide gels containing cell walls from M. luteus (Telkov et al 2006). In the case of E. coli, when it expresses the rpf gene, Rpf accumulates in its periplasm and causes cell lysis (Mukamolova et al 2006). In this work, a decreased absorbance for the E. coli suspension mixed with supernatant as compared with the boiled supernatant mixture was also observed (table 1). To better determine the lytic activity of the supernatant, its ability to lyse MUF triNAG was measured. Fluorescence increased in MUF tri-NAG solutions in the presence of chicken egg white lysozyme (positive control) and M. luteus supernatant, whereas in the presence of boiled supernatant it did not (figure 4). The fluorescence increase in the presence of supernatant was smaller compared to that of the positive control (figure 4), but reported specific activity of M. luteus Rpf is 50-fold less than that for chicken egg white lysozyme 53 (Mukamolova et al 2006). Boiling seems again to impair the lytic activity of the supernatant (figures 4 and 6). Telkov et al (2006) determined the dynamics of Rpf accumulation in M. luteus culture medium. They observed that Rpf accumulation increases after the onset of the exponential phase, reaches a maximum at the late exponential phase, and decreases dramatically before the start of the stationary phase (Telkov et al 2006). Results of this work were similar to those observed for the supernatant activity at different growth stages of M. luteus (figure 5). Activity increased markedly at the onset of the exponential phase, and decreased once the culture entered the stationary phase. Since the observed activity trend along the M. luteus growth stages (figure 5) matched the reported Rpf accumulation in liquid medium (Telkov et al 2006), it is very likely that Rpf was responsible for the lytic activity of the supernatant against MUF tri-NAG. 6.2 Enhanced extraction experiments The medium chosen for culturing bacteria from the enhanced extraction experiments, the modified R2 agar, has three important features: (1) it contains a wide variety of compounds, to allow the growth of a broad range of soil microorganisms; (2) it has a low nutrient concentration to resemble rather oligotrophic conditions in the environment, thus enabling the growth of oligotrophs; and (3) it contains sodium pyruvate, whose presence can reduce the formation of oxidizing agents such as H2O2 (Bogosian et al 2000) and thus enable also the growth of injured bacteria present in the soil. Compared to the CFU numbers observed in the extraction with PBS (control), the SRpf-enhanced extraction increased the number of colonies observed on solid media about one order of magnitude (figures 9 and 10). This increase must have been promoted by the presence of a M. luteus growth factor, most likely Rpf (Mukamolova et al 1998), not by any low molecular metabolites present in the LMM medium after cultivation as the supernatant was deprived of its low molecular weight fraction, and the LMM salts were included in the control and the cell-wall-enhanced extraction. In the case of the cell wall-enhanced extraction, a slight increase of CFU numbers on media was observed compared with the control extraction. Nevertheless, the cell wallenhanced extraction approach needs to be improved by testing the effect of different cell wall concentrations on the extraction efficiency. In a Mycobacterium tuberculosis and 54 Mycobacterium smegmatis resuscitation experiments with peptidoglycan fragments from their cell walls, Nikitushkin et al (2013) determined a peptidoglycan concentration at which the resuscitation of these bacteria was maximum (0.1 g ml-1). Reducing the concentration 5 times or increasing it 2 times decreased the number of resuscitated cells. A reduction in the CFU number observed for the boiled supernatant extraction compared to the SRpf-enhanced extraction indicated that the effectiveness of M. luteus growth factors to promote cell division, at least for some bacteria, was linked to its enzymatic activity. This favors the resuscitation model (section 2.3.2) in which M. luteus Rpf hydrolyzes bacterial cell wall and, in consequence, triggers resuscitation (Pinto et al 2015). Boiling the supernatant reduced its lytic activity against the cell wall suspensions and the MUF tri-NAG substrate (sections 5.2 and 5.3), so it can also reduce its ability to hydrolyze the bacterial cell wall of dormant cells, reducing the number of resuscitated CFUs observed on solid medium (Telkov et al 2006). Nevertheless, not all bacteria must have benefited from the presence of supernatant in the extraction approach in the same way. Although the presence of M. luteus supernatant enhanced the growth of CFU on medium (figures 9 and 10), more research is needed to correctly ascertain how M. luteus growth factors promote resuscitation and bacterial growth. The diversity screening was necessary to prove that the enhanced extractions improved the efficiency of cultivation in general. Based on the OTUs which were seen only in the supernatant-enhanced extraction (figure 11), the total OTU numbers for each extraction and the diversity indices (table 2, figure 12), it is clear that a higher diversity was observed in the enhanced extractions compared to the control. Importantly, the enhanced extraction increased the number of different bacteria that appeared in culture. The Simpson’s diversity index differences among extractions are very small. The Simpson’s diversity index was higher for the cell wall-enhanced extraction than for the SRpfenhanced extraction. The Simpson index (which is subtracted from 1 to get the Simpson index of diversity) gives more weight to the more plentiful species (or OTUs) in the samples (Hill 1973). The success of the extraction to bring difficult-to-culture bacteria to culture must be evaluated in terms of the increased number of “rarities”, rather than the enrichment of evidently predominant bacteria. In this sense, it is better to consider the Chao1 estimator to address the differences in the diversity among different extractions (Chao 1984). The SRpfenhanced extraction was thus the extraction with the highest observed diversity (table 2, figure 12). 55 The increased diversity for the SRpf-enhanced extraction as compared to the cell wallenhanced extraction also evidences an important opportunity area of the former enhanced extraction: the supernatant can be promoting the growth and detection of “rarities”, but also of otherwise predominant representatives. Under this scenario, predominant bacterial representatives could still outcompete slow-growers, and thus thwart an increase in observed diversity. Higher dilutions could be beneficial to increase the number of “rarities” and to avoid that copiotrophs overgrow oligotrophs. It is possible that the “rare cells”, most likely oligotrophs, exist in higher numbers in soil than the fast growers observed, but because of their slow-growing nature they are easily overgrown by the fast growers (Button et al 1993). Some OTUs appeared only in the PBS (control) extraction. It is possible that due to the lytic activity of Rpf, some actively growing bacteria can be lysed in the presence of supernatant (like E. coli when Rpf accumulates in its periplasm). Though unlikely, this can include also yet-uncultured representatives. It is also worth to question why bacteria which only appeared in the PBS extraction did not respond to the M. luteus growth factors or were inhibited by them. An example is OTU012 (appendix), which appeared several times in the control extraction, but did not appear either in the supernatant-enhanced or the cell wallenhanced extractions. The identity of all the OTUs found in this work will be subjected to identity based on the 16S rRNA gene. It is expected that some of these OTUs can result in new bacterial species, cultured in the laboratory for the first time. Although this work does not adjudge the lytic effect of supernatant to be a result of its Rpf content, and by the date of publication of this work its identification was pending (4.2.10), several hints suggest that this is the case. These hints include the reduced enzymatic activity of the supernatant after boiling (figures 2, 4 and 6) and the dynamics of supernatant lytic activity at different growth stages of M. luteus (figure 5). The enhanced extraction was also caused by a product of the bacterium M. luteus, and not by some medium component (section 5.5). In the SDS-PAGE gel, two bands were found which corresponded to the reported size of Rpf (section 5.4, figure 8). 56 Chapter 7.0. Conclusion Several strategies have been developed in recent years to bring yet-uncultured bacteria to culture. Notwithstanding the important efforts already made in the field, due to the amount of uncultured microorganisms, there is still a very long path for researchers to travel. Efforts are also directed to understand the nature of the VBNC state. The molecular mechanisms which trigger the entrance into this state are still blurry, and hopefully they will become clearer in forthcoming years. Two enhanced approaches for the isolation of difficult-to-culture bacteria were tested in this work. One of them, the SRpf-enhanced extraction, proved to increase the yield of cultivation both in number of CFU (figures 9 and 10) and diversity (figures 11 and 12, table 2). A lot of unique OTUs were cultured by enriching the extraction buffer with SRpf (figure 11), and some of these OTUs can be previously-uncultured soil microorganisms. As described by Telkov et al (2006), and as suggested by the reduced CFU numbers observed for the extraction with boiled supernatant, Rpf resuscitation potential, at least for some cells, is likely linked to its enzymatic activity. The diversity increase for the SRpf-enhanced extraction also suggests that SRpf is useful to increase the culturability of a wide range of different bacteria, both gram positives and gram negatives. Dormancy and unculturability may be more intrinsically related than what research has recognized in recent years. There is a need to address both issues more closely to each other. Novel media which truly resemble natural environments, as well as high throughput cultivation techniques have already been developed. In this work, bacteria extracted with the SRpf-enhanced buffer grew on R2 modified agar. These bacteria which grew on the laboratory medium may also have been present in the control extraction; although the medium was suitable for them, they did not grow. The combination of the resuscitation potential of SRpf, together with novel cultivation media which resemble natural environments and other cultivation approaches (2.2) can result powerful to culture bacteria which have resisted cultivation in the laboratory. The VBNC state must be explained as fast as novel laboratory media have been developed. If VBNC cells exhibit generalized physiological features, like their thickened cell wall (Signoretto et al 2000, Zengler et al 2002), then they can also share universal resuscitation approaches. Knowledge on the molecular background of the VBNC state can also uncover new strategies to culture bacteria more efficiently. Difficult-to-culture bacteria are like the 57 Briar-Rose, they might already be sleeping in majestic castles. Now the question is, how to give them the wake-up kiss? 58 Bibliography Alain K, Zbinden M, Le Bris N, Lesongeur F, Quérellou J, Gaill F, Cambon-Bonavita M-A (2004). Early steps in microbial colonization processes at deep-sea hydrothermal vents. Environmental Microbiology 6: 227-241. Alain K, Querellou J (2009). Cultivating the uncultured: limits, advances and future challenges. Extremophiles 13: 583-594. Almiron M, Link AJ, Furlong D, Kolter R (1992). A novel DNA-binding protein with regulatory and protective roles in starved Escherichia coli. Genes Dev 6: 2646-2654. Ammerman J, Azam F (1981). Dissolved cyclic adenosine monophosphate (cAMP) in the sea and uptake of cAMP by marine bacteria. Mar Ecol Prog Ser 5: 85-89. Anderson KL, Roberts C, Disz T, Vonstein V, Hwang K, Overbeek R, Olson PD, Projan SJ, Dunman PM (2006). Characterization of the Staphylococcus aureus heat shock, cold shock, stringent, and SOS responses and their effects on log-phase mRNA turnover. J Bacteriol 188: 6739-6756. Asakura H, Ishiwa A, Arakawa E, Makino S-i, Okada Y, Yamamoto S, Igimi S (2007). Gene expression profile of Vibrio cholerae in the cold stress-induced viable but nonculturable state. Environmental Microbiology 9: 869-879. Ayrapetyan M, Williams TC, Oliver JD (2015). Bridging the gap between viable but nonculturable and antibiotic persistent bacteria. Trends in Microbiology 23: 7-13. Bakken LR, Olsen RA (1987). The relationship between cell size and viability of soil bacteria. Microbial Ecology 13: 103-114. Bassler BL (1999). How bacteria talk to each other: regulation of gene expression by quorum sensing. Current Opinion in Microbiology 2: 582-587. Ben-Dov E, Kramarsky-Winter E, Kushmaro A (2009). An in situ method for cultivating microorganisms using a double encapsulation technique. FEMS Microbiology Ecology 68: 363-371. Bogosian G, Aardema ND, Bourneuf EV, Morris PJL, O'Neil JP (2000). Recovery of Hydrogen Peroxide-Sensitive Culturable Cells of Vibrio vulnificus Gives the Appearance of Resuscitation from a Viable but Nonculturable State. Journal of Bacteriology 182: 5070-5075. Bogosian G, Bourneuf EV (2001). A matter of bacterial life and death. EMBO reports 2: 770774. 59 Bollmann A, Lewis K, Epstein SS (2007). Incubation of Environmental Samples in a Diffusion Chamber Increases the Diversity of Recovered Isolates. Applied and Environmental Microbiology 73: 6386-6390. Bomar L, Maltz M, Colston S, Graf J (2011). Directed Culturing of Microorganisms Using Metatranscriptomics. mBio 2. Bovill RA, Mackey BM (1997). Resuscitation of ‘non-culturable’ cells from aged cultures of Campylobacter jejuni. Microbiology 143: 1575-1581. Button DK, Schut F, Quang P, Martin R, Robertson BR (1993). Viability and Isolation of Marine Bacteria by Dilution Culture: Theory, Procedures, and Initial Results. Applied and Environmental Microbiology 59: 881-891. Casova K, Cerny J, Szakova J, Balik J, Tlustos P (2009). Cadmium balance in soils under different fertilization managements including sewage sludge application. Plant Soil Environ 55: 353-361. Cohen-Gonsaud M, Keep NH, Davies AP, Ward J, Henderson B, Labesse G (2004). Resuscitation-promoting factors possess a lysozyme-like domain. Trends in Biochemical Sciences 29: 7-10. Cole JJ (1999). Aquatic Microbiology for Ecosystem Scientists: New and Recycled Paradigms in Ecological Microbiology. Ecosystems 2: 215-225. Colwell RR (2000). Viable but nonculturable bacteria: a survival strategy. Journal of Infection and Chemotherapy 6: 121-125. Chao A (1984). Nonparametric Estimation of the Number of Classes in a Population. Scandinavian Journal of Statistics 11: 265-270. Chen RF (1968). Fluorescent pH Indicator. Spectral Changes of 4-Methylumbelliferone. Analytical Letters 1: 423-428. Choi JW, Sherr EB, Sherr BF (1996). Relation between presence-absence of a visible nucleoid and metabolic activity in bacterioplankton cells. Limnology and Oceanography 41: 1161-1168. D'Onofrio A, Crawford JM, Stewart EJ, Witt K, Gavrish E, Epstein S, Clardy J, Lewis K (2010). Siderophores from Neighboring Organisms Promote the Growth of Uncultured Bacteria. Chemistry & Biology 17: 254-264. Davis KER, Joseph SJ, Janssen PH (2005). Effects of Growth Medium, Inoculum Size, and Incubation Time on Culturability and Isolation of Soil Bacteria. Applied and Environmental Microbiology 71: 826-834. 60 Denich TJ, Beaudette LA, Lee H, Trevors JT (2003). Effect of selected environmental and physico-chemical factors on bacterial cytoplasmic membranes. Journal of Microbiological Methods 52: 149-182. Dewi Puspita I, Kamagata Y, Tanaka M, Asano K, Nakatsu CH (2012). Are Uncultivated Bacteria Really Uncultivable? Microbes and Environments 27: 356-366. Dukan S, Nyström T (1999). Oxidative Stress Defense and Deterioration of GrowtharrestedEscherichia coli Cells. Journal of Biological Chemistry 274: 26027-26032. Durbin RD (1961). Techniques for the observation and isolation of soil microorganisms. The Botanical Review 27: 522-560. Dworkin J, Shah IM (2010). Exit from dormancy in microbial organisms. Nat Rev Micro 8: 890-896. Epstein S (2009). Microbial awakenings. Nature 457: 1083. Epstein S (2013). The phenomenon of microbial uncultivability. Current Opinion in Microbiology 16: 636-642. Ferrari BC, Winsley T, Gillings M, Binnerup S (2008). Cultivating previously uncultured soil bacteria using a soil substrate membrane system. Nat Protocols 3: 1261-1269. Gibb S, Strimmer K (2012). MALDIquant: a versatile R package for the analysis of mass spectrometry data. Bioinformatics 28: 2270-2271. Giovannoni S, Stingl U (2007). The importance of culturing bacterioplankton in the 'omics' age. Nat Rev Micro 5: 820-826. Gorshkov V, Petrova O, Gogoleva N, Gogolev Y (2010). Cell-to-cell communication in the populations of enterobacterium Erwinia carotovora ssp. atroseptica SCRI1043 during adaptation to stress conditions. FEMS Immunology & Medical Microbiology 59: 378385. Heim S, Del Mar Lleo M, Bonato B, Guzman CA, Canepari P (2002). The Viable but Nonculturable State and Starvation Are Different Stress Responses of Enterococcus faecalis, as Determined by Proteome Analysis. Journal of Bacteriology 184: 67396745. Hett EC, Chao MC, Steyn AJ, Fortune SM, Deng LL, Rubin EJ (2007). A partner for the resuscitation-promoting factors of Mycobacterium tuberculosis. Molecular Microbiology 66: 658-668. Hill MO (1973). Diversity and Evenness: A Unifying Notation and Its Consequences. Ecology 54: 427-432. 61 Ingham CJ, Sprenkels A, Bomer J, Molenaar D, van den Berg A, van Hylckama Vlieg JET, de Vos WM (2007). The micro-Petri dish, a million-well growth chip for the culture and high-throughput screening of microorganisms. Proceedings of the National Academy of Sciences 104: 18217-18222. Jones SE, Lennon JT (2010). Dormancy contributes to the maintenance of microbial diversity. Proceedings of the National Academy of Sciences 107: 5881-5886. Kaeberlein T, Lewis K, Epstein SS (2002). Isolating "Uncultivable" Microorganisms in Pure Culture in a Simulated Natural Environment. Science 296: 1127-1129. Kana BD, Mizrahi V (2010). Resuscitation-promoting factors as lytic enzymes for bacterial growth and signaling. FEMS Immunology & Medical Microbiology 58: 39-50. Kaprelyants AS, Kell DB (1992). Rapid assessment of bacterial viability and vitality by rhodamine 123 and flow cytometry. Journal of Applied Bacteriology 72: 410-422. Kaprelyants AS, Gottschal JC, Kell DB (1993). Dormancy in non-sporulating bacteria. FEMS Microbiology Letters 104: 271-286. Kaprelyants AS, Kell DB (1993). Dormancy in Stationary-Phase Cultures of Micrococcus luteus: Flow Cytometric Analysis of Starvation and Resuscitation. Applied and Environmental Microbiology 59: 3187-3196. Kaprelyants AS, Mukamolova GV, Kell DB (1994). Estimation of dormant Micrococcus luteus cells by penicillin lysis and by resuscitation in cell-free spent culture medium at high dilution. FEMS Microbiology Letters 115: 347-352. Kaprelyants AS, Mukamolova GV, Kormer SS, Weichart DH, Young M, Kell DB (1999). Intercellular signalling and the multiplication of prokaryotes: bacterial cytokines. Symp Soc Gen Microbiol 57: 33-69. Keep NH, Ward JM, Robertson G, Cohen-Gonsaud M, Henderson B (2006). Bacterial resuscitation factors: revival of viable but non-culturable bacteria. Cellular and Molecular Life Sciences CMLS 63: 2555-2559. Kell DB, Kaprelyants AS, Weichart DH, Harwood CR, Barer MR (1998). Viability and activity in readily culturable bacteria: a review and discussion of the practical issues. Antonie van Leeuwenhoek 73: 169-187. Kell DB, Young M (2000). Bacterial dormancy and culturability: the role of autocrine growth factors. Curr Opin Microbiol 3: 238-243. Kikuchi Y (2009). Endosymbiotic Bacteria in Insects: Their Diversity and Culturability. Microbes and Environments 24: 195-204. 62 Koch AL (1971). The Adaptive Responses of Escherichia coli to a Feast and Famine Existence. In: Rose AH, Wilkinson JF (eds). Advances in Microbial Physiology. Academic Press. pp 147-217. Kogure K, Simidu U, Taga N (1979). A tentative direct microscopic method for counting living marine bacteria. Canadian Journal of Microbiology 25: 415-420. Koltunov V, Greenblatt CL, Goncharenko AV, Demina GR, Klein BY, Young M, Kaprelyants AS (2010). Structural Changes and Cellular Localization of Resuscitation-Promoting Factor in Environmental Isolates of Micrococcus luteus. Microbial Ecology 59: 296-310. Laemmli UK (1970). Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4. Nature 227: 680-685. Lai C-J, Chen S-Y, Lin IH, Chang C-H, Wong H-c (2009). Change of protein profiles in the induction of the viable but nonculturable state of Vibrio parahaemolyticus. International Journal of Food Microbiology 135: 118-124. Lam H, Oh D-C, Cava F, Takacs CN, Clardy J, de Pedro MA, Waldor MK (2009). D-Amino Acids Govern Stationary Phase Cell Wall Remodeling in Bacteria. Science 325: 15521555. Lewis K (2007). Persister cells, dormancy and infectious disease. Nat Rev Micro 5: 48-56. Lewis K (2010). Persister Cells. Annual Review of Microbiology 64: 357-372. Li L, Mendis N, Trigui H, Oliver JD, Faucher SP (2014). The importance of the viable but non-culturable state in human bacterial pathogens. Front Microbiol 5: 258. Marouga R, Kjelleberg S (1996). Synthesis of immediate upshift (Iup) proteins during recovery of marine Vibrio sp. strain S14 subjected to long-term carbon starvation. Journal of Bacteriology 178: 817-822. Martinez A, Kolter R (1997). Protection of DNA during oxidative stress by the nonspecific DNA-binding protein Dps. Journal of Bacteriology 179: 5188-5194. Morita RY (1988). Bioavailability of energy and its relationship to growth and starvation survival in nature. Canadian Journal of Microbiology 34: 436-441. Morita RY (1993). Bioavailability of Energy and the Starvation State. In: Kjelleberg S (ed). Starvation in Bacteria. Springer US: Boston, MA. pp 1-23. Muela A, Seco C, Camafeita E, Arana I, Orruño M, López JA, Barcina I (2008). Changes in Escherichia coli outer membrane subproteome under environmental conditions inducing the viable but nonculturable state. FEMS Microbiology Ecology 64: 28-36. 63 Mukamolova GV, Kaprelyants AS, Young DI, Young M, Kell DB (1998). A bacterial cytokine. Proceedings of the National Academy of Sciences 95: 8916-8921. Mukamolova GV, Turapov OA, Kazarian K, Telkov M, Kaprelyants AS, Kell DB, Young M (2002). The rpf gene of Micrococcus luteus encodes an essential secreted growth factor. Molecular Microbiology 46: 611-621. Mukamolova GV, Kaprelyants AS, Kell DB, Young M (2003). Adoption of the transiently non-culturable state — a bacterial survival strategy? Advances in Microbial Physiology. Academic Press. pp 65-129. Mukamolova GV, Murzin AG, Salina EG, Demina GR, Kell DB, Kaprelyants AS, Young M (2006). Muralytic activity of Micrococcus luteus Rpf and its relationship to physiological activity in promoting bacterial growth and resuscitation. Molecular Microbiology 59: 84-98. Nichols D (2007). Cultivation gives context to the microbial ecologist. FEMS Microbiology Ecology 60: 351-357. Nichols D, Lewis K, Orjala J, Mo S, Ortenberg R, O'Connor P, Zhao C, Vouros P, Kaeberlein T, Epstein SS (2008). Short Peptide Induces an “Uncultivable” Microorganism To Grow In Vitro. Applied and Environmental Microbiology 74: 4889-4897. Nikitushkin VD, Demina GR, Shleeva MO, Kaprelyants AS (2013). Peptidoglycan fragments stimulate resuscitation of “non-culturable” mycobacteria. Antonie van Leeuwenhoek 103: 37-46. Nilsson H-O, Blom J, Al-Soud WA, Ljungh Å, Andersen LP, Wadström T (2002). Effect of Cold Starvation, Acid Stress, and Nutrients on Metabolic Activity of Helicobacter pylori. Applied and Environmental Microbiology 68: 11-19. Nyström T (2001). Not quite dead enough: on bacterial life, culturability, senescence, and death. Archives of Microbiology 176: 159-164. Nyström T (2003). Nonculturable bacteria: programmed survival forms or cells at death's door? BioEssays 25: 204-211. Oliver JD (2005). The viable but nonculturable state in bacteria. J Microbiol 43: 93-100. Pagnier I, Raoult D, La Scola B (2008). Isolation and identification of amoeba-resisting bacteria from water in human environment by using an Acanthamoeba polyphaga coculture procedure. Environmental Microbiology 10: 1135-1144. Pfeffer JM, Strating H, Weadge JT, Clarke AJ (2006). Peptidoglycan O Acetylation and Autolysin Profile of Enterococcus faecalis in the Viable but Nonculturable State. Journal of Bacteriology 188: 902-908. 64 Pham VHT, Kim J (2012). Cultivation of unculturable soil bacteria. Trends in Biotechnology 30: 475-484. Pinto D, Almeida V, Almeida Santos M, Chambel L (2011). Resuscitation of Escherichia coli VBNC cells depends on a variety of environmental or chemical stimuli. Journal of Applied Microbiology 110: 1601-1611. Pinto D, Santos MA, Chambel L (2015). Thirty years of viable but nonculturable state research: Unsolved molecular mechanisms. Critical Reviews in Microbiology 41: 6176. Postgate JR (1969). Chapter XVIII Viable counts and Viability. In: Norris JR, Ribbons DW (eds). Methods in Microbiology. Academic Press. pp 611-628. Rao SPS, Alonso S, Rand L, Dick T, Pethe K (2008). The protonmotive force is required for maintaining ATP homeostasis and viability of hypoxic, nonreplicating Mycobacterium tuberculosis. Proceedings of the National Academy of Sciences 105: 11945-11950. Ravagnani A, Finan CL, Young M (2005). A novel firmicute protein family related to the actinobacterial resuscitation-promoting factors by non-orthologous domain displacement. BMC Genomics 6: 1-14. Rickman L, Scott C, Hunt DM, Hutchinson T, Menéndez MC, Whalan R, Hinds J, Colston MJ, Green J, Buxton RS (2005). A member of the cAMP receptor protein family of transcription regulators in Mycobacterium tuberculosis is required for virulence in mice and controls transcription of the rpfA gene coding for a resuscitation promoting factor. Molecular Microbiology 56: 1274-1286. Rittershaus Emily SC, Baek S-H, Sassetti Christopher M (2013). The Normalcy of Dormancy: Common Themes in Microbial Quiescence. Cell Host & Microbe 13: 643651. Rollins DM, Colwell RR (1986). Viable but nonculturable stage of Campylobacter jejuni and its role in survival in the natural aquatic environment. Applied and Environmental Microbiology 52: 531-538. Rozen Y, LaRossa RA, Templeton LJ, Smulski DR, Belkin S (2002). Gene expression analysis of the response by Escherichia coli to seawater. Antonie van Leeuwenhoek 81: 15-25. Rustad TR, Minch KJ, Brabant W, Winkler JK, Reiss DJ, Baliga NS, Sherman DR (2012). Global analysis of mRNA stability in Mycobacterium tuberculosis. Nucleic Acids Research. 65 Sachidanandham R, Yew-Hoong Gin K (2008). A dormancy state in nonspore-forming bacteria. Applied Microbiology and Biotechnology 81: 927-941. Schultz JE, Latter GI, Matin A (1988). Differential regulation by cyclic AMP of starvation protein synthesis in Escherichia coli. Journal of Bacteriology 170: 3903-3909. Senoh M, Ghosh-Banerjee J, Ramamurthy T, Hamabata T, Kurakawa T, Takeda M, Colwell RR, Nair GB, Takeda Y (2010). Conversion of viable but nonculturable Vibrio cholerae to the culturable state by co-culture with eukaryotic cells. Microbiology and Immunology 54: 502-507. Shaikh AS, Tang YJ, Mukhopadhyay A, Martín HG, Gin J, Benke PI, Keasling JD (2010). Study of stationary phase metabolism via isotopomer analysis of amino acids from an isolated protein. Biotechnology Progress 26: 52-56. Signoretto C, del Mar Lleò M, Tafi MC, Canepari P (2000). Cell Wall Chemical Composition ofEnterococcus faecalis in the Viable but Nonculturable State. Applied and Environmental Microbiology 66: 1953-1959. Smith B, Oliver JD (2006). In Situ and In Vitro Gene Expression by Vibrio vulnificus during Entry into, Persistence within, and Resuscitation from the Viable but Nonculturable State. Applied and Environmental Microbiology 72: 1445-1451. Song J, Oh H-M, Cho J-C (2009). Improved culturability of SAR11 strains in dilution-toextinction culturing from the East Sea, West Pacific Ocean. FEMS Microbiology Letters 295: 141-147. Staley JT, Konopka A (1985). Measurement of in Situ Activities of Nonphotosynthetic Microorganisms in Aquatic and Terrestrial Habitats. Annual Review of Microbiology 39: 321-346. Stevenson BS, Eichorst SA, Wertz JT, Schmidt TM, Breznak JA (2004). New Strategies for Cultivation and Detection of Previously Uncultured Microbes. Applied and Environmental Microbiology 70: 4748-4755. Stevenson LH (1977). A case for bacterial dormancy in aquatic systems. Microbial Ecology 4: 127-133. Stewart EJ (2012). Growing Unculturable Bacteria. Journal of Bacteriology 194: 4151-4160. Stingl U, Cho J-C, Foo W, Vergin KL, Lanoil B, Giovannoni SJ (2008). Dilution-toExtinction Culturing of Psychrotolerant Planktonic Bacteria from Permanently Icecovered Lakes in the McMurdo Dry Valleys, Antarctica. Microbial Ecology 55: 395405. 66 Sturm A, Dworkin J (2015). Phenotypic Diversity as a Mechanism to Exit Cellular Dormancy. Current Biology 25: 2272-2277. Suzina NE, Mulyukin AL, Dmitriev VV, Nikolaev YA, Shorokhova AP, Bobkova YS, Barinova ES, Plakunov VK, El-Registan GI, Duda VI (2006). The structural bases of long-term anabiosis in non-spore-forming bacteria. Advances in Space Research 38: 1209-1219. Svenning MM, Wartiainen I, Hestnes AG, Binnerup SJ (2003). Isolation of methane oxidising bacteria from soil by use of a soil substrate membrane system. FEMS Microbiology Ecology 44: 347-354. Takayama K, Kjelleberg S (2000). The role of RNA stability during bacterial stress responses and starvation. Environmental Microbiology 2: 355-365. Telkov MV, Demina GR, Voloshin SA, Salina EG, Dudik TV, Stekhanova TN, Mukamolova GV, Kazaryan KA, Goncharenko AV, Young M, Kaprelyants AS (2006). Proteins of the Rpf (resuscitation promoting factor) family are peptidoglycan hydrolases. Biochemistry (Moscow) 71: 414-422. Trevors JT (2011). Viable but non-culturable (VBNC) bacteria: Gene expression in planktonic and biofilm cells. Journal of Microbiological Methods 86: 266-273. Tripp HJ, Kitner JB, Schwalbach MS, Dacey JWH, Wilhelm LJ, Giovannoni SJ (2008). SAR11 marine bacteria require exogenous reduced sulphur for growth. Nature 452: 741-744. Vartoukian SR, Palmer RM, Wade WG (2010). Strategies for culture of ‘unculturable’ bacteria. FEMS Microbiology Letters 309: 1-7. Ventura M, Canchaya C, Tauch A, Chandra G, Fitzgerald GF, Chater KF, van Sinderen D (2007). Genomics of Actinobacteria: Tracing the Evolutionary History of an Ancient Phylum. Microbiology and Molecular Biology Reviews 71: 495-548. Wai SN, Moriya T, Kondo K, Misumi H, Amako K (1996). Resuscitation of Vibrio cholerae O1 strain TSI-4 from a viable but nonculturable state by heat shock. FEMS Microbiology Letters 136: 187-191. Wang Y, Hammes F, Boon N, Chami M, Egli T (2009). Isolation and characterization of low nucleic acid (LNA)-content bacteria. ISME J 3: 889-902. Watve M, Shejval V, Sonawane C, Rahalkar M, Matapurkar A, Shouche Y, Patole M, Phadnis N, Champhenkar A, Damle K (2000). The ‘K’selected oligophilic bacteria: a key to uncultured diversity. Curr Sci 78: 1535-1542. 67 Xu H-S, Roberts N, Singleton FL, Attwell RW, Grimes DJ, Colwell RR (1982). Survival and viability of nonculturableEscherichia coli andVibrio cholerae in the estuarine and marine environment. Microbial Ecology 8: 313-323. Yasumoto-Hirose M, Nishijima M, Ngirchechol MK, Kanoh K, Shizuri Y, Miki W (2006). Isolation of Marine Bacteria by In Situ Culture on Media-Supplemented Polyurethane Foam. Marine Biotechnology 8: 227-237. Zengler K, Toledo G, Rappé M, Elkins J, Mathur EJ, Short JM, Keller M (2002). Cultivating the uncultured. Proceedings of the National Academy of Sciences 99: 15681-15686. Zhou X, Cegelski L (2012). Nutrient-Dependent Structural Changes in S. aureus Peptidoglycan Revealed by Solid-State NMR Spectroscopy. Biochemistry 51: 81438153. 68 Appendix. OTU Table 69 70 Appendix 1. First 100 OTU (total number 241). NA = not reliable identification. Cell Wall-, Supernatant-enhanced extractions. Control (PBS). The last 141 OTUs are mainly non-identified (NA) singletons within the Supernatant treatment (not shown). Pg Ctrl Sup Taxonomy OTU001 18 24 53 Bacillus pumilus;NA OTU002 14 16 34 NA;Paenibacillus azoreducens Arthrobacter pascens;NA;Arthrobacter polychromogenes;Arthrobacter aurescens;Arthrobacter oxydans; OTU003 10 0 63 Arthrobacter globiformis;Arthrobacter ureafaciens;Arthrobacter nicotinovorans;Pseudomonas syringae OTU004 6 0 16 NA;Bacillus simplex;Bacillus muralis OTU005 2 0 38 Arthrobacter polychromogenes;Arthrobacter oxydans;NA;Arthrobacter sulfonivorans OTU006 14 2 8 NA OTU007 1 3 12 NA OTU008 8 7 0 NA OTU009 2 0 12 Pseudomonas koreensis;Pseudomonas chlororaphis;Pseudomonas corrugate;Pseudomonas thivervalensis Arthrobacter pascens;Arthrobacter polychromogenes;NA;Arthrobacter oxydans; Arthrobacter aurescens; OTU010 5 6 10 Arthrobacter globiformis OTU011 5 4 5 NA Pseudomonas corrugata;Pseudomonas chlororaphis;Pseudomonas jessenii; Pseudomonas thivervalensis; OTU012 0 12 0 Pseudomonas koreensis OTU013 1 0 17 Arthrobacter polychromogenes;Arthrobacter oxydans;Arthrobacter pascens;NA OTU014 3 1 11 NA OTU015 0 0 12 NA;Arthrobacter aurescens;Arthrobacter nicotinovorans;Arthrobacter ureafaciens OTU016 5 0 9 NA OTU018 5 4 0 Bacillus drentensis;NA OTU019 0 1 8 Bacillus megaterium OTU020 5 0 8 NA OTU022 0 0 9 Arthrobacter globiformis;NA;Arthrobacter oxydans OTU024 3 0 4 Bacillus niacini;NA OTU026 3 2 1 NA;Bacillus megaterium OTU027 0 1 5 Arthrobacter aurescens;NA OTU029 0 0 6 Bacillus firmus OTU031 2 2 7 NA OTU032 OTU033 OTU035 OTU037 OTU038 OTU039 OTU040 OTU042 OTU043 OTU045 OTU046 OTU048 OTU049 OTU050 OTU051 OTU052 OTU054 OTU056 OTU057 OTU058 OTU059 OTU060 OTU061 OTU063 OTU064 OTU065 OTU067 OTU068 Pg Ctrl Sup Taxonomy 1 0 6 NA 0 0 6 NA;Paenibacillus macerans 2 3 0 Bacillus simplex;Bacillus muralis 0 6 0 NA;Arthrobacter pascens Pseudomonas chlororaphis;Pseudomonas corrugata;Pseudomonas umsongensis;Pseudomonas thivervalensis; Pseudomonas 0 0 5 kilonensis;Pseudomonas jessenii 0 0 5 Pseudomonas umsongensis;Pseudomonas kilonensis 0 0 5 Pseudomonas corrugata;Pseudomonas brassicacearum;Pseudomonas koreensis;Pseudomonas chlororaphis 3 1 0 Bacillus marisflavi 0 4 0 NA;Cupriavidus campinensis 1 1 2 NA 1 3 2 NA 6 1 1 NA 0 4 0 NA 2 0 2 NA;Bacillus indicus 1 0 3 Arthrobacter nicotinovorans;Arthrobacter aurescens;Arthrobacter ureafaciens 1 0 3 NA;Bacillus niacini Pseudomonas vancouverensis;Pseudomonas corrugata;Pseudomonas jessenii; 4 0 0 Pseudomonas thivervalensis 0 0 4 Arthrobacter polychromogenes;Arthrobacter oxydans;NA 0 0 4 Arthrobacter polychromogenes;Arthrobacter oxydans;NA 0 0 4 NA;Arthrobacter sulfonivorans;Arthrobacter oxydans 0 0 4 NA;Arthrobacter polychromogenes;Arthrobacter oxydans 0 0 4 NA;Arthrobacter oxydans 0 2 3 NA;Arthrobacter polychromogenes;Arthrobacter sulfonivorans 3 0 0 Cupriavidus campinensis 1 2 0 Arthrobacter histidinolovorans;Arthrobacter sulfonivorans;NA 1 0 2 NA 1 0 2 Bacillus indicus;Bacillus idriensis;NA 1 1 1 Arthrobacter polychromogenes;NA 72 OTU069 OTU070 OTU071 OTU072 OTU073 OTU074 OTU075 OTU077 OTU078 OTU079 OTU080 OTU081 OTU082 OTU083 OTU084 OTU085 OTU087 OTU088 OTU090 OTU091 OTU092 OTU093 OTU094 OTU095 OTU096 OTU097 OTU098 OTU100 Pg Ctrl Sup Taxonomy 1 2 0 NA 0 1 2 NA 1 0 2 NA 1 0 4 Arthrobacter sulfonivorans;Arthrobacter oxydans;Arthrobacter pascens 0 0 3 NA 0 0 3 NA;Variovorax paradoxus 0 0 3 NA 0 0 3 NA 1 0 3 Bacillus pumilus 3 2 0 NA 2 0 3 NA 2 0 0 NA 2 0 0 NA 1 1 0 Arthrobacter polychromogenes;Arthrobacter oxydans 1 1 0 Arthrobacter polychromogenes 1 1 0 Arthrobacter polychromogenes;Arthrobacter scleromae 1 1 0 Arthrobacter polychromogenes;Arthrobacter oxydans 1 1 2 Bacillus megaterium;NA 1 1 0 NA 2 0 0 NA 1 1 0 Arthrobacter oxydans;Arthrobacter polychromogenes;NA 1 1 0 NA 0 2 0 NA 0 2 0 NA 1 0 1 NA;Arthrobacter globiformis 0 0 2 Pseudomonas kilonensis 0 0 2 NA 0 0 2 NA 73