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Critical Reviews in Microbiology, 31:55–67, 2005 c Taylor & Francis Inc. Copyright ISSN: 1040-841X print / 1549-7828 online DOI: 10.1080/10408410590899228 Ecological and Agricultural Significance of Bacterial Polyhydroxyalkanoates Daniel Kadouri, Edouard Jurkevitch, and Yaacov Okon Department of Plant Pathology and Microbiology, and The Otto Warburg Minerva Center for Agricultural Biotechnology, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, Rehovot, Israel Susana Castro-Sowinski Department of Plant Pathology and Microbiology, and The Otto Warburg Minerva Center for Agricultural Biotechnology, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, Rehovot, Israel and Departamento de Bioquı́mica, Instituto Clemente Estable (IIBCE), Unidad Asociada de Bioquı́mica, Instituto de Biologı́a, Facultad de Ciencias, Universidad de la República. Av. Italia, 3318, 11600, Montevideo, Uruguay noates (PHAs), and are homopolymers or copolymers containing different alkyl groups at the β position (Anderson & Dawes 1990). PHAs are structurally simple macromolecules accumulated as discrete granules to levels as high as 90% of the cell dry weight and are generally believed to play a role as a sink for carbon and reducing equivalents when other nutrient supplies are limited, and when the population is not growing exponentially in batch culture (Senior & Dawes 1973; Williams & Peoples 1996; Madison & Huisman 1999). Alternatively, PHAs are accumulated when growing in a continuous culture at constant dilution rate under nitrogen limitation (Dawes 1986). These molecules exhibit material properties that are similar to those of some common plastics such as polypropylene (Byrom 1987). The many different PHAs that have been identified to date are primarily linear; head-to-tail polyesters composed of 3-hydroxy fatty acid monomers. In these polymers, the carboxyl group of one monomer forms an ester bond with the hydroxyl group of the neighboring monomer. The chemical diversity of PHAs is large, and includes short, medium, and long chain polymers, homoand heteropolymers, and many types of substituted groups. For a detailed review see Kim and Lenz (2001). These variations are the basis for the diversity of the PHA polymer family and for their vast array of potential applications (Byrom 1987; Williams & Peoples 1996; Madison & Huisman 1999). The industrial interest in PHAs as plastics products has been extensively reviewed and is not in the scope of this review (van der Walle et al. 2001). We will mainly focus on the ecological and agricultural significance of PHAs. The biosynthesis and degradation of PHA is a cyclic mechanism that has been earlier proposed in Azotobacter beijerinckii and Hydrogenomonas eutropha (Senior & Dawes 1973; Oeding & Schlegel 1993). Polyhydroxyalkanoates (PHAs) are a group of carbon and energy storage compounds that are accumulated during suboptimal growth by many bacteria, and intracellularly deposited in the form of inclusion bodies. Accumulation of PHAs is thought to be used by bacteria to increase survival and stress tolerance in changing environments, and in competitive settings where carbon and energy sources may be limited, such as those encountered in the soil and the rhizosphere. Understanding the role that PHAs play as internal storage polymers is of fundamental importance in microbial ecology, and holds great potential for the improvement of bacterial inoculants for plants and soils. This review summarizes the current knowledge on the ecological function of PHAs, and their strategic role as survival factors in microorganisms under varying environmental stress is emphasized. It also explores the phylogeny of the PHA cycle enzymes, PHA synthase, and PHA depolymerase, suggesting that PHA accumulation was earlier acquired and maintained during evolution, thus contributing to microbial survival in the environment. Keywords Polyhydroxyalkanoates (PHA); Poly-β-Hydroxybutyrate (PHB); Bacterial Survival; PHA Metabolism; PHA Synthase; PHA Depolymerase; Phylogeny 1. INTRODUCTION A wide variety of taxonomically different groups of microorganisms are known to produce intracellular energy and carbon storage compounds, generally described as polyhydroxyalka- Received 29 September 2004; accepted 13 October 2004. Address correspondence to Yaacov Okon, Department of Plant Pathology and Microbiology, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, P.O. Box 12, Rehovot 76100, Israel. E-mail: [email protected] 55 56 D. KADOURI ET AL. 1996; Jendrossek & Handrick 2002). In contrast, intracellular PHA depolymerases degrade intracellular PHA granules in the accumulating strain, in order to mobilize PHA in the absence of a suitable exogenous carbon source or energy source (Saegusa et al. 2001). Therefore, the former can be seen as “scavenging” enzymes, not necessarily found in PHA-accumulating organisms (e.g., fungi), while the latter are truly engaged in cellular PHA cycling. 2. FIG. 1. Key enzyme reactions of the anabolism and catabolism of PHA in Azospirillum brasilense (produces only PHB). The synthesis and utilization of PHAs are mediated by PHA synthases (PhaC), intracellular PHA depolymerases (PhaZ), phasins (PhaP), and regulators (PhaR). The PhaR regulator/PhaP phasin system plays an important role in promoting PHA production (York et al. 2001, 2002). PHA accumulation is a widely distributed prokaryotic phenotype. Since the identification and characterization of the first enzyme involved in PHA synthesis (For Azospirillum brasilense see Figure 1) more than 60 genes from a wide range of bacteria have been cloned or identified as putative PHA biosynthetic genes (see section on phylogeny). Within these enzymes, PhaC is the only one exclusively involved in PHA biosynthesis and it is responsible for the polymerization of β-hydroxyalkanoyl-CoA monomers into poly-βhydroxyalkanoate. Bacterial PHA synthases can be classified in three classes depending on the number of carbon atoms in the monomers (Rehm & Steinbüchel 1999): (1) Class I PhaCs, are also called short-chain-length (SCL) PHA synthases, are composed of one type of subunit, and preferentially use three to five carbon monomers as substrates; (2) Class II PhaCs, called medium-chain-length (MCL) PHA synthases, are also composed of one type of subunits and use monomers with 6 to 14 carbons as substrates; and, (3) Class III PhaCs that preferentially use three to five carbon-long substrates and are composed of two different subunits (Steinbüchel et al. 1992). The PHA degradation pathway as described in most bacteria, begins with the depolymerization of PHA to D-β-hydroxybutyrate monomers by PHA depolymerase (encoded by phaZ) (For Azospirillum brasilense see Figure 1). PHA degradation can whether proceed intracellularly or extracellularly (Jendrossek & Handrick 2002). Extracellular PHA depolymerases are secreted by many bacteria from very varied environments under aerobic and anaerobic conditions, for the utilization of PHAs left in the environment after the lysis of cells in which the compounds were accumulated (Tanio et al. 1982; Jensrossek et al. WITH A LITTLE HELP FROM MY PHA DURING ENVIRONMENTAL STRESS The function of PHAs as intracellular carbon storage compounds that can be mobilized and used when carbon acts as a limiting resource has been the subject of most of the research in assigning a role for these compounds (Macrae & Wilkinson 1958; Sierra & Gibbons 1962; Hippe 1967; Hippe & Schlegel 1967). Under certain circumstances, free-living bacterial cells with a higher content of PHA may survive longer than those with a lower PHA content, either because they are not subjected to some additional adverse factors, or because they can utilize their reserve material longer and more efficiently (Dawes & Senior 1973; Matin et al. 1979). It has been shown that PHA accumulation plays a major role in some bacteria that live in close association with eukaryotes. PHA accumulation was involved in promoting long-term survival under starvation conditions of the intracellular parasite of amoebae, Legionella pneumophila (James et al. 1999). Interestingly, PHA accumulation was promoted during bacterial growth under iron-limitation into the amoeba host which therefore may induce a PHA-rich phenotype, rendering the bacteria more fit for extracellular survival in low-nutrient environments. In rhizobia, bacteria that are characterized by a free-living and a symbiotic stage with a eukaryotic host, the impact of the bacteria-host relationship on PHA accumulation is diverse (see below). Soil is a heterogeneous, discontinuous, and structured environment with a high diversity of microhabitats in which conditions can change rapidly (Postma et al. 1989). Bacterial cells in soil face different stresses, such as low nutrient availability and detrimental physical, chemical, or biological factors, all fluctuating in time and space. To cope with this changing, often oligotrophic environment, soil bacteria have developed various survival strategies (van Elsas & van Overbeek 1992). It has been proposed that the accumulation and degradation of PHA is one such strategy by which bacteria can improve establishment, proliferation, and survival in competitive settings such as soil and rhizosphere (Okon & Itzigsohn 1992). Additionally, the conditions for bacterial PHA production can be met in soil when a high C:N ratio prevails, as is mostly the case in the rhizosphere, where it is estimated to be at about 20. Nitrogen availability may become a limiting factor for bacterial growth, especially in some nitrogen-poor sites. These conditions of suboptimal growth are conducive to the production of PHAs (Madison & Huisman 1999). SIGNIFICANCE OF BACTERIAL POLYHYDROXYALKANOATES Supporting data for PHA production in telluric environments was provided by Wang and Bakken (1998) who screened 63 soil bacteria samples for PHA production and assessed that PHA accumulation is an aid in the survival of starving soil bacteria. They found that soil bacteria within the pseudomonads, coryneform and bacilli produce PHA. In addition, Azospirillum and Azotobacter, as well as other rhizosphere bacteria where shown to produce PHA in culture under a C:N ratio of 20 (Itzigsohn et al. 1995). The free-living Gram-negative nitrogen-fixing rhizobacteria belonging to the genus Azospirillum are extensively used as plant growth promoting rhizobacteria (PGPR) (Okon & Vanderleyden 1997). These bacteria, which are of agronomical importance, are also models for understanding the physiology and ecology of inoculants, and for deciphering traits important for survival, colonization, and effectiveness. One such trait appears to be the secretion of plant-like growth promoting substances such as auxins, gibberellins, and cytokinins by the bacteria, leading to an increase in root surface area, and to an enhancement of water and mineral uptake (Okon & Kapulnik 1986; Fallik et al. 1994; Okon & Vanderleyden 1997; Burdman et al. 2000; Steenhoudt & Vanderleyden 2000). Production and accumulation of PHA in A. brasilense, which was examined by Tal and Okon (1985) and Tal et al. (1990a), may also be one such trait. Under appropriate conditions, A. brasilense cells may accumulate above 75% of their dry weight exclusively as polyβ-hydroxybutyrate (PHB) (Tal & Okon 1985; Tal et al. 1990a; Itzigsohn et al. 1995). It was shown that the C:N ratio in the medium, and the oxygen partial pressure are controlling factors for PHA production in Azospirillum (Nur et al. 1981; Paul et al. 1990; Tal et al. 1990b). Increased survival and respiration under starvation conditions were detected in A. brasilense containing higher amount of PHA (Tal & Okon 1985). In addition, A. brasilense mutants defective in their capability to produce PHA (phaC− ) (Kadouri et al. 2002) or to degrade PHA (phaZ − ) (Kadouri et al. 2003a) survived shorter than the wild-type strain. As with Ralstonia eutropha (Handrick et al. 2000), accumulated PHA in A. brasilense can also support cell multiplication in the absence of an exogenous carbon source (Kadouri et al. 2002). The term “survival” sensus stricto may therefore not precisely cover the impact of PHA because it may imply the arrest of multiplication. “Stress alleviation” may be more appropriate. In addition, PHA as a sole energy source was shown to support nitrogenase activity and aerotaxis, two physiological features that are extremely energy consuming (Tal & Okon 1985). The production of cell types, such as spore and cyst production is yet another survival strategy but some evidence has been provided that spore formation and germination may be linked with PHA biosynthesis and utilization. In Bacillus cereus cells that had accumulated PHA, the polymer disappeared after sporulation, while the degradation products were incorporated into the spore. In that case PHA may serve as a carbon and energy reserve for sporulation (Kominek & Halvorson 1965; Nakata 1965). Moreover, López et al. (1995) 57 observed that in a PHA negative mutant of Bacillus megaterium, sporulation occurred immediately after exposure to river water, while survival of vegetative cells was clearly decreased as compared to wild type, pointing that in an oligotrophic environment, cells depleted of intracellular carbon source may be committed to earlier sporulation than normal cells. The mutant spores needed a heat shock for germination, suggesting that PHA or its degradation products are involved in this process (López et al. 1995). Similar results were found in Azotobacter vinelandii in which PHA was utilized as a carbon and energy source during encystment (Lin & Sadoff 1968; Segura et al. 2003). However, mutations in the phaB and phaC genes in A. vinelandii had no impact on encystment or cyst viability under laboratory conditions, but the possibility that under natural conditions, PHA metabolism does have such an impact cannot be ruled out (Segura et al. 2003). Several other works, in which different PHA-producing bacteria were incubated under starvation condition in natural oligotrophic environments, or under conditions mimicking natural settings, showed that wild-type strains containing PHAs survived starvation better than either PHA-polymerase or PHAdepolymerase mutants (López et al. 1995; Ruiz et al. 1999). Under such conditions, morphological changes associated with starvation were largely delayed in the wild-type strains of Pseudomonas oleovorans (Ruiz et al. 2001). Changes from rod-like to coccus-like shapes are often part of the starvation response, a major regulator of which is the rpoS gene (Lange & HengerAronis 1991; Gentry et al. 1993). In summary, PHA production is a widespread trait, lending support to the hypothesis that this is a central feature of survival physiology when cells are faced with starvation and in response to sporulation and encysment. However, the ability to produce PHA is apparently not absolute for improved survival ability during this type of stress: Non-PHA producing bacterial strains isolated from soil survived starvation periods equally as well as strains capable of PHA accumulation (Wang & Bakken 1998), suggesting that PHA enhances the survival of some, but not all, of the bacteria, which must then rely on alternative strategies. In PHA-producing bacteria, PHA is a major determinant for overcoming periods of carbon and energy starvation, and may represent a basic feature for so-called “environmental bacteria.” This point will be further developed in the section dealing with phylogeny. Besides its defined role in starvation alleviation, PHA compounds appear to endow the producing cells with increased endurance to other types of environmental stresses, as was shown when P. oleovorans and a PHA depolymerization-minus mutant strain were used to assess the effect of PHA on survival and resistance to various stress agents. When exposed to 20% ethanol, the wild-type strain survived better than the PHA depolymeraseminus strain, with 23% and 9% of the cells surviving the challenge, for the wild-type and mutant strains, respectively. Also, a 47◦ C heat shock was less damaging to the wild type than to 58 D. KADOURI ET AL. the mutant strain (Ruiz et al. 2001). Similar observations were made for psychrotrophic strains of Rhizobium under cold stress (Sardesai et al. 2001). An extensive analysis of the role of PHA in the protection of A. brasilense cells exposed to physical and chemical stresses was performed, with the aim of understanding how central parameters such as quality, longevity, reliability, and efficacy of commercial bacterial inoculants for agricultural uses can be improved. After exposure of A. brasilense to stress and adverse conditions such as UV-irradiation, desiccation, and osmotic pressure, the survival of PHA-rich bacteria was higher than that of PHA-poor ones (Tal & Okon 1985). Similar results were obtained working with phaC (Kadouri et al. 2002) and phaZ mutants of A. brasilense (Kadouri et al. 2003a; Kadouri et al. 2003b). The ability of these mutants to tolerate various stresses, such as ultraviolet irradiation, heat, osmotic pressure, osmotic shock, desiccation, and to grow in the presence of hydrogen peroxide, was significantly affected. Thus, as the two PHA mutants, one blocked in the anabolic (phaC mutant), the second in the catabolic route (phaZ mutant), are similarly altered, increased resistance to the described stresses can be traced to a normal functioning of the PHA cycle, and not exclusively to the presence of the polymer. The mechanisms by which the PHA cycle favors stress alleviation are not yet fully understood. Early work on R. eutropha suggested an association of PHA utilization with respiration and oxidative phosphorylation (Hippe & Schlegel 1967). It was found that a rise in ATP and guanosine tetraphosphate (ppGpp) levels was concomitant with PHA degradation. This phenomenon was only observed in wild type P. oleovorans and not in a PHA depolymerase-deficient strain unable to degrade the polymer (Ruiz et al. 2001). The effector ppGpp was shown to increase mRNA translation of the central stationary phase regulator rpoS (Brown et al. 2002), which upregulates resistance to environmental insults such as ethanol, H2 O2 , high temperature, or high salt concentration (Lange & Henger-Aronis 1991; Samiguet et al. 1995; Ramos-González and Molin 1998; Ruiz et al. 2001). The sigma subunit RpoS activates the expression of genes involved in cell survival in cessation of growth and provides cross-protection to various stresses. Recently, it was found that the enhanced cross-tolerance to different stress agents during PHA-depolymerization in P. oleovorans is related to an increase in the intracellular concentration of RpoS (Ruiz et al. 2004). Ruiz et al. (2004) suggested that there is an association between PHA depolymerization and the stress tolerance phenotype, controlled by RpoS. In addition, Peralta-Gil et al. (2002) showed that one of the promoters that control PHA synthesis in A. vinelandii is regulated by RpoS. It seems that to respond properly to diverse stresses, PHA-producing bacteria require the rpoS gene product, this product induces expression of many genes, including the ones responsible for PHA depolymerization, and allows the organism to mediate changes in cellular physiology and structure and to adapt, resist, and survive under stress conditions. The data gathered in these various studies suggest a complex role for PHA in stress alleviation. However, the evidence clearly shows a relationship between PHA metabolism and cell alleviation under stress, mainly environmental stress. The granules may offer physical protection in stresses such as UV radiation, protecting DNA from damage, and increase resistance to desiccation, but normally functioning PHA anabolic and catabolic pathways seem to be essential to provide increased stress protection. While stress response players such as rpoS and ppGpp (or their functional homologues in different organisms) are central in determining the type and the strength of the response by redirecting cell resources to the synthesis of the appropriate effectors, the PHA cycle could be responsible for providing the fuel necessary for this response, and therefore could also determine its intensity. 3. ENERGY FLOW AND PHA METABOLISM PHA is generally overproduced and accumulated when more reducing power is being generated than consumed, due to the limitation for other syntheses (Dawes 1986; Babel et al. 2001). On the other side of the cycle, PHAs can serve as sources of NADH and ultimately, ATP. This energy-generation capacity can be used to fuel various energy-consuming pathways which in the absence of PHA would be slowed down or blocked. For example, Rothermich et al. (2000) reported the PHA accumulation during the night and degradation during the day of PHA from a variety of photosynthetic benthic microbial mats obtained from soil, compost, and sewage sludge. The authors suggest that the accumulation of PHA at night appears to be related to routine dark energy metabolism and is not influenced by the availability of organic nutrients. Among others, energy taxis are involved in chemotaxis to a carbon source and it appears to be important in plant-microbe interaction. It has been shown that A. brasilense exhibited stronger chemotaxis toward attractants such as fructose, malate, or sweet corn seeds exudates in a semi-solid agar test, than the phaC mutant (Kadouri et al. 2003b). Nevertheless, the mutant exhibited a three-time increase in swimming velocity over the wild-type strain (Kadouri et al. 2002; Kadouri et al. 2003b). Under the tested conditions (carbon-free medium), this increased motility did not provide the mutant with an elevated chemotactic response, probably because of the absence of PHA as a storage compound that could be utilized during the starvation period. Increase motility was not observed in a phaZ mutant that accumulated high PHA levels but was incapable of degrading it (Kadouri et al. 2003a). The reduced power produced during PHA degradation likely energized the chemotactic process in the environment, where sources of reducing power are low. In A. brasilense, PHA oxidation involves a specific NADH-dependent dehydrogenase, which competes for TCA-cycle intermediates in the electron transport system (Tal et al. 1990a, 1990b). When PHA accumulation is disrupted, more resources are accessible to the TCA cycle, resulting in an increased motility in the phaC mutant as SIGNIFICANCE OF BACTERIAL POLYHYDROXYALKANOATES compared to the wild type. Therefore, a similar motility of the phaZ mutant and of the wild-type strains is probably due to the inability of the mutant to generate excess reducing power, as was likely the case in the phaC mutant when PHA polymerization was disrupted. Several lines of evidence suggest that taxis in plant-associated bacteria is metabolism dependent (Grishanin et al. 1991), which explains that chemotaxis is dependent to PHA metabolism. The redox state of the rhizosphere is one of the most important parameters for maintaining this ecological system. Thus the energy taxis, driven by PHA catabolism, toward metabolizable substrates in plant root exudates may play a major role in plant-microbe interactions. Another feature of phaC mutants in A. brasilense, is a considerable increase in excreted exopolysaccharide (EPS), when grown under a high C:N ratio over the wild-type strain. In such mutants, EPS production may act as a sink for carbon and reducing equivalents which are diverted from the blocked PHAsynthesis pathway. Azospirilla are known for their capacity to aggregate and flocculate under diverse stress conditions, and some studies have suggested that EPS are involved in cell aggregation and in root adhesion (Burdman et al. 2000). Recently, Bahat-Samet et al. (2004) showed that the arabinose content of A. brasilense EPS plays a role in cell aggregation. The phaC mutant was more aggregative, and exhibited an increased (five to six times) ability to adhere to roots relative to the wild type (Kadouri et al. 2002; Kadouri et al. 2003b). In contrast, EPS production in the wild-type strain, as well as its aggregation capability was higher than that of the phaZ mutant under the same conditions (Kadouri et al. 2003a). Burdman et al. (2000) suggested that cell aggregation could increase survival of Azospirillum cells under diverse stress conditions. The attachment of the bacteria to plant roots, within bacterial aggregates and flocs, is a requirement for the establishment of the bacterial-root association. Thus, both PHA accumulation and cell aggregation could constitute a protected model of growth that allows survival in a hostile environment. This phenomenon of cell aggregation—PHA metabolism—may also be important during root colonization where cell aggregation is commonly observed. In Rhizobium spp.,PHA formation decreases the amount of reducing equivalents that could otherwise be used for nitrogen fixation during the symbiosis of the bacteria with their host plants, as observed when a PHA− strain of Rhizobium etli exhibited a higher and prolonged nitrogenase activity in nodules (Cevallos et al. 1996). As a consequence, plants inoculated with the PHA− mutants had higher nitrogen content. Along similar lines, it can be mentioned that mutations in the nitrogenaseencoding genes that resulted in the absence of nitrogen fixation by Bradyrhizobium japonicum, brought about an increase in PHA synthesis. It therefore seems that PHA synthesis can serve as an alternative pathway for the regeneration of reducing equivalents in the nodule (Hahn et al. 1984). Like R. etli, Azorhizobium caulinodans also accumulates PHA in both symbiotic and freeliving state, but A. caulinodans phaC mutant was totally devoid of nitrogenase activity ex plant and induced nodules devoid of 59 bacteria (Mandon et al. 1998). Interestingly, nitrogenase activity of the mutant was partially restored by constitutive expression of the nifA gene. Mandon et al. (1998) suggested that PHA is required for maintaining the reducing power of the cell and that nifA expression adapts nitrogen fixation to the carbon and the reducing equivalents available in the nodule. Also Vassileva and Ignatov (2002) studied the relationship between PHA formation and nitrogenase activity in Galega orientalis—Rhizobium galegae symbiosis. They reported high acetylene reduction activity parallel to PHA degradation when applying low concentrations of plant growth promoters and polyamine modulators. In contrast to R. etli, A. caulinodans, and R. galegae that forms PHA in both the free-living and bacteroid states, S. meliloti bacteroids typically do not deposit PHA. S. meliloti strains defective in PHA formation were not affected in their ability to induce nodule formation. Nodules developed at the same timing and had the same appearance on alfalfa plants (Medicago sativa) inoculated with the wild type or the mutant strains. Additionally, the acetylenereducing activity, a measure of the nitrogenase activity, was not affected in these PHA defective mutants when compared to the wild-type strain. It was concluded that an effective symbiosis between S. meliloti and alfalfa is not affected by PHA formation, and that a reduced competition for reducing equivalent may be a reason for the efficiency of nitrogen fixation in alfalfa nodules (Hahn et al. 1984). The data herein suggest that a sensitive regulatory mechanism that controls the synthesis or degradation of PHA in bacteroids is present and point its importance to maintain an effective symbiosis. A well-regulated PHA cycle therefore appears to be central both for a balanced use of the available energy (as seen with the swimming velocity in A. brasilense and nitrogen fixation in Rhizobium spp., R. galegae, and A. caulinodans) and for a balanced distribution of the carbon resources (as seen with EPS production and aggregation), at least when a high C:N ratio prevails. 4. PHA: AN ECOLOGICAL EDGE? Although single strain inoculation experiments done with S. meliloti PHA− mutants and wild type strains suggested that competent symbiosis between S. meliloti and alfalfa is not affected by PHA formation (Povolo et al. 1994), results from co-inoculation experiments suggested that the wild-type bacteria outnumbered PHA mutant bacteria by more than 200 to 1 (Willis & Walker 1998). This result indicates that the phaC mutant strains were less competitive, and that PHA production may provide an advantage to the strain during nodule initiation or invasion. Although in the case of S. meliloti, the mutation was not disadvantageous after the establishment of symbiosis, pleiotropic effects may have reduced competitiveness of the free-living bacteria in the rhizosphere. On the other hand, rhizobia are positively chemotactic toward a variety of amino acids, dicarboxylic acids, and sugars, toward the nodulation gene-inducing flavonoids secreted by the roots of its host. Mutants defective in motility or chemotaxis are impaired in their ability to compete for sites of 60 D. KADOURI ET AL. nodule initiation on the host root (Caetano-Anollés et al. 1988). If like A. brasilense, S. meliloti PHA catabolism is involved in energy supply to the chemotaxis, it is reasonable that phaC mutant strains are less competitive than the wild type. No differences were detected in a range of carbon sources utilized by a wild type and a phaC mutant of A. brasilense, but the generation time of the former was always shorter than that of the later (Kadouri et al. 2003b). Similar findings were reported with A. brasilense phaZ (Kadouri et al. 2003a) and R. etli phaC mutants (Cevallos et al. 1996). It was observed that a mutation in the phaC gene severely impaired the ability of R. etli to grow in minimal media supplemented with glucose or pyruvate. Such consequences may further hinder the competitiveness in the environment of mutants defective in PHA accumulation or utilization (Okon & Itzigsohn 1992). The possession of relatively high growth rates seems to be an obviously important factor for rhizosphere competence. Although it was suggested that carbon is not a limiting factor for microorganisms in the rhizosphere (Cheng et al. 1996), the ability of r-type soil inhabitants to grow quickly, that is, to attain short growth rates on the available compounds in the rhizosphere, probably affect their capacity for root colonization (Simons et al. 1996; Jjemba & Alexander 1999). However, the impact of a functional PHA cycle on root colonization is not always evident as shown in a study in which plant root colonization by wild type and phaC mutant strains of A. brasilense were evaluated under sterile and non-sterile conditions in soil. Both root colonization and plant growth promoting effects were similar under each of the tested conditions (Kadouri et al. 2002). While the lack for influence of the mutation on these parameters may stem from the optimal plant growth conditions as well as the high inoculum level used in the study (which can reduce the plant growth promoting effect and blur differences in colonization efficiency, respectively), it is left to be seen if under inoculation and growth conditions found in the field, the impaired stress resistance and physiological changes observed in cells with a disrupted PHA cycle are translated in reduced colonization and growth promotion. Studies carried out with the potential bioremediation and biocontrol agent, B. megaterium, on sterile and non-sterile microcosms soil showed that in non-sterile soil the total cell number (vegetative cells plus spores) of a PHA accumulating strain was higher than that of the PHA-negative mutant (López et al. 1998). Similar result was obtained in homogeneous aquatic microcosms (López et al. 1995). These results suggest that PHA accumulation contributed to the survival capabilities and spore quality in homogeneous and heterogeneous environments enriched with organic matter such as crop field soil; thus contributing in the ecological edge of B. megaterium in its natural environment. 5. INOCULANT CARRIERS—AN ULTIMATE MODEL FOR STRESS SURVIVAL Inoculant preparations for agricultural uses can constitute particularly stressful environments. Bacterial cells may have to be stored for long periods, and should also survive desiccation and possibly hot conditions. A major feature inoculants should possess is a high capacity to maintain high survival rates of the bacterial cells within the carrier itself. Most of the research done in this field is based on improving carrier properties by adding elements that can prolong survival, such as nutrients, or other synthetic products (López et al. 1998). The vast amount of information gathered on Azospirilla throughout the years suggests that for an inoculant to be successful—to provide efficient root colonization—the type of carrier material is not the only important parameter, but the metabolic state of the cells and their capability to use intercellular storage material can be of significance for survival within the carrier itself. This knowledge originated from studies showing that while the carriers may vary, plant growth promotion effects were more consistent with A. brasilense inoculants containing high amount of PHA (Fallik & Okon 1996). Confirmation of these results was obtained in field experiments carried out in Mexico with maize and wheat, where better consistency in increasing crop yield was obtained using peat inoculants prepared with PHA-rich Azospirillum cells (Dobbelaere et al. 2001). In addition, studies carried out with the A. brasilense phaC mutant showed that among different inoculant carriers (peat, sianic sand, perlite), peat sustained the highest populations of inoculated bacteria and perlite the lowest. In all cases, the wild-type strain survived better than the mutant but the variations between the carriers were very large. It was thus concluded that the production of PHA is of critical importance for improving the shelf life, efficiency, and reliability of commercial inoculants (Kadouri et al. 2003b). 6. A PHYLOGENETIC OVERVIEW OF PHA SYNTHESIS AND DEGRADATION A BLASTP search enabled us to identified more than 170 and 60 PhaC and PhaZ homologous proteins, respectively. For further phylogenetic studies, a subset of 67 PhaC (61 from Proteobacteria, 3 from Gram positive bacteria and 3 sequences from Cyanobacteria) and 46 PhaZ (all proteobacterial in origin) representative proteins annotated as known or putative were chosen. Environmental sequences and sequences with E-values worse than 10−3 were not included in this study. In addition, 16S rDNA gene sequences were used to infer phylogenetic trees for the species and for comparative (congruence) purposes. Multiple alignments of protein sequences were performed using the CLUSTAL W program (Thompson et al. 1994) and edited for graphic presentation with GeneDoc (Nicholas et al. 1997). Phylogenetic trees were inferred with maximum-parsimony (heuristic search factor of 2) and neighbor-joining (p-distance matrix) analyses, using MEGA2 (Kumar et al. 2001). In almost all cases, the PhaC protein trees were congruent with the 16S rDNA data, clustering as phylogenetic taxa (Figures 2 and 3), suggesting the existence of “genotypic” clusters that corresponded to traditional species designations. The PhaC tree showed a topology in agreement to that reported by SIGNIFICANCE OF BACTERIAL POLYHYDROXYALKANOATES 61 FIG. 2. Phylogenetic tree of PhaC generated using the Neighbor-Joining (NJ) distance method. Similar topology was generated using Maximum Parsimony (MP) method (data not shown). Accession Numbers are given between parentheses. Numbers in brackets identify clusters. An asterisk labels sequences showing discrepancies with the 16S rDNA phylogeny of the cluster. 62 D. KADOURI ET AL. FIG. 3. Phylogenetic relationship based on the analysis of the 16S rRNA gene among the bacterial groups used in this analysis generated using the NeighborJoining (NJ) distance method. SIGNIFICANCE OF BACTERIAL POLYHYDROXYALKANOATES Steinbüchel and Hein (2001) and Rehm (2003). However, the tree presented in our work includes 67 instead of 36 and 58 sequences, respectively. Six clusters were evident (Figure 2). The first monophyletic cluster exclusively contained members of the Pseudomonadaceae, with Class II synthases (Cluster 1) and was constituted of two divergent groups. The other γ -Proteobacteria belonged to a different, separated cluster (Cluster 3). Within this cluster, Aeromonas caviae that synthesizes copolyesters of MCL-PHAs was found (Shimamura et al. 1994; Doi et al. 1995). Although the PhaC belonging to α-Proteobacteria, was found in two, well separated and defined clusters (4 and 5), their relative positions remain uncertain due to low bootstrap values. The larger cluster (Cluster 4) included a PhaC from A. brasilense that exclusively synthesizes homopolymers of polyhydroxyburyrate (Itzigsohn et al. 1995). Within the smaller cluster 5 was found Rhodobacter sphaeroides that only produces SCL-PHA (Clemente et al. 2000). A group of PhaCs originating from β-Proteobacteria appeared to form cluster 2. This cluster included R. eutropha, Azotobacter spp., and Chromobacterium violaceum. R. eutropha’s PhaC is a representative member of the Class I PhaCs and the most intensively studied synthase. R. eutropha (Chua et al. 1998) and C. violaceum (Kolibachuk et al. 1999) synthesize copolyesters of 3-hydroxybutyrate-co-3hydroxyvalerate (3HB-3HV), but it has also been reported that R. eutropha produces 3-hydroxybutyrate-co-3-mercaptopropionic acid or co-3,3’-thiodipropionic acid (Lutke-Eversloh and Steinbüchel 2003). In addition, Azotobacter PhaC has also been identified as a Class I PhaC (Pettinari et al. 2001). Cluster 2 and the two Pseudomonadaceae branches appeared to derive from a common ancestor, with a high degree of confidence. The clustering of Azotobacter with the β-Proteobacteria is unambiguous. Cluster 6 was used as an outgroup and included PhaCs from cyanobacteria, Bacillus species as well as from two γ -Proteobacteria, Allochromatium vinosum (formally called Chromatium), and Thiocystis violacea, all member of Class III PhaC (Hein et al. 1998; McCool & Cannon 2001; Yuan et al. 2001). To summarize, while clusters 1 and 6 PhaCs were class I and class III PHAs, respectively, the PhaCs from the other clusters were involved in the synthesis of widely different types of PHAs. Although the PhaC and 16S rDNA data were congruent for individual clusters, the branching orders were not necessarily identical. The clear separation of the Pseudomonaceae’s PhaCs from those of the other γ -Proteobacteria, and the large distance between the clusters formed by organisms belonging to this group—which also reflects their phylogenetic grouping, as well as the presence of Azotobacter within cluster 2, support a very ancient split, and most probably a duplication event within cluster 1. The few incongruence found between the PhaC and the 16S rDNA phylogenies could be explained by very large distances separating the organisms and low bootstrap values (R. prowazekii), or possibly, horizontal gene transfer (HGT) (P. putida in β-Proteobacteria). In the case of R. prowazekii, the GC content of the gene matches the average of the genome, so HGT 63 does not appear to have taken place, or it was a very ancient event. Interestingly, this is the only occurrence of a phaC-like gene in a pathogen, all the others originating from “environmental bacteria,” such as soil or waterborne bacteria. Moreover, no such function seems to be present in Rickettsia conorii, a closely related pathogen. Whether R. prowazekii indeed produces PHAs is unknown. To survive and grow under fluctuating environmental conditions, including periods of unbalanced nutrient and energy supply, microorganisms may use several strategies, including PHA accumulation, which can provide the energy and carbon necessary to maintain metabolic activities and protein synthesis under detrimental situations. The PhaC protein appears to be highly conserved, it is widely distributed within the Bacteria domain (we can suppose that it is present in the many known PHA producing bacterial species), even though to the best of our knowledge, no PHA production by δ- or ε-Proteobacteria has been reported. We also have presented a large body of data supporting a role for PHA in alleviating environmental stresses. We believe these data lend strong support to the hypothesis that PHA metabolism is an essential feature for survival of many bacteria in the environment, or in other words, endows the cells with an ecological advantage. The mechanism and regulation of PHA mobilization is poorly understood, but a clear distinction exists between intracellular and extracellular PHA degradation by way of the PhaZ enzymes (Jendrossek & Handrick 2002). Herein, we focus on the intracellular PhaZ (i-PhaZ). The i-PhaZ of P. oleovorans, R. rubrum, R. eutropha, and Paracoccus denitrificans are the best studied (Huisman et al. 1991; Merrick et al. 1999; Gao et al. 2001; Saegusa et al. 2001; Ueda et al. 2002; York et al. 2003). Most of the clusters of the phylogenetic tree for the PhaZ enzyme presented here clearly show well-supported internal structures, but their orders are often undefined (Figure 4). Moreover, taking exception from the Pseudomonadaceae’s PhaZs, which along with those of two β-Proteobacteria were only very distantly related to the other PhaZs, and from two other clusters containing few sequences, none of the clusters were homogeneous, that is, they all contained one or more sequences not congruent at the class level with the 16S rDNA data. It has been reported that R. eutropha possesses three different PhaZs (York et al. 2003). In addition, we detected two copies of PhaZ in the genome of Raltonia metallidurans and Burkholderia fungorum. R. eutropha PhaZ2 and PhaZ3 clustered independently, but were associated with the PhaZ of R. metallidurans. R. eutropha PhaZ1 and PhaZ3 clustered with PhaZs of other β-Proteobacteria, but the PhaZ3 sequence diverged from the PhaZ1 one. This multiplication of depolymerases in one organism may reflect the diversity of the PHAs it is able to produce. The incongruence within the mentioned cluster and within the rest of the tree maybe signs of HGT, duplication events, or of parallel evolution of proteins originally performing various functions. In other words, while the synthetic process appeared to be stringently conserved, a number of options for using the polymer as a substrate may have developed in parallel or were laterally acquired. 64 D. KADOURI ET AL. FIG. 4. Phylogenetic tree of PhaZ generated using the Neighbor-Joining (NJ) distance method. Similar topology was generated using Maximum Parsimony (MP) method (data not shown). Accession Numbers are given between parentheses. SIGNIFICANCE OF BACTERIAL POLYHYDROXYALKANOATES TABLE 1 PHA in bacterial environmental fitness and agriculture Features Cell survival under starvation in batch and continuous culture (days) Cell survival under nutrient limitation in water, soil, rhizosphere and phyllosphere Cell survival in inoculant carriers Establishment of inoculum in soil and plant surfaces Energy source and flow for cell motility, chemotaxis, aerotaxis and biological nitrogen fixation Sporulation, cyst formation and germination Control of exopolysaccharide production Endurance under environmental stress: heat, UV-irradiation, desiccation, osmotic stress, osmotic shock, ethanol and H2 O2 Balanced use of available energy and distribution of carbon resources 7. Selected references Tal and Okon 1985; Anderson and Dawes 1990; James et al. 1999. Okon and Itzigsohn 1992; López et al. 1995; Ruiz et al. 1999. Fallik and Okon 1996; Dobbelaere et al. 2001; Kadouri et al. 2003b. Kadouri et al. 2002; Kadouri et al. 2003b. Tal and Okon 1985; Cevallos et al. 1996; Kadouri et al. 2002. Kominek and Halvorson 1965; Segura et al. 2003. Kadouri et al. 2002. Tal and Okon 1985; Kadouri et al. 2003a; Kadouri et al. 2003b. Dawes 1986; Rothermich et al. 2000; Babel et al. 2001. CONCLUDING REMARKS PHA-related features influencing bacterial fitness are summarized in Table 1. In order to survive in nutrient-poor ecosystems, bacteria use different strategies, among them, the use of PHAs as intracellular carbon storage compounds. Many works have revealed that cells with higher content of PHA can survive longer than those with lower amounts, and that PHA degraded elements can be used rapidly for numerous metabolic needs. Accumulation of PHA can provide the cell with the ability to endure a variety of harmful physical and chemical stresses, either directly linked to the presence of the polyester itself (PHA granules) or through a cascade of events concomitant with PHA degradation and the expression of genes involved in protection against damaging agents. The first type of events may be hypothesized to occur when PHAs offer physical protection to damage and may include the use of PHAs as carbon source under nutrient deficiency. In the second type of event, PHAs function as a source of power (reducing equivalents) that can be mobilized to enact reaction to the stress (maybe such as ATP-dependent repair mechanisms, osmoregulation). In both cases, a normally 65 functioning PHA cycle may enable the maintenance of optimal metabolic capabilities (including nitrogen fixation and chemotaxis) under sub-optimal conditions, permitting not only survival but also cell proliferation. The capability of PHA to provide the cell with an endogenous carbon reservoir to aid in stress endurance, and to support quick growth on the nutrients accessible, is an important factor for soil competence, which can probably affect their capacity to colonize plants. The growth rate (replication times, generation times) of bacteria in the soil, rhizosphere, phyllosphere, and inoculants is apparently much slower than in batch culture under laboratory conditions. While in the latter PHAs are utilized and depleted after several days under starvation, in nature the PHAs could be aiding bacterial division and survival for much longer periods. In addition, the phylogenetic analysis of PHA metabolism proteins evidences that PHA accumulation is an essential feature for survival of many bacteria in the environment, or in other words, endows the cells with an ecological advantage. 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