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RESEARCH LETTER Isolation and characterization of a Nocardiopsis sp. from honeybee guts Preeti B. Patil, Yu Zeng, Tami Coursey, Preston Houston, Iain Miller & Shawn Chen Molecular and Cellular Biology Program, Department of Biological Sciences, Ohio University, Athens, OH, USA Correspondence: Shawn Chen, Molecular and Cellular Biology Program, Department of Biological Sciences, Ohio University, Athens, OH 45701, USA. Tel.: 11 740 597 3112; fax: 11 740 593 0300; e-mail: [email protected] Received 21 April 2010; revised 24 June 2010; accepted 23 August 2010. Final version published online 16 September 2010. DOI:10.1111/j.1574-6968.2010.02104.x Editor: Michael Bidochka MICROBIOLOGY LETTERS Keywords actinomycetes; honeybee gut microbiota; Nocardiopsis; antagonistic activities; phenazines. Abstract Although actinomycetes are the plant-associated environmental bacteria best known for producing thousands of antibiotics, their presence in the guts of flower-feeding honeybees has rarely been reported. Here, we report on the selective isolation of actinomycetes from the gut microbiota of healthy honeybees, and their inhibitory activity against honeybee indigenous bacteria. More than 70% of the sampled honeybees (N 4 40) in a season carried at least one CFU of actinomycete. The isolates from bees of one location produced inhibitory bioactivities that were almost exclusively against several bee indigenous Bacillus strains and Grampositive human pathogens but not Escherichia coli. An antibiotic-producing actinomycete closely related to Nocardiopsis alba was isolated from the guts in every season of the year. A DNA fragment encoding a homologous gene (phzD) involved in phenazine biosynthesis was identified in the isolate. Expression of the phzD detected by reverse transcription-PCR can explain the survival of this organism in anaerobic environments as some redox-active extracellular phenazines are commonly regarded as respiratory electron acceptors. The results raise important questions concerning the roles of the antibiotic-producing actinomycetes and the phenazine-like molecules in honeybee guts and honey. Introduction Insect digestive tracts support communities of symbiotic and transient microorganisms that are increasingly the subjects of studies of microbial diversity and novel bioactive microbial products (Breznak, 2004; Evans & Armstrong, 2006). In general, insect gut microbiota make significant contributions to the nutrition of the insect host, as demonstrated in well-studied examples such as termites, cockroaches, wood-feeding beetles and aphids (Douglas, 1998; Dillon & Dillon, 2004). With the advancement of new sequencing methods, gut microbial communities have been analyzed in an even wider range of insects (Broderick et al., 2004; Xiang et al., 2006; Sen et al., 2009). Honeybees, Apis mellifera, are an interesting model for studies of gut microorganisms because they have a complex digestive tract. Workers collect nectar (carbohydrate source) and pollen (source of protein, fatty acids, sterols, vitamins and minerals) and bring them back to hives to feed larvae and house bees by oral regurgitation. The nectar and pollen mixed with water are temporarily stored in the crop (honey stomach), an enlargement of the esophagus. The ventriculus (midgut) 2010 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved c is the functional stomach followed by an anterior intestine and rectum. Recent metagenomic surveys have shown diverse bacteria in this insect host (Jeyaprakash et al., 2003; Mohr & Tebbe, 2006; Cox-Foster et al., 2007). Understanding their specific contributions to the physiology of honeybees requires isolation of the microorganisms and subsequent biochemical and genetic characterizations. The sporulating actinomycetes are ubiquitous in terrestrial habitats and include common genera such as Streptomyces, Frankia, Nocardia and Micromonospora (Ventura et al., 2007). They are well known for their metabolic capabilities, with Streptomyces being the major producers of thousands of antibiotics (Berdy, 2005). Actinomycetes, as one of the rhizosphere bacteria, also produce a wide range of hydrolytic exoenzymes (e.g. chitinases, cellulase, etc.), and are therefore primary contributors to the cycling of carbon in organic matter derived from fungi and plants. Because of the importance and potential growth advantages of these bacteria, several studies have focused on the isolation and visualization of actively growing actinomycetes in the guts of beetles, termites and millipedes (Bignell et al., 1979; Gozev & Byzov, 2006; Scott et al., 2008). Previously, nonpathogenic FEMS Microbiol Lett 312 (2010) 110–118 111 Actinomycetes in honeybee guts microbiota associated with honeybees have mostly been examined using classical culture-based techniques, and chemotaxonomic characterization of the isolates, which have described a group of Gram-variable pleomorphic bacteria in honeybee guts but not in adequate detail (Gilliam, 1997). Although data from the latest pyrosequencing technology applied to honeybee gut microbiota are yet to be published, few metagenomic studies have revealed the presence of actinomycetes in this environment (Cox-Foster et al., 2007). Also, it is known that PCR amplification of bacterial 16S rRNA genes with universal primers could have dramatically underestimated the population of high-GC Actinobacteria in a complex community (Stach et al., 2003). However, one culture-based report indicated that Streptomyces sometimes could become dominant in bee guts (Mohr & Tebbe, 2007). To our knowledge, no antibiotic-producing actinomycetes from the guts of honeybees have ever been characterized, though Streptomyces are among the microorganisms found in honey (Snowdon & Cliver, 1996) and honey products have well-known antimicrobial properties (Kwakman et al., 2008). Honey has been a popular folk medicine for healing wound and soothing sore throat since ancient times. In this report, selective media were used to isolate actinomycetes from the digestive tract of adult honeybees. The antibiotic activities produced under laboratory conditions were evaluated against bee indigenous Bacillus strains, Escherichia coli and two drug-resistant human pathogens. One frequently encountered isolate identified as a species of Nocardiopsis was further characterized and the expression of an antibiotic biosynthetic gene was analyzed. Materials and methods Isolation of actinomycetes from honeybee guts and the growth media Adult worker honeybees were collected from six locations, most of which have o 10 isolated hives. Within 12 h of capture, bees were externally sterilized with 70–100% alcohol and dissected under sterile conditions. The digestive tracts, from crop to rectum, were pooled, lightly homogenized and suspended in saline and plated on selective agar plates. The gut contents from each bee were spread on one plate. To better investigate the actinomycete diversity in the complex microbial milieu of the insect gut, different selective media were used for the colony isolation. Four out of 10 selective media tested yielded better results in that the growth of actinomycetes was favored over other bacteria, fungi and molds (Bredholdt et al., 2007; Babalola et al., 2009; Maldonado et al., 2009; Qin et al., 2009). They were as follows: actinomycete isolation agar (AIA) supplemented with cycloheximide (50 mg mL1) and rifamycin (5 mg mL1) (sodium caseinate 2 g; asparagine 0.1 g; sodium propionate 4 g; K2HPO4 0.5 g; MgSO4 0.1 g; FeSO4 0.001 g; glycerol 10 g and agar 15 g L1 distilled water), MSM agar (microcrystalline cellulose 10 g; casein 0.3 g; KNO3 0.2 g; K2HPO4 0.5 g; CaCO3 0.02 g; FeSO4 0.01 g; NaCl 5 g; MgCl2 6H2O 30 g; KCl 20 g; agar 15 g L1 distilled water), IM5 agar (humic acid 1.0 g; K2HPO4 0.5 g, FeSO4 7H2O 1 mg, vitamin B solution 1 mL, agar 20 g L1 distilled water, adjusted to pH 8.2) and IM7 agar (similar to IM5 but the humic acid is replaced with chitin 2.0 g L1). After incubation at 30 1C for 3–7 days, filamentous bacterial colonies that appeared powdery, fuzzy or leathery were selected and purified (Fig. 1a). Gram stain followed by examination under light microscope confirmed that isolates had the morphology of actinomycetes. Spores of actinomycete isolates were scraped off the agar and mixed with 20% glycerol to be stored in 80 1C. To make duplicates for long-term storage, the spores of each strain were also suspended in 5% nonfat dry milk and lyophilized. The solid growth media for BE74 were AIA and mannitol soya flour (MS) agar (Kieser et al., 2000). The liquid growth media for BE74 were AIB (broth with the ingredients same as AIA without agar) and ISP1 (Shirling & Gottlieb, 1966). (a) (b) Fig. 1. (a) An example of an actinomycete isolation agar plate that selectively allowed the growth of actinomycetes from the guts of one honeybee. The white colonies have identical characteristic colonial morphology of actinomycete. (b) An example of a MH plate used in the agar diffusion bioassay. The test organism was a bee indigenous Bacillus marisflavi strain. The overlaying agar plugs were taken from separate agar plates pre-grown with individual actinomycete isolates. FEMS Microbiol Lett 312 (2010) 110–118 2010 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved c 112 Agar diffusion assay of antagonistic activity Actinomycete isolates were individually cultured on Petri dishes that have four sections or 24-well tissue culture plates for 3–6 days. Two agar media, Müller–Hinton (MH) agar (Difco) and diagnostic sensitivity test (DST) agar (Oxoid), were used to grow the test organisms. Most test organisms here could grow to a full lawn on MH agar plate within 12 h but the Enterococcus grew better on DST agar. In the assay, a fresh culture of the test organisms (at OD600 nm 0.04–0.08) was swiped across an MH agar plate with a cotton Q-tip. A sterile 200 mL pipette tip was used with its wide-opening end to bore through the agar plate (0.5 cm thickness) grown with an actinomycete lawn. The agar plug (estimated 0.11 cm3) lifted out was overlaid on the seeded MH agar plate. Two plugs were separated about 1.5 cm in distance. About 15–18 plugs could be arrayed on the surface area of a plate of 100 mm diameter and about 30–40 plugs on a 150 mm plate (Fig. 1b). After incubation at 30 1C overnight, a clearing zone (Z2 mm) surrounding the agar plug indicated that the actinomycete produced a level of diffusible substance that inhibited the growth of the test organism. Genetic identification of microorganisms and phylogenetic analysis Genomic DNA isolation followed a salting-out procedure (Kieser et al., 2000), but started with 2–3 mL liquid culture and the volume of the solution used was one-tenth of that used in the standard procedure. Mycelia were lysed by bead beating (Biospec) with 0.1-mm-glass beads. PCR amplification (with SuperTaq, Ambion) of the partial 16S rRNA gene of the interested colony was attempted with universal bacterial primers (27F, 50 -AGAGTTTGATCMTGGCTCAG; 63F, 50 -CAGGCCTA ACACATGCAAGTC; 907R, 50 -CCGTCAATTCMTTTRAGT TT; 1378R, 5 0 -ACGGGCGGTGTGTACAAG and 1492R, 5 0 AAGGAGGTGATCCAGCC) as well as Actinobacteria classspecific primers (Stach et al., 2003). Sequences of the PCR products were analyzed by BLAST search, and the most closely related species were determined. DNA or protein sequences were aligned with CLUSTALW algorithm implemented in BIOEDIT software using the default parameters. The aligned and trimmed sequence regions were used as the input files to infer phylogenetic trees based on neighbor joining of genetic distance with bootstrapping in MOLECULAR EVOLUTIONARY GENETICS ANALYSIS (MEGA) software version 4.0.2. The accession numbers for the partial sequences of BE74 16S rRNA gene and phzD genes are HM588007 and HM588008. Amplification of partial phzD from BE74, the RNA isolation and reverse transcription (RT)-PCR The primers used for amplifying the 340-bp phzD fragment were as follows: PhzD254-282F, AAC AGC GCG GYC 2010 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved c P.B. Patil et al. TSC TCA AGG ACT TCT GG and PhzD571-592R, SSG CRC AGC GCT CGG CGG CGT A. Mycelia of BE74 were collected from one agar plate or 1 mL liquid culture for RNA isolation using an RNA isolation kit (RibopureBacteria, Ambion). Total RNAs were treated with DNase for half an hour and extracted using the standard phenol– chloroform method. Reverse transcription (RT) was performed with 200 ng RNA, SuperScript II reverse transcriptase (Invitrogen) and random hexamers. Two microliters of the RT reaction were subjected to a PCR reaction with the primers designed for an 162-nt fragment within the phzD gene of BE74: NocPhzD_F1, AAC AGC GCG GCC TCC TCA AGG ACT TCT GG and NocPhzD_R2, TTG GTG AGC AGG AGG TCC TCA CCG TCG. The annealing temperature was 64 1C and there were 30 PCR cycles. Results Isolation of antibiotic-producing actinomycetes from honeybee guts Initially, a small number of adult worker honeybees (N = 6) were collected in September 2008 from hives at six locations (separated by 3–20 miles) in southeastern Ohio. After the processing and selective isolation of actinomycetes with the AIA, the purified actinomycete colonies were analyzed using morphology (colors of aerial and substrate mycelia, pigments and starch lysis zone, etc.) and sequences of the amplified partial 16S rRNA gene. The results confirmed the presence of actinomycetes, mainly a diverse group of Streptomyces, in the guts of honeybees. Three to eight different Streptomyces species could be identified, with six bees from each of five locations. Bees of the remaining one location did not yield any actinomycete-like colonies on the AIA, but did produce a large number of nonactinomycete colonies. DNA typing showed that these nonactinomycete isolates were related to at least five Bacillus species (identity of the 16S rRNA gene 4 97%). They were Bacillus cereus, Bacillus gibsonii, Bacillus pumilus, Bacillus firmus and Bacillus marisflavi. Some of them are known to be used as biocontrol agents to inhibit the growth of pathogenic microorganisms such as foulbrood in bees and fungi on plant roots (Alippi & Reynaldi, 2006; Choudhary & Johri, 2009). It is unclear whether the predominance of one or more Bacillus species in this bee yard is related to the lower actinomycete diversity in the guts of the bees. One location that has a few isolated beehives was chosen to continue monitoring of actinomycetes diversity every 3 months in a year. Antibiotic activity against the bee indigenous Bacillus strains or E. coli was measured using an agar diffusion assay. The details were described in the methods. Positive results were interpreted as defensive rather than as nutritional interactions between the microorganisms because the FEMS Microbiol Lett 312 (2010) 110–118 113 Actinomycetes in honeybee guts At least one actinomycete CFU At least one actinomycete CFU producing antibiotics 90% Fig. 2. Percent of the sampled honeybees (N 4 40) that carried at least one actinomycete in the gut microbial communities, the number of actinomycetes producing detectable bioactivities over the 1 year sampling period and the number of total actinomycete isolates from honeybee guts. The honeybees were from a single bee yard in Athens, OH. Five other bee yards were also sampled (see Results). Percent of the sampled honeybeesm (N > 40) 80% 70% 60% 50% 40% 30% 20% 10% 0% Bioactive isolates Seasonal total isolates actinomycetes were already in late growth stage when used in the assay and the test organisms were microorganisms with shorter doubling times under the assay conditions. Potential competitive growth disadvantage of the test organisms like the Bacillus strains and E. coli can thus be ruled out with confidence. Also, it has been argued that actinomycetes in insects are predisposed toward engaging in defensive antagonism (Kaltenpoth, 2009). The B. marisflavi isolate identified in the initial experiment was used as a Gram-positive organism for the primary screening in the following survey because it seemed to be the most sensitive to the antibiotic activities produced by the actinomycete isolates. For understanding the seasonal changes in actinomycete diversity in honeybee guts, at least 40 bees were collected from the chosen bee yard four times during the year. At the times of December 5th (winter), April 21st (spring), July 16th (summer) and September 30th (fall) from 2008 to 2009, the gut microbial communities were assumed to be most influenced by the seasonal changes. AIA with supplements was used as the main selective medium (see Materials and methods). Over 70% of the bees in any one of the four seasons carried at least one CFU of actinomycete in their guts (Fig. 2). In some cases, thousands of conspicuous actinomycete colonies were found in a single honeybee (Fig. 1a). Between 28% and 58% of the bees at this location produced at least one actinomycete isolate with detectable bioactivities (Fig. 2). The highest diversity of actinomycetes was found in honeybees collected in the summer, and the lowest in the winter (Fig. 2). Of the 401 actinomycete isolates obtained, 163 isolates exhibited bioactivity against the bee indigenous B. marisflavi strain (Fig. 2). All except four of the 163 bioactive isolates had no observable effect on the growth of E. coli. Only one of the total 401 isolates showed exclusive antagonism against E. coli. Therefore, FEMS Microbiol Lett 312 (2010) 110–118 Winter Spring Summer Fall Total 27 58 45 110 60 120 31 113 163 401 there appeared to be a specificity of the bioactivities produced by the actinomycetes from honeybee guts. Actinomycetes producing anti-Gram-positive activities are enriched in the honeybee guts To investigate whether the actinomycete isolates can stably produce the antimicrobial activities, or whether the antagonisms are more broadly apparent against different Bacillus species and other Gram-positive pathogens, the actinomycete spores were revived after being frozen for 4–16 months in storage. They were grown on AIA and used in the bioassay with the test microorganisms listed (Table 1). One hundred and fifteen strains were able to grow well and showed consistent inhibitory activity against the B. marisflavi strain. In addition, nearly one-third of them were active against the previously isolated bee indigenous B. pumilus and B. cereus strains. The growth of Bacillus subtilis was inhibited by about the same number of the actinomycete strains. Two human pathogens, vancomycin-resistant Enterococcus faecium and methicillin-resistant Staphylococcus aureus, were also used as the test organisms in screening for the antibacterial activities produced by the actinomycete isolates. More than one quarter of the isolates clearly produced antibacterial substances that inhibited the growth of the two human pathogens under the assay conditions. We also attempted to test the inhibition of Paenibacillus larvae, the causative agent of hive disease American foulbrood. Because of its much slower growth rate on the MH agar in the bioassay, the agar diffusion assay method failed to show a clear antagonism between an actinomycete isolate and the Paenibacillus strain. Nonetheless, the important confirmation in the second-round screening was that none of the revived strains produced anti-E. coli activities. 2010 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved c 114 P.B. Patil et al. Table 1. Microorganisms tested in the agar diffusion bioassay Microorganisms tested Number of antagonistic actinomycete isolates Bacillus marisflavi,w Bacillus pumilus Bacillus cereus Bacillus subtilis ATCC6051 Enterococcus faecium ATCC51559 Staphylococcus aureus ATCC43300 Paenibacillus larvae ATCC13537 Escherichia coli ATCC25922w 163 35 38 37 30 33 –z 4 Isolated from honeybee guts and identified by partial 16S rRNA gene sequences ( 4 97% identity). w Used as test organisms in the primary screening with 401 actinomycete isolates. The rest test organisms were used with 115 isolates that were revived and reproducibly active against Bacillus marisflavi. z Attempted but inconclusive with the agar diffusion assay. The frequent occurrence of anti-Bacillus bioactivity from insect gut actinomycetes is especially notable in comparison to the bioactivities produced by actinomycetes from soil samples collected in this geographic region. When the same procedures were used to isolate soil actinomycetes, we found more colonies producing anti-Gram-negative and broadspectrum antibiotics (together 60–70%) than anti-Bacillusspecific producers (30–40%) (S. Chen, unpublished data). Therefore, there appeared to be an unusual enrichment of actinomycetes producing anti-Gram-positive bacteria activities in honeybee guts. This observation is reminiscent of early reports that the bioactivities of Streptomyces isolates from earthworm guts were all against Gram-positive bacteria (Kristufek et al., 1993). It could also explain a number of reports indicating that the antimicrobial activity of honey products is mainly against Gram-positive bacteria [reviewed in Viuda-Martos et al. (2008)]. A Nocardiopsis alba strain was one of the actinomycetes frequently isolated from the honeybee guts One actinomycete isolate named BE74, which consistently produced inhibitory activities against the B. marisflavi strain, was noticed because of its distinctive colonial morphology on the AIA – it appeared waxy, and only started to generate very thin aerial mycelia and poorly sporulate after 5–7 days of incubation. It produced abundant white aerial mycelia and spores when growing on MS agar, but exhibited much less exuberant growth on ISP1 agar. The substrate mycelia on MS agar are brown to yellow. Diffusible brown and light yellow pigments were observed on MS and AIA agars. Scanning electron micrographs of BE74 grown on AIA showed long, unbranched and not-fragmented mycelia that can twist to form spore chains (Fig. 3). The spores have smooth surfaces and the chains are spiral in shape. 2010 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved c Fig. 3. Scanning electron micrograph of the spiral hyphae of a rare actinomycete from the guts of the sampled honeybees. The actinomycete (BE74) identified as Nocardiopsis alba was grown on AIA for 7 days. A BLAST search with partial 16S rRNA gene sequences (1252 bp) of BE74 showed similarity (93–99%) to members of the genus Nocardiopsis in the Nocardiopsaceae family. In a phylogenetic tree based on the neighbor-joining algorithm, BE74 is clustered with all Nocardiopsis typing species (Tamura et al., 2008). The closest strain to BE74 is N. alba DSM 43377 (99% identity). The two formed a clade that was strongly supported by a high bootstrap value (100%). The N. alba strain BE74 was susceptible to rifamycin (2 mg mL1) on AIA. It was isolated from bee guts in all four seasons. However, we can only ascertain that 23% of the sampled bees (N = 40) at this location in the winter carried the N. alba strain. The isolate produced medium levels of antagonism (clearing zones 3–7 mm) against the B. marisflavi strain. It showed no activities against other organisms in Table 1 except B. cereus. Identification of a putative phenazine biosynthetic gene (phzD) in the Nocardiopsis strain Nocardiopsis species have been isolated from marine sediments (Engelhardt et al., 2010). Antibiotic biosynthetic genes were searched in a draft of the genome of Nocardiopsis dassonvillei DSM 43111 (Wu et al., 2009). One gene cluster proposed for an involvement in phenazine biosynthesis has been identified in this organism (Mentel et al., 2009). Phenazines are a family of nitrogen-containing tricyclic pigments produced by rhizosphere bacteria including Pseudomonas and Streptomyces (Pierson & Pierson, 2010). Interestingly, it has been shown that some secreted phenazines of Pseudomonas aeruginosa can promote the anaerobic survival of the producer itself via extracellular electron transfer FEMS Microbiol Lett 312 (2010) 110–118 115 Actinomycetes in honeybee guts (a) Fig. 4. (a) Alignment of partial PhzD protein sequences, each of which contains 112 continuous amino acids. M.r., Microbispora rosea; N.d., Nocardiopsis dassonvillei; S.a., Streptomyces anulatus; S.c., Streptomyces cinnamonens; S.l., Streptomyces lomondensis; P.a., Pseudomonas aeruginosa; P.f., P. fluorescens 2–79. The amino acid residues marked with asterisks are conserved and involved in binding of the substrate (Parsons et al., 2003). (b) Evolutionary relationships of the eight PhzD proteins inferred using the neighborjoining method in the MEGA 4.0.2 software. The accession numbers are in the parentheses. The bootstrap consensus tree inferred from 1000 replicates is presented. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test are shown next to the branches. (c) RT-PCR analysis of phzD gene expression in BE74 with total RNAs isolated from mycelia under three growth conditions: lanes 1 and 2, MS agar; lanes 3 and 4, AIA; lanes 5 and 6, AIB broth. Samples in odd-numbered lanes are the negative controls (no reverse transcriptase in the RT reactions) for the samples in the following evennumbered lanes. M, 100 bp DNA marker. The arrow indicates the amplified phzD DNA fragment. BE74 M.r. N d N.d. S.l. S.a. S.c. P.a. P.f. BE74 M.r. N.d. S.l. S.a. S.c. P.a. P.f. ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| 10 20 30 40 50 60 QRGLLKDFWG AGMKAVAEHT DIVPELAPDG EDLLLTKWRY SAFAQTDLAE RMAAQGRDQI QRGLLKDFWG PGMRTDAADR EVVAELTPAE GDWVLTKWRY SAFFRSDLLE RMRAAGRDQL QRGLLKDFWG PGMRRSPEDR LVVDELAPSP DDWMFTKLRY SAFHKSDLLE RMRAAGRDQL RMRAAGRDQ QRGLLKDFWG PGMRTDAADR EVVAELTPAP GDWVLTKWRY SASFRSDLLE RMRAAGRDQL QRGLLKDFWG PGMRPEPEDR QVVDALAPAE QDWMLTKWRY SAFFKTDLLR RMRAAGRDQL QRGLLKDFWG PGMRPAPEDR QVVDALAPTE QDWLLTKWRY SAFFKTDLLE RMRAAGRDQL QRGLLKDFWG PGMRASPADR EVVEELAPGP DDWLLTKWRY SAFFHSDLLQ RMRAAGRDQL QRGLLKDFWG PGMKASPTDR EVVDALAPQP GDWLLTKWRY SAFFNSDLLQ RLHASGRDQL * * * * ....|....| ....|....| ....|....| ....|....| ....|....| .. 70 80 90 100 110 VVTGVYAHIG CQLTAADAFM RDIRPFLIAD ALADFNADYH RMAVRYAAER CA VLCGVYAHVG VLATALEAFT NDIQTFLAAD ALGSFSEAHH RLALDYAAER CA VVCGVYAHVG VLMTAVEAYT NDIQTFLVAD AVADFNADYH RMAVRYAAER CA VLCGVYAHVG VLATALEAFT NDIQTFLAAD ALGDFSEAHH RLALDYAAER CA VLCGVYAHVG VLATAVEAFT HDIQPFFVAD ATADFSEHYH RSALTYAAER CA ILCGVYAHVG VLATAVDAFT HDIQPFFVAD ATADFSQDYH RSALTYAAER CA VLCGVYAHVG VLISTVDAYS NDIQPFLVAD AIADFSEAHH RMALEYAASR CA ILCGVYAHVG VLISSVDAYS NDIQPFLVAD AIADFSKEHH WMAMEYAASR CA ** * (b) M (c) 2 3 4 5 6 phzD 100 bp (Wang et al., 2010). Therefore, we were interested in whether N. alba from the honeybee gut has the phenazine biosynthetic genes and whether they are expressed. The phenazine biosynthetic pathway is branched from the shikimate pathway in bacteria (Mentel et al., 2009). Five genes, phzB, phzD, phzE, phzF and phzG, are required for biosynthesis of the core structure, and they are highly conserved in all known phenazine biosynthetic gene clusters. phzF has been used as a genetic marker for analyzing the diversity and evolution of phenazine biosynthetic pathways in many Gram-negative bacteria, most of which are pseudomonads (Mavrodi et al., 2010). The PCR primers for phzF were tested with BE74 genomic DNA but the reactions did not yield products under the suggested conditions. Instead, PCR primers based on the alignments of phzD genes encoding an isochorismatase from Streptomcyes cinnamonensis DSM 1042, Streptomyces anulatus LU9663 and N. dassonvillei DSM 43111 yielded an 340-bp fragment with the BE74 DNA. Putative protein sequences encoded by this DNA fragment showed the highest homology to a part of PhzD from N. dassonvillei DSM 43111 and other FEMS Microbiol Lett 312 (2010) 110–118 1 homologs (similarity 70–90%) involved in isochorismate metabolism. The protein is unlikely a member of the hydrolase family of primary metabolism that substantially differ from the PhzDs. The major amino acid residues of PhzD involved in binding an isochorismate substrate were found to be encoded in the sequences (Fig. 4a) (Parsons et al., 2003). The two primers were also used to amplify the same region of PhzD homologs from the genomes of two other actinomycetes, Streptomyces lomondensis ATCC25299 and Microbispora rosea ATCC15738, previously known to produce phenazines. Alignments of the partial sequences (112 out of total 207 amino acids) of six actinomycete PhzD proteins allowed the construction of phylogenetic trees (Fig. 4a). The trees constructed with several algorithms have the same topology. Streptomyces lomondensis and M. rosea PhzDs are more closely associated with each other compared with the PhzDs of other two Streptomcyes. Nocardiopsis PhzDs also form their own group, although the sequence of BE74 PhzD is somewhat divergent from that of N. dassonvillei (Fig. 4b). This observation is in contrast to the higher homology (98%) 2010 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved c 116 of the 16S rRNA genes between the two species, which suggests that the two biosynthetic genes in Nocardiopsis species may have evolved differently. To preliminarily investigate the expression of the putative phzD gene, RT-PCR was used to detect the phzD transcript. Total RNAs were isolated from mycelia harvested from MS and AIA agar plates and actinomycete isolation broth (AIA without agar). Cells grown with these media should be in significantly different physiological states. Nonetheless, the phzD gene was always expressed under the three conditions (Fig. 4c). Although regulation of phz gene expression in actinomycetes is unknown, the result herein suggests that the phz mRNAs might be expressed in the Nocardiopsis BE74 cells in various environments. Discussion The gut microbiota of insects is an interesting source of microbial diversity and study of the interactions within an ecological context. Small molecules naturally produced by some environmental bacteria are expected to influence the microbial community as well as the physiology of an insect host, especially when the insects are reared in the wild. In this report, we focused on the selective isolation of actinomycetes from honeybee guts. The majority of the bioactivities produced by the actinomycete isolates were specific against several bee indigenous Bacillus strains and two drugresistant Gram-positive human pathogens. One rare-actinomycete isolate from the honeybee gut identified as a strain of N. alba was preliminarily characterized. Production of phenazine-like redox-active molecules by this isolate could contribute to its ability to temporarily survive the anoxic or anaerobic conditions that may occur in honeybee guts (Andreas et al., 2000; Johnson & Barbehenn, 2000). It was thereafter observed that one type of the modified phenazines, so-called endophenazines, was previously detected as the metabolites of S. anulatus. Four strains of this species producing endophenazines were isolated from the intestines of leaf beetles, millipedes, woodlice and other arthropods collected in various countries over Europe during a search for symbiotic actinomycetes of the animals (Gebhardt et al., 2002). Furthermore, the antimicrobial spectra of endophenazines were reported as having good activity against several Gram-positive bacteria but no activity against Gram-negative bacteria (Gebhardt et al., 2002). Preliminary analysis with the 16S rRNA genes of some isolates in our collection revealed the presence of S. anulatus in honeybee guts, which supports our finding here that similar redox-active molecules are produced by the Nocardiopsis isolate from honeybee guts. Although the relationship between the actinomycetes and insects needs to be further characterized, production of endophenazines might be a first step toward establishing or evolving a symbiotic relationship. It would be interesting to investigate the fre2010 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved c P.B. Patil et al. quency of occurrence of actinomycete phenazine producers in honeybee guts. Various gene-centric pangenomic or multilocus sequence typing approaches could be used. Naturally occurring phenazines are redox-active compounds, traditionally thought as antimicrobials that include over 100 structures (Laursen & Nielsen, 2004). In several Pseudomonas models, the biological roles of phenazines have recently been expanded with implications in microbial interaction processes such as shuttling electron, intracellular signaling, contributing to form biofilm and enhancing anaerobic survival (Pierson & Pierson, 2010). These roles are also expected for phenazines produced by actinomycetes, with possibly additional functions beyond antibiotic because the structural diversity of actinomycete phenazines is even greater and the lifecycle of actinomycetes is generally complex. Phenazines produced by the actinomycetes from honeybee guts probably have structural commonalities even though the producers can be quite different (e.g. Nocardiopsis vs. Streptomyces). Indeed, more actinomycete isolates in our study displayed specific antagonism against a B. marisflavi strain than against other Bacillus strains (Table 1). On the other hand, other microbial metabolites that share an anthranilic acid structural moiety with phenazines, such as actinomycins and quinolones, also have widely known electrochemical properties. In addition, thiols, quinones and coumarins of microbial origins have noticeable electron transfer capabilities. Voltammetric measurements of the purified compounds will shed light on the proposed biological functions of these secondary metabolites. Lastly, some actinomycetes carry numerous stressresponsive genes for maintaining viability in anaerobiosis (van Keulen et al., 2007). Using the extracellular redoxactive secondary metabolites as respiratory electron acceptors could be another survival strategy of actinomycetes. In summary, studying the actinomycetes isolated from honeybee guts and the metabolites produced will yield many insights into the fundamental biology and chemistry of these microorganisms. From a practical standing point, the health and well-being of honeybees is of considerable concern as they are the important agricultural resources. Actinomycete-produced organic compounds have been marketed or are being investigated as insecticides (e.g. spinosad). Given the specificity of the actinomycetes that honeybees retain in their guts and bring back to hives, several important questions have arisen: Are they beneficial bacteria or opportunistic pathogens to the honeybees? Are phenazines virulence factors or contributors to a healthy gut microbial community? Are phenazines present in raw honey and do they contribute to its antimicrobial properties? Phenazines are often produced in large quantities in situ and can be directly detected in the soil or the human tissues colonized with the microorganisms (Wilson et al., 1988; Thomashow et al., 1990). 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