Figures
Abstract
Many Streptomyces strains are known to produce valinomycin (VLM) antibiotic and the VLM biosynthetic gene cluster (vlm) has been characterized in two independent isolates. Here we report the phylogenetic relationships of these strains using both parsimony and likelihood methods, and discuss whether the vlm gene cluster shows evidence of horizontal transmission common in natural product biosynthetic genes. Eight Streptomyces strains from around the world were obtained and sequenced for three regions of the two large nonribosomal peptide synthetase genes (vlm1 and vlm2) involved in VLM biosynthesis. The DNA sequences representing the vlm gene cluster are highly conserved among all eight environmental strains. The geographic distribution pattern of these strains and the strict congruence between the trees of the two vlm genes and the housekeeping genes, 16S rDNA and trpB, suggest vertical transmission of the vlm gene cluster in Streptomyces with no evidence of horizontal gene transfer. We also explored the relationship of the sequence of vlm genes to that of the cereulide biosynthetic genes (ces) found in Bacillus cereus and found them highly divergent from each other at DNA level (genetic distance values≥95.6%). It is possible that the vlm gene cluster and the ces gene cluster may share a relatively distant common ancestor but these two gene clusters have since evolved independently.
Citation: Matter AM, Hoot SB, Anderson PD, Neves SS, Cheng Y-Q (2009) Valinomycin Biosynthetic Gene Cluster in Streptomyces: Conservation, Ecology and Evolution. PLoS ONE 4(9): e7194. https://doi.org/10.1371/journal.pone.0007194
Editor: Mukund Thattai, Tata Institute of Fundamental Research, India
Received: June 7, 2009; Accepted: August 13, 2009; Published: September 29, 2009
Copyright: © 2009 Matter et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This research was supported by funds from the University of Wisconsin-Milwaukee. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Microbial natural products mediate a wide range of biological and biochemical interactions both among microbes and between microbes and higher organisms [1], [2]. Information embedded in natural product biosynthetic genes and gene clusters includes not only biochemical codes for the production of diverse chemical molecules by enzyme catalysts, but also evolutionary and ecological clues about the origin, transmission and distribution of the genetic determinants of natural product biosynthesis. All genes are subject to evolution via vertical transmission (VT), through such processes as point mutation, gene duplication or deletion, recombination,or swapping. In addition to VT, natural product biosynthetic genes and gene clusters are often found to transmit intra- or inter-generically through lateral or horizontal gene transfer (HGT) as well [2], [3]. HGT origins are typically identified by criteria including incongruence between phylogenetic trees constructed with genes involved in primary metabolism [i.e. housekeeping genes such as 16S rDNA, trpB (encoding tryptophan synthetase β-subunit), rpoC1 (encoding RNA polymerase β-chain)] and the genes in question, atypical sequence composition, the presence or absence of genes in closely related genera, and the flanking of genes by insertion elements such as transposons [4].
A classic example of HGT is the transmission of nonribosomal peptide synthetase (NRPS) genes responsible for the production of β-lactam antibiotics (e.g., penicillins and cephalosporins), evidently from bacteria to bacteria and from bacteria to fungi [5], [6]. Two recent surveys of polyketide synthase genes revealed the independent evolution of bacterial speciation and natural product biosynthetic genes, supporting the theory of HGT [7], [8]. In contrast, a study of microcystin biosynthetic genes by Rantala et al. [9] concluded that the microcystin biosynthetic genes from distantly related cyanobacteria appear to share an ancient common ancestor, and have co-evolved along with housekeeping genes for the entire history of toxin production. Furthermore, analysis of the conserved cyanopeptolin biosynthetic genes from three genera of cyanobacteria by Rounge at al. [10] identified independent evolution traces of those genes within each genus, disfavoring an origin by HGT.
Valinomycin (VLM; Figure S1) is a cyclic depsipeptide natural product with a wide range of reported biological activities including insecticidal, nematocidal, antibacterial, antiviral and apoptosis-inducing/cytotoxic/anticancer activities ([11] and references cited in [12]). Eleven Streptomyces strains isolated from around the world were reported to produce variable levels of VLM. The VLM biosynthetic gene cluster (vlm), cloned independently from S. tsusimaensis ATCC 15141 by us [12] and from S. levoris A9 by others [13], consists of two large NRPS genes (vlm1 and vlm2, 10,286 bp and 7,967 bp, respectively) and a few functionally less defined small ORFs. Fifteen distinctive domains in the deduced NRPS mega-enzymes (Vlm1 and Vlm2) are organized into four modules (Figure S1), responsible for the incorporation of four substrate monomers: D-hydroxy-isovaleric acid, D-valine, L-lactic acid, and L-valine, respectively [13]. A C-terminal thioesterase domain on Vlm2 is postulated to mediate the oligomerization of three 4-unit intermediates to a linear full-length precursor and subsequently the cleavage and cyclization of precursor into the final product VLM [12].
Cereulide (Figure S1), an emetic toxin produced by a large number of Bacillus cereus strains [14], [15], is a natural analog of VLM. The cereulide biosynthetic gene cluster (ces), which is located on a mega virulence plasmid related to the B. anthracis toxin plasmid pXO1 [16], [17], shares a high degree of organizational similarity to that of the vlm gene cluster, and contains two large NRPS genes, cesA and cesB, which are highly homologous to vlm1 and vlm2 [13] (Figure S1). These observations prompt one to hypothesize an evolutionary relationship between the ces gene cluster and the vlm gene cluster.
Here we report a survey of the vlm gene cluster among diverse Streptomyces isolates and a phylogenetic comparison to the ces gene cluster. The objectives of this study were: 1) To determine whether all 10 strains of Streptomyces isolates in our sampling, including eight strains reported to produce VLM, and two non-producer strains, actually produce VLM; 2) To determine the placement of VLM-producing Streptomyces strains within an overall phylogenetic analysis of 16S rDNA data for 47 Streptomyces strains and two outgroups (Mycobacterium tuberculosis and Nocardia farcinica); 3) To explore whether the vlm gene cluster of diverse Streptomyces isolates from around the world display any evidence of HGT (as is common in other natural product biosynthetic genes) by comparing phylogenies based on the vlm DNA sequences with those based on the housekeeping genes, 16S rDNA and trpB; and 4) To determine how similar a representative ces gene cluster in B. cereus AH187 type strain is to the vlm gene cluster in Streptomyces at DNA level.
Results
Verification and Quantification of VLM Production
All 10 Streptomyces strains (Table 1) were subjected to unified fermentation conditions and their levels of VLM production were determined by liquid chromatography-mass spectrometry (LC-MS) (Figure S2). Positive ion signals were detected for multiple VLM adducts: [VLM+H]+ = 1111.6 m/z, [VLM+NH4]+ = 1128.7 m/z, [VLM+Na]+ = 1133.6 m/z, and [VLM+K]+ = 1149.8 m/z. Together, these signals form a unique, consistent, four-peaked fingerprint corresponding to VLM, which was detected in each of the eight Streptomyces strains previously reported to produce VLM, eluting at about 14 min. Peak areas were used to estimate the concentrations of VLM detected in each sample, in comparison to a commercially available standard (Sigma Aldrich, St. Louis, MO). VLM production ranges from 4.25 (S. fulvissimus) to 32.8 mg L−1 (S. exfoliatus). The VLM fingerprint was not detected in the negative control strain S. coelicolor or S. hawaiiensis.
Sampling of VLM Biosynthetic Gene Fragments
Of the nine regions (amplicons A to I) targeted for PCR amplification based on homology to the vlm gene cluster of reference strain S. tsusimaensis [12] (Fig. 1a), five regions were successfully amplified from all eight VLM-producing strains; none was amplified from either negative control strains (Fig. 1b). The five amplified regions, corresponding to the reference gene cluster, include: ORF11 (encoding a putative necrosis-inducing factor) (amplicon A), the 3′ region of ORF14 (function unknown) (amplicon D); the 5′ region of vlm1 (1,113 bp, encoding an adenylation domain in Vlm1 NRPS module 1), designated “vlm1” (amplicon G; also see Figure S1); the region spanning the 3′ region of vlm1 (766 bp, encoding part of a condensation domain in Vlm1 NRPS module 3), a short intergenic region (23 bp) and the 5′ region of vlm2 (1,038, encoding part of an adenylation domain in Vlm2 NRPS module 1), designated “vlm1/2” (amplicon H; also see Figure S1), and the 3′ region of vlm2 (668 bp, encoding a terminal thioesterase domain), designated “vlm2” (amplicon I; also see Figure S1). Since the functions of the putative ORFs are not yet established, only the DNA sequences of last three amplicons (G, H and I) were used for phylogenetic analyses and Southern hybridization. DNAs of near-complete 16S rDNA and trpB were successfully amplified from all strains used in this study.
(a) The vlm gene cluster from S. tsusimaensis including two critical nonribosomal peptide synthetase (NRPS) genes (vlm1 and vlm2) and five ORFs [12], and the position of nine regions targeted by DNA amplification (amplicons A to I; also see Figure S1). Amplicons G, H and I were used as molecular probes for downstream experiments. (b) Checkerboard of amplicons that were either amplified successfully by PCR (+, white background,) or not (−, grey background) from test strains (by acronyms. See Table 1 for full names and other information). (c) Southern hybridization patterns of Streptomyces genomic DNAs probed by Amplicon G, H or I, respectively. Strain S. COELI (by acronym) serves as a negative control. Strains identified in a clade during phylogenetic analyses (Fig. 3) are labeled in one color. Approximately 2 µg of total DNA from each strain was digested with PstI. (d) Geographic distribution of the VLM-producing strains around the world with the same color-coded phylogenetic relatedness.
Detection of VLM Biosynthetic Gene Homology by Southern Hybridization
All eight VLM-producing strains (including reference strain S. tsusimaensis) tested positive for genes vlm1 and vlm2, as well as the “vlm1/2” intergenic region; the control strain, S. coelicolor, tested negative (S. hawaiiensis not included due to lack of gel space) (Fig. 1c). Hybridized restriction fragment lengths evidently clustered among the vlm-positive strains. Probe G hybridized to two clear classes of fragment, 5.5 kb and 9.0 kb in size; Probe H, which spans the 3′ region of vlm1 through the 5′ region of vlm2, hybridized to fragments with three size ranges: 6.0 kb, 9.0 kb and >10 kb; and Probe I hybridized to two or three classes of fragment ranging in size from 3.0 kb to >10 kb. Those hybridization patterns are color-labeled and have been found largely in agreement with the following phylogenetic data (see Phylogenetic Analyses), except for strain S. exfoliatus (Malaysia) which deserves more discussion later. Those results suggest that the vlm gene cluster is highly conserved among all VLM-producing strains isolated from a wide range of geographic locations, as probes hybridized during high stringency washes (2×15 min in 0.2×SSC, 0.1% SDS at 68°C), indicating significant sequence identity. Pairwise sequence comparison indicated that vlm amplicons of the same region from all eight VLM-producing strains have greater than 86% of actual sequence identity (Figure S3).
Phylogenetic Analyses
Streptomyces 16S rDNAs.
The 16S rDNA sequences of a total of 49 taxa, including 10 strains obtained by this study, 37 randomly selected Streptomyces strains and two outgroups, were analyzed using MP and BI in an effort to determine how the VLM-producing strains are distributed throughout Streptomyces (Fig. 2; also see Table S3 for detailed parameters). The alignment length is 1413 bases with five indels that were treated as missing data and not scored. Individual sequences range in length from 1376 bp (S. chromogenus NBRC 13374) to 1393 bp (M. tuberculosis H37Rv). MP analysis resulted in 99 equally parsimonious trees; the MP strict consensus and bootstrap trees (not presented) were largely congruent with the BI majority rule consensus tree, especially where bootstrap (BS) or posterior probability (PP) values were moderate to high. While the topology of the non-VLM-producing Streptomyces differs somewhat between the MP and BI generated trees, the clade containing the VLM-producers is identical and moderately supported as monophyletic (BS = 76, PP = 98).
A sampling of 47 randomly selected Streptomyces strains and two outgroups are analyzed by 16S rDNA sequences. Each supported node is labeled with its posterior probability. Strains used in this study are in bold-face type, including negative controls (*).
Within this clade, S. tsusimaensis and S. anulatus (Malaysia) are sister to a largely unresolved clade consisting of all remaining VLM-producers and two additional Streptomyces strains (S. atroolivaceus LMG 19306 and S. microflavus NBRC 13062) not known to produce VLM.
VLM biosynthetic genes.
The data for vlm1, vlm1/2 and vlm2, were analyzed separately, using MP. The trees produced had very similar topologies (Fig. 3 and Table S3). All trees were midpoint rooted since an appropriate outgroup is not available for vlm data. In all three trees, grouping of strains (BP = 100) are consistent: S. tsusimaensis and S. anulatus (Malaysia) formed a clade sister to the remaining Streptomyces species, S. exfoliatus are sister to S. sp. PRL1642 and S. griseus 10/ppi, and the rest group together with slightly variable topologies.
Analysis of vlm1, vlm1/2 and vlm2 sequences from all eight VLM-producing strains generated almost identical phylogenetic trees, indicating the representability of short gene fragments for not-so-easily accessible sequences of large natural product biosynthetic genes (e.g. NRPS genes).
The partition homogeneity test for vlm1, vlm1/2, and vlm2 supported the congruence and subsequent combination of the three gene segments (P = 0.259). Also, the trees from different regions of the vlm genes are similar in topology (Fig. 3); therefore the data sets were combined for MP and BI analyses. Both model test algorithms chose GTR+G [18], [19] as the best fitting model for individual vlm sequences; therefore, there was no need to partition data when running BI on the combined data set. The overall topology of both the MP and BI trees produced from the combined vlm data are congruent with the trees produced from individual data sets, recognizing the same three clades (Fig. 4B, left half). All support values of the combined vlm data were extremely high (BS≥98, PP = 100).
(A) ML phylogram resulting from analysis of vlm/ces data for Streptomyces strains and a Bacillus cereus type strain AH187. (B) Strict consensus trees resulting from maximum parsimony (MP) analyses of combined vlm (left half) data and combined 16S rDNA/trpB (right half) data. The vlm data set is mid-point rooted; the 16S rDNA/trpB data set is outgroup rooted with Nocardia farcinica. Branch lengths appear above the branches; bootstrap values (1000 replicates) and Bayesian inference (BI) analysis posterior probability values (parentheses) appear below. Species acronyms are as in Fig. 4A.
The translated protein sequences of the vlm data sets (Vlm1, Vlm1/2, Vlm2 and Vlm-combined) were also analyzed by MP in order to confirm the robustness of the nucleotide MP analysis and to identify a potential outgroup for rooting purposes. The topologies of the resultant trees are similar to those from the nucleotide data (trees not presented). Specifically, MP analysis of the combined Vlm protein data set produced two equally parsimonious trees with low homoplasy (see Table S3 for detailed parameters). When the tree was outgroup rooted, the topology of the combined Vlm protein data set was the same as for the nucleotide data, except for the third clade, which collapsed in the strict consensus tree to form a trichotomy. Bootstrap values for supported branches are all 100%, not including the S. anulatus (USA)/S. griseus 1/k branch, which is weakly supported (BS = 58).
Assessing similarity of the vlm (Streptomyces) and the ces (Bacillus cereus) gene clusters.
The F81 pairwise analysis of genetic distances demonstrated that the vlm sequences of all Streptomyces strains are relatively distant from the representative ces sequence of B. cereus AH187 (Table S4). All vlm sequences sampled had genetic distances ranging from 0.82–12.8%; whereas the distances between vlm and ces sequences ranged between 95.6–98.9%, suggesting that the vlm gene cluster and the ces gene clusters evolved independently for a relatively long period of time. Aligning the Bacillus ces sequence with Streptomyces vlm sequences was actually very difficult and presented many homology issues. Because of this, we included the ces sequence of B. cereus in our ML analyses only to give some idea of the long branch that separates the ces data from all Streptomyces vlm sequences (Fig. 4A) and did not employ ces sequence as an outgroup for vlm analyses (Fig. 3 and Fig. 4B).
16S rDNA and trpB of the VLM-producing strains.
The 16S rDNA and trpB gene sequences of eight VLM-producing strains, along with those of negative control strains, were analyzed separately using MP to determine the phylogeny reflective of Streptomyces, to assess its congruence with that of the vlm gene fragments. All phylogenies are outgroup rooted with Nocardia farcinica. In the MP tree of 16S rDNA data set (tree not presented), the VLM-producing strains form a well supported clade (BS = 96), with S. tsusimaensis sister to all remaining strains. Within this grouping, S. anulatus (Malaysia) is sister to a basal polytomy consisting of S. sp. PRL 1642, S. exfoliatus, S. griseus 10/ppi and an unresolved clade consisting of the three remaining VLM-producing strains.
The MP analysis of the trpB data resulted in seven equally parsimonious trees (Table S3). In the MP strict consensus tree, all of the Streptomyces strains, non-VLM and VLM-producers group in a well supported (BS = 100), unresolved polytomy (tree not presented). Within this large polytomy the following group as sister species: S. coelicolor with S. hawaiiensis (non-VLM producers); S. tsusimaensis with S. anulatus (Malaysia); S. sp PRL 1642 with S. griseus 10/ppi; and S. anulatus (USA) sister to S. fulvissimus and S. exfoliatus.
Because both the 16S rDNA and trpB data sets produced unresolved MP trees, the data sets were then combined even though the P-score for the partition homogeneity test was low (P = 0.01; see Table S3) in order to increase resolution. A known weakness of the partition homogeneity test is incorrect rejection of the null hypothesis when the number of informative sites is low [20]. Furthermore where individual trees are resolved, especially with moderate to excellent support (BS≥70), the topology is similar between the 16S rDNA and the trpB trees. MP analysis of the combined data resulted in five equally parsimonious trees. In the MP strict consensus tree, all of the VLM-producers form a monophyletic group with S. tsusimaensis and S. anulatus (Malaysia) forming a basal trichotomy with a clade consisting of all remaining VLM-producers. S. exfoliatus, the sister species S. sp. PRL 1642 and S. griseus 10/ppi, and a clade consisting of S. anulatus (Malaysia), S. fulvissimus and S. griseus 1/k, form a trichotomy as well (Fig. 4B, right half).
BI analysis was also performed on the 16S rDNA/trpB combined data set to compare with the results of MP analysis. The best fitting nucleotide substitution model chosen by both hLRTs and AIC for the 16S rDNA data set is GTR+I+G, while the best fitting model for trpB is GTR+I [18], [19]. Therefore, the combined data set was partitioned to reflect these differing models. The majority rule consensus tree has a similar topology to that of the MP tree (tree not presented), but S. tsusimaensis and S. anulatus (Malaysia) are included in a weakly supported clade (PP = 0.56) with S. coelicolor and S. hawaiiensis.
Combined vlm/16S rDNA/trpB of the VLM-producing strains.
All three vlm data sets were combined with 16S rDNA and trpB data for MP and BI analysis, supported by a strong P-score of 0.405 for the partition homogeneity test (Table S3). This was done to substantiate congruence between the vlm data with that of the housekeeping genes, 16S rDNA and trpB. MP and BI analyses produced unrooted trees (not presented) with the same topology as the combined vlm data (Fig. 4B) and with highly supported branches (BS≥99 and PP = 1.00).
Other Analyses
The vlm1 and vlm2 datasets have a slightly lower G+C content (69 and 67%, respectively) than that of vlm1/2 (71%), but the G+C content (70%) of the combined vlm sequence is in close proximity to what is typical for Streptomyces (e.g. 72% in S. coelicolor [21]) (Table S5). Furthermore, the vlm data sets were analyzed for non-synonymous versus synonymous substitution rates using the SNAP program [22]. Calculations of ds (synonymous) to dn (non-synonymous) substitutions included: 0.60 (vlm1), 1.72 (vlm1/2), 1.34 (vlm2) and 1.57 (vlm-combined).
Pulse field gel electrophoresis and subsequent Southern hybridization with the amplicon G probe indicates that the vlm gene cluster is on the chromosome of every VLM-producer and not on a plasmid (data not shown). This is in agreement with the findings by [23] who likewise reported that the putative vlm gene cluster of S. levoris A9 is present on the chromosome and not on a plasmid.
Discussion
The vlm gene cluster (Fig. 1a, also see Figure S1), represented by the three DNA regions surveyed in this study, is highly conserved among all eight VLM-producing strains. Strong similarity is indicated by the ability to amplify all three regions of the vlm genes from all taxa (except negative control strains) by PCR using primers based on the sequences of reference strain S. tsusimaensis (Fig. 1b). Strong similarity among the vlm genes of VLM-producing strains is also supported by the positive results of Southern analysis. Each of the three regions was detected in all VLM-producing strains under conditions of high stringency (Fig. 1c). Restriction fragment length patterns also generally correlate with the three clades: 1) S. tsusimaensis and S. anulatus (Malaysia), 2) S. sp. PRL1642 and S. griseus 10/ppi, and 3) S. anulatus (USA), S. fulvissimus and S. griseus 1/k. The remaining one, S. exfoliatus (Malaysia), groups variably with others; its pattern is similar to the clades 2) and 3) when probed for vlm1 (amplicon G), close to the clade 1) when probed for vlm1/2 (amplicon H), but more similar to the clades 1) and 3) when probed for vlm2 (amplicon I). Variations of the restriction fragment length patterns may attribute to very recent point mutations that have altered certain restriction enzyme sites.
VLM was detected by LC-MS from all strains verified to contain the vlm gene cluster, thus indicating that the gene cluster is intact and was actively expressed in every strain under the conditions tested. Fermentation conditions were not optimized for individual strains; therefore, the levels of VLM production were relative. The active production of this antibiotic by all strains containing the gene cluster implies that VLM must confer certain selective advantages in nature. Nevertheless, the vlm gene cluster is not essential. Our previous studies have generated vlm–gene insertion mutants which completely lost VLM production but had no apparent defect in growth or differentiation [12]. VLM was repeatedly discovered for its agricultural and potential pharmaceutical applications ([11] and references cited in [12]). It remains intriguing yet difficult to probe the indigenous function of VLM in Streptomyces.
Despite a significant structural similarity between VLM and cereulide and an organizational similarity between the vlm gene cluster and the ces gene cluster [13] (Figure S1), our analyses did not identify a close relationship between these two gene clusters. The fact that the overall G+C content of the vlm gene cluster of Streptomyces is about 70% [12] and that of the ces gene cluster of B. cereus is about 36% [16], [17], each of which falling within the typical G+C content range of the organism that harbors the gene cluster, suggests that the codon usage in these two groups of bacteria is very different. It is possible that the vlm gene cluster and the ces gene cluster may share a relatively distant common ancestor but these two gene clusters have since evolved independently.
Combined analysis of the nucleotide sequence from the three vlm gene regions, vlm1, vlm1/2 and vlm2, resulted in a more robust phylogeny than individual data sets using MP and BI analyses. The differing relationships of S. griseus species, S. fulvissimus, and S. anulatus (USA) among the various gene regions are weakly supported, probably due to a low number of evolutionary changes between the taxa. Within the Vlm1 protein data set in particular, zero to two character states define the S. anulatus (USA)/S. fulvissimus relationship. The amount of parsimony informative characters is substantially increased during combined analysis, thus improving the overall accuracy of the phylogenetic inference.
MP and BI analyses of the individual and combined 16S rDNA and trpB data sets resulted in less resolved phylogenies, although the consensus trees are congruent with the combined vlm sequence MP and BI trees where resolved (Fig. 4B). 16S rDNA has been known to be too conserved to resolve relationships among closely related strains [24]. Concatenating 16S rDNA sequence with other housekeeping genes such as trpB had previously resolved relationships between Streptomyces species with success [25]. Nevertheless, the additional character information from trpB is not enough to completely resolve the relationships between the highly related strains in this study, but it does provide independent support about the correct root of the Streptomyces tree and additional support for clades.
Surprisingly, the VLM-producers do not group by geographic origin (Fig. 1d). Most notably, S. griseus 1/k and 10/ppi, which were isolated from the same area [26], occur in separate clades. Based on MP analysis of the combined vlm sequences, these two strains differ by at least 322 characters, as opposed to less than 100 characters between them and their nearest neighbors (both different species). A similar situation is seen between the S. anulatus strains (Malaysia and USA). As microbial distribution is a result of both environmental and dispersal processes [27], it remains unclear how these taxonomically closely related bacterial strains dispersed around the globe while two isolates from the same area are phylogenetically distinct from each other based on both housekeeping genes and secondary metabolic genes. These observations warrant revisiting the taxonomic definition of some of the VLM-producers, as many of the strains were defined decades ago.
Evolutionary patterns within natural product biosynthetic genes and gene clusters generally show strong evidence of HGT, supported by certain signatures such as incongruence between phylogenetic trees of housekeeping genes versus the gene in question [4]. Although the resolution of the phylogenetic tree produced from the 16S rDNA/trpB combined data set is not as robust as that produced from the combined vlm data set, the tree reveals complete congruence with the phylogenetic tree of the vlm sequences (Fig. 4B). Furthermore, the partition homogeneity test between the vlm data sets and the housekeeping genes resulted in a P-value of 0.405, indicating statistical congruence [28]. Both results suggest that the vlm genes represent organismal evolution by VT not HGT. In other words, the vlm genes have co-evolved along with the housekeeping genes, at least in a more recent evolutionary timeframe.
A similar, but less resolved topology with the VLM-producers was found when sampling the 16S rDNA of many more Streptomyces taxa (Fig. 2). This larger 16S rDNA tree was constructed to represent the distribution of Streptomyces species throughout the genus. The tree reveals that the VLM-producing Streptomyces are not randomly distributed, but rather found in a well-supported monophyletic clade (BS = 76, PP = 98) that includes two additional Streptomyces strains. Because of their presence in this grouping, S. atroolivaceus LMG 19306 and S. microflavus NBRC 13062, were recently obtained from public stock centers but tested negative for both VLM production and the presence of vlm gene fragments (data not shown). It is not clear whether these two strains have never had the gene cluster, or if they have completely lost the vlm gene cluster. The monophyletic grouping of the vlm gene cluster within the Streptomyces species is highly suggestive of inheritance from a common ancestry by VT rather than by HGT, which would typically result in a more sporadic distribution throughout the trees, amongst less highly related strains [29].
Materials and Methods
Sampling, Bacterial Strains and Cultivation Conditions
Our sampling includes eight available VLM-producing Streptomyces strains (out of 11 strains reported in the literature) isolated from diverse geographic locations around the world, and two non-producer control strains (Table 1; Fig. 1d). These Streptomyces strains were obtained either from public culture collections or from the Pettit laboratory. Strains were grown from spore suspensions (stored at −80°C) in tryptic soy broth (TSB) supplemented with 34% (w/v) glucose and 0.5% (w/v) glycine in shake flasks containing glass beads to break apart mycelia. Cultures were incubated at 30°C with agitation at 150 rpm for approximately 60 hr before harvested for total DNA preparation by Kirby mix procedure described in [30].
Valinomycin Detection
VLM production was detected and quantified by LC-MS, similarly to previously described [12]. All 10 Streptomyces strains were grown in 50 ml of fermentation medium at 30°C for 6 days under constant agitation (150 rpm). Cells and resins (Diaion HP-20 from Supelco, Bellefonte, PA) were then collected together by centrifugation at 4,000×g for 20 min at ambient temperature and lyophilized to dryness. Crude VLM preparation was obtained by extracting the dried cell debris and resins with 25 ml methanol. Twenty µl of this preparation was injected into an 1100 Series LC/MSD Trap mass spectrometer (Agilent, Santa Clara, CA) for detection of the positive ion signals of VLM. The LC program included a 5-min linear gradient from buffer A (50% acetonitrile with 0.1% formic acid) to buffer B (99.9% acetonitrile with 0.1% formic acid), a constant elution in buffer B for 10 min, followed by a linear gradient return to buffer A in 5 min. Samples were fractionated by a 2.1×50 mm Eclipse Plus-C18 column (Agilent) with a flow rate of 0.5 ml min−1.
Primer Design and Sequence Analyses
Primers (see Table S1 for details) were designed to amplify and sequence vlm gene fragments based on conserved regions of protein sequences. Protein homologies were identified by protein-protein BLAST (blastp), position-specific iterated and pattern-hit initiated BLAST (PSI- and PHI-BLAST) using the published vlm gene cluster sequence (GenBank accession no. DQ174261) of S. tsusimaensis as reference. Subsequent protein sequences were aligned using ClustalW [31] as part of the LaserGene software package (DNAStar, Madison, WI). Sequences for individual isolates were aligned using SeqMan. Individual chromatograms were inspected for integrity and ambiguous bases were corrected when there was a sequence overlap. Consensus sequences for individual isolates were exported, edited with SeqEdit (when concatenated to another data set), and imported into MegAlign for alignment under the criteria set by ClustalW. Alignments were manually inspected before exporting in the Nexus format [32]. Additional corrections were made using McClade 4, v. 4.01 [33], including end trimming to remove areas of missing data, and further alignment of indels (insertions and deletions) before phylogenetic analysis.
PCR, DNA Sequencing and Southern Analysis
A typical PCR reaction contained 50–100 ng of total DNA, 7.5% DMSO, 100 µM of each dNTP, 1×ThermoPol buffer, 2.5 units of Taq DNA polymerase (New England BioLabs, Ipswich, MA), and 0.5 µM of each primer (Operon Biotechnologies, Huntsville, AL) in a total volume of 50 µl. PCR was carried out at multiple program conditions with multiple sets of primers in order to optimize amplification for each gene fragment. Weakly amplified DNAs were cloned into pGEM–T Easy vector (Promega, Madison, WI) for propagation. DNA sequencing was performed at the University of Wisconsin-Madison Biotechnology Center. Three probes were prepared from purified PCR products of S. tsusimaensis that include amplicons G, H and I (Fig. 1a). Labeling of DNA as probe, Southern blotting, hybridization and detection were performed per manufacturer's instructions, using the DIG High Priming DNA Labeling and Detection Starter Kit II (Roche Diagnostics, Indianapolis, IN).
Phylogenetic Analyses
The 16S rDNA sequences of the eight VLM-producing strains obtained by this study, together with those of two negative control strains [S. hawaiiensis NRRL 15010 and S. coelicolor A3(2)], and 37 randomly selected Streptomyces strains and two outgroups (of Mycobacterium tuberculosis H37Rv and Nocardia farcinica IFM 10152) that were obtained from The Ribosomal Database Project (RDP-II) [34] or the GenBank database (see Table S2 for GenBank accession numbers), were analyzed using MP and BI in an effort to determine how the VLM-producing strains are distributed throughout Streptomyces.
Data sets were analyzed independently and in combination for phylogenetic reconstruction using the analytical methods maximum parsimony (MP) and Bayesian inference (BI). MP analyses were performed using PAUP* 4.0b10 [35], employing the branch and bound search option for all data sets except for those containing greater than 20 taxa; for these a heuristic search using 100 random addition sequence replicates was employed. Since indels were not plentiful and the additional information did not improve tree resolution, gaps were treated as missing data and not scored. Outgroups were chosen based on homology and ease of sequence alignment in order to root trees. In cases where outgroups were unavailable (for vlm gene nucleotide data), trees were midpoint rooted. If multiple best trees were produced, a strict consensus tree was calculated. Bootstrapping calculations were performed with 1000 replicates and a branch and bound search for the Streptomyces 16S rDNA data set, for which a heuristic search with 100 replicates was used due to the large size of the data set (40+ taxa, 1300 bp each). Consistency indices (measuring degree of homoplasy) were calculated excluding uninformative characters to prevent artificially inflated values [36].
Before combining any data sets, a partition homogeneity test (otherwise known as the incongruence length difference test) [37], was implemented in PAUP* 4.0b10 using 1000 branch and bound search replications to determine compatibility. This test measures the character congruence of two or more data sets. The null hypothesis of congruence is accepted (P≥0.05) by calculating the probability that the sum of the most-parsimonious tree lengths derived from random partitions of the data sets is equal to or lower than the sum of the tree lengths from the individual data sets. If the probability is low (P≤0.05), i.e. the random partitions have more homoplasy than the individual data sets, then the null hypothesis of congruence is rejected. When the null hypothesis is rejected, the data sets are considered incongruent and are not necessarily suitable for combination.
BI analyses were conducted using MrBayes 3.1 [38]. Best-fit substitution models for individual data sets were identified using the hierarchical likelihood ratio test (hLRT) and the Akaike Information Criterion (AIC) implemented in MrModelTest v2.2 [39]. Data were partitioned if different models were identified for individual data sets before combination. Parameters were set for the general model of DNA substitution [general time reversible (GTR); nst = 6] with different rate variations (as determine by hLRT and AIC), and otherwise flat priors. Markov chains (four, three heated) were run for one or two million generations, sampling trees every 100 generations for a total of 10,001 or 20,001 samples. Stationarity was determined to have been reached at 10% of the total sample size; therefore the first 1,000 or 2,000 trees were discarded as the burn-in phase. The standard deviation of split frequencies and parameter values were checked [potential scale reduction factor (PSRF) = 1.0] to verify that the analyses had been run long enough. Majority rule consensus trees were summarized and exported, showing posterior probabilities and average branch lengths.
To assess the similarity of a representative ces gene cluster in B. cereus AH187 type strain to the vlm gene cluster in Streptomyces, concatenated sequence regions of vlm genes were compared to those of ces genes. Alignments were accomplished using ClustalW [31], with subsequent manual alignment by eye in McClade 4, v. 4.01 [33]. Genetic distances were calculated using PAUP* and the F81 algorithm [40], which accounts for the unequal base frequencies of our data [41]. To show branch lengths via a phylogram, an ML analysis was performed with Garli vers. 0.95 [42], using the default parameters with “save every improved topology” unchecked.
Other Analyses
G+C content and sequence divergence calculations were performed using shareware programs respectively: GCUA (General Codon Usage Analysis) [43] and SNAP (Synonymous Non-synonymous Analysis Program; www.hiv.lanl.gov) [22]. Pulse field gel electrophoresis was performed on all VLM-producing strains according to the manufacturer's instructions (Bio-Rad, Hercules, CA).
Supporting Information
Table S1.
Primers used to amplify the VLM biosynthetic gene (vlm) fragments, 16S rDNA and trpB.
https://doi.org/10.1371/journal.pone.0007194.s001
(0.08 MB DOC)
Table S2.
Strains and GenBank accession numbers of DNA sequences used for phylogenetic analyses.
https://doi.org/10.1371/journal.pone.0007194.s002
(0.14 MB DOC)
Table S3.
Primary data of phylogenetic analyses.
https://doi.org/10.1371/journal.pone.0007194.s003
(0.09 MB DOC)
Table S4.
Genetic distances calculated between concatenated vlm (Streptomyces) and ces (Bacillus cereus) DNA sequences.
https://doi.org/10.1371/journal.pone.0007194.s004
(0.05 MB DOC)
Table S5.
G+C content and substitution rates for the vlm data sets.
https://doi.org/10.1371/journal.pone.0007194.s005
(0.04 MB DOC)
Figure S1.
Structures of valinomycin and cereulide, and the domain/module organization and substrate specificity of their respective NRPSs.
https://doi.org/10.1371/journal.pone.0007194.s006
(0.40 MB TIF)
Figure S2.
Verification and quantification of VLM production.
https://doi.org/10.1371/journal.pone.0007194.s007
(0.13 MB TIF)
Figure S3.
Percentage identity and divergence of pairwise comparison among the vlm sequences.
https://doi.org/10.1371/journal.pone.0007194.s008
(1.32 MB TIF)
Acknowledgments
We thank Robin Pettit for providing three Streptomyces isolates. We also appreciate anonymous reviewers' helpful comments.
Author Contributions
Conceived and designed the experiments: AMM YQC. Performed the experiments: AMM PDA YQC. Analyzed the data: AMM SBH SSN YQC. Contributed reagents/materials/analysis tools: SBH PDA. Wrote the paper: AMM SBH YQC.
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