Screening and identification of actinobacteria from marine sediments: Investigation of potential producers for antimicrobial agents and type I polyketides

Marine actinobacteria were isolated from the sediment samples collected in Xinghai Bay, Xiaoping Island and Changhai in Dalian, China. Fifteen selective media were employed, of which Humic acid-Vitamin medium recovered the highest number of isolates. Eleven of the 239 isolates obtained from the selective media were selected for further investigations based on colony morphology and pigment formation. Phylogenetic analysis of their 16S rRNA sequences showed that these strains belong to the genera of Streptomyces and Micromonospora. One of these strains identified is a new species of Streptomyces. Type I polyketide synthase (PKSI) gene fragments were amplified from three strains. The PKSI sequence of one of these strains (S187) showed high homology to the KS gene involved in meridamycin biosynthesis. Based on this result, the neurotrophic activity of S187 was further investigated. Culture broth of S187 was applied to rat pheochromocytoma (PC12) cells, and a 2.3-fold increase in growth over control cells was observed by the 3-(4,5-dimethylthiazolyl)-2,5-diphenyltetrazolium bromide assay. These results indicated the importance of further exploration of the marine actinobacteria in Dalian sea area for antimicrobial agents and type I polyketides.     

Evolutionary evidence on suitability of SecD as a target for development of antibacterial agents against Staphylococcus aureus

Staphylococcus aureus causes many infections and its drug resistance is a worrying challenge for medical care. The SecD subunit of Sec secretion system in methicillin‐resistant S. aureus is an attractive target because SecD dysfunction leads to the death of bacteria and SecD as a target is more efficient than SecA and SecF. Evolution could have made SecD to become insensitive to antibacterial agents although the drugs directly against SecD have yet to develop. So far, no detailed information on SecD evolution has been available, thus 2686 SecD sequences with full taxonomic information from kingdom to species were analyzed. First, the variance of pairwise p‐distance was evaluated for each taxonomic group. Second, the variance was further partitioned into intergroup and intragroup variances for quantification of horizontal and vertical gene transfer. Third, phylogenetic tree was built to trace the evolutionary pathway. The results showed that overall evolution of SecDs appears to have undergone horizontal and vertical gene transfer. Only 0.5% horizontal transfers were found between any two SecDs in S. aureus, 6.8% and 8.8% horizontal transfers were found between any two Staphylococcus SecDs from different and the same species, and only one SecD from S. aureus was located far away from its sister cluster. Thus, statistic and evolutionary analyses demonstrate that the SecDs from staphylococcus species have a small chance of mutating, and provide taxonomic evidence to use the SecD as a potential target for new generation of antibacterial agents against S. aureus.     

Naturally mosaic operons for secondary metabolite biosynthesis: variability and putative horizontal transfer of discrete catalytic domains of the epothilone polyketide synthase locus

A putative instance of horizontal gene transfer (HGT) involving adjacent, discrete -ketoacyl synthase (KS), acyl carrier protein (ACP) and nonribosomal peptide synthase (NRPS) domains of the epothilone Type I polyketide biosynthetic gene cluster from the myxobacterium Sorangium cellulosom was identified using molecular phylogenetics and sequence analyses. The specific KS domain of the module EPO B fails to cluster phylogenetically with other epothilone KS sequences present at this locus, in contrast to what is typically observed in many other Type I polyketide synthase (PKS) biosynthetic loci. Furthermore, the GC content of the epoB KS, epoA ACP and NRPS domains differs significantly from the base composition of other epothilone domain sequences. In addition, the putatively transferred epothilone loci are located near previously identified transposon-like sequences. Lastly, comparison with other KS loci revealed another possible case of horizontal transfer of secondary metabolite genes in the genus Pseudomonas. This study emphasizes the use of several lines of concordant evidence (phylogenetics, base composition, transposon sequences) to infer the evolutionary history of particular gene and enzyme sequences, and the results support the idea that genes coding for adaptive traits, e.g. defensive natural products, may be prone to transposition between divergent prokaryotic taxa and genomes.Communicated by W. Arber     

Phylogenetic analysis of polyketide synthase I domains from soil metagenomic libraries allows selection of promising clones

The metagenomic approach provides direct access to diverse unexplored genomes, especially from uncultivated bacteria in a given environment. This diversity can conceal many new biosynthetic pathways. Type I polyketide synthases (PKSI) are modular enzymes involved in the biosynthesis of many natural products of industrial interest. Among the PKSI domains, the ketosynthase domain (KS) was used to screen a large soil metagenomic library containing more than 100,000 clones to detect those containing PKS genes. Over 60,000 clones were screened, and 139 clones containing KS domains were detected. A 700-bp fragment of the KS domain was sequenced for 40 of 139 randomly chosen clones. None of the 40 protein sequences were identical to those found in public databases, and nucleic sequences were not redundant. Phylogenetic analyses were performed on the protein sequences of three metagenomic clones to select the clones which one can predict to produce new compounds. Two PKS-positive clones do not belong to any of the 23 published PKSI included in the analysis, encouraging further analyses on these two clones identified by the selection process.     

Horizontal Transfer of Two Operons Coding for Hydrogenases Between Bacteria and Archaea

Using a phylogenetic approach, we discovered three putative horizontal transfers between bacterial and archaeal species involving large clusters of genes. One transfer involves an operon of 13 genes, called mbx, wich probably was transferred into the genome of Thermotoga maritima from a species belonging or close to the Pyrococcus genus. The two others implied an operon of six genes, called ech, transferred independently to the genomes of Thermoanaerobacter tengcongensis and Desulfovibrio gigas, from a species belonging or close to the Methanosarcina genus. All these transfers affected operons coding for multisubunit membrane-bound (NiFe) hydrogenases involved in the energy metabolism of the donor genomes. The functionality of the transferred operons has not been experimentally demonstrated for T. maritima, whereas in D. gigas and T. tengcongensis the encoded multisubunit hydrogenase could have a role in energy conservation. This report adds several cases of horizontal gene transfers among hydrogenases already described.Reviewing Editor: Dr. Siv Andersson     

Phylogenetic Analysis of Polyketide Synthase I Domains from Soil Metagenomic Libraries Allows Selection of Promising Clones

The metagenomic approach provides direct access to diverse unexplored genomes, especially from uncultivated bacteria in a given environment. This diversity can conceal many new biosynthetic pathways. Type I polyketide synthases (PKSI) are modular enzymes involved in the biosynthesis of many natural products of industrial interest. Among the PKSI domains, the ketosynthase domain (KS) was used to screen a large soil metagenomic library containing more than 100,000 clones to detect those containing PKS genes. Over 60,000 clones were screened, and 139 clones containing KS domains were detected. A 700-bp fragment of the KS domain was sequenced for 40 of 139 randomly chosen clones. None of the 40 protein sequences were identical to those found in public databases, and nucleic sequences were not redundant. Phylogenetic analyses were performed on the protein sequences of three metagenomic clones to select the clones which one can predict to produce new compounds. Two PKS-positive clones do not belong to any of the 23 published PKSI included in the analysis, encouraging further analyses on these two clones identified by the selection process.     

Population genomics and speciation in yeasts

The advent of efficient whole genome sequencing and the large molecular and genetic toolbox available for studies in Saccharomyces cerevisiae and related species have allowed unprecedented analysis of big issues such as: what causes reproductive isolation and eventual speciation? The species complex encompassing S. cerevisiae and relatives consists of six species and several naturally occurring hybrids, which have nearly collinear genomes. They fit the biological species definition of within species fertility and between species sterility. There are examples of chromosome rearrangements and of genetic incompatibilities between species of the complex, which contribute to reproductive isolation but these are not universally present. In addition, simple sequence divergence has been shown to cause reproductive isolation via the action of the mismatch repair system. Although all three of these mechanisms contribute to extant reproductive isolation, which if any, drive the speciation process is still an open question. Population genomic surveys of whole genome sequences reveal introgressions and horizontal gene transfers between species, indicating that the species barriers are not complete. This gene flow between species, although infrequent, brings into question the nature of yeast species.     

Horizontal Gene Transfers in prokaryotes show differential preferences for metabolic and translational genes

Background  

Horizontal gene transfer (HGT) is an important process, which contributes in bacterial pathogenesis and drug resistance. A number of methods have been proposed for detection of horizontal gene transfer. One successful approach to the detection of HGT events is due to Novichkov et al. (J. Bacteriology 186, 6575–85), who rely on comparing phylogenetic distances within a gene family with genomic distances of the source organisms. Building on their approach, we introduce outlier detection in the correlation between those two sets of distances. This approach is designed to detect horizontal transfers of core set of genes present in many bacteria. The principle behind method allows detection of xenologous gene displacements as well as acquisition of novel genes.     

Birth,death and horizontal transfer of the fumonisin biosynthetic gene cluster during the evolutionary diversification of Fusarium

Fumonisins are a family of carcinogenic secondary metabolites produced by members of the Fusarium fujikuroi species complex (FFSC) and rare strains of Fusarium oxysporum. In Fusarium, fumonisin biosynthetic genes (FUM) are clustered, and the cluster is uniform in gene organization. Here, sequence analyses indicated that the cluster exists in five different genomic contexts, defining five cluster types. In FUM gene genealogies, evolutionary relationships between fusaria with different cluster types were largely incongruent with species relationships inferred from primary‐metabolism (PM) gene genealogies, and FUM cluster types are not trans‐specific. In addition, synonymous site divergence analyses indicated that three FUM cluster types predate diversification of FFSC. The data are not consistent with balancing selection or interspecific hybridization, but they are consistent with two competing hypotheses: (i) multiple horizontal transfers of the cluster from unknown donors to FFSC recipients and (ii) cluster duplication and loss (birth and death). Furthermore, low levels of FUM gene divergence in F. bulbicola, an FFSC species, and F. oxysporum provide evidence for horizontal transfer of the cluster from the former, or a closely related species, to the latter. Thus, uniform gene organization within the FUM cluster belies a complex evolutionary history that has not always paralleled the evolution of Fusarium.     

Molecular evolution in the gnd locus of Salmonella enterica

The gnd gene, the structural gene for 6-phosphogluconate dehydrogenase, was sequenced and analyzed in 34 isolates from different serovars of the seven subspecies of Salmonella enterica to provide comparative information on the evolution in this gene, which has been studied extensively in Escherichia coli. The gene tree obtained by the neighbor- joining method in general gave separate branches for each subspecies, with the few exceptions readily explained by recombination. There is evidence of recombination involving transfer of long (more than 400 bp) and short (30-150 bp) segments of DNA. Four of the six long-segment transfers detected are at the 5' end of the gene, and in all four cases a variant of the chi sequence is located close to the recombination junction and appears to have mediated the recombination events. We suggest that in these four cases and in a fifth case with intersubspecies transfer of the whole gnd gene, the adjacent rfb (O antigen) locus may have been transferred in the same event. The estimates of the number of synonymous substitutions per synonymous site, KS, and the number of nonsynonymous substitutions per nonsynonymous site, KA, within the E. coli and S. enterica gnd genes, and also between the two species show an interesting distribution, with KS being lower toward the ends of the gene and KA in particular being lower in the first than in the second domain. In S. enterica, synonymous sites also seem to be subjected to negative selection. The ratio of KA to KS was higher within S. enterica and E. coli than between them, which may indicate that intraspecies variation is essentially between clones and that mildly deleterious mutations can be fixed within clones, which would thus raise KA within species.      

Habitat-specific type I polyketide synthases in soils and street sediments

Actinomycetes produce many pharmaceutically useful compounds through type I polyketide biosynthetic pathways. Soil has traditionally been an important source for these actinomycete-derived pharmaceuticals. As the rate of antibiotic discovery has decreased and the incidence of antibiotic resistance has increased, researchers have looked for alternatives to soil for bioprospecting. Street sediment, where actinomycetes make up a larger fraction of the bacterial population than in soil, is one such alternative environment. To determine if these differences in actinomycetal community structure are reflected in type I polyketide synthases (PKSI) distribution, environmental DNA from soils and street sediments was characterized by sequencing amplicons of PKSI-specific PCR primers. Amplicons covered two domains: the last 80 amino acids of the ketosynthase (KS) domain and the first 240 amino acids of the acyltransferase (AT) domain. One hundred and ninety clones from ten contrasting soils from six regions and nine street sediments from six cities were sequenced. Twenty-five clones from two earthworm-affected samples were also sequenced. UniFrac lineage-specific analysis identified two clades that clustered with actinomycetal GenBank matches that were street sediment-specific, one similar to the PKSI segment of the mycobactin siderophore involved in mycobacterial virulence. A clade of soil-specific sequences clustered with GenBank matches from the ambruticin and jerangolid pathways of Sorangium cellulosum. All three of these clades were found in sites >700 km apart. Street sediments are enriched in actinomycetal PKSIs. Non-actinomycetal PKSI pathways may be more chemically diverse than actinomycetal PKSIs. Common soil and street sediment PKIs are globally distributed.     

Functional divergence and horizontal transfer of type IV secretion systems

The type IV secretion system (TFSSs) is a multifunctional family of translocation pathways that mediate the transfer of DNA among bacteria and deliver DNA and proteins to eukaryotic cells during bacterial infections. Horizontal transmission has dominated the evolution of the TFSS, as demonstrated here by a lack of congruence between the tree topology inferred from components of the TFSS and the presumed bacterial species divergence pattern. A parsimony analysis suggests that conjugation represents the ancestral state and that the divergence from conjugation to secretion of effector molecules has occurred independently at multiple sites in the tree. The result shows that the nodes at which functional shifts have occurred coincide with those of horizontal gene transfers among distantly related bacteria. We suggest that it is the transfer between species that paved the way for the divergence of the TFSSs and discuss the general role of horizontal gene transfers for the evolution of novel gene functions.     

The Repetitive DNA Elements Called CRISPRs and Their Associated Genes: Evidence of Horizontal Transfer Among Prokaryotes

We have found direct DNA repeats 21–47 bp in length interspersed with nonrepetitive sequences of similar length, or clustered regularly interspaced short palindromic repeats (CRISPRs) in a wide range of diverse prokaryotes, including many Archaeal and Eubacterial species. A number of cas, CRISPR-associated genes have also been characterized in many of the same organisms. Phylogenetic analysis of these cas genes suggests that the CRISPR loci have been propagated via HGT, horizontal gene transfer. We suggest a mechanism by which this HGT has occurred, namely, that the CRISPR loci can be carried between cells on megaplasmids ≥40 kb in length. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Stuart Newfeld]     

Characterization of <Emphasis Type="Italic">Hymenobacter</Emphasis> isolates from Victoria Upper Glacier,Antarctica reveals five new species and substantial non-vertical evolution within this genus

We isolated several Hymenobacter-like strains from Victoria Upper Glacier, Antarctica, basal ice that diverged substantially from currently defined Hymenobacter species according to their 16S rRNA and gyrB gene phylogenies. All strains were psychrotolerant, heterotrophic aerobes which grew preferentially on low salt and low nutrient strength agar. Further phenotypic and chemotaxonomic characterization of these isolates supported their assignment as five novel species: H. algoricola sp. nov., H. antarcticus sp. nov., H. elongatus sp. nov., H. fastidiosus sp. nov., and H. glaciei sp. nov. Remarkable among these data was the prevalence of horizontal gene transfers and phenotypic variation, even between apparently closely related strains. These results suggest extensive non-vertical evolution within the genus Hymenobacter, and may reflect evolutionary trajectories resulting from dormancy, e.g., during interment in glacial ice.     

Novel Group I Introns Encoding a Putative Homing Endonuclease in the Mitochondrial <Emphasis Type="Italic">cox1</Emphasis> Gene of Scleractinian Corals

Analyses of mitochondrial sequences revealed the existence of a group I intron in the cytochrome oxidase subunit 1 (cox1) gene in 13 of 41 genera (20 out of 73 species) of corals conventionally assigned to the suborder Faviina. With one exception, phylogenies of the coral cox1 gene and its intron were concordant, suggesting at most two insertions and many subsequent losses. The coral introns were inferred to encode a putative homing endonuclease with a LAGLI-DADG motif as reported for the cox1 group I intron in the sea anemone Metridium senile. However, the coral and sea anemone cox1 group I introns differed in several aspects, such as the intron insertion site and sequence length. The coral cox1 introns most closely resemble the mitochondrial cox1 group I introns of a sponge species, which also has the same insertion site. The coral introns are also more similar to the introns of several fungal species than to that of the sea anemone (although the insertion site differs in the fungi). This suggests either a horizontal transfer between a sponge and a coral or independent transfers from a similar fungal donor (perhaps one with an identical insertion site that has not yet been discovered). The common occurrence of this intron in corals strengthens the evidence for an elevated abundance of group I introns in the mitochondria of anthozoans. [Reviewing Editor: Dr. Niles Lehman]     

Multiple origins of fungal group I introns located in the same position of nuclear SSU rRNA gene

The archiascomycetous fungus Protomyces pachydermus has two group I introns within the nuclear small subunit (nSSU) rRNA gene. One of these introns has an internal open reading frame (ORF) that encodes a predicted protein of 228 amino acid residues. On the other hand, Protomyces macrosporus has two group I introns that insert at the same positions as P. pachydermus, which have no ORF. Each alignment was constructed with Protomyces group I introns located in the same position and other introns retrieved by the BLAST Search. Each phylogenetic tree based on the alignment shows that Protomyces introns are monophyletic but the relationships among fungal introns do not reflect on the fungal phylogeny. Therefore, it is suggested that two different horizontal transfers of group I introns occurred at the early stage of Protomyces species diversification. Received: 11 June 1997 / Accepted: 2 September 1997     

Evolution of <Emphasis Type="Italic">Pleopsidium</Emphasis> (Lichenized Ascomycota) S943 Group I Introns and the Phylogeography of an Intron-Encoded Putative Homing Endonuclease

The sporadic distribution of nuclear group I introns among different fungal lineages can be explained by vertical inheritance of the introns followed by successive losses, or horizontal transfers from one lineage to another through intron homing or reverse splicing. Homing is mediated by an intron-encoded homing endonuclease (HE) and recent studies suggest that the introns and their associated HE gene (HEG) follow a recurrent cyclical model of invasion, degeneration, loss, and reinvasion. The purpose of this study was to compare this model to the evolution of HEGs found in the group I intron at position S943 of the nuclear ribosomal DNA of the lichen-forming fungus Pleopsidium. Forty-eight S943 introns were found in the 64 Pleopsidium samples from a worldwide screen, 22 of which contained a full-length HEG that encodes a putative 256-amino acid HE, and 2 contained HE pseudogenes. The HEGs are divided into two closely related types (as are the introns that encode them) that differ by 22.6% in their nucleotide sequences. The evolution of the Pleopsidium intron-HEG element shows strong evidence for a cyclical model of evolution. The intron was likely acquired twice in the genus and then transmitted via two or three interspecific horizontal transfers. Close geographical proximity plays an important role in intron-HEG horizontal transfer because most of these mobile elements were found in Europe. Once acquired in a lineage, the intron-HEG element was also vertically transmitted, and occasionally degenerated or was lost. [Reviewing Editor: Dr. Manyuan Long]     

The evolutionary genetics of thehobo transposable element in theDrosophila melanogaster complex

Hobo elements are a family of transposable elements found inDrosophila melanogaster and its three sibling species:D. simulans, D. mauritiana andD. sechellia. Studies inD. melanogaster have shown thathobo may be mobilized, and that the genetic effects of such mobilizations included the general features of hybrid dysgenesis: mutations, chromosomal rearrangements and gonadal dysgenis in F1 individuals. At the evolutionary level somehobo-hybridizing sequences have also been found in the other members of themelanogaster subgroup and in many members of the relatedmontium subgroup. Surveys of older collected strains ofD. melanogaster suggest that completehobo elements were absent prior to 50 years ago and that they have recently been introduced into this species by horizontal transfer. In this paper we review our findings and those of others, in order to precisely describe the geographical distribution and the evolutionary history ofhobo in theD. melanogaster complex. Studies of the DNA sequences reveal a different level of divergence between the groupD. melanogaster, D. simulans andD. mauritiana and the fourth speciesD. sechellia. The hypothesis of multiple transfers in the recent past into theD. melanogaster complex from a common outside source is discussed.     

Naturally mosaic operons for secondary metabolite biosynthesis: variability and putative horizontal transfer of discrete catalytic domains of the epothilone polyketide synthase locus

A putative instance of horizontal gene transfer (HGT) involving adjacent, discrete beta-ketoacyl synthase (KS), acyl carrier protein (ACP) and nonribosomal peptide synthase (NRPS) domains of the epothilone Type I polyketide biosynthetic gene cluster from the myxobacterium Sorangium cellulosom was identified using molecular phylogenetics and sequence analyses. The specific KS domain of the module EPO B fails to cluster phylogenetically with other epothilone KS sequences present at this locus, in contrast to what is typically observed in many other Type I polyketide synthase (PKS) biosynthetic loci. Furthermore, the GC content of the epoB KS, epoA ACP and NRPS domains differs significantly from the base composition of other epothilone domain sequences. In addition, the putatively transferred epothilone loci are located near previously identified transposon-like sequences. Lastly, comparison with other KS loci revealed another possible case of horizontal transfer of secondary metabolite genes in the genus Pseudomonas. This study emphasizes the use of several lines of concordant evidence (phylogenetics, base composition, transposon sequences) to infer the evolutionary history of particular gene and enzyme sequences, and the results support the idea that genes coding for adaptive traits, e.g. defensive natural products, may be prone to transposition between divergent prokaryotic taxa and genomes.     

Phylogenetic analysis of 70 kD heat shock protein sequences suggests a chimeric origin for the eukaryotic cell nucleus

Background: The evolutionary relationships between archaebacteria, eubacteria and eukaryotic cells are of central importance in biology. The current view is that each of these three groups of organisms constitutes a monophyletic domain, and that eukaryotic cells have evolved from an archaebacterial ancestor. Recent studies on a number of highly conserved protein sequences do not, however, support this view and raise important questions concerning the evolutionary relationships between all extant organisms, particularly regarding the origin of eukaryotic cells.Results We have used sequences of 70 kD heat shock protein (hsp70) — the most conserved protein found to date in all species — to examine the evolutionary relationship between various species. We have obtained two new archaebacterial hsp70 sequences from the species, Thermoplasma acidophilum and Halobacterium cutirubrum. A global comparison of hsp70 sequences, including our two new sequences, shows that all known archaebacterial homologs share a number of sequence signatures with the Gram-positive group of bacteria that are not found in any other prokaryotic or eukaryotic species. In contrast, the eukaryotic homologs are shown to share a number of unique sequence features with the Gram-negative bacteria that are not present in any archaebacteria. Detailed phylogenetic analyses of hsp70 sequences strongly support a specific evolutionary relationship between archaebacteria and Gram-positive bacteria on the one hand, and Gram-negative bacteria and eukaryotes on the other. The phylogenetic analyses also indicate a polyphyletic branching of archaebacteria within the Gram-positive species. The possibility that the observed relationships are due to horizontal gene transfers can be excluded on the basis of sequence characteristics of different groups of homologs.Conclusion Our results do not support the view that archaebacteria constitute a monophyletic domain, but instead suggest a close evolutionary linkage between archaebacteria and Gram-positive bacteria. Furthermore, in contrast to the presently accepted view, eukaryotic hsp70s show a close and specific relationship to those from Gram-negative species. To explain the phylogenies based on different gene sequences, a chimeric model for the origin of the eukaryotic cell nucleus involving fusion between an archaebacterium and a Gram-negative eubacterium is proposed. Several predictions from the chimeric model are discussed.