Medically Relevant Acinetobacter Species Require a Type II Secretion System and Specific Membrane-Associated Chaperones for the Export of Multiple Substrates and Full Virulence

Acinetobacter baumannii, A. nosocomialis, and A. pittii have recently emerged as opportunistic human pathogens capable of causing severe human disease; however, the molecular mechanisms employed by Acinetobacter to cause disease remain poorly understood. Many pathogenic members of the genus Acinetobacter contain genes predicted to encode proteins required for the biogenesis of a type II secretion system (T2SS), which have been shown to mediate virulence in many Gram-negative organisms. Here we demonstrate that Acinetobacter nosocomialis strain M2 produces a functional T2SS, which is required for full virulence in both the Galleria mellonella and murine pulmonary infection models. Importantly, this is the first bona fide secretion system shown to be required for virulence in Acinetobacter. Using bioinformatics, proteomics, and mutational analyses, we show that Acinetobacter employs its T2SS to export multiple substrates, including the lipases LipA and LipH as well as the protease CpaA. Furthermore, the Acinetobacter T2SS, which is found scattered amongst five distinct loci, does not contain a dedicated pseudopilin peptidase, but instead relies on the type IV prepilin peptidase, reinforcing the common ancestry of these two systems. Lastly, two of the three secreted proteins characterized in this study require specific chaperones for secretion. These chaperones contain an N-terminal transmembrane domain, are encoded adjacently to their cognate effector, and their disruption abolishes type II secretion of their cognate effector. Bioinformatic analysis identified putative chaperones located adjacent to multiple previously known type II effectors from several Gram-negative bacteria, which suggests that T2SS chaperones constitute a separate class of membrane-associated chaperones mediating type II secretion.     

Isolation and characterization of crude-oil-degrading bacteria from oil-water mixture in Dagang oilfield,China

Isolating novel crude-oil-degrading bacteria from oil-water mixture of oil production well and evaluating their degradation capacities are vitally important in the remediation of oil-polluted environments and crude oil exploitation. According to the molecular screening with degenerate primers of alkane hydroxylase gene (alk B), a strain Acinetobacter sp. LS-1 with alk B gene was isolated. This strain exhibited a 99.9% similarity with genus Acinetobacter. This alk B gene which is one of the key enzymes of metabolic process was identified. This gene sequence showed 98% similarity of its nucleotide and related amino acids to the genus Marinobacter but exhibited below 70% similarity to the genus Acinetobacter. This phylogenetic analysis indicated that alk B may have been transferred horizontally between bacteria. The isolated strain could utilize crude oil as the sole carbon, achieving a high degradation (70.3%) in 7 days. Microcalorimetric analysis of the metabolic processes for hexadecane degradation also demonstrated the ability of this strain to utilize hydrocarbons. Thus, this strain enables to degrade hydrocarbons as the sole carbon source from the gene level, combined with material and energy metabolism. These findings will benefit this strain in the remediation of oil-polluted environments and oil exploitation.     

Phylogenetic conservatism of functional traits in microorganisms

A central question in biology is how biodiversity influences ecosystem functioning. Underlying this is the relationship between organismal phylogeny and the presence of specific functional traits. The relationship is complicated by gene loss and convergent evolution, resulting in the polyphyletic distribution of many traits. In microorganisms, lateral gene transfer can further distort the linkage between phylogeny and the presence of specific functional traits. To identify the phylogenetic conservation of specific traits in microorganisms, we developed a new phylogenetic metric—consenTRAIT—to estimate the clade depth where organisms share a trait. We then analyzed the distribution of 89 functional traits across a broad range of Bacteria and Archaea using genotypic and phenotypic data. A total of 93% of the traits were significantly non-randomly distributed, which suggested that vertical inheritance was generally important for the phylogenetic dispersion of functional traits in microorganisms. Further, traits in microbes were associated with a continuum of trait depths (τ_D), ranging from a few deep to many shallow clades (average τ_D: 0.101–0.0011 rRNA sequence dissimilarity). Next, we demonstrated that the dispersion and the depth of clades that contain a trait is correlated with the trait''s complexity. Specifically, complex traits encoded by many genes like photosynthesis and methanogenesis were found in a few deep clusters, whereas the ability to use simple carbon substrates was highly phylogenetically dispersed. On the basis of these results, we propose a framework for predicting the phylogenetic conservatism of functional traits depending on the complexity of the trait. This framework enables predicting how variation in microbial composition may affect microbially-mediated ecosystem processes as well as linking phylogenetic and trait-based patterns of biogeography.     

Genomic functional annotation using co-evolution profiles of gene clusters

Background  

The current speed of sequencing already exceeds the capability of annotation, creating a potential bottleneck. A large proportion of the genes in microbial genomes remains uncharacterized. Here we propose a new method for functional annotation using the conservation patterns of gene clusters. If several gene clusters show the same coevolution pattern across different genomes it is reasonable to infer they are functionally related. The gene cluster phylogenetic profile integrates chromosomal proximity information and phylogenetic profile information and allows us to infer functional dependences between the gene clusters even at great distance on the chromosome.     

The complete mitochondrial genome of Plesiastrea versipora (Scleractinia,Plesiastreidae) sheds light on its phylogeny and taxonomy of the family Plesiastreidae

The genus Plesiastrea used to be a member of the traditional family Faviidae, falling into the challenging ‘Bigmessidae’ clade, and was re-established until recent molecular phylogenies published. The entire mitogenome of the symbiotic coral Plesiastrea versipora (Lamarck, 1816), the type species of the family Plesiastreidae, was sequenced. The length of the mitochondrial genome is 15,320 bp and it includes thirteen protein-coding genes (PCGs), two rRNAs and two tRNAs. The nucleotide composition of GC is 32%. We perform phylogenetic reconstruction based on maximum likelihood (ML) and Bayesian analysis(BI) using all PCGs. Our result indicates that P. versipora clusters closely with species which belong to Mussidae, Merulinidae and Lobophylliidae. Our phylogenetic analyses provide solid evidence for phylogenetic placement of P. versipora and the evolutionary relationships among different families within the traditional robust clade of Scleractinia. In addition, the mitogenome data provide useful information for further molecular systematic investigations on Plesiastreidae as well as conservation biology research of P. versipora.     

Phylogenetic Co-Occurrence of ExoR,ExoS, and ChvI,Components of the RSI Bacterial Invasion Switch,Suggests a Key Adaptive Mechanism Regulating the Transition between Free-Living and Host-Invading Phases in Rhizobiales

Both bacterial symbionts and pathogens rely on their host-sensing mechanisms to activate the biosynthetic pathways necessary for their invasion into host cells. The Gram-negative bacterium Sinorhizobium meliloti relies on its RSI (ExoR-ExoS-ChvI) Invasion Switch to turn on the production of succinoglycan, an exopolysaccharide required for its host invasion. Recent whole-genome sequencing efforts have uncovered putative components of RSI-like invasion switches in many other symbiotic and pathogenic bacteria. To explore the possibility of the existence of a common invasion switch, we have conducted a phylogenomic survey of orthologous ExoR, ExoS, and ChvI tripartite sets in more than ninety proteobacterial genomes. Our analyses suggest that functional orthologs of the RSI invasion switch co-exist in Rhizobiales, an order characterized by numerous invasive species, but not in the order’s close relatives. Phylogenomic analyses and reconstruction of orthologous sets of the three proteins in Alphaproteobacteria confirm Rhizobiales-specific gene synteny and congruent RSI evolutionary histories. Evolutionary analyses further revealed site-specific substitutions correlated specifically to either animal-bacteria or plant-bacteria associations. Lineage restricted conservation of any one specialized gene is in itself an indication of species adaptation. However, the orthologous phylogenetic co-occurrence of all interacting partners within this single signaling pathway strongly suggests that the development of the RSI switch was a key adaptive mechanism. The RSI invasion switch, originally found in S. meliloti, is a characteristic of the Rhizobiales, and potentially a conserved crucial activation step that may be targeted to control host invasion by pathogenic bacterial species.     

A Gene Transfer Agent and a Dynamic Repertoire of Secretion Systems Hold the Keys to the Explosive Radiation of the Emerging Pathogen Bartonella

Gene transfer agents (GTAs) randomly transfer short fragments of a bacterial genome. A novel putative GTA was recently discovered in the mouse-infecting bacterium Bartonella grahamii. Although GTAs are widespread in phylogenetically diverse bacteria, their role in evolution is largely unknown. Here, we present a comparative analysis of 16 Bartonella genomes ranging from 1.4 to 2.6 Mb in size, including six novel genomes from Bartonella isolated from a cow, two moose, two dogs, and a kangaroo. A phylogenetic tree inferred from 428 orthologous core genes indicates that the deadly human pathogen B. bacilliformis is related to the ruminant-adapted clade, rather than being the earliest diverging species in the genus as previously thought. A gene flux analysis identified 12 genes for a GTA and a phage-derived origin of replication as the most conserved innovations. These are located in a region of a few hundred kb that also contains 8 insertions of gene clusters for type III, IV, and V secretion systems, and genes for putatively secreted molecules such as cholera-like toxins. The phylogenies indicate a recent transfer of seven genes in the virB gene cluster for a type IV secretion system from a cat-adapted B. henselae to a dog-adapted B. vinsonii strain. We show that the B. henselae GTA is functional and can transfer genes in vitro. We suggest that the maintenance of the GTA is driven by selection to increase the likelihood of horizontal gene transfer and argue that this process is beneficial at the population level, by facilitating adaptive evolution of the host-adaptation systems and thereby expansion of the host range size. The process counters gene loss and forces all cells to contribute to the production of the GTA and the secreted molecules. The results advance our understanding of the role that GTAs play for the evolution of bacterial genomes.     

Combined Analysis of Variation in Core,Accessory and Regulatory Genome Regions Provides a Super-Resolution View into the Evolution of Bacterial Populations

The use of whole-genome phylogenetic analysis has revolutionized our understanding of the evolution and spread of many important bacterial pathogens due to the high resolution view it provides. However, the majority of such analyses do not consider the potential role of accessory genes when inferring evolutionary trajectories. Moreover, the recently discovered importance of the switching of gene regulatory elements suggests that an exhaustive analysis, combining information from core and accessory genes with regulatory elements could provide unparalleled detail of the evolution of a bacterial population. Here we demonstrate this principle by applying it to a worldwide multi-host sample of the important pathogenic E. coli lineage ST131. Our approach reveals the existence of multiple circulating subtypes of the major drug–resistant clade of ST131 and provides the first ever population level evidence of core genome substitutions in gene regulatory regions associated with the acquisition and maintenance of different accessory genome elements.     

A study of the distribution of phylogenetically conserved blocks within clusters of mammalian homeobox genes

Genome sequencing efforts of the last decade have produced a large amount of data, which has enabled whole-genome comparative analyses in order to locate potentially functional elements and study the overall patterns of phylogenetic conservation. In this paper we present a statistically based method for the characterization of these patterns in mammalian DNA sequences. We have applied this approach to the study of exceptionally well conserved homeobox gene clusters (Hox), based on an alignment of six species, and we have constructed a map of Hox cataloguing the conserved fragments, along with their locations in relation to the genes and other landmarks, sometimes showing unexpected layouts.     

Detection of quorum sensing signal molecules and identification of an autoinducer synthase gene among biofilm forming clinical isolates of Acinetobacter spp

Background

Quorum sensing is a term that describes an environmental sensing system that allows bacteria to monitor their own population density which contributes significantly to the size and development of the biofilm. Many gram negative bacteria use N-acyl-homoserine lactones as quorum sensing signal molecules. In this study, we sought to find out if the biofilm formation among clinical isolates of Acinetobacter spp. is under the control of autoinducing quorum sensing molecules.

Methodology/Principal Findings

Biofilm formation among clinical isolates of Acinetobacter spp. was assessed and the production of signal molecules were detected with Chromobacterium violaceum CV026 biosensor system. Characterisation of autoinducers was carried out by mass spectrometric analysis. We have also reported the identification of an autoinducer synthase gene, abaΙ among the isolates that produce quorum sensing signal molecules and have reported that the mutation in the abaI gene influences their biofilm forming capabilities. Using a microtitre-plate assay it was shown that 60% of the 50 Acinetobacter spp. isolates significantly formed biofilms. Further detection with the biosensor strain showed that some of these isolates produced long chain signal molecules. Mass spectrometric analysis revealed that five of these isolates produced N-decanoyl homoserine lactone and two isolates produced acyl-homoserine lactone with a chain length equal to C₁₂. The abaΙ gene was identified and a tetracycline mutant of the abaΙ gene was created and the inhibition in biofilm formation in the mutant was shown.

Conclusions/Significance

These data are of great significance as the signal molecules aid in biofilm formation which in turn confer various properties of pathogenicity to the clinical isolates including drug resistance. The use of quorum sensing signal blockers to attenuate bacterial pathogenicity is therefore highly attractive, particularly with respect to the emergence of multi antibiotic resistant bacteria.     

Molecular and Evolutionary Analysis of NEAr-Iron Transporter (NEAT) Domains

Iron is essential for bacterial survival, being required for numerous biological processes. NEAr-iron Transporter (NEAT) domains have been studied in pathogenic Gram-positive bacteria to understand how their proteins obtain heme as an iron source during infection. While a 2002 study initially discovered and annotated the NEAT domain encoded by the genomes of several Gram-positive bacteria, there remains a scarcity of information regarding the conservation and distribution of NEAT domains throughout the bacterial kingdom, and whether these domains are restricted to pathogenic bacteria. This study aims to expand upon initial bioinformatics analysis of predicted NEAT domains, by exploring their evolution and conserved function. This information was used to identify new candidate domains in both pathogenic and nonpathogenic organisms. We also searched metagenomic datasets, specifically sequence from the Human Microbiome Project. Here, we report a comprehensive phylogenetic analysis of 343 NEAT domains, encoded by Gram-positive bacteria, mostly within the phylum Firmicutes, with the exception of Eggerthella sp. (Actinobacteria) and an unclassified Mollicutes bacterium (Tenericutes). No new NEAT sequences were identified in the HMP dataset. We detected specific groups of NEAT domains based on phylogeny of protein sequences, including a cluster of novel clostridial NEAT domains. We also identified environmental and soil organisms that encode putative NEAT proteins. Biochemical analysis of heme binding by a NEAT domain from a protein encoded by the soil-dwelling organism Paenibacillus polymyxa demonstrated that the domain is homologous in function to NEAT domains encoded by pathogenic bacteria. Together, this study provides the first global bioinformatics analysis and phylogenetic evidence that NEAT domains have a strong conservation of function, despite group-specific differences at the amino acid level. These findings will provide information useful for future projects concerning the structure and function of NEAT domains, particularly in pathogens where they have yet to be studied.     

The genome of the truffle-parasite Tolypocladium ophioglossoides and the evolution of antifungal peptaibiotics

Background

Two major mycoparasitic lineages, the family Hypocreaceae and the genus Tolypocladium, exist within the fungal order, Hypocreales. Peptaibiotics are a group of secondary metabolites almost exclusively described from Trichoderma species of Hypocreaceae. Peptaibiotics are produced by nonribosomal peptide synthetases (NRPSs) and have antibiotic and antifungal activities. Tolypocladium species are mainly truffle parasites, but a few species are insect pathogens.

Results

The draft genome sequence of the truffle parasite Tolypocladium ophioglossoides was generated and numerous secondary metabolite clusters were discovered, many of which have no known putative product. However, three large peptaibiotic gene clusters were identified using phylogenetic analyses. Peptaibiotic genes are absent from the predominantly plant and insect pathogenic lineages of Hypocreales, and are therefore exclusive to the largely mycoparasitic lineages. Using NRPS adenylation domain phylogenies and reconciliation of the domain tree with the organismal phylogeny, it is demonstrated that the distribution of these domains is likely not the product of horizontal gene transfer between mycoparasitic lineages, but represents independent losses in insect pathogenic lineages. Peptaibiotic genes are less conserved between species of Tolypocladium and are the product of complex patterns of lineage sorting and module duplication. In contrast, these genes are more conserved within the genus Trichoderma and consistent with diversification through speciation.

Conclusions

Peptaibiotic NRPS genes are restricted to mycoparasitic lineages of Hypocreales, based on current sampling. Phylogenomics and comparative genomics can provide insights into the evolution of secondary metabolite genes, their distribution across a broader range of taxa, and their possible function related to host specificity.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1777-9) contains supplementary material, which is available to authorized users.     

Phylogenetic Analysis of Burkholderia Species by Multilocus Sequence Analysis

Burkholderia comprises more than 60 species of environmental, clinical, and agro-biotechnological relevance. Previous phylogenetic analyses of 16S rRNA, recA, gyrB, rpoB, and acdS gene sequences as well as genome sequence comparisons of different Burkholderia species have revealed two major species clusters. In this study, we undertook a multilocus sequence analysis of 77 type and reference strains of Burkholderia using atpD, gltB, lepA, and recA genes in combination with the 16S rRNA gene sequence and employed maximum likelihood and neighbor-joining criteria to test this further. The phylogenetic analysis revealed, with high supporting values, distinct lineages within the genus Burkholderia. The two large groups were named A and B, whereas the B. rhizoxinica/B. endofungorum, and B. andropogonis groups consisted of two and one species, respectively. The group A encompasses several plant-associated and saprophytic bacterial species. The group B comprises the B. cepacia complex (opportunistic human pathogens), the B. pseudomallei subgroup, which includes both human and animal pathogens, and an assemblage of plant pathogenic species. The distinct lineages present in Burkholderia suggest that each group might represent a different genus. However, it will be necessary to analyze the full set of Burkholderia species and explore whether enough phenotypic features exist among the different clusters to propose that these groups should be considered separate genera.     

“Methanoplasmatales,” Thermoplasmatales-Related Archaea in Termite Guts and Other Environments,Are the Seventh Order of Methanogens

The Euryarchaeota comprise both methanogenic and nonmethanogenic orders and many lineages of uncultivated archaea with unknown properties. One of these deep-branching lineages, distantly related to the Thermoplasmatales, has been discovered in various environments, including marine habitats, soil, and also the intestinal tracts of termites and mammals. By comparative phylogenetic analysis, we connected this lineage of 16S rRNA genes to a large clade of unknown mcrA gene sequences, a functional marker for methanogenesis, obtained from the same habitats. The identical topologies of 16S rRNA and mcrA gene trees and the perfect congruence of all branches, including several novel groups that we obtained from the guts of termites and cockroaches, strongly suggested that they stem from the same microorganisms. This was further corroborated by two highly enriched cultures of closely related methanogens from the guts of a higher termite (Cubitermes ugandensis) and a millipede (Anadenobolus sp.), which represented one of the arthropod-specific clusters in the respective trees. Numerous other pairs of habitat-specific sequence clusters were obtained from the guts of other termites and cockroaches but were also found in previously published data sets from the intestinal tracts of mammals (e.g., rumen cluster C) and other environments. Together with the recently described Methanomassiliicoccus luminyensis isolated from human feces, which falls into rice cluster III, the results of our study strongly support the idea that the entire clade of “uncultured Thermoplasmatales” in fact represents the seventh order of methanogenic archaea, for which the provisional name “Methanoplasmatales” is proposed.     

The molecular phylogenetic signature of Bali cattle revealed by maternal and paternal markers

Bali cattle is a domestic cattle breed that can be found in Malaysia. It is a domestic cattle that was purely derived from a domestication event in Banteng (Bos javanicus) around 3,500 BC in Indonesia. This research was conducted to portray the phylogenetic relationships of the Bali cattle with other cattle species in Malaysia based on maternal and paternal lineage. We analyzed the cytochrome c oxidase I (COI) mitochondrial gene and SRY of Y chromosome obtained from five species of the Bos genus (B. javanicus, Bos gaurus, Bos indicus, Bos taurus, and Bos grunniens). The water buffalo (Bubalus bubalis) was used as an outgroup. The phylogenetic relationships were observed by employing several algorithms: Neighbor-Joining (PAUP version 4.0), Maximum parsimony (PAUP version 4.0) and Bayesian inference (MrBayes 3.1). Results from the maternal data showed that the Bali cattle formed a monophyletic clade, and together with the B. gaurus clade formed a wild cattle clade. Results were supported by high bootstrap and posterior probability values together with genetic distance data. For the paternal lineage, the sequence variation is low (with parsimony informative characters: 2/660) resulting an unresolved Neighbor-Joining tree. However, Bali cattle and other domestic cattle appear in two monophyletic clades distinct from yak, gaur and selembu. This study expresses the potential of the COI gene in portraying the phylogenetic relationships between several Bos species which is important for conservation efforts especially in decision making since cattle is highly bred and hybrid breeds are often formed. Genetic conservation for this high quality beef cattle breed is important by maintaining its genetic characters to prevent extinction or even decreased the genetic quality.     

Horizontal Gene Acquisition of Liberibacter Plant Pathogens from a Bacteriome-Confined Endosymbiont of Their Psyllid Vector

he Asian citrus psyllid Diaphorina citri is a notorious agricultural pest that transmits the phloem-inhabiting alphaproteobacterial ‘Candidatus Liberibacter asiaticus’ and allied plant pathogens, which cause the devastating citrus disease called Huanglongbing or greening disease. D. citri harbors two distinct bacterial mutualists in the symbiotic organ called bacteriome: the betaproteobacterium ‘Candidatus Profftella armatura’ in the syncytial cytoplasm at the center of the bacteriome, and the gammaproteobacterium ‘Candidatus Carsonella ruddii’ in uninucleate bacteriocytes. Here we report that a putative amino acid transporter LysE of Profftella forms a highly supported clade with proteins of L. asiaticus, L. americanus, and L. solanacearum. L. crescens, the most basal Liberibacter lineage currently known, lacked the corresponding gene. The Profftella-Liberibacter subclade of LysE formed a clade with proteins from betaproteobacteria of the order Burkholderiales, to which Profftella belongs. This phylogenetic pattern favors the hypothesis that the Liberibacter lineage acquired the gene from the Profftella lineage via horizontal gene transfer (HGT) after L. crescens diverged from other Liberibacter lineages. K _A/K _S analyses further supported the hypothesis that the genes encoded in the Liberibacter genomes are functional. These findings highlight the possible evolutionary importance of HGT between plant pathogens and their insect vector’s symbionts that are confined in the symbiotic organ and seemingly sequestered from external microbial populations.     

Comparative Phylogenomics and Evolution of the Brucellae Reveal a Path to Virulence

Brucella species include important zoonotic pathogens that have a substantial impact on both agriculture and human health throughout the world. Brucellae are thought of as “stealth pathogens” that escape recognition by the host innate immune response, modulate the acquired immune response, and evade intracellular destruction. We analyzed the genome sequences of members of the family Brucellaceae to assess its evolutionary history from likely free-living soil-based progenitors into highly successful intracellular pathogens. Phylogenetic analysis split the genus into two groups: recently identified and early-dividing “atypical” strains and a highly conserved “classical” core clade containing the major pathogenic species. Lateral gene transfer events brought unique genomic regions into Brucella that differentiated them from Ochrobactrum and allowed the stepwise acquisition of virulence factors that include a type IV secretion system, a perosamine-based O antigen, and systems for sequestering metal ions that are absent in progenitors. Subsequent radiation within the core Brucella resulted in lineages that appear to have evolved within their preferred mammalian hosts, restricting their virulence to become stealth pathogens capable of causing long-term chronic infections.