Characterizing proteases in an Antarctic Janthinobacterium sp. isolate: Evidence of a protease horizontal gene transfer event

We report the isolation of a cold-adapted bacterium belonging to the genus Janthinobacterium （named AU 11）, from a water sample collected in Lake Uruguay （King George Island, South Shetlands）. AUI 1 （growth between 4℃ and 30℃） produces a single cold-active extracellular protease （ExPAU11）, differentially expressed at low temperature. ExPAU11 was identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry （MALDI-ToF MS） as an alkaline metallo-protease （70% coverage with an extracellular protease of Janthinobacterium sp. PI12）, and by protease-inhibitor screening identified as a serine-protease. To the best of our knowledge this is the first experimental evidence of a cold-active extracellular protease produced by Janthinobacterium. Furthermore, we identified a serine-protease gene （named JSP8A） showing 60% identity （98% query coverage） to subtilisin peptidases belonging to the $8 family （S8A subfamily） of many cyanobacteria. A phylogenetic analysis of the JSP8A protease, along with related bacterial protein sequences, confirms that JSP8A clusters with S8A subtilisin sequences from different cyanobacteria, and is clearly separated from SSA bacterial sequences of other phyla （including its own）. An analysis of the genomic organization around JSP8A suggests that this protease gene was acquired in an event that duplicated a racemase gene involved in transforming L- to D-amino acids. Our results suggest that AU11 probably acquired this subtilisin-like protease gene by horizontal gene transfer （HGT） from a cyanobacterittrn. We discuss the relevance of a bacterial protease-HGT in the Antarctic environment in light of this hypothesis.

Evolution of PE35 and PPE68 Gene Families in Mycobacterium: Roles of Horizontal Gene Transfer and Evolutionary Constraints

Mycobacterium is a genus of bacteria with over a hundred non-pathogenic and pathogenic species, best recognized for certain members known to cause diseases such as tuberculosis and leprosy. Two novel protein families important in the pathogenesis of Mycobacterium species are the PE and PPE families. These two protein families affect the antigenic profiles, disturbing host immunity. To better understand the origin and evolution of these gene families and the differences in their composition between pathogenic and non-pathogenic strains, several bioinformatic analyses were conducted both among Mycobacterium and closely related species that contain PE35 and PPE68 gene homologs. The methods included protein homology searches (BLASTP), horizontal gene transfer analysis (IslandViewer), phylogenetic analysis, gene cluster analysis and structural and functional constraints. Results revealed that PE and PPE gene homologs were not only limited to Mycobacterium, but also existed in three other non-mycobacterial genera, Rhodococcus, Tsukamurella and Segniliparus, and were possibly initially acquired from non-mycobacterial microorganisms by multiple horizontal gene transfers. Results also demonstrated that PE and PPE genes were more diverse and more rapidly evolving in pathogenic Mycobacterium as compared with non-pathogenic Mycobacterium and other non-mycobacterial species. These findings possibly shed light on the diverse functions and origins of the PE/PPE proteins among these organisms.

Horizontal gene transfer of a syp homolog contributes to the virulence of Burkholderia glumae

Horizontal gene transfer(HGT)has been proved a major driving force in prokaryotic evolution.However,the molecular functions of these transferred genes in pathogenic bacteria especially plant pathogenic bacteria are still not fully investigated.In this study,the whole-genome in silico analysis was performed and found a syringopeptin synthetase(syp)homolog in Burkholderia glumae,which can cause bacterial panicle blight in rice,was predicted to be horizontally transferred from Pseudomonas ancestor with solid confidence by phylogenetic analysis.The comprehensive molecular experiments were performed to study the potential role of this gene in B.glumae.Inoculation of rice panicles with the syp mutant resulted in 60%lower disease index compared with the wild type(WT)parent strain,suggesting the requirement of syp for the full virulence of B.glumae.Chromatography analysis of exudates from B.glumae showed suppression of synthesis of metabolites analogous to syringopeptin in the mutants.All these data raise the possibility of HGT phenomenon in shaping the virulence and adaptation of B.glumae over evolutionary time.

Horizontal gene transfer of plant-specific leucine-rich repeats between plants and bacteria

Leucine rich repeats (LRRs) are present in over 14,000 proteins that have been identified in viruses, bacteria, archaea, and eukaryotes. Two to sixty-two LRRs occur in tandem forming an overall arc shaped domain. There are eight classes of LRRs. Plant specific LRRs (class: PS-LRR) had previously been recognized in only plant proteins. However, we find that PS-LRRs are also present in proteins from bacteria. We investigated the origin of bacterial PS-LRR domains. PSLRR proteins are widely distributed in most plants;they are found in only a few bacterial species. There are no PS-LRR proteins from archaea. Bacterial PS-LRRs in twenty proteins from eleven bacterial species (in the three phyla: Proteobacteria, Cyanobacteria, and Bacteroidetes) are significantly more similar to the PS-LRR class than to the other seven classes of LRR proteins. Not only amino acid sequences but also nucleotide sequences of the bacterial PS-LRR domains show highly significant similarity with those of many plant proteins. The program, EGID (Ensemble algorithm for Genomic Island Detection), predicts that Synechococcus sp. CYA_ 1022 came from another organism. Four bacterial PS-LRR proteins contain AhpC-TSA, IgA peptidase M64, the immunoglobulin domain, the Calx-b domain, and the He_PIG domain;these domains show no similarity with any eukaryotic (plant) proteins, in contrast to the similarities of their respective PS-LRRs. The present results indicate that horizontal gene transfer (HGT) of genes/gene fragments encoding PS-LRR domains occurred between bacteria and plants, and HGT among the eleven bacterial species, of the three phyla, as opposed to descent from a common ancestor. There is the possibility of the occurrence of one HGT event from plant to bacteria. A series of HGTs might then have occurred recently and rapidly among these eleven species of bacteria.

An acyltransferase gene that putatively functions in anthocyanin modification was horizontally transferred from Fabaceae into the genus Cuscuta

Horizontal gene transfer(HGT) refers to the flow of genetic materials to non-offspring,and occasionally HGT in plants can improve the adaptation of organisms in new niches due to expanded metabolic capability.Anthocyanins are an important group of water-soluble red,purple,or blue secondary metabolites,whose diversity results from modification after the main skeleton biosynthesis.Cuscuta is a stem holoparasitic genus,whose members form direct connection with hosts to withdraw water,nutrients,and macromolecules.Such intimate association is thought to increase the frequency of HGT.By transcriptome screening for foreign genes in Cuscuta australis,we discovered that one gene encoding a putative anthocyanin acyltransferase gene of the BAHD family,which is likely to be involved in anthocyanin modification,was acquired by C.australis from Fabaceae through HGT.The anthocyanin acyltransferase-like(AT-like) gene was confirmed to be present in the genome assembly of C.australis and the transcriptomes of Cuscuta pentagona.The higher transcriptional level in old stems is consistent with its putative function in secondary metabolism by stabilizing anthocyanin at neutral pH and thus HGT of this AT-like gene may have improved biotic and abiotic resistance of Cuscuta.

环境中污染物降解基因的水平转移(HGT)及其在生物修复中的作用

水平基因转移是不同于垂直基因转移的遗传物质的交流方式。在污染环境这一特异生态环境中,降解基因的水平转移有着独特的功能与作用。研究环境中污染物降解基因在微生物间的水平转移,更深入地了解微生物种群适应污染环境的机理,对于评价污染物的环境毒理、生物可降解性以及污染环境的可修复潜力具有重要参考价值。在污染物生物修复实践中,可以通过调控降解基因的水平转移,增强污染环境中微生物的降解能力,更有效地发挥生物修复作用。文章将对环境中细菌间基因交流的机制,污染物降解基因的水平转移对微生物适应污染环境的机理、水平基因转移对代谢途径的进化及其对污染物生物修复作用的影响等方面的研究进展做一综述。

Phylogenetic diversity of dimethylsulfoniopropionate-dependent demethylase gene dmdA in distantly related bacteria isolated from Arctic and Antarctic marine environments

Dimethylsulfoniopropionate(DMSP) is mainly produced by marine phytoplankton as an osmolyte, antioxidant,predator deterrent, or cryoprotectant. DMSP is also an important carbon and sulfur source for marine bacteria.Bacteria may metabolize DMSP via the demethylation pathway involving the DMSP demethylase gene(dmdA) or the cleavage pathway involving several different DMSP lyase genes. Most DMSP released into seawater is degraded by bacteria via demethylation. To test a hypothesis that the high gene frequency of dmdA among major marine taxa results in part from horizontal gene transfer(HGT) events, a total of thirty-one bacterial strains were isolated from Arctic Kongsfjorden seawater in this study. Analysis of 16S rRNA gene sequences showed that,except for strains BSw22118, BSw22131 and BSw22132 belonging to the genera Colwellia, Pseudomonas and Glaciecola, respectively, all bacteria fell into the genus Pseudoalteromonas. DmdA genes were detected in five distantly related bacterial strains, including four Arctic strains(Pseudoalteromonas sp. BSw22112, Colwellia sp.BSw22118, Pseudomonas sp. BSw22131 and Glaciecola sp. BSw22132) and one Antarctic strain(Roseicitreum antarcticum ZS2–28). Their dmdA genes showed significant similarities(97.7%–98.3%) to that of Ruegeria pomeroyi DSS–3, which was originally isolated from temperate coastal seawater. In addition, the sequence of the gene transfer agent(GTA) capsid protein gene(g5) detected in Antarctic strain ZS2–28 exhibited a genetically closely related to that of Ruegeria pomeroyi DSS–3. Among the five tested strains, only Pseudomonas sp. BSw22131 could grow using DMSP as the sole carbon source. The results of this study support the hypothesis of HGT for dmdA among taxonomically heterogeneous bacterioplankton, and suggest a wide distribution of functional gene(i.e., dmdA) in global marine environments.

Horizontal and vertical gene transfer drive sediment antibiotic resistome in an urban lagoon system

Rapid urbanization has resulted in pervasive occurrence of antibiotic resistance genes(ARGs)in urban aquatic ecosystems.However,limited information is available concerning the ARG profiles and the forces responsible for their assembly in urban landscape lagoon systems.Here,we employed high-throughput quantitative PCR(HT-q PCR)to characterize the spatial variations of ARGs in surface and core sediments of Yundang Lagoon,China.The results indicated that the average richness and absolute abundance of ARGs were 11 and 53 times higher in the lagoon sediments as compared to pristine reference Tibetan lake sediments,highlighting the role of anthropogenic activities in ARG pollution.Co-occurrence network analysis indicated that various anaerobic prokaryotic genera belonging to Alpha-,Deltaproteobacteria,Bacteroidetes,Euryarchaeota,Firmicutes and Synergistetes were the potential hosts of ARGs.The partial least squares-path modeling(PLS-PM)analysis revealed positive and negative indirect effects of physicochemical factors and heavy metals on the lagoon ARG profiles,via biotic factors,respectively.The horizontal(mediated by mobile genetic elements)and vertical(mediated by prokaryotic communities)gene transfer may directly contribute the most to drive the abundance and composition of ARGs,respectively.Furthermore,the neutral community model demonstrated that the assembly of sediment ARG communities was jointly governed by deterministic and stochastic processes.Overall,this study provides novel insights into the diversity and distribution of ARGs in the benthic habitat of urban lagoon systems and underlying mechanisms for the spread and proliferation of ARGs.

Horizontal transfer and evolution of the biosynthetic gene cluster for benzoxazinoids in plants

Benzoxazinoids are a class of protective and allelopathic plant secondary metabolites that have been identified in multiple grass species and are encoded by the Bx biosynthetic gene cluster(BGC)in maize.Data mining of 41 high-quality grass genomes identified complete Bx clusters(containing genes Bx1–Bx5 and Bx8)in three genera(Zea,Echinochloa,and Dichanthelium)of Panicoideae and partial clusters in Triticeae.The Bx cluster probably originated from gene duplication and chromosomal translocation of native homologs of Bx genes.An ancient Bx cluster that included additional Bx genes(e.g.,Bx6)is presumed to have been present in ancestral Panicoideae.The ancient Bx cluster was putatively gained by the Triticeae ancestor via horizontal transfer(HT)from the ancestral Panicoideae and later separated into multiple segments on different chromosomes.Bx6 appears to have been under less constrained selection compared with the Bx cluster during the evolution of Panicoideae,as evidenced by the fact that it was translocated away from the Bx cluster in Zea mays,moved to other chromosomes in Echinochloa,and even lost in Dichanthelium.Further investigations indicate that purifying selection and polyploidization have shaped the evolutionary trajectory of Bx clusters in the grass family.This study provides the first candidate case of HT of a BGC between plants and sheds new light on the evolution of BGCs.

VERTICAL HEREDITY VS. HORIZONTAL GENE TRANSFER: A CHALLENGE TO BACTERIAL CLASSIFICATION

The diversity and classification of microbes has been a long-standing issue.Molecular phylogeny of the prokaryotes based on comparison of the 16S rRNA sequences of the small ribosomal subunit has led to a reasonable tree of life in the late 1970s. How-ever, the availability of more and more complete bacterial genomes has brought about complications instead of refinement of the tree. In particular, it turns out that different choice of genes may tell different history. This might be caused by possible horizontal gene transfer (HGT) among species. There is an urgent need to develop phylogenetic methods that make use of whole genome data. We describe a new approach in molecular phylogeny,namely, tree construction based on K-tuple frequency analysis of the genomic sequences.Putting aside the technicalities, we emphasize the transition from randomness to determin-ism when the string length K increases and try to comment on the challenge mentioned in the title.

Phage-mediated horizontal gene transfer and its implications for the human gut microbiome

Horizontal gene transfer(HGT)in the microbiome has profound consequences for human health and disease.The spread of antibiotic resistance genes,virulence,and pathogenicity determinants predominantly occurs by way of HGT.Evidence exists of extensive horizontal transfer in the human gut microbiome.Phage transduction is a type of HGT event in which a bacteriophage transfers non-viral DNA from one bacterial host cell to another.The abundance of tailed bacteriophages in the human gut suggests that transduction could act as a significant mode of HGT in the gut microbiome.Here we review in detail the known mechanisms of phage-mediated HGT,namely specialized and generalized transduction,lateral transduction,gene-transfer agents,and molecular piracy,as well as methods used to detect phage-mediated HGT,and discuss its potential implications for the human gut microbiome.

Extracellular Micro-RNAs in Health and Disease: Basic Science, Biogenesis and Release

Small non-protein coding micro-RNAs are regularly exported out of cells, both in health and disease. More than ninety percent of extracellular miRNAs are associated with lower-molecular-mass complexes bound to Argonaute 2 (Ago2), nucleophosmin-1 (NPM1) and high density lipoproteins (HDL), whereas the rest (~10%) are membrane-vesicle-encapsulated within exosomes, shedding microvesicles and apoptotic bodies. Regardless of the debate of the nature of circulating miRNA as byproducts of routine cell activities or mediators of cell-cell communication, proper understanding of the molecular behaviors of miRNA in health and disease, is expected to open a new gate for the discovery of new diagnostic tools and possibly therapeutic implementation in the near future.

In silico Analysis of the Potential Infection Mechanisms of Magnaporthe grisea from Horizontal Gene Transfer Hypothesis

Horizontal gene transfer （HGT） has long been considered as a principal force for an organism to gain novel genes in genome evolution. Homology search, phylogenetic analysis and nucleotide composition analysis are three major objective approaches to arguably determine the occurrence and directionality of HGT. Here, 21 genes that possess the potential to horizontal transfer were acquired from the whole genome of Magnaporthe grisea according to annotation, among which three candidate genes （corresponding protein accession numbers are EAA55123, EAA47200 and EAA52136） were selected for further analysis. According to BLAST homology results, we subsequently conducted phylogenetic analysis of the three candidate HGT genes. Moreover, nucleotide composition analysis was conducted to further validate these HGTs. In addition, the functions of the three candidate genes were searched in COG database. Consequently, we conclude that the gene encoding protein EAA55123 is transferred from Clostridium perfringens. Another HGT event is between EAA52136 and a certain metazoan＇s corresponding gene, but the direction remains uncertain. Yet, EAA47200 is not a transferred gene.

Evolution of Xanthomonas Gene Content:Gene Gain/Loss History and Species Divergence

Horizontal gene transfer （HGT） plays key roles in the evolution of pathogenetic bacteria, especially in pathogenetic associated genes. In this study, the evolutionary dynamics of Xanthomonas at species level were determined by the comparative analysis of the complete genomes of 15 Xanthomonas strains. A concatenated multiprotein phyletic pattern and a dataset with Xanthomonas clusters of orthologous genes were constructed. Mathematical extrapolation estimates that the core genome will reach a minimum of about 1 547 genes while the pan-genome will increase up to 22 624 genes when sequencing 1 000 genomes. The HGT extent in this genus was assessed by using a Markov-based probabilistic method. The reconstructed gene gain/loss history, which contained several features consistent with biological observations, showed that nearly 60% of the Xanthomonas genes were acquired by HGT. A large fraction of variability was in the clade ancestor nodes and ＂leaves of the tree＂. Coexpression analysis suggested that the pathogenic and metabolic variation between Xanthomonas oryzae pv. oryzicola and Xanthomonas oryzae pv. oryzae might due to recently-transferred genes. Our results strongly supported that the gene gain/loss may play an important role in divergence and pathogenicity variation of Xanthomonas species.

Strand-biased Gene Distribution in Bacteria Is Related to both Horizontal Gene Transfer and Strand-biased Nucleotide Composition

Although strand-biased gene distribution （SGD） was described some two decades ago, the underlying molecular mechanisms and their relationship remain elusive. Its facets include, but are not limited to, the degree of biases, the strand-preference of genes, and the influence of background nucleotide composition variations. Using a dataset composed of 364 non-redundant bacterial genomes, we sought to illus- trate our current understanding of SGD. First, when we divided the collection of bacterial genomes into non-polC and polC groups according to their possession of DnaE isoforms that correlate closely with taxonomy, the SGD of the polC group stood out more sig- nificantly than that of the non-polC group. Second, when examining horizontal gene transfer, coupled with gene functional conservation （essentiality） and expressivity （level of expression）, we realized that they all contributed to SGD. Third, we further demonstrated a weaker G-dominance on the leading strand of the non-polC group but strong purine dominance （both G and A） on the leading strand of the polC group. We propose that strand-biased nucleotide composition plays a decisive role for SGD since the polC-bearing genomes are not only AT-rich but also have pronounced purine-rich leading strands, and we believe that a special mutation spectrum that leads to a strong purine asymmetry and a strong strand-biased nucleotide composition coupled with functional selections for genes and their functions are both at work.

Organization, expression and evolution of flagellar genesin <i>Rhodobacter sphaeroides </i>2.4.1

Rhodobacter sphaeroides 2.4.1 belongs to the?-3 subdivision of the Proteobacteria. It possesses a multipartite genome structure consisting of two circular chromosomes, andit displays a wide range of metabolic diversity.Approximately 40 flagellar proteins are required for structure, assembly, and regulation of the flagellum formation in most bacterial species. R. sphaeroidescontains two flagellar gene clusters (fla1 and fla2),which encode 38 and 21 proteins, respectively. Thirty-six of these genes exist in duplicate gene-pairs.A combination of genome analysis, phylogenetic analysis and mRNA expression analysis were employed to examine the conservation of structure, function and evolution of fla1 and fla2 in R. sphaeroides. The results demonstrated that fla2, which was shared among members of ?-Proteobacteria, is native toR. sphaeroides, while fla1 was horizontally transferred from a member of ?-Proteobacteria.In addition, genes located in fla1 are expressed over several growth conditions, but those in fla2 are barely expressed.

Multiple Chromosomes in Bacteria: Low Level of Evolutionary Constraint Drives the Rapid Genetic Divergence of Chromosome II

Multiple chromosomes in bacteria are designated as a larger primary chromosome (CI) and smaller accessory chromosomes (CII and CIII). Although previous studies examined multiple chromosomes in several bacterial species, the evolutionary mechanisms for the origin of CIIs still remain unclear. In this study, the four following hypotheses were tested. 1) CIIs exhibit lower sequence conservation and sequence divergence compared to their corresponding CIs across species of Proteobacteria. 2) The differential sequence divergence of CI and CII depends on pathogenic and non-pathogenic lifestyles. 3) CIIs harbor a higher level of horizontal gene transfers (HGTs) than CIs. 4) Orthologs located on CIIs experience less purifying selection than their corresponding orthologs on CIs. Results reveal a higher level of sequence conservation of CIs than the sequence conservation of CIIs. There is no significant difference in HGT estimates between CIs and CIIs. A majority of orthologous genes of CIs and CIIs experience purifying selection;however, genes on CIIs were significantly less constrained than the corresponding ones on CIs. This finding is true for both pathogenic and non-pathogenic bacteria, but the selective constraints for non-pathogenic bacteria are relatively less constrained. It was concluded that the differential selective constraint is a potent driving force for the rapid evolution of CII. Therefore, gene expression analysis at the transcriptome and proteome levels may shed light on the gene regulation mechanisms that might affect the sequence divergence between CI and CII.

Unexpected complex horizontal gene transfer in teleost fish

Horizontal gene transfer(HGT)is a common occurrence across all domains of life.However,most HGT events were reported between single-celled organisms or parasites and hosts(Van Etten and Bhattacharya 2020).A type II antifreeze protein(AFP)gene was the first and sole evidence of HGT direct vertebrate-to-vertebrate DNA transmission.AFP is only found in 3 widely separated branches of teleost fishes(herring,sea raven,and smelts),sharing amino acid similarity up to 80%(Graham et al.2008).

The statistical power of k-mer based aggregative statistics for alignment-free detection of horizontal gene transfer

Alignment-based database search and sequence comparison are commonly used to detect horizontal gene transfer(HGT).However,with the rapid increase of sequencing depth,hundreds of thousands of contigs are routinely assembled from metagenomics studies,which challenges alignment-based HGT analysis by overwhelming the known reference sequences.Detecting HGT by k-mer statistics thus becomes an attractive alternative.These alignment-free statistics have been demonstrated in high performance and efficiency in wholegenome and transcriptome comparisons.To adapt k-mer statistics for HGT detection,we developed two aggregative statistics T^(S)_(sum ) and T^(*)_(sum),which subsample metagenome contigs by their representative regions,and summarize the regional D^(S) _(2) and D^(*)_(2)metrics by their upper bounds.We systematically studied the aggregative statistics’power at different k-mer size using simulations.Our analysis showed that,in general,the power of T^(S)_(sum) and T^(*)_(sum) increases with sequencing coverage,and reaches a maximum power>80%at k=6,with 5%Type-I error and the coverage ratio>0.2x.The statistical power ofT^(S)_(sum) and T^(*)_(sum) was evaluated with realistic simulations of HGT mechanism,sequencing depth,read length,and base error.We expect these statistics to be useful distance metrics for identifying HGT in metagenomic studies.

新兴污染物对抗生素抗性基因水平转移的影响

抗生素抗性基因(antibiotic resistance genes,ARGs)引起的抗生素耐药性问题被列为新兴环境污染问题,对生态环境和人类健康构成严重威胁。当前备受关注的环境新兴污染物(如内分泌干扰物、重金属、微塑料、纳米塑料等)都可以通过水平基因转移单独或协同促进ARGs在环境中的传播、转移和扩散。新兴污染物如何影响ARGs的水平转移过程已成为备受关注的研究热点。本文全面综述了ARGs的来源及水平基因转移的3种经典方式(接合、自然转化和转导),总结了新兴污染物影响ARGs水平转移的规律及潜在机制,以充分认识ARGs在生态系统中的传播动态与最终命运,有助于全面了解新兴污染物在ARGs传播中的作用。最后提出了目前ARGs水平转移的阻断措施和研究的局限性,并为未来研究提出了相关建议,以便制定有效策略防控并阻断ARGs在环境中的传播过程。