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September 22, 2019  |  

Identification of the DNA methyltransferases establishing the methylome of the cyanobacterium Synechocystis sp. PCC 6803.

DNA methylation in bacteria is important for defense against foreign DNA, but is also involved in DNA repair, replication, chromosome partitioning, and regulatory processes. Thus, characterization of the underlying DNA methyltransferases in genetically tractable bacteria is of paramount importance. Here, we characterized the methylome and orphan methyltransferases in the model cyanobacterium Synechocystis sp. PCC 6803. Single molecule real-time (SMRT) sequencing revealed four DNA methylation recognition sequences in addition to the previously known motif m5CGATCG, which is recognized by M.Ssp6803I. For three of the new recognition sequences, we identified the responsible methyltransferases. M.Ssp6803II, encoded by the sll0729 gene, modifies GGm4CC, M.Ssp6803III, encoded by slr1803, represents the cyanobacterial dam-like methyltransferase modifying Gm6ATC, and M.Ssp6803V, encoded by slr6095 on plasmid pSYSX, transfers methyl groups to the bipartite motif GGm6AN7TTGG/CCAm6AN7TCC. The remaining methylation recognition sequence GAm6AGGC is probably recognized by methyltransferase M.Ssp6803IV encoded by slr6050. M.Ssp6803III and M.Ssp6803IV were essential for the viability of Synechocystis, while the strains lacking M.Ssp6803I and M.Ssp6803V showed growth similar to the wild type. In contrast, growth was strongly diminished of the ?sll0729 mutant lacking M.Ssp6803II. These data provide the basis for systematic studies on the molecular mechanisms impacted by these methyltransferases.


September 22, 2019  |  

Sequencing of pT5282-CTXM, p13190-KPC and p30860-NR, and comparative genomics analysis of IncX8 plasmids.

This study proposes a replicon-based scheme for typing IncX plasmids into nine separately clustering subgroups, including IncX1a, IncX1ß and IncX2-8. The complete nucleotide sequences of three IncX8 plasmids, namely pT5282-CTXM and p30860-NR from Enterobacter cloacae and p13190-KPC from Klebsiella pneumoniae, were determined and were compared with two other previously sequenced IncX8 plasmids (pCAV1043-58 and pCAV1741-16). These five plasmids possessed conserved IncX8 backbones with limited genetic variation with respect to gene content and organisation, and each of them carried one or three accessory modules that harboured resistance markers and metabolic gene clusters as well as transposons, insertion sequence (IS)-based transposition units and miniature inverted repeat transposable elements (MITEs), indicating that the relatively small IncX8 backbones were able to integrate various foreign genetic contents. The resistance genes blaCTX-M-3 and blaTEM-1 (ß-lactam resistance), blaKPC-2 (carbapenem resistance) and ?blaTEM-1, and tet(A) (tetracycline resistance) and mph(E) (macrolide resistance) were found in pT5282-CTXM, p13190-KPC and pCAV1741-16, respectively, whilst p30860-NR and pCAV1043-58 carried no resistance genes. The data presented here provide an insight into the diversification and evolution history of IncX8 plasmids. Copyright © 2018 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.


September 22, 2019  |  

Tracing genomic divergence of Vibrio bacteria in the Harveyi clade.

The mechanism of bacterial speciation remains a topic of tremendous interest. To understand the ecological and evolutionary mechanisms of speciation in Vibrio bacteria, we analyzed the genomic dissimilarities between three closely related species in the so-called Harveyi clade of the genus Vibrio, V. campbellii, V. jasicida, and V. hyugaensis The analysis focused on strains isolated from diverse geographic locations over a long period of time. The results of phylogenetic analyses and calculations of average nucleotide identity (ANI) supported the classification of V. jasicida and V. hyugaensis into two species. These analyses also identified two well-supported clades in V. campbellii; however, strains from both clades were classified as members of the same species. Comparative analyses of the complete genome sequences of representative strains from the three species identified higher syntenic coverage between genomes of V. jasicida and V. hyugaensis than that between the genomes from the two V. campbellii clades. The results from comparative analyses of gene content between bacteria from the three species did not support the hypothesis that gene gain and/or loss contributed to their speciation. We also did not find support for the hypothesis that ecological diversification toward associations with marine animals contributed to the speciation of V. jasicida and V. hyugaensis Overall, based on the results obtained in this study, we propose that speciation in Harveyi clade species is a result of stochastic diversification of local populations, which was influenced by multiple evolutionary processes, followed by extinction events.IMPORTANCE To investigate the mechanisms underlying speciation in the genus Vibrio, we provided a well-assembled reference of genomes and performed systematic genomic comparisons among three evolutionarily closely related species. We resolved taxonomic ambiguities and identified genomic features separating the three species. Based on the study results, we propose a hypothesis explaining how species in the Harveyi clade of Vibrio bacteria diversified. Copyright © 2018 American Society for Microbiology.


September 22, 2019  |  

Linking genotype and phenotype in an economically viable propionic acid biosynthesis process

Propionic acid (PA) is used as a food preservative and increasingly, as a precursor for the synthesis of monomers. PA is produced mainly through hydrocarboxylation of ethylene, also known as the `oxo-process’; however, Propionibacterium species are promising biological PA producers natively producing PA as their main fermentation product. However, for fermentation to be competitive, a PA yield of at least 0.6 g/g is required.


September 22, 2019  |  

The complete methylome of an entomopathogenic bacterium reveals the existence of loci with unmethylated adenines.

DNA methylation can serve to control diverse phenomena in eukaryotes and prokaryotes, including gene regulation leading to cell differentiation. In bacteria, DNA methylomes (i.e., methylation state of each base of the whole genome) have been described for several species, but methylome profile variation during the lifecycle has rarely been studied, and only in a few model organisms. Moreover, major phenotypic changes have been reported in several bacterial strains with a deregulated methyltransferase, but the corresponding methylome has rarely been described. Here we report the first methylome description of an entomopathogenic bacterium, Photorhabdus luminescens. Eight motifs displaying a high rate of methylation (>94%) were identified. The methylome was strikingly stable over course of growth, but also in a subpopulation responsible for a critical step in the bacterium’s lifecycle: successful survival and proliferation in insects. The rare unmethylated GATC motifs were preferentially located in putative promoter regions, and most of them were methylated after Dam methyltransferase overexpression, suggesting that DNA methylation is involved in gene regulation. Our findings bring key insight into bacterial methylomes and encourage further research to decipher the role of loci protected from DNA methylation in gene regulation.


September 22, 2019  |  

Large scale changes in host methylation patterns induced by IncA/C plasmid transformation in Vibrio cholerae

DNA methylation is a central epigenetic modification and has diverse biological functions in eukaryotic and prokaryotic organisms alike. The IncA/C plasmid genomes are approximately 150kb in length and harbour three methylase genes, two of which demonstrate cytosine specificity. Transformation of the Vibrio cholerae strain C6706 with the IncA/C plasmid pVC211 resulted in a significant relabelling of the methylation patterns on the host chromosomes. The new methylation patterns induced by transformation with IncA/C plasmid were accepted by the restriction enzymes of the hosttextquoterights restriction modification (RM) system. These data uncover a novel mechanism by which plasmids can be compatible with a hosttextquoterights RM system and suggest a possible reason that plasmids of the IncA/C family are broad-host-range.


September 22, 2019  |  

Discovery of multi-drug resistant, MCR-1 and ESBL-coproducing ST117 Escherichia coli from diseased chickens in Northeast China

An endemic multi-drug resistant ST117 E. coli isolate coproducing MCR-1 and 3 ESBL loci was, for the first time, detected from diseased chicken, Liaoning Province, in Northeast China, from 2011 to 2012. Whole-genome sequencing revealed 5 unique plasmids, namely pHXH-1, pHXH-2, pHXH-3, pHXH-4 and pHXH-5). Among them, pHXH1 and pHXH4 encode ESBL, and pHXH-5 mediates MCR-1 colistin resistance. The results indicate that the potentially-national dissemination of MCR-1-positive pathogens with pan-drug resistance proceeds via food chains.


September 22, 2019  |  

A comparison of genotypic and phenotypic methods for analyzing the susceptibility to sulfamethoxazole and trimethoprim in Edwardsiella piscicida.

In a study of 39 isolates of Edwardsiella piscicida made from Korean aquaculture sites, sul genes were detected in 16 isolates and dfr genes in 19. Ten isolates were shown to contain both sul and dfr genes. MIC and disc diffusion zones assays were performed to measure the phenotypic susceptibilities of the 39 isolates. Normalized resistance interpretation was applied to these data to categorize isolates as either fully susceptible or as manifesting reduced susceptibility. The standard CLSI protocols specify the use of a mixture of sulfamethoxazole/trimethoprim (20:1) in both MIC and disc diffusion tests. Using the CLSI MIC protocol, 100% of the isolates containing dfr genes, but only 75% of the isolates containing sul genes, were categorized as manifesting reduced susceptibility. Using the CLSI disc diffusion protocol, only 58% of the isolates containing dfr genes and 69% of those containing sul genes were categorized as manifesting reduced susceptibility. When the single agent trimethoprim was substituted for the combined mixture in both the MIC and disc diffusion protocols, 100% of the dfr-positive isolates were categorized as NWT. When the single-agent sulfamethoxazole was substituted, the analysis of the MIC characterized 100% and the disc zone data 94% of the sul-positive isolates as manifesting reduced susceptibility. It is argued that the use of trimethoprim and sulfamethoxazole as single agents in phenotypic susceptibility tests would provide more meaningful data than the currently recommended use of these two agents combined.


September 22, 2019  |  

Comparative analyses of CTX prophage region of Vibrio cholerae seventh pandemic wave 1 strains isolated in Asia.

Vibrio cholerae O1 causes cholera, and cholera toxin, the principal mediator of massive diarrhea, is encoded by ctxAB in the cholera toxin (CTX) prophage. In this study, the structures of the CTX prophage region of V. cholerae strains isolated during the seventh pandemic wave 1 in Asian countries were determined and compared. Eighteen strains were categorized into eight groups by CTX prophage region-specific restriction fragment length polymorphism and PCR profiles and the structure of the region of a representative strain from each group was determined by DNA sequencing. Eight representative strains revealed eight distinct CTX prophage regions with various combinations of CTX-1, RS1 and a novel genomic island on chromosome I. CTX prophage regions carried by the wave 1 strains were diverse in structure. V. cholerae strains with an area specific CTX prophage region are believed to circulate in South-East Asian countries; additionally, multiple strains with distinct types of CTX prophage region are co-circulating in the area. Analysis of a phylogenetic tree generated by single nucleotide polymorphism differences across 2483 core genes revealed that V. cholerae strains categorized in the same group based on CTX prophage region structure were segregated in closer clusters. CTX prophage region-specific recombination events or gain and loss of genomic elements within the region may have occurred at much higher frequencies and contributed to producing a panel of CTX prophage regions with distinct structures among V. cholerae pathogenic strains in lineages with close genetic backgrounds in the early wave 1 period of the seventh cholera pandemic.© 2018 The Authors. Microbiology and Immunology published by The Societies and John Wiley & Sons Australia, Ltd.


September 22, 2019  |  

Therapeutic potential of a new jumbo phage that infects Vibrio coralliilyticus, a widespread coral pathogen.

Biological control using bacteriophages is a promising approach for mitigating the devastating effects of coral diseases. Several phages that infect Vibrio coralliilyticus, a widespread coral pathogen, have been isolated, suggesting that this bacterium is permissive to viral infection and is, therefore, a suitable candidate for treatment by phage therapy. In this study, we combined functional and genomic approaches to evaluate the therapeutic potential of BONAISHI, a novel V. coralliilyticus phage, which was isolated from the coral reef in Van Phong Bay (Vietnam). BONAISHI appears to be strictly lytic for several pathogenic strains of V. coralliilyticus and remains infectious over a broad range of environmental conditions. This candidate has an unusually large dsDNA genome (303 kb), with no genes that encode known toxins or implicated in lysogeny control. We identified several proteins involved in host lysis, which may offer an interesting alternative to the use of whole bacteriophages for controlling V. coralliilyticus. A preliminary therapy test showed that adding BONAISHI to an infected culture of Symbiodinium sp. cells reduced the impact of V. coralliilyticus on Symbiodinium sp. photosynthetic activity. This study showed that BONAISHI is able to mitigate V. coralliilyticus infections, making it a good candidate for phage therapy for coral disease.


September 22, 2019  |  

pYR4 from a Norwegian isolate of Yersinia ruckeri is a putative virulence plasmid encoding both a type IV pilus and a type IV secretion system

Enteric redmouth disease caused by the pathogen Yersinia ruckeri is a significant problem for fish farming around the world. Despite its importance, only a few virulence factors of Y. ruckeri have been identified and studied in detail. Here, we report and analyze the complete DNA sequence of pYR4, a plasmid from a highly pathogenic Norwegian Y. ruckeri isolate, sequenced using PacBio SMRT technology. Like the well-known pYV plasmid of human pathogenic Yersiniae, pYR4 is a member of the IncFII family. Thirty-one percent of the pYR4 sequence is unique compared to other Y. ruckeri plasmids. The unique regions contain, among others genes, a large number of mobile genetic elements and two partitioning systems. The G+C content of pYR4 is higher than that of the Y. ruckeri NVH_3758 genome, indicating its relatively recent horizontal acquisition. pYR4, as well as the related plasmid pYR3, comprises operons that encode for type IV pili and for a conjugation system (tra). In contrast to other Yersinia plasmids, pYR4 cannot be cured at elevated temperatures. Our study highlights the power of PacBio sequencing technology for identifying mis-assembled segments of genomic sequences. Comparative analysis of pYR4 and other Y. ruckeri plasmids and genomes, which were sequenced by second and the third generation sequencing technologies, showed errors in second generation sequencing assemblies. Specifically, in the Y. ruckeri 150 and Y. ruckeri ATCC29473 genome assemblies, we mapped the entire pYR3 plasmid sequence. Placing plasmid sequences on the chromosome can result in erroneous biological conclusions. Thus, PacBio sequencing or similar long-read methods should always be preferred for de novo genome sequencing. As the tra operons of pYR3, although misplaced on the chromosome during the genome assembly process, were demonstrated to have an effect on virulence, and type IV pili are virulence factors in many bacteria, we suggest that pYR4 directly contributes to Y. ruckeri virulence.


September 22, 2019  |  

Functional metagenomics identifies an exosialidase with an inverting catalytic mechanism that defines a new glycoside hydrolase family (GH156).

Exosialidases are glycoside hydrolases that remove a single terminal sialic acid residue from oligosaccharides. They are widely distributed in biology, having been found in prokaryotes, eukaryotes, and certain viruses. Most characterized prokaryotic sialidases are from organisms that are pathogenic or commensal with mammals. However, in this study, we used functional metagenomic screening to seek microbial sialidases encoded by environmental DNA isolated from an extreme ecological niche, a thermal spring. Using recombinant expression of potential exosialidase candidates and a fluorogenic sialidase substrate, we discovered an exosialidase having no homology to known sialidases. Phylogenetic analysis indicated that this protein is a member of a small family of bacterial proteins of previously unknown function. Proton NMR revealed that this enzyme functions via an inverting catalytic mechanism, a biochemical property that is distinct from those of known exosialidases. This unique inverting exosialidase defines a new CAZy glycoside hydrolase family we have designated GH156.© 2018 Chuzel et al.


September 22, 2019  |  

Microevolution of Neisseria lactamica during nasopharyngeal colonisation induced by controlled human infection.

Neisseria lactamica is a harmless coloniser of the infant respiratory tract, and has a mutually-excluding relationship with the pathogen Neisseria meningitidis. Here we report controlled human infection with genomically-defined N. lactamica and subsequent bacterial microevolution during 26 weeks of colonisation. We find that most mutations that occur during nasopharyngeal carriage are transient indels within repetitive tracts of putative phase-variable loci associated with host-microbe interactions (pgl and lgt) and iron acquisition (fetA promotor and hpuA). Recurrent polymorphisms occurred in genes associated with energy metabolism (nuoN, rssA) and the CRISPR-associated cas1. A gene encoding a large hypothetical protein was often mutated in 27% of the subjects. In volunteers who were naturally co-colonised with meningococci, recombination altered allelic identity in N. lactamica to resemble meningococcal alleles, including loci associated with metabolism, outer membrane proteins and immune response activators. Our results suggest that phase variable genes are often mutated during carriage-associated microevolution.


September 22, 2019  |  

The enterococcus cassette chromosome, a genomic variation enabler in enterococci.

Enterococcus faecium has a highly variable genome prone to recombination and horizontal gene transfer. Here, we have identified a novel genetic island with an insertion locus and mobilization genes similar to those of staphylococcus cassette chromosome elements SCCmec This novel element termed the enterococcus cassette chromosome (ECC) element was located in the 3′ region of rlmH and encoded large serine recombinases ccrAB similar to SCCmec Horizontal transfer of an ECC element termed ECC::cat containing a knock-in cat chloramphenicol resistance determinant occurred in the presence of a conjugative reppLG1 plasmid. We determined the ECC::cat insertion site in the 3′ region of rlmH in the E. faecium recipient by long-read sequencing. ECC::cat also mobilized by homologous recombination through sequence identity between flanking insertion sequence (IS) elements in ECC::cat and the conjugative plasmid. The ccrABEnt genes were found in 69 of 516 E. faecium genomes in GenBank. Full-length ECC elements were retrieved from 32 of these genomes. ECCs were flanked by attR and attL sites of approximately 50?bp. The attECC sequences were found by PCR and sequencing of circularized ECCs in three strains. The genes in ECCs contained an amalgam of common and rare E. faecium genes. Taken together, our data imply that ECC elements act as hot spots for genetic exchange and contribute to the large variation of accessory genes found in E. faeciumIMPORTANCEEnterococcus faecium is a bacterium found in a great variety of environments, ranging from the clinic as a nosocomial pathogen to natural habitats such as mammalian intestines, water, and soil. They are known to exchange genetic material through horizontal gene transfer and recombination, leading to great variability of accessory genes and aiding environmental adaptation. Identifying mobile genetic elements causing sequence variation is important to understand how genetic content variation occurs. Here, a novel genetic island, the enterococcus cassette chromosome, is shown to contain a wealth of genes, which may aid E. faecium in adapting to new environments. The transmission mechanism involves the only two conserved genes within ECC, ccrABEnt, large serine recombinases that insert ECC into the host genome similarly to SCC elements found in staphylococci. Copyright © 2018 Sivertsen et al.


September 22, 2019  |  

Functionality of two origins of replication in Vibrio cholerae strains with a single chromosome.

Chromosomal inheritance in bacteria usually entails bidirectional replication of a single chromosome from a single origin into two copies and subsequent partitioning of one copy each into daughter cells upon cell division. However, the human pathogen Vibrio cholerae and other Vibrionaceae harbor two chromosomes, a large Chr1 and a small Chr2. Chr1 and Chr2 have different origins, an oriC-type origin and a P1 plasmid-type origin, respectively, driving the replication of respective chromosomes. Recently, we described naturally occurring exceptions to the two-chromosome rule of Vibrionaceae: i.e., Chr1 and Chr2 fused single chromosome V. cholerae strains, NSCV1 and NSCV2, in which both origins of replication are present. Using NSCV1 and NSCV2, here we tested whether two types of origins of replication can function simultaneously on the same chromosome or one or the other origin is silenced. We found that in NSCV1, both origins are active whereas in NSCV2 ori2 is silenced despite the fact that it is functional in an isolated context. The ori2 activity appears to be primarily determined by the copy number of the triggering site, crtS, which in turn is determined by its location with respect to ori1 and ori2 on the fused chromosome.


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