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September 22, 2019  |  

Nonmutational mechanism of inheritance in the Archaeon Sulfolobus solfataricus.

Epigenetic phenomena have not yet been reported in archaea, which are presumed to use a classical genetic process of heritability. Here, analysis of independent lineages of Sulfolobus solfataricus evolved for enhanced fitness implicated a non-Mendelian basis for trait inheritance. The evolved strains, called super acid-resistant Crenarchaeota (SARC), acquired traits of extreme acid resistance and genome stability relative to their wild-type parental lines. Acid resistance was heritable because it was retained regardless of extensive passage without selection. Despite the hereditary pattern, in one strain, it was impossible for these SARC traits to result from mutation because its resequenced genome had no mutation. All strains also had conserved, heritable transcriptomes implicated in acid resistance. In addition, they had improved genome stability with absent or greatly decreased mutation and transposition relative to a passaged control. A mechanism that would confer these traits without DNA sequence alteration could involve posttranslationally modified archaeal chromatin proteins. To test this idea, homologous recombination with isogenic DNA was used to perturb native chromatin structure. Recombination at up-regulated loci from the heritable SARC transcriptome reduced acid resistance and gene expression in the majority of recombinants. In contrast, recombination at a control locus that was not part of the heritable transcriptome changed neither acid resistance nor gene expression. Variation in the amount of phenotypic and expression changes across individuals was consistent with Rad54-dependent chromatin remodeling that dictated crossover location and branch migration. These data support an epigenetic model implicating chromatin structure as a contributor to heritable traits.

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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.

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September 22, 2019  |  

N6-methyladenine DNA modification in Xanthomonas oryzae pv. oryzicola genome.

DNA N6-methyladenine (6mA) modifications expand the information capacity of DNA and have long been known to exist in bacterial genomes. Xanthomonas oryzae pv. Oryzicola (Xoc) is the causative agent of bacterial leaf streak, an emerging and destructive disease in rice worldwide. However, the genome-wide distribution patterns and potential functions of 6mA in Xoc are largely unknown. In this study, we analyzed the levels and global distribution patterns of 6mA modification in genomic DNA of seven Xoc strains (BLS256, BLS279, CFBP2286, CFBP7331, CFBP7341, L8 and RS105). The 6mA modification was found to be widely distributed across the seven Xoc genomes, accounting for percent of 3.80, 3.10, 3.70, 4.20, 3.40, 2.10, and 3.10 of the total adenines in BLS256, BLS279, CFBP2286, CFBP7331, CFBP7341, L8, and RS105, respectively. Notably, more than 82% of 6mA sites were located within gene bodies in all seven strains. Two specific motifs for 6?mA modification, ARGT and AVCG, were prevalent in all seven strains. Comparison of putative DNA methylation motifs from the seven strains reveals that Xoc have a specific DNA methylation system. Furthermore, the 6?mA modification of rpfC dramatically decreased during Xoc infection indicates the important role for Xoc adaption to environment.

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September 22, 2019  |  

Impacts of horizontal gene transfer on the compact genome of the clavulanic acid-producing Streptomyces strain F613-1.

Mobile genetic elements involved in mediating horizontal transfer events contribute to bacterial evolution, and bacterial genomic plasticity and instability result in variation in functional genetic information in Streptomyces secondary metabolism. In a previous study, we reported the complete genome sequence of the industrial Streptomyces strain F613-1, which produces high yields of clavulanic acid. In this study, we used comparative genomics and bioinformatics to investigate the unique genomic features of this strain. Taken together, comparative genomics were used to systematically investigate secondary metabolism capabilities and indicated that frequent exchange of genetic materials between Streptomyces replicons may shape the remarkable diversities in their secondary metabolite repertoires. Moreover, a 136.9-kb giant region of plasticity (RGP) was found in the F613-1 chromosome, and the chromosome and plasmid pSCL4 are densely packed with an exceptionally large variety of potential secondary metabolic gene clusters, involving several determinants putatively accounting for antibiotic production. In addition, the differences in the architecture and size of plasmid pSCL4 between F613-1 and ATCC 27064 suggest that the pSCL4 plasmid could evolve from pSCL4-like and pSCL2-like extrachromosomal replicons. Furthermore, the genomic analyses revealed that strain F613-1 has developed specific genomic architectures and genetic patterns that are well suited to meet the requirements of industrial innovation processes.

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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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September 22, 2019  |  

Conjugative transfer of a novel Staphylococcal plasmid encoding the biocide resistance gene, qacA.

Staphylococcus aureus is the leading cause of skin and soft tissue infections (SSTI). Some S. aureus strains harbor plasmids that carry genes that affect resistance to biocides. Among these genes, qacA encodes the QacA Multidrug Efflux Pump that imparts decreased susceptibility to chlorhexidine, a biocide used ubiquitously in healthcare facilities. Furthermore, chlorhexidine has been considered as a S. aureus decolonization strategy in community settings. We previously conducted a chlorhexidine-based SSTI prevention trial among Ft. Benning Army trainees. Analysis of a clinical isolate (C02) from that trial identified a novel qacA-positive plasmid, pC02. Prior characterization of qacA-containing plasmids is limited and conjugative transfer of those plasmids has not been demonstrated. Given the implications of increased biocide resistance, herein we characterized pC02. In silico analysis identified genes typically associated with conjugative plasmids. Moreover, pC02 was efficiently transferred to numerous S. aureus strains and to Staphylococcus epidermidis. We screened additional qacA-positive S. aureus clinical isolates and pC02 was present in 27% of those strains; other unique qacA-harboring plasmids were also identified. Ten strains were subjected to whole genome sequencing. Sequence analysis combined with plasmid screening studies suggest that qacA-containing strains are transmitted among military personnel at Ft. Benning and that strains carrying qacA are associated with SSTIs within this population. The identification of a novel mechanism of qacA conjugative transfer among Staphylococcal strains suggests a possible future increase in the prevalence of antiseptic tolerant bacterial strains, and an increase in the rate of infections in settings where these agents are commonly used.

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September 22, 2019  |  

A mcr-1-carrying conjugative IncX4 plasmid in colistin-resistant Escherichia coli ST278 strain isolated from dairy cow feces in Shanghai, China.

Enterobacteriaceae, including Escherichia coli, has been shown to acquire the colistin resistance gene mcr-1. A strain of E. coli, EC11, which is resistant to colistin, polymyxin B and trimethoprim-sulfamethoxazole, was isolated in 2016 from the feces of a dairy cow in Shanghai, China. Strain EC11 identifies with sequence type ST278 and is susceptible to 19 frequently used antibiotics. Whole genome sequencing of strain EC11 showed that this strain contains a 31-kb resistance plasmid, pEC11b, which belongs to the IncX4 group. The mcr-1 gene was shown to be inserted into a 2.6-kb mcr-1-pap2 cassette of pEC11b. Plasmid pEC11b also contained putative conjugal transfer components, including an oriT-like region, relaxase, type IV coupling protein, and type IV secretion system. We were successful in transferring pEC11b to E. coli C600 with an average transconjugation efficiency of 4.6 × 10-5. Additionally, a MLST-based analysis comparing EC11 and other reported mcr-positive E. coli populations showed high genotypic diversity. The discovery of the E. coli strain EC11 with resistance to colistin in Shanghai emphasizes the importance of vigilance in detecting new threats like mcr genes to public health. Detection of mcr genes helps in tracking, slowing, and responding to the emergence of antibiotic resistance in Chinese livestock farming.

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September 22, 2019  |  

Acquired interbacterial defense systems protect against interspecies antagonism in the human gut microbiome

The genomes of bacteria derived from the gut microbiota are replete with pathways that mediate contact-dependent interbacterial antagonism. However, the role of direct interactions between co-resident microbes in driving microbiome composition is not well understood. Here we report the widespread occurrence of acquired interbacterial defense (AID) gene clusters in the human gut microbiome. These clusters are found on predicted mobile elements and encode arrays of immunity genes that confer protection against interbacterial toxin-mediated antagonism in vitro and in gnotobiotic mice. We find that Bacteroides ovatus strains containing AID systems that inactivate B. fragilis toxins delivered between cells by the type VI secretion system are enriched in samples lacking detectable B. fragilis. Moreover, these strains display significantly higher abundance in gut metagenomes than strains without AID systems. Finally, we identify a recombinase-associated AID subtype present broadly in Bacteroidales genomes with features suggestive of active gene acquisition. Our data suggest that neutralization of contact-dependent interbacterial antagonism via AID systems plays an important role in shaping human gut microbiome ecology.

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September 22, 2019  |  

Analysis of structural variants in four African cichlids highlights an association with developmental and immune related genes

African Lakes Cichlids are one of the most impressive example of adaptive radiation. Independently in Lake Victoria, Tanganyika, and Malawi, several hundreds of species arose within the last 10 million to 100,000 years. Whereas most analyses in cichlids focused on nucleotide substitutions across species to investigate the genetic bases of this explosive radiation, to date, no study has investigated the contribution of structural variants (SVs) to speciation events (through a reduction of gene flow) and adaptation to different ecological niches. Here, we annotate and characterize the repertoires and evolutionary potential of different SV classes (deletion, duplication, inversion, insertions and translocations) in five cichlid species (Astatotilapia burtoni, Metriaclima zebra, Neolamprologus brichardi, Pundamilia nyererei and Oreochromis niloticus). We investigate the patterns of gain/loss evolution across the phylogeny for each SV type enabling the identification of both lineage specific events and a set of conserved SVs, common to all four species in the radiation. Both deletion and inversion events show a significant overlap with SINE elements, while inversions additionally show a limited, but significant association with DNA transposons. Genes lying inside inverted regions are enriched for genes regulating behaviour, or involved in skeletal and visual system development. Moreover, we find that duplicated genes show enrichment for textquoterightantigen processing and presentationtextquoteright (GO:0019882) and other immune related categories. Altogether, we provide the first, comprehensive overview of rearrangement evolution in East African Cichlids, and some initial insights into their possible contribution to adaptation.

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September 22, 2019  |  

Genome sequences of two diploid wild relatives of cultivated sweetpotato reveal targets for genetic improvement

Sweetpotato [Ipomoea batatas (L.) Lam.] is a globally important staple food crop, especially for sub-Saharan Africa. Agronomic improvement of sweetpotato has lagged behind other major food crops due to a lack of genomic and genetic resources and inherent challenges in breeding a heterozygous, clonally propagated polyploid. Here, we report the genome sequences of its two diploid relatives, I. trifida and I. triloba, and show that these high-quality genome assemblies are robust references for hexaploid sweetpotato. Comparative and phylogenetic analyses reveal insights into the ancient whole-genome triplication history of Ipomoea and evolutionary relationships within the Batatas complex. Using resequencing data from 16 genotypes widely used in African breeding programs, genes and alleles associated with carotenoid biosynthesis in storage roots are identified, which may enable efficient breeding of varieties with high provitamin A content. These resources will facilitate genome-enabled breeding in this important food security crop.

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September 22, 2019  |  

DNA Methylation by Restriction Modification Systems Affects the Global Transcriptome Profile in Borrelia burgdorferi.

Prokaryote restriction modification (RM) systems serve to protect bacteria from potentially detrimental foreign DNA. Recent evidence suggests that DNA methylation by the methyltransferase (MTase) components of RM systems can also have effects on transcriptome profiles. The type strain of the causative agent of Lyme disease, Borrelia burgdorferi B31, possesses two RM systems with N6-methyladenosine (m6A) MTase activity, which are encoded by the bbe02 gene located on linear plasmid lp25 and bbq67 on lp56. The specific recognition and/or methylation sequences had not been identified for either of these B. burgdorferi MTases, and it was not previously known whether these RM systems influence transcript levels. In the current study, single-molecule real-time sequencing was utilized to map genome-wide m6A sites and to identify consensus modified motifs in wild-type B. burgdorferi as well as MTase mutants lacking either the bbe02 gene alone or both bbe02 and bbq67 genes. Four novel conserved m6A motifs were identified and were fully attributable to the presence of specific MTases. Whole-genome transcriptome changes were observed in conjunction with the loss of MTase enzymes, indicating that DNA methylation by the RM systems has effects on gene expression. Genes with altered transcription in MTase mutants include those involved in vertebrate host colonization (e.g., rpoS regulon) and acquisition by/transmission from the tick vector (e.g., rrp1 and pdeB). The results of this study provide a comprehensive view of the DNA methylation pattern in B. burgdorferi, and the accompanying gene expression profiles add to the emerging body of research on RM systems and gene regulation in bacteria.IMPORTANCE Lyme disease is the most prevalent vector-borne disease in North America and is classified by the Centers for Disease Control and Prevention (CDC) as an emerging infectious disease with an expanding geographical area of occurrence. Previous studies have shown that the causative bacterium, Borrelia burgdorferi, methylates its genome using restriction modification systems that enable the distinction from foreign DNA. Although much research has focused on the regulation of gene expression in B. burgdorferi, the effect of DNA methylation on gene regulation has not been evaluated. The current study characterizes the patterns of DNA methylation by restriction modification systems in B. burgdorferi and evaluates the resulting effects on gene regulation in this important pathogen. Copyright © 2018 American Society for Microbiology.

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September 22, 2019  |  

Novel energy conservation strategies and behaviour of Pelotomaculum schinkii driving syntrophic propionate catabolism.

Under methanogenic conditions, short-chain fatty acids are common byproducts from degradation of organic compounds and conversion of these acids is an important component of the global carbon cycle. Due to the thermodynamic difficulty of propionate degradation, this process requires syntrophic interaction between a bacterium and partner methanogen; however, the metabolic strategies and behaviour involved are not fully understood. In this study, the first genome analysis of obligately syntrophic propionate degraders (Pelotomaculum schinkii HH and P. propionicicum MGP) and comparison with other syntrophic propionate degrader genomes elucidated novel components of energy metabolism behind Pelotomaculum propionate oxidation. Combined with transcriptomic examination of P. schinkii behaviour in co-culture with Methanospirillum hungatei, we found that formate may be the preferred electron carrier for P. schinkii syntrophy. Propionate-derived menaquinol may be primarily re-oxidized to formate, and energy was conserved during formate generation through newly proposed proton-pumping formate extrusion. P. schinkii did not overexpress conventional energy metabolism associated with a model syntrophic propionate degrader Syntrophobacter fumaroxidans MPOB (i.e., CoA transferase, Fix and Rnf). We also found that P. schinkii and the partner methanogen may also interact through flagellar contact and amino acid and fructose exchange. These findings provide new understanding of syntrophic energy acquisition and interactions.© 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.

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September 22, 2019  |  

Meiotic drive of female-inherited supernumerary chromosomes in a pathogenic fungus.

Meiosis is a key cellular process of sexual reproduction that includes pairing of homologous sequences. In many species however, meiosis can also involve the segregation of supernumerary chromosomes, which can lack a homolog. How these unpaired chromosomes undergo meiosis is largely unknown. In this study we investigated chromosome segregation during meiosis in the haploid fungus Zymoseptoria tritici that possesses a large complement of supernumerary chromosomes. We used isogenic whole chromosome deletion strains to compare meiotic transmission of chromosomes when paired and unpaired. Unpaired chromosomes inherited from the male parent as well as paired supernumerary chromosomes in general showed Mendelian inheritance. In contrast, unpaired chromosomes inherited from the female parent showed non-Mendelian inheritance but were amplified and transmitted to all meiotic products. We concluded that the supernumerary chromosomes of Z. tritici show a meiotic drive and propose an additional feedback mechanism during meiosis, which initiates amplification of unpaired female-inherited chromosomes.© 2018, Habig et al.

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September 22, 2019  |  

Genome-scale analysis of Acetobacterium bakii reveals the cold adaptation of psychrotolerant acetogens by post-transcriptional regulation.

Acetogens synthesize acetyl-CoA via CO2 or CO fixation, producing organic compounds. Despite their ecological and industrial importance, their transcriptional and post-transcriptional regulation has not been systematically studied. With completion of the genome sequence of Acetobacterium bakii (4.28-Mb), we measured changes in the transcriptome of this psychrotolerant acetogen in response to temperature variations under autotrophic and heterotrophic growth conditions. Unexpectedly, acetogenesis genes were highly up-regulated at low temperatures under heterotrophic, as well as autotrophic, growth conditions. To mechanistically understand the transcriptional regulation of acetogenesis genes via changes in RNA secondary structures of 5′-untranslated regions (5′-UTR), the primary transcriptome was experimentally determined, and 1379 transcription start sites (TSS) and 1100 5′-UTR were found. Interestingly, acetogenesis genes contained longer 5′-UTR with lower RNA-folding free energy than other genes, revealing that the 5′-UTRs control the RNA abundance of the acetogenesis genes under low temperature conditions. Our findings suggest that post-transcriptional regulation via RNA conformational changes of 5′-UTRs is necessary for cold-adaptive acetogenesis.© 2018 Shin et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

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September 22, 2019  |  

Investigation of inter- and intraspecies variation through genome sequencing of Aspergillus section Nigri.

Aspergillus section Nigri comprises filamentous fungi relevant to biomedicine, bioenergy, health, and biotechnology. To learn more about what genetically sets these species apart, as well as about potential applications in biotechnology and biomedicine, we sequenced 23 genomes de novo, forming a full genome compendium for the section (26 species), as well as 6 Aspergillus niger isolates. This allowed us to quantify both inter- and intraspecies genomic variation. We further predicted 17,903 carbohydrate-active enzymes and 2,717 secondary metabolite gene clusters, which we condensed into 455 distinct families corresponding to compound classes, 49% of which are only found in single species. We performed metabolomics and genetic engineering to correlate genotypes to phenotypes, as demonstrated for the metabolite aurasperone, and by heterologous transfer of citrate production to Aspergillus nidulans. Experimental and computational analyses showed that both secondary metabolism and regulation are key factors that are significant in the delineation of Aspergillus species.

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