The Dawning Era of Comprehensive Transcriptome Analysis in Cellular Microbiology

Bacteria rapidly change their transcriptional patterns during infection in order to adapt to the host environment. To investigate host–bacteria interactions, various strategies including the use of animal infection models, in vitro assay systems and microscopic observations have been used. However, these studies primarily focused on a few specific genes and molecules in bacteria. High-density tiling arrays and massively parallel sequencing analyses are rapidly improving our understanding of the complex host–bacterial interactions through identification and characterization of bacterial transcriptomes. Information resulting from these high-throughput techniques will continue to provide novel information on the complexity, plasticity, and regulation of bacterial transcriptomes as well as their adaptive responses relative to pathogenecity. Here we summarize recent studies using these new technologies and discuss the utility of transcriptome analysis

Airborne Microbial Communities at High-Altitude and Suburban Sites in Toyama, Japan Suggest a New Perspective for Bioprospecting

Airborne microorganisms, especially those at high altitude, are exposed to hostile conditions, including ultraviolet (UV) radiation, desiccation, and low temperatures. This study was conducted to compare the composition and abundance of airborne microorganisms at a high-altitude site, Mt. Jodo [2,839 m above mean sea level (AMSL)] and a suburban site (23 m AMSL) in Toyama, Japan. To our knowledge, this is the first study to investigate microbial communities in air samples collected simultaneously at two sites in relatively close proximity, from low and high altitude. Air samples were collected over a period of 3 years during 2009–2011. We then examined the bacterial and eukaryotic communities and estimated the abundance of bacteria and fungi with real-time TaqMan PCR. The airborne bacterial and eukaryotic communities differed between high-altitude and suburban sites on each sampling day. Backward trajectory analysis of air masses that arrived at high-altitude and suburban sites on each sampling day displayed almost the same paths. The bacterial communities were dominated by Actinobacteria, Firmicutes, and Proteobacteria, while the eukaryotic communities included Ascomycota, Basidiomycota, and Streptophyta. We also predicted some application of such microbial communities. The airborne bacterial and fungal abundance at the high-altitude site was about two times lower than that at the suburban site. These results showed that each airborne microbial communities have locality even if they are collected close location

Interplay of a non-conjugative integrative element and a conjugative plasmid in the spread of antibiotic resistance via suicidal plasmid transfer from an aquaculture Vibrio isolate

The capture of antimicrobial resistance genes (ARGs) by mobile genetic elements (MGEs) plays a critical role in resistance acquisition for human-associated bacteria. Although aquaculture environments are recognized as important reservoirs of ARGs, intra- and intercellular mobility of MGEs discovered in marine organisms is poorly characterized. Here, we show a new pattern of interspecies ARGs transfer involving a ‘non-conjugative’ integrative element. To identify active MGEs in a Vibrio ponticus isolate, we conducted whole-genome sequencing of a transconjugant obtained by mating between Escherichia coli and Vibrio ponticus. This revealed integration of a plasmid (designated pSEA1) into the chromosome, consisting of a self-transmissible plasmid backbone of the MOBH group, ARGs, and a 13.8-kb integrative element Tn6283. Molecular genetics analysis suggested a two-step gene transfer model. First, Tn6283 integrates into the recipient chromosome during suicidal plasmid transfer, followed by homologous recombination between the Tn6283 copy in the chromosome and that in the newly transferred pSEA1. Tn6283 is unusual among integrative elements in that it apparently does not encode transfer function and its excision barely generates unoccupied donor sites. Thus, its movement is analogous to the transposition of insertion sequences rather than to that of canonical integrative and conjugative elements. Overall, this study reveals the presence of a previously unrecognized type of MGE in a marine organism, highlighting diversity in the mode of interspecies gene transfer

Interplay of a non-conjugative integrative element and a conjugative plasmid in the spread of antibiotic resistance via suicidal plasmid transfer from an aquaculture Vibrio isolate

<div><p>The capture of antimicrobial resistance genes (ARGs) by mobile genetic elements (MGEs) plays a critical role in resistance acquisition for human-associated bacteria. Although aquaculture environments are recognized as important reservoirs of ARGs, intra- and intercellular mobility of MGEs discovered in marine organisms is poorly characterized. Here, we show a new pattern of interspecies ARGs transfer involving a ‘non-conjugative’ integrative element. To identify active MGEs in a <i>Vibrio ponticus</i> isolate, we conducted whole-genome sequencing of a transconjugant obtained by mating between <i>Escherichia coli</i> and <i>Vibrio ponticus</i>. This revealed integration of a plasmid (designated pSEA1) into the chromosome, consisting of a self-transmissible plasmid backbone of the MOB_H group, ARGs, and a 13.8-kb integrative element Tn<i>6283</i>. Molecular genetics analysis suggested a two-step gene transfer model. First, Tn<i>6283</i> integrates into the recipient chromosome during suicidal plasmid transfer, followed by homologous recombination between the Tn<i>6283</i> copy in the chromosome and that in the newly transferred pSEA1. Tn<i>6283</i> is unusual among integrative elements in that it apparently does not encode transfer function and its excision barely generates unoccupied donor sites. Thus, its movement is analogous to the transposition of insertion sequences rather than to that of canonical integrative and conjugative elements. Overall, this study reveals the presence of a previously unrecognized type of MGE in a marine organism, highlighting diversity in the mode of interspecies gene transfer.</p></div

Comparative genomic analyses of Streptococcus mutans provide insights into chromosomal shuffling and species-specific content

<p>Abstract</p> <p>Background</p> <p><it>Streptococcus mutans </it>is the major pathogen of dental caries, and it occasionally causes infective endocarditis. While the pathogenicity of this species is distinct from other human pathogenic streptococci, the species-specific evolution of the genus <it>Streptococcus </it>and its genomic diversity are poorly understood.</p> <p>Results</p> <p>We have sequenced the complete genome of <it>S. mutans </it>serotype <it>c </it>strain NN2025, and compared it with the genome of UA159. The NN2025 genome is composed of 2,013,587 bp, and the two strains show highly conserved core-genome. However, comparison of the two <it>S. mutans </it>strains showed a large genomic inversion across the replication axis producing an X-shaped symmetrical DNA dot plot. This phenomenon was also observed between other streptococcal species, indicating that streptococcal genetic rearrangements across the replication axis play an important role in <it>Streptococcus </it>genetic shuffling. We further confirmed the genomic diversity among 95 clinical isolates using long-PCR analysis. Genomic diversity in <it>S. mutans </it>appears to occur frequently between insertion sequence (IS) elements and transposons, and these diversity regions consist of restriction/modification systems, antimicrobial peptide synthesis systems, and transporters. <it>S. mutans </it>may preferentially reject the phage infection by clustered regularly interspaced short palindromic repeats (CRISPRs). In particular, the CRISPR-2 region, which is highly divergent between strains, in NN2025 has long repeated spacer sequences corresponding to the streptococcal phage genome.</p> <p>Conclusion</p> <p>These observations suggest that <it>S. mutans </it>strains evolve through chromosomal shuffling and that phage infection is not needed for gene acquisition. In contrast, <it>S. pyogenes </it>tolerates phage infection for acquisition of virulence determinants for niche adaptation.</p

Enantioselective utilization of D-amino acids by deep-sea microorganisms

Microorganisms that utilize various D-amino acids (DAAs) were successfully isolated from deep-sea sediments. The isolates were phylogenetically assigned to Alphaproteobacteria, Gammmaproteobacteria, and Bacilli. Some of the isolates exhibited high enantioselective degradation activities to various DAAs. In particular, the Alphaproteobacteria Nautella sp. strain A04V exhibited robust growth in minimal medium supplemented with D-Val as a sole carbon and nitrogen source, whereas its growth was poor on minimal medium supplemented with L-Val instead of D-Val. Its growth was facilitated most when racemic mixtures of valine were used. In contrast, the Nautella strains isolated from shallow-sea grew only with L-Val. No significant differences were found among the strains in the genome sequences including genes possibly related to DAA metabolisms.http://www.godac.jamstec.go.jp/darwin/cruise/yokosuka/yk05-15/ehttp://www.godac.jamstec.go.jp/darwin/cruise/natsushima/nt06-17/ehttp://www.godac.jamstec.go.jp/darwin/cruise/natsushima/nt08-24/

Persistent colonization of non-lymphoid tissue-resident macrophages by Stenotrophomonas maltophilia

Accumulating evidence has revealed that lymphoid tissue-resident commensal bacteria (e.g. Alcaligenes spp.) survive within dendritic cells. We extended our previous study by investigating microbes that persistently colonize colonic macrophages. 16S rRNA-based metagenome analysis using DNA purified from murine colonic macrophages revealed the presence of Stenotrophomonas maltophilia. The in situ intracellular colonization by S. maltophilia was recapitulated in vitro by using bone marrow-derived macrophages (BMDMs). Co-culture of BMDMs with clinically isolated S. maltophilia led to increased mitochondrial respiration and robust IL-10 production. We further identified a 25-kDa protein encoded by the gene assigned as smlt2713 (recently renamed as SMLT_RS12935) and secreted by S. maltophilia as the factor responsible for enhanced IL-10 production by BMDMs. IL-10 production is critical for maintenance of the symbiotic condition, because intracellular colonization by S. maltophilia was impaired in IL-10-deficient BMDMs, and smlt2713-deficient S. maltophilia failed to persistently colonize IL-10-competent BMDMs. These findings indicate a novel commensal network between colonic macrophages and S. maltophilia that is mediated by IL-10 and smlt2713

Complete Genome Sequence of the Nonheterocystous Cyanobacterium Pseudanabaena sp. ABRG5-3

We report here the complete sequences of the main genome (4.8 Mb) and seven plasmids of the semifilamentous, nonheterocystous cyanobacterium Pseudanabaena sp. ABRG5-3, a strain isolated from a pond in Japan. These data are expected to enhance our understanding of the Pseudanabaena subclade near the root of cyanobacterial diversity

CRISPR Inhibition of Prophage Acquisition in Streptococcus pyogenes

Streptococcus pyogenes, one of the major human pathogens, is a unique species since it has acquired diverse strain-specific virulence properties mainly through the acquisition of streptococcal prophages. In addition, S. pyogenes possesses clustered regularly interspaced short palindromic repeats (CRISPR)/Cas systems that can restrict horizontal gene transfer (HGT) including phage insertion. Therefore, it was of interest to examine the relationship between CRISPR and acquisition of prophages in S. pyogenes. Although two distinct CRISPR loci were found in S. pyogenes, some strains lacked CRISPR and these strains possess significantly more prophages than CRISPR harboring strains. We also found that the number of spacers of S. pyogenes CRISPR was less than for other streptococci. The demonstrated spacer contents, however, suggested that the CRISPR appear to limit phage insertions. In addition, we found a significant inverse correlation between the number of spacers and prophages in S. pyogenes. It was therefore suggested that S. pyogenes CRISPR have permitted phage insertion by lacking its own spacers. Interestingly, in two closely related S. pyogenes strains (SSI-1 and MGAS315), CRISPR activity appeared to be impaired following the insertion of phage genomes into the repeat sequences. Detailed analysis of this prophage insertion site suggested that MGAS315 is the ancestral strain of SSI-1. As a result of analysis of 35 additional streptococcal genomes, it was suggested that the influences of the CRISPR on the phage insertion vary among species even within the same genus. Our results suggested that limitations in CRISPR content could explain the characteristic acquisition of prophages and might contribute to strain-specific pathogenesis in S. pyogenes