Construction of multiple metagenome assembled genomes containing carbon monoxide dehydrogenases from anaerobic carbon monoxide enrichment cultures.

Despite its toxicity to many organisms, including most prokaryotes, carbon monoxide (CO) is utilized by some aerobic and anaerobic prokaryotes. Hydrogenogenic CO utilizers employ carbon monoxide dehydrogenase (CODH) and energy-converting hydrogenase (ECH) to oxidize CO and reduce protons to produce H2. Those prokaryotes constitute a rare biosphere and are difficult to detect even with PCR amplification and with metagenomic analyses. In this study, anaerobic CO-enrichment cultures followed by construction of metagenome assembled genomes (MAGs) detected high-quality MAGs from potential hydrogenogenic CO utilizers. Of 32 MAGs constructed, 5 were potential CO utilizer harboring CODH genes. Of the five MAGs, two were classified into the genus Thermolithobacter on the basis of 16S rRNA sequence identity, related to Carboxydocella tharmautotrophica 41, with an average nucleotide identity (ANI) of approximately 72%. Additionally, two were related to Geoglobus acetivorans with ANI values ranging from 75 to 77% to G. acetivorans SBH6, and one MAG was identified as Desulfotomaculum kuznetsovii with an ANI > 96% to D. kuznetsovii DSM 6115. The two Thermolithobacter MAGs identified in this study contained CODH-ECH gene clusters, and were therefore identified as potential hydrogenogenic CO utilizers. However, these MAGs harbored three CODH gene clusters that showed distinct physiological functions in addition to CODH-ECH gene clusters. In total, the five potential CO utilizer MAGs contained sixteen CODH genes. Among those CODHs, four sets did not cluster with any known CODH protein sequences (with an identity of > 90%), and the CODH database was expanded.

Isolation, Genomic Sequence and Physiological Characterization of Parageobacillus sp. G301, an Isolate Capable of Both Hydrogenogenic and Aerobic Carbon Monoxide Oxidation.

Prokaryotes that can oxidize carbon monoxide (CO oxidizers) can use this gas as a source of carbon or energy. They oxidize carbon monoxide with carbon monoxide dehydrogenases (CODHs): these are divided into nickel-containing CODH (Ni-CODH), which are sensitive to O2, and molybdenum-containing CODH (Mo-CODH), which can function aerobically. The oxygen conditions required for CO oxidizers to oxidize CO may be limited, as those which have been isolated and characterized so far contain either Ni- or Mo-CODH. Here, we report a novel CO oxidizer, Parageobacillus sp. G301, which is capable of CO oxidation using both types of CODH based on genomic and physiological characterization. This thermophilic, facultatively anaerobic Bacillota bacterium was isolated from the sediments of a freshwater lake. Genomic analyses revealed that strain G301 possessed both Ni-CODH and Mo-CODH. Genome-based reconstruction of its respiratory machinery and physiological investigations indicated that CO oxidation by Ni-CODH was coupled with H2 production (proton reduction), whereas CO oxidation by Mo-CODH was coupled with O2 reduction under aerobic conditions and nitrate reduction under anaerobic conditions. G301 would thus be able to thrive via CO oxidation under a wide range of conditions, from aerobic environments to anaerobic environments, even with no terminal electron acceptors other than protons. Comparative genome analyses revealed no significant differences in genome structures and encoded cellular functions, except for CO oxidation between CO oxidizers and non-CO oxidizers in the genus Parageobacillus; CO oxidation genes are retained exclusively for CO metabolism and related respiration. IMPORTANCE Microbial CO oxidation has received much attention because it contributes to global carbon cycling in addition to functioning as a remover of CO, which is toxic to many organisms. Some microbial CO oxidizers, including both bacteria and archaea, exhibit sister relationships with non-CO oxidizers even in genus-level monophyletic groups. In this study, we demonstrated that a new isolate, Parageobacillus sp. G301, is capable of both anaerobic (hydrogenogenic) and aerobic CO oxidation, which has not been previously reported. The discovery of this new isolate, which is versatile in CO metabolism, will accelerate research on CO oxidizers with diverse CO metabolisms, expanding our understanding of microbial diversity. Through comparative genomic analyses, we propose that CO oxidation genes are not essential genetic elements in the genus Parageobacillus, providing insights into the factors which shape the punctate distribution of CO oxidizers in the prokaryote tree, even in genus-level monophyletic groups.

Ecological Dynamics of Broad- and Narrow-Host-Range Viruses Infecting the Bloom-Forming Toxic Cyanobacterium Microcystis aeruginosa.

Microcystis aeruginosa is predicted to interact and coexist with diverse broad- and narrow-host-range viruses within a bloom; however, little is known about their affects on Microcystis population dynamics. Here, we developed a real-time PCR assay for the quantification of these viruses that have different host ranges. During the sampling period, total Microcystis abundance showed two peaks in May and August with a temporary decrease in June. The Microcystis population is largely divided into three phylotypes based on internal transcribed sequences (ITS; ITS types I to III). ITS I was the dominant phylotype (66% to 88%) except in June. Although the ITS II and III phylotypes were mostly less abundant, these phylotypes temporarily increased to approximately equivalent abundances of the ITS I population in June. During the same sampling period, the abundances of the broad-host-range virus MVGF_NODE331 increased from April to May and from July to October with a temporary decrease in June, in which its dynamics were in proportion to the increase of total Microcystis abundances regardless of changes in host ITS population composition. In contrast, the narrow-host-range viruses MVG_NODE620 and Ma-LMM01 were considerably less abundant than the broad-host-range virus and generally did not fluctuate in the environment. Considering that M. aeruginosa could increase the abundance and sustain the bloom under the prevalence of the broad-host-range virus, host abundant and diverse antiviral mechanisms might contribute to coexistence with its viruses. IMPORTANCE The bloom-forming toxic cyanobacterium Microcystis aeruginosa interacts with diverse broad- and narrow-host-range viruses. However, the dynamics of the Microcystis population (at the intraspecies level) and viruses with different host ranges remain unknown. Our real-time PCR assays unveiled that the broad-host-range virus gradually increased in abundance over the sampling period, in proportion to the increase in total Microcystis abundance regardless of changes in genotypic composition. The narrow-host-range viruses were considerably less abundant than the broad-host-range virus and did not generally fluctuate in the environment. The expansion and maintenance of the Microcystis bloom even under the increased infection by the broad-host-range virus suggested that highly abundant and diverse antiviral mechanisms allowed them to coexist with viruses under selective pressure. This paper expands our knowledge about the ecological dynamics of Microcystis viruses and provides potential insights into their coexistence with their host.

Biome-specific distribution of Ni-containing carbon monoxide dehydrogenases.

Ni-containing carbon monoxide dehydrogenase (Ni-CODH) plays an important role in the CO/CO2-based carbon and energy metabolism of microbiomes. Ni-CODH is classified into distinct phylogenetic clades, A-G, with possibly distinct cellular roles. However, the types of Ni-CODH clade used by organisms in different microbiomes are unknown. Here, we conducted a metagenomic survey of a protein database to determine the relationship between the phylogeny and biome distribution of Ni-CODHs. Clustering and phylogenetic analyses showed that the metagenome assembly-derived Ni-CODH sequences were distributed in ~ 60% Ni-CODH clusters and in all Ni-CODH clades. We also identified a novel Ni-CODH clade, clade H. Biome mapping on the Ni-CODH phylogenetic tree revealed that Ni-CODHs of almost all the clades were found in natural aquatic environmental and engineered samples, whereas those of specific subclades were found only in host-associated samples. These results are comparable with our finding that the diversity in the phylum-level taxonomy of host-associated Ni-CODH owners is statistically different from those of the other biomes. Our findings suggest that while Ni-CODH is a ubiquitous enzyme produced across diverse microbiomes, its distribution in each clade is biased and mainly affected by the distinct composition of microbiomes.

Diversity analysis of thermophilic hydrogenogenic carboxydotrophs by carbon monoxide dehydrogenase amplicon sequencing using new primers.

The microbial H2-producing (hydrogenogenic) carbon monoxide (CO)-oxidizing activity by the membrane-associated CO dehydrogenase (CODH)/energy-converting hydrogenase (ECH) complex is an important metabolic process in the microbial community. However, the studies on hydrogenogenic carboxydotrophs had to rely on inherently cultivation and isolation methods due to their rare abundance, which was a bottleneck in ecological study. Here, we provided gene-targeted sequencing method for the diversity estimation of thermophilic hydrogenogenic carboxydotrophs. We designed six new degenerate primer pairs which effectively amplified the coding regions of CODH genes forming gene clusters with ECH genes (CODHech genes) in Firmicutes which includes major thermophilic hydrogenogenic carboxydotrophs in terrestrial thermal habitats. Amplicon sequencing by these primers using DNAs from terrestrial hydrothermal sediments and CO-gas-incubated samples specifically detected multiple CODH genes which were identical or phylogenetically related to the CODHech genes in Firmictes. Furthermore, we found that phylogenetically distinct CODHech genes were enriched in CO-gas-incubated samples, suggesting that our primers detected uncultured hydrogenogenic carboxydotrophs as well. The new CODH-targeted primers provided us with a fine-grained (~ 97.9% in nucleotide sequence identity) diversity analysis of thermophilic hydrogenogenic carboxydotrophs by amplicon sequencing and will bolster the ecological study of these microorganisms.

Genetic Engineering of Carbon Monoxide-dependent Hydrogen-producing Machinery in Parageobacillus thermoglucosidasius.

The metabolic engineering of carbon monoxide (CO) oxidizers has the potential to create efficient biocatalysts to produce hydrogen and other valuable chemicals. We herein applied markerless gene deletion to CO dehydrogenase/energy-converting hydrogenase (CODH/ECH) in the thermophilic facultative anaerobe, Parageobacillus thermoglucosidasius. We initially compared the transformation efficiency of two strains, NBRC 107763T and TG4. We then disrupted CODH, ECH, and both enzymes in NBRC 107763T. The characterization of growth in all three disruptants under 100% CO demonstrated that both enzymes were essential for CO-dependent growth with hydrogen production in P. thermoglucosidasius. The present results will become a platform for the further metabolic engineering of this organism.

Draft Genome Sequence of Thermanaeromonas sp. Strain C210, Isolated in the Presence of Carbon Monoxide.

The genus Thermanaeromonas comprises two species of thermophilic, strictly anaerobic, spore-forming bacteria. Here, we report the draft genome sequence of Thermanaeromonas sp. strain C210, which was first isolated in the presence of carbon monoxide. The genome sequence provides insight into carbon monoxide-dependent metabolism for members of the genus Thermanaeromonas.

Anaerobic and hydrogenogenic carbon monoxide-oxidizing prokaryotes: Versatile microbial conversion of a toxic gas into an available energy.

Carbon monoxide (CO) is a gas that is toxic to various organisms including humans and even microbes; however, it has low redox potential, which can fuel certain microbes, namely, CO oxidizers. Hydrogenogenic CO oxidizers utilize an energy conservation system via a CO dehydrogenase/energy-converting hydrogenase complex to produce hydrogen gas, a zero emission fuel, by CO oxidation coupled with proton reduction. Biochemical and molecular biological studies using a few model organisms have revealed their enzymatic reactions and transcriptional response mechanisms using CO. Biotechnological studies for CO-dependent hydrogen production have also been carried out with these model organisms. In this chapter, we review recent advances in the studies of these microbes, which reveal their unique and versatile metabolic profiles and provides future perspectives on ecological roles and biotechnological applications. Over the past decade, the number of isolates has doubled (37 isolates in 5 phyla, 20 genera, and 32 species). Some of the recently isolated ones show broad specificity to electron acceptors. Moreover, accumulating genomic information predicts their unique physiologies and reveals their phylogenomic relationships with novel potential hydrogenogenic CO oxidizers. Combined with genomic database surveys, a molecular ecological study has unveiled the wide distribution and low abundance of these microbes. Finally, recent biotechnological applications of hydrogenogenic CO oxidizers have been achieved via diverse approaches (e.g., metabolic engineering and co-cultivation), and the identification of thermophilic facultative anaerobic CO oxidizers will promote industrial applications as oxygen-tolerant biocatalysts for efficient hydrogen production by genomic engineering.

Carbon monoxide-dependent transcriptional changes in a thermophilic, carbon monoxide-utilizing, hydrogen-evolving bacterium Calderihabitans maritimus KKC1 revealed by transcriptomic analysis.

Calderihabitans maritimus KKC1 is a thermophilic, carbon monoxide (CO)-utilizing, hydrogen-evolving bacterium that harbors seven cooS genes for anaerobic CO dehydrogenases and six hyd genes for [NiFe] hydrogenases and capable of using a variety of electron acceptors coupled to CO oxidation. To understand the relationships among these unique features and the transcriptional adaptation of the organism to CO, we performed a transcriptome analysis of C. maritimus KKC1 grown under 100% CO and N2 conditions. Of its 3114 genes, 58 and 32 genes were significantly upregulated and downregulated in the presence of CO, respectively. A cooS-ech gene cluster, an "orphan" cooS gene, and bidirectional hyd genes were upregulated under CO, whereas hydrogen-uptake hyd genes were downregulated. Transcriptional changes in anaerobic respiratory genes supported the broad usage of electron acceptors in C. maritimus KKC1 under CO metabolism. Overall, the majority of the differentially expressed genes were oxidoreductase-like genes, suggesting metabolic adaptation to the cellular redox change upon CO oxidation. Moreover, our results suggest a transcriptional response mechanism to CO that involves multiple transcription factors, as well as a CO-responsive transcriptional activator (CooA). Our findings shed light on the diverse mechanisms for transcriptional and metabolic adaptations to CO in CO-utilizing and hydrogen-evolving bacteria.

Draft Genome Sequences of Two Thermophilic Moorella sp. Strains, Isolated from an Acidic Hot Spring in Japan.

The thermophilic Moorella sp. strains E308F and E306M were isolated from an acidic hot spring in Japan. Here, we report the draft genome sequences of E308F (3.06 Mbp; G+C content, 54.0%) and E306M (2.99 Mbp; G+C content, 54.4%), to advance the genomic information available on the genus Moorella.

Cooccurrence of Broad- and Narrow-Host-Range Viruses Infecting the Bloom-Forming Toxic Cyanobacterium Microcystis aeruginosa.

Viruses play important roles in regulating the abundance and composition of bacterial populations in aquatic ecosystems. The bloom-forming toxic cyanobacterium Microcystis aeruginosa is predicted to interact with diverse cyanoviruses, resulting in Microcystis population diversification. However, current knowledge of the genomes from these viruses and their infection programs is limited to those of Microcystis virus Ma-LMM01. Here, we performed a time series sampling at a small pond in Japan during a Microcystis bloom and then investigated the genomic information and transcriptional dynamics of Microcystis-interacting viruses using metagenomic and metatranscriptomic approaches. We identified 15 viral genomic fragments classified into three groups, groups I (including Ma-LMM01), II (high abundance and transcriptional activity), and III (new lineages). According to the phylogenetic distribution of Microcystis strains possessing spacers against each viral group, the group II-original viruses interacted with all three phylogenetically distinct Microcystis population types (phylotypes), whereas the groups I and III-original viruses interacted with only one or two phylotypes, indicating the cooccurrence of broad- (group II) and narrow (groups I and III)-host-range viruses in the bloom. These viral fragments showed the highest transcriptional levels during daytime regardless of their genomic differences. Interestingly, M. aeruginosa expressed antiviral defense genes in the environment, unlike what was seen with an Ma-LMM01 infection in a previous culture experiment. Given that broad-host-range viruses often induce antiviral responses within alternative hosts, our findings suggest that such antiviral responses might inhibit viral multiplication, mainly that of broad-host-range viruses like those in group II.IMPORTANCE The bloom-forming toxic cyanobacterium Microcystis aeruginosa is thought to have diversified its population through the interactions between host and viruses in antiviral defense systems. However, current knowledge of viral genomes and infection programs is limited to those of Microcystis virus Ma-LMM01, which was a narrow host range in which it can escape from the highly abundant host defense systems. Our metagenomic approaches unveiled the cooccurrence of narrow- and broad-host-range Microcystis viruses, which included fifteen viral genomic fragments from Microcystis blooms that were classified into three groups. Interestingly, Microcystis antiviral defense genes were expressed against viral infection in the environment, unlike what was seen in a culture experiment with Ma-LMM01. Given that viruses with a broad host range often induce antiviral responses within alternative hosts, our findings suggest that antiviral responses inhibit viral reproduction, especially that of broad-range viruses like those in group II. This paper augments our understanding of the interactions between M. aeruginosa and its viruses and fills an important knowledge gap.

Diversity and distribution of thermophilic hydrogenogenic carboxydotrophs revealed by microbial community analysis in sediments from multiple hydrothermal environments in Japan.

In hydrothermal environments, carbon monoxide (CO) utilisation by thermophilic hydrogenogenic carboxydotrophs may play an important role in microbial ecology by reducing toxic levels of CO and providing H2 for fuelling microbial communities. We evaluated thermophilic hydrogenogenic carboxydotrophs by microbial community analysis. First, we analysed the correlation between carbon monoxide dehydrogenase (CODH)-energy-converting hydrogenase (ECH) gene cluster and taxonomic affiliation by surveying an increasing genomic database. We identified 71 genome-encoded CODH-ECH gene clusters, including 46 whose owners were not reported as hydrogenogenic carboxydotrophs. We identified 13 phylotypes showing > 98.7% identity with these taxa as potential hydrogenogenic carboxydotrophs in hot springs. Of these, Firmicutes phylotypes such as Parageobacillus, Carboxydocella, Caldanaerobacter, and Carboxydothermus were found in different environmental conditions and distinct microbial communities. The relative abundance of the potential thermophilic hydrogenogenic carboxydotrophs was low. Most of them did not show any symbiotic networks with other microbes, implying that their metabolic activities might be low.

Transcriptome analysis of a thermophilic and hydrogenogenic carboxydotroph Carboxydothermus pertinax.

A thermophilic and hydrogenogenic carboxydotroph, Carboxydothermus pertinax, performs hydrogenogenic CO metabolism in which CODH-II couples with distally encoded ECH. To enhance our knowledge of its hydrogenogenic CO metabolism, we performed whole transcriptome analysis of C. pertinax grown under 100% CO or 100% N2 using RNA sequencing. Of the 2577 genes, 36 and 64 genes were differentially expressed genes (DEGs) with false discovery rate adjusted P value < 0.05 when grown under 100% CO or 100% N2, respectively. Most of the DEGs were components of 23 gene clusters, suggesting switch between metabolisms via intensive expression changes in a relatively low number of gene clusters. Of the 9 significantly expressed gene clusters under 100% CO, CODH-II and ECH gene clusters were found. Only the ECH gene cluster was regulated by the CO-responsive transcriptional factor CooA, suggesting that others were separately regulated in the same transcriptional cascade as the ECH gene cluster. Of the 14 significantly expressed gene clusters under 100% N2, ferrous iron transport gene cluster involved in anaerobic respiration and prophage region were found. Considering that the expression of the temperate phage was strictly repressed under 100% CO, hydrogenogenic CO metabolism might be stable for C. pertinax.

Draft Genome Sequence of Parageobacillus thermoglucosidasius Strain TG4, a Hydrogenogenic Carboxydotrophic Bacterium Isolated from a Marine Sediment.

Parageobacillus thermoglucosidasius possesses biotechnological potential for fuel generation. Here, we report the draft genome sequence of P. thermoglucosidasius strain TG4, which was first isolated from a marine sediment. The genome sequence provides insight into the plasmid diversity and carbon monoxide-dependent hydrogen production capacity of P. thermoglucosidasius.

Correction: Locality and diel cycling of viral production revealed by a 24 h time course cross-omics analysis in a coastal region of Japan.

The original version of this Article contained an error in the main text citations and reference list. These errors have now been corrected in both the PDF and HTML versions of the Article.

LexA Binds to Transcription Regulatory Site of Cell Division Gene ftsZ in Toxic Cyanobacterium Microcystis aeruginosa.

Previously, we showed that DNA replication and cell division in toxic cyanobacterium Microcystis aeruginosa are coordinated by transcriptional regulation of cell division gene ftsZ and that an unknown protein specifically bound upstream of ftsZ (BpFz; DNA-binding protein to an upstream site of ftsZ) during successful DNA replication and cell division. Here, we purified BpFz from M. aeruginosa strain NIES-298 using DNA-affinity chromatography and gel-slicing combined with gel electrophoresis mobility shift assay (EMSA). The N-terminal amino acid sequence of BpFz was identified as TNLESLTQ, which was identical to that of transcription repressor LexA from NIES-843. EMSA analysis using mutant probes showed that the sequence GTACTAN3GTGTTC was important in LexA binding. Comparison of the upstream regions of lexA in the genomes of closely related cyanobacteria suggested that the sequence TASTRNNNNTGTWC could be a putative LexA recognition sequence (LexA box). Searches for TASTRNNNNTGTWC as a transcriptional regulatory site (TRS) in the genome of M. aeruginosa NIES-843 showed that it was present in genes involved in cell division, photosynthesis, and extracellular polysaccharide biosynthesis. Considering that BpFz binds to the TRS of ftsZ during normal cell division, LexA may function as a transcriptional activator of genes related to cell reproduction in M. aeruginosa, including ftsZ. This may be an example of informality in the control of bacterial cell division.

Insight into Energy Conservation via Alternative Carbon Monoxide Metabolism in Carboxydothermus pertinax Revealed by Comparative Genome Analysis.

Carboxydothermus species are some of the most studied thermophilic carboxydotrophs. Their varied carboxydotrophic growth properties suggest distinct strategies for energy conservation via carbon monoxide (CO) metabolism. In this study, we used comparative genome analysis of the genus Carboxydothermus to show variations in the CO dehydrogenase-energy-converting hydrogenase gene cluster, which is responsible for CO metabolism with H2 production (hydrogenogenic CO metabolism). Indeed, the ability or inability to produce H2 with CO oxidation is explained by the presence or absence of this gene cluster in Carboxydothermus hydrogenoformans, Carboxydothermus islandicus, and Carboxydothermus ferrireducens Interestingly, despite its hydrogenogenic CO metabolism, Carboxydothermus pertinax lacks the Ni-CO dehydrogenase catalytic subunit (CooS-I) and its transcriptional regulator-encoding genes in this gene cluster, probably due to inversion. Transcriptional analysis in C. pertinax showed that the Ni-CO dehydrogenase gene (cooS-II) and distantly encoded energy-converting-hydrogenase-related genes were remarkably upregulated with 100% CO. In addition, when thiosulfate was available as a terminal electron acceptor in 100% CO, the maximum cell density and maximum specific growth rate of C. pertinax were 3.1-fold and 1.5-fold higher, respectively, than when thiosulfate was absent. The amount of H2 produced was only 62% of the amount of CO consumed, less than expected according to hydrogenogenic CO oxidation (CO + H2O → CO2 + H2). Accordingly, C. pertinax would couple CO oxidation by Ni-CO dehydrogenase II with simultaneous reduction of not only H2O but also thiosulfate when grown in 100% CO.IMPORTANCE Anaerobic hydrogenogenic carboxydotrophs are thought to fill a vital niche by scavenging potentially toxic CO and producing H2 as an available energy source for thermophilic microbes. This hydrogenogenic carboxydotrophy relies on a Ni-CO dehydrogenase-energy-converting hydrogenase gene cluster. This feature is thought to be common to these organisms. However, the hydrogenogenic carboxydotroph Carboxydothermus pertinax lacks the gene for the Ni-CO dehydrogenase catalytic subunit encoded in the gene cluster. Here, we performed a comparative genome analysis of the genus Carboxydothermus, a transcriptional analysis, and a cultivation study in 100% CO to prove the hydrogenogenic CO metabolism. Results revealed that C. pertinax could couple Ni-CO dehydrogenase II alternatively to the distal energy-converting hydrogenase. Furthermore, C. pertinax represents an example of the functioning of Ni-CO dehydrogenase that does not always correspond to its genomic context, owing to the versatility of CO metabolism and the low redox potential of CO.

Incomplete Selective Sweeps of Microcystis Population Detected by the Leader-End CRISPR Fragment Analysis in a Natural Pond.

The freshwater cyanobacterium Microcystis aeruginosa frequently forms toxic massive blooms and exists in an arms race with its infectious phages in aquatic natural environments, and as a result, has evolved extremely diverse and elaborate antiviral defense systems, including the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated genes (Cas) system. Here, to assess Microcystis population dynamics associated with exogenous mobile genetic elements such as phages and plasmids, we examined the temporal variation in CRISPR genotypes (CTs) by analyzing spacer sequences detected in a natural pond between June and October 2013 when a cyanobacterial bloom occurred. A total of 463,954 high-quality leader-end CRISPR sequences were obtained and the sequences containing spacers were classified into 31 previously reported CTs and 68 new CTs based on the shared order of the leader-end spacers. CT19 was the most dominant genotype (32%) among the 16 most common CTs, followed by CT52 (14%) and CT58 (9%). Spacer repertoires of CT19 showed mainly two different types; CT19origin, which was identical to the CT19 spacer repertoire of previously isolated strains, and CT19new+, which contained a new spacer at the leader-end of the CRISPR region of CT19origin, which were present in almost equal abundance, accounting for up to 99.94% of CT19 sequences. Surprisingly, we observed the spacer repertoires of the second to tenth spacers of CT19origin at the most leader-end of proto-genotype sequences of CT19origin. These were observed during the sampling in this study and our previous study at the same ecosystem in 2010 and 2011, suggesting these CTs persisted from 2011 to 2013 in spite of phage pressure. The leader-end variants were observed in other CT genotypes. These findings indicated an incomplete selective sweep of Microcystis populations. We explained the phenomenon as follow; the abundance of Microcystis varied seasonally and drastically, resulting that Microcystis populations experience a bottleneck once a year, and thereby founder effects following a bottleneck mean that older CTs have an equal chance of increasing in prevalence as the CTs generated following acquisition of newer spacers.

Anodic and Cathodic Extracellular Electron Transfer by the Filamentous Bacterium Ardenticatena maritima 110S.

Ardenticatena maritima strain 110S is a filamentous bacterium isolated from an iron-rich coastal hydrothermal field, and it is a unique isolate capable of dissimilatory iron or nitrate reduction among the members of the bacterial phylum Chloroflexi. Here, we report the ability of A. maritima strain 110S to utilize electrodes as a sole electron acceptor and donor when coupled with the oxidation of organic compounds and nitrate reduction, respectively. In addition, multicellular filaments with hundreds of cells arranged end-to-end increased the extracellular electron transfer (EET) ability to electrodes by organizing filaments into bundled structures, with the aid of microbially reduced iron oxide minerals on the cell surface of strain 110S. Based on these findings, together with the attempt to detect surface-localized cytochromes in the genome sequence and the demonstration of redox-dependent staining and immunostaining of the cell surface, we propose a model of bidirectional electron transport by A. maritima strain 110S, in which surface-localized multiheme cytochromes and surface-associated iron minerals serve as a conduit of bidirectional EET in multicellular filaments.

Transcriptome Analysis of a Bloom-Forming Cyanobacterium Microcystis aeruginosa during Ma-LMM01 Phage Infection.

Microcystis aeruginosa forms massive blooms in eutrophic freshwaters, where it is constantly exposed to lytic cyanophages. Unlike other marine cyanobacteria, M. aeruginosa possess remarkably abundant and diverse potential antiviral defense genes. Interestingly, T4-like cyanophage Ma-LMM01, which is the sole cultured lytic cyanophage infecting M. aeruginosa, lacks the host-derived genes involved in maintaining host photosynthesis and directing host metabolism that are abundant in other marine cyanophages. Based on genomic comparisons with closely related cyanobacteria and their phages, Ma-LMM01 is predicted to employ a novel infection program that differs from that of other marine cyanophages. Here, we used RNA-seq technology and in silico analysis to examine transcriptional dynamics during Ma-LMM01 infection to reveal host transcriptional responses to phage infection, and to elucidate the infection program used by Ma-LMM01 to avoid the highly abundant host defense systems. Phage-derived reads increased only slightly at 1 h post-infection, but significantly increased from 16% of total cellular reads at 3 h post-infection to 33% of all reads by 6 h post-infection. Strikingly, almost none of the host genes (0.17%) showed a significant change in expression during infection. However, like other lytic dsDNA phages, including marine cyanophages, phage gene dynamics revealed three expression classes: early (host-takeover), middle (replication), and late (virion morphogenesis). The early genes were concentrated in a single ∼5.8-kb window spanning 10 open reading frames (gp054-gp063) on the phage genome. None of the early genes showed homology to the early genes of other T4-like phages, including known marine cyanophages. Bacterial RNA polymerase (σ70) recognition sequences were also found in the upstream region of middle and late genes, whereas phage-specific motifs were not found. Our findings suggest that unlike other known T4-like phages, Ma-LMM01 achieves three sequential gene expression patterns with no change in host promoter activity. This type of infection that does not cause significant change in host transcriptional levels may be advantageous in allowing Ma-LMM01 to escape host defense systems while maintaining host photosynthesis.