Shewanella Piezotolerans WP3 Research Articles

Impact of metabolism and temperature on 2H ∕ 1H fractionation in lipids of the marine bacterium Shewanella piezotolerans WP3

Abstract. Compound-specific hydrogen isotopes have increasingly been used as a powerful proxy for investigating biogeochemical cycles and climate change over the past 2 decades. Understanding the hydrogen isotope in extant organisms is fundamental for us to interpret such isotope signals preserved in natural environmental samples. Here, we studied the controls on hydrogen isotope fractionation between fatty acids and growth water by an Fe-reducing heterotrophic marine bacterium Shewanella piezotolerans WP3 growing on different organic substrates, including N-acetyl-D-glucosamine (GlcNac), glucose, acetate, pyruvate, L-alanine, and L-glutamate. Meanwhile, we also evaluated the impact of growth temperature on the hydrogen isotope composition of fatty acids using GlcNac as the sole organic substrate. Our results show that the abundance-weighted mean fatty-acid / water fractionations (εFA/water) display considerable variations for cultures grown on different substrates. Specifically, WP3 yielded the most 2H-enriched fatty acids growing on L-glutamate and pyruvate with an εFA/water of 52 ± 14 ‰ and 44 ± 4 ‰, respectively, and exhibited 2H depletion using GlcNac (−76 ± 1 ‰) and glucose (−67 ± 35 ‰) as sole carbon sources and relatively small fractionations on acetate (23 ± 3 ‰) and L-alanine (−4 ± 9 ‰). Combined with metabolic model analysis, our results indicate that the central metabolic pathways exert a fundamental effect on the hydrogen isotope composition of fatty acids in heterotrophs. Temperature also has an obvious influence on the δ2H values of fatty acids, with strong 2H depletion at an optimal growth temperature (−23 ± 2 ‰ and −23 ‰ growing at 15 and 20 ∘C, respectively) and relatively small fractionations at non-optimal temperatures (4 ± 5 ‰, −4 ± 12 ‰, and 15 ± 41 ‰ at 4, 10, and 25 ∘C, respectively). We hypothesized that this may be associated with temperature-induced enzyme activity for nicotinamide adenine dinucleotide phosphate (NADPH) production. This study helps understand the controlling factors of hydrogen isotope fractionation by marine bacteria, laying the foundation for further interpreting the hydrogen isotope signatures of lipids as an important proxy to decode the biogeochemical cycles and ecological changes in marine sediments.

The effects of metabolism and temperature on carbon isotope composition of lipids in marine bacterium Shewanella piezotolerans WP3

The carbon isotope composition of lipids has been widely used to decode the biogeochemical cycle and reconstruct paleo-environment and paleo-ecological changes over geologic time. However, up to now, little is known about the controls on the carbon isotope signatures of heterotrophic microbes with ecological functions in the ocean. Here, we investigate the carbon isotope fractionation between fatty acids and carbon sources by a representative marine bacterium Shewanella piezotolerans WP3 growing on different organic substrates, including N-acetyl-D-glucosamine (GlcNac), glucose, acetate, pyruvate, L-alanine, and L-glutamate. The effect of growth temperature on the carbon isotope fractionation is also examined using GlcNac as sole carbon source. Our results show that the carbon isotope fractionations vary obviously between different carbon sources: fatty acids are strongly depleted in 13C growing on GlcNac and glucose with abundance-weighted average 13C/12C fractionations ranging from −7.7 to −3.8‰, slightly depleted in 13C on acetate and L-alanine (−0.8 to −0.4‰), and enriched in 13C on pyruvate and L-glutamate (0.6–2.1‰). More importantly, we found a considerable variation of fractionations between different growth temperatures with strongly 13C-depleted at optimal growth temperature (15–20 °C) with abundance-weighted average 13C/12C fractionations ranging from −5.2 to −4.8‰ and relatively small fractionations on 4 °C, 10 °C and 25 °C (−2.1 to −3.4‰). We hypothesize that the controls on carbon isotope fractionation are associated with decarboxylation and oxidation-related dehydrogenase complex under different growth temperatures. This study proves that carbon isotope fractionation in heterotrophs is related to which pathway for carbon sources enter into the central metabolism, and may be served as useful “fingerprints” for interpreting carbon isotope signatures in sedimentary lipids, and inferring ecological functions of S. piezotolerans WP3 in certain environments, especially in Fe-reducing oceanic sediments.

The origin and impeded dissemination of the DNA phosphorothioation system in prokaryotes

Phosphorothioate (PT) modification by the dnd gene cluster is the first identified DNA backbone modification and constitute an epigenetic system with multiple functions, including antioxidant ability, restriction modification, and virus resistance. Despite these advantages for hosting dnd systems, they are surprisingly distributed sporadically among contemporary prokaryotic genomes. To address this ecological paradox, we systematically investigate the occurrence and phylogeny of dnd systems, and they are suggested to have originated in ancient Cyanobacteria after the Great Oxygenation Event. Interestingly, the occurrence of dnd systems and prophages is significantly negatively correlated. Further, we experimentally confirm that PT modification activates the filamentous phage SW1 by altering the binding affinity of repressor and the transcription level of its encoding gene. Competition assays, concurrent epigenomic and transcriptomic sequencing subsequently show that PT modification affects the expression of a variety of metabolic genes, which reduces the competitive fitness of the marine bacterium Shewanella piezotolerans WP3. Our findings strongly suggest that a series of negative effects on microorganisms caused by dnd systems limit horizontal gene transfer, thus leading to their sporadic distribution. Overall, our study reveals putative evolutionary scenario of the dnd system and provides novel insights into the physiological and ecological influences of PT modification.

Condition-Specific Molecular Network Analysis Revealed That Flagellar Proteins Are Involved in Electron Transfer Processes of Shewanella piezotolerans WP3.

Because of the ability to metabolize a large number of electron acceptors such as nitrate, nitrite, fumarate, and metal oxides, Shewanella species have attracted much attention in recent years. Generally, the use of these electron acceptors is mainly achieved through electron transfer proteins and their interactions which will dynamically change across different environmental conditions in cells. Therefore, functional analysis of condition-specific molecular networks can reveal biological information on electron transfer processes. By integrating expression data and molecular networks, we constructed condition-specific molecular networks for Shewanella piezotolerans WP3. We then identified condition-specific key genes and studied their potential functions with an emphasis on their roles in electron transfer processes. Functional module analysis showed that different flagellar assembly modules appeared under these conditions and suggested that flagellar proteins are important for these conditions. We also identified the electron transfer modules underlying these various environmental conditions. The present results could help with screening electron transfer genes and understanding electron transfer processes under various environmental conditions for the Shewanella species.

The thermo-regulated genetic switch of deep-sea filamentous phage SW1 and its distribution in the Pacific Ocean.

Viruses, especially bacteriophages, are thought to have important functions in the deep-sea ecosystem, but little is known about the induction mechanism of benthic phages in response to environmental change. Our prior work characterized a cold-active filamentous phage SW1 that infects the deep-sea bacterium Shewanella piezotolerans WP3; however, the underlying mechanism of the putative thermo-regulated genetic switch of SW1 is still unclear. In this study, the DNA copy number and mRNA abundance of the deep-sea phage SW1 were quantified in the whole life cycle of its host S. piezotolerans WP3 at different temperatures. Our results demonstrated that the induction of SW1 is dependent on a threshold temperature (4°C), but this dependency is not proportional to temperature gradient. RNA-Seq analyses revealed two highly transcribed regions at 4°C and verified the presence of a long 3' untranslated region (UTR) in the SW1 genome. Interestingly, recruitment analysis showed that SW1-like inoviruses prevail in deep sea (depth >1000m) and photic epipelagic and mesopelagic zones (depth <1000m), which suggested that the thermo-regulated genetic switch revealed in SW1 may be widely distributed in the ocean.

Aerobically incubated bacterial biomass-promoted formation of disordered dolomite and implication for dolomite formation

With the realization that the formation of low-temperature dolomite could involve the microbial activity, a new pathway to understand the origin and mechanism of dolomite is emerging. Although microbially mediated CaMg carbonates and dolomite occur in some aerobic and high-salinity conditions, little information about the exact role of aerobic microbes is available. Herein, a strain of moderately halophilic bacteria, Shewanella piezotolerans WP3, was selected to study the involvement of aerobically incubated bacterial biomass in the CaMg carbonate mineralization. Different biomass components were isolated from the bacterial cultures by a set of separation techniques, and used to mediate CaMg carbonate mineralization under a carbon dioxide diffused system. The experimental results showed that bacterial cells play a dominant role in the formation of disordered dolomite. And the mineralization of the disordered dolomite can be attributed to the bare cells and bound extracellular polymeric substances (bound EPS) isolated from the bacterial cells, most likely due to the promotion of high concentration and density of carboxyl and phosphoryl groups on the bacterial biomass (i.e., bare cells and bound EPS). Hence, it does imply that bacterial biomass, even without the active microbial activities, can also play an important role in the formation of dolomite. Current results can not only extend the insight into the biologically influenced mineralization of CaMg carbonates, but also shed light on the precipitation of disordered dolomite/dolomite in modern settings and geological records.

Pressure-Regulated Gene Expression and Enzymatic Activity of the Two Periplasmic Nitrate Reductases in the Deep-Sea Bacterium Shewanella piezotolerans WP3.

Shewanella species are widely distributed in marine environments, from the shallow coasts to the deepest sea bottom. Most Shewanella species possess two isoforms of periplasmic nitrate reductases (NAP-α and NAP-β) and are able to generate energy through nitrate reduction. However, the contributions of the two NAP systems to bacterial deep-sea adaptation remain unclear. In this study, we found that the deep-sea denitrifier Shewanella piezotolerans WP3 was capable of performing nitrate respiration under high hydrostatic pressure (HHP) conditions. In the wild-type strain, NAP-β played a dominant role and was induced by both the substrate and an elevated pressure, whereas NAP-α was constitutively expressed at a relatively lower level. Genetic studies showed that each NAP system alone was sufficient to fully sustain nitrate-dependent growth and that both NAP systems exhibited substrate and pressure inducible expression patterns when the other set was absent. Biochemical assays further demonstrated that NAP-α had a higher tolerance to elevated pressure. Collectively, we report for the first time the distinct properties and contributions of the two NAP systems to nitrate reduction under different pressure conditions. The results will shed light on the mechanisms of bacterial HHP adaptation and nitrogen cycling in the deep-sea environment.

Insights into the formation mechanism of vaterite mediated by a deep-sea bacterium Shewanella piezotolerans WP3

Abstract Microbially mediated carbonate mineralization plays a crucial role in biogeochemical cycling of carbon, as microbes can efficiently transform atmospheric CO2 and organic carbon into carbonate cements which represent a significant sink in the global carbon cycle. It is well established that diverse bacteria from various natural habitats can induce the precipitation of calcium carbonate, and an increasing number of bacteria were found to be able to mediate the mineralization of vaterite. However, the precise mechanisms for the formation of bacterial vaterite are not fully understood. To better understand the effect of bacterial activity on vaterite formation, Shewanella piezotolerans WP3 was selected as a model microbe to induce calcium carbonate mineralization. A combination of bacterial and biomimetic mineralization experiments was adopted. Different bacterial components including native cells, EPS-free cells, cell-bound EPS, soluble EPS, and small molecule organics were isolated from the cultures and used to influence calcium carbonate crystallization and growth. The identification and characterization of the mineralized products were done using field emission scanning electron microscopy (FESEM), X-ray powder diffraction (XRD), Raman spectroscopy, Fourier transform infrared spectroscopy (FT-IR), thermogravimetry and differential thermal analysis (TG-DTA), X-ray photoelectron spectroscopy (XPS), transmission electron microscopy (TEM) and selected area electron diffraction (SAED). Bacterial in situ mineralization experiments demonstrate that S. piezotolerans WP3 can not only promote the mineralization of calcium carbonate, but also mediate polymorph selection of vaterite. Biomimetic mineralization experiments involving individual bacterial components reveal that different bacterial components have different effects on calcium carbonate polymorphism, and low molecular-weight amino acids secreted by the bacteria play a dominant role on vaterite formation and stabilization. Our observations also suggest that soluble EPS can induce the formation of aragonite while bound EPS facilitate calcite erosion. Current results can provide a deeper insight into bacterially mediated mineralization of vaterite and calcium carbonate polymorphism problem, and help evaluate the significance of biogenic carbonate in the diagenetic carbonate sinks.

Enhancing the Adaptability of the Deep-Sea Bacterium Shewanella piezotolerans WP3 to High Pressure and Low Temperature by Experimental Evolution under H2O2 Stress.

Oxidative stresses commonly exist in natural environments, and microbes have developed a variety of defensive systems to counteract such events. Although increasing evidence has shown that high hydrostatic pressure (HHP) and low temperature (LT) induce antioxidant defense responses in cells, there is no direct evidence to prove the connection between antioxidant defense mechanisms and the adaptation of bacteria to HHP and LT. In this study, using the wild-type (WT) strain of a deep-sea bacterium, Shewanella piezotolerans WP3, as an ancestor, we obtained a mutant, OE100, with an enhanced antioxidant defense capacity by experimental evolution under H2O2 stress. Notably, OE100 exhibited better tolerance not only to H2O2 stress but also to HHP and LT (20 MPa and 4°C, respectively). Whole-genome sequencing identified a deletion mutation in the oxyR gene, which encodes the transcription factor that controls the oxidative stress response. Comparative transcriptome analysis showed that the genes associated with oxidative stress defense, anaerobic respiration, DNA repair, and the synthesis of flagella and bacteriophage were differentially expressed in OE100 compared with the WT at 20 MPa and 4°C. Genetic analysis of oxyR and ccpA2 indicated that the OxyR-regulated cytochrome c peroxidase CcpA2 significantly contributed to the adaptation of WP3 to HHP and LT. Taken together, these results confirmed the inherent relationship between antioxidant defense mechanisms and the adaptation of a benthic microorganism to HHP and LT.IMPORTANCE Oxidative stress exists in various niches, including the deep-sea ecosystem, which is an extreme environment with conditions of HHP and predominantly LT. Although previous studies have shown that HHP and LT induce antioxidant defense responses in cells, direct evidence to prove the connection between antioxidant defense mechanisms and the adaptation of bacteria to HHP and LT is lacking. In this work, using the deep-sea bacterium Shewanella piezotolerans WP3 as a model, we proved that enhancement of the adaptability of WP3 to HHP and LT can benefit from its antioxidant defense mechanism, which provided useful insight into the ecological roles of antioxidant genes in a benthic microorganism and contributed to an improved understanding of microbial adaptation strategies in deep-sea environments.

Deep-Sea Bacterium Shewanella piezotolerans WP3 Has Two Dimethyl Sulfoxide Reductases in Distinct Subcellular Locations.

Dimethyl sulfoxide (DMSO) acts as a substantial sink for dimethyl sulfide (DMS) in deep waters and is therefore considered a potential electron acceptor supporting abyssal ecosystems. Shewanella piezotolerans WP3 was isolated from west Pacific deep-sea sediments, and two functional DMSO respiratory subsystems are essential for maximum growth of WP3 under in situ conditions (4°C/20 MPa). However, the relationship between these two subsystems and the electron transport pathway underlying DMSO reduction by WP3 remain unknown. In this study, both DMSO reductases (type I and type VI) in WP3 were found to be functionally independent despite their close evolutionary relationship. Moreover, immunogold labeling of DMSO reductase subunits revealed that the type I DMSO reductase was localized on the outer leaflet of the outer membrane, whereas the type VI DMSO reductase was located within the periplasmic space. CymA, a cytoplasmic membrane-bound tetraheme c-type cytochrome, served as a preferential electron transport protein for the type I and type VI DMSO reductases, in which type VI accepted electrons from CymA in a DmsE- and DmsF-independent manner. Based on these results, we proposed a core electron transport model of DMSO reduction in the deep-sea bacterium S. piezotolerans WP3. These results collectively suggest that the possession of two sets of DMSO reductases with distinct subcellular localizations may be an adaptive strategy for WP3 to achieve maximum DMSO utilization in deep-sea environments.IMPORTANCE As the dominant methylated sulfur compound in deep oceanic water, dimethyl sulfoxide (DMSO) has been suggested to play an important role in the marine biogeochemical cycle of the volatile anti-greenhouse gas dimethyl sulfide (DMS). Two sets of DMSO respiratory systems in the deep-sea bacterium Shewanella piezotolerans WP3 have previously been identified to mediate DMSO reduction under in situ conditions (4°C/20 MPa). Here, we report that the two DMSO reductases (type I and type VI) in WP3 have distinct subcellular localizations, in which type I DMSO reductase is localized to the exterior surface of the outer membrane and type VI DMSO reductase resides in the periplasmic space. A core electron transport model of DMSO reduction in WP3 was constructed based on genetic and physiological data. These results will contribute to a comprehensive understanding of the adaptation mechanisms of anaerobic respiratory systems in benthic microorganisms.

Genome-Wide Detection of Small Regulatory RNAs in Deep-Sea Bacterium Shewanella piezotolerans WP3.

Shewanella are one of the most abundant Proteobacteria in the deep-sea and are renowned for their versatile electron accepting capacities. The molecular mechanisms involved in their adaptation to diverse and extreme environments are not well understood. Small non-coding RNAs (sRNAs) are known for modulating the gene expression at transcriptional and posttranscriptional levels, subsequently playing a key role in microbial adaptation. To understand the potential roles of sRNAs in the adaptation of Shewanella toward deep-sea environments, here an in silico approach was utilized to detect the sRNAs in the genome of Shewanella piezotolerans WP3, a piezotolerant and psychrotolerant deep-sea iron reducing bacterium. After scanning 3673 sets of 5′ and 3′ UTRs of orthologous genes, 209 sRNA candidates were identified with high confidence in S. piezotolerans WP3. About 92% (193 out of 209) of these putative sRNAs belong to the class trans-encoded RNAs, suggesting that trans-regulatory RNAs are the dominant class of sRNAs in S. piezotolerans WP3. The remaining 16 cis-regulatory RNAs were validated through quantitative polymerase chain reaction. Five cis-sRNAs were further shown to act as cold regulated sRNAs. Our study provided additional evidence at the transcriptional level to decipher the microbial adaptation mechanisms to extreme environmental conditions.

Characterisation of the regulatory function of the H-NS protein in the benthic bacterium Shewanella piezotolerans WP3 under cold conditions.

The histone-like nucleoid structuring (H-NS) protein is conserved in Gram-negative bacteria and plays important roles as a multifunctional bacterial modulator. However, its roles in deep-sea microorganisms warrant investigation. In this study, the relationship between temperature and the regulatory function of H-NS was characterised in the benthic bacterium Shewanella piezotolerans WP3. Comparative microarray analysis of the hns knockout mutant and wild-type strain identified a total of 264 differentially expressed genes at 4°C, which is significantly less than that at 20°C. Notably, H-NS was proved to be involved in the cold-mediated induction of lateral flagellar gene transcription. We further showed that the binding affinity of H-NS is higher at 4°C than 20°C, thus explaining the significant difference in the expression pattern at different temperatures.

Characterization of the relationship between polar and lateral flagellar structural genes in the deep-sea bacterium Shewanella piezotolerans WP3.

Bacteria with a dual flagellar system, which consists of a polar flagellum (PF) and several lateral flagella (LF), have been identified in diverse environments. Nevertheless, whether and how these two flagellar systems interact with each other is largely unknown. In the present study, the relationship between the structural genes for the PF and LF of the deep-sea bacterium Shewanella piezotolerans WP3 was investigated by genetic, phenotypic and phylogenetic analyses. The mutation of PF genes induced the expression of LF genes and the production of LF in liquid medium, while the defective LF genes led to a decrease in PF gene transcription. However, the level of PF flagellin remained unchanged in LF gene mutants. Further investigation showed that the flgH2 gene (encoding LF L-ring protein) can compensate for mutations of the flgH1 gene (encoding PF L-ring protein), but this compensation does not occur between the flagellar hook-filament junction proteins (FlgL1, FlgL2). Swarming motility was shown to specifically require LF genes, and PF genes cannot substitute for the LF genes in the lateral flagella synthesis. Considering the importance of flagella-dependent motility for bacterial survival in the abyssal sediment, our study thus provided a better understanding of the adaptation strategy of benthic bacteria.

A transcriptome resource for the deep-sea bacterium Shewanella piezotolerans WP3 under cold and high hydrostatic pressure shock stress

Low temperature and high hydrostatic pressure (HHP) are two of the most remarkable environmental factors influencing deep-sea ecosystem. The adaptive mechanisms of microorganisms which live in these extreme environments to low temperature and high pressure warrant investigation. In this study, the global gene expression patterns of the deep-sea bacterium Shewanella piezotolerans WP3 in response to cold (0 °C) and HHP (50 MPa) shock were evaluated through DNA microarray analysis. Results revealed that 22, 66, and 106 genes were differentially expressed after WP3 was respectively exposed to cold shock for 30, 60, and 90 min. Of these genes, 16 genes were identified as common differentially expressed genes (DEGs). After 30 min and 120 min of HHP shock, 5 and 10 genes were respectively identified as DEGs. The hierarchical clustering analysis of the DEG pattern indicated that WP3 may employ different adaptive strategies to cope with cold and HHP shock stress. Taken together, our study provided a transcriptome resource for deep-sea bacterial responses to cold and HHP stress. This study also established a basis for further investigations on environmental adaptive mechanisms utilized by benthic bacteria.

The Two Sets of DMSO Respiratory Systems of Shewanella piezotolerans WP3 Are Involved in Deep Sea Environmental Adaptation.

Dimethyl sulfoxide (DMSO) is an abundant methylated sulfur compound in deep sea ecosystems. However, the mechanism underlying DMSO-induced reduction in benthic microorganisms is unknown. Shewanella piezotolerans WP3, which was isolated from a west Pacific deep sea sediment, can utilize DMSO as the terminal electron acceptor. In this study, two putative dms gene clusters [type I (dmsEFA1B1G1H1) and type II (dmsA2B2G2H2)] were identified in the WP3 genome. Genetic and physiological analyses demonstrated that both dms gene clusters were functional and the transcription of both gene clusters was affected by changes in pressure and temperature. Notably, the type I system is essential for WP3 to thrive under in situ conditions (4°C/20 MPa), whereas the type II system is more important under high pressure or low temperature conditions (20°C/20 MPa, 4°C/0.1 MPa). Additionally, DMSO-dependent growth conferred by the presence of both dms gene clusters was higher than growth conferred by either of the dms gene clusters alone. These data collectively suggest that the possession of two sets of DMSO respiratory systems is an adaptive strategy for WP3 survival in deep sea environments. We propose, for the first time, that deep sea microorganisms might be involved in global DMSO/DMS cycling.

Global transcriptome analysis of the heat shock response of the deep-sea bacterium Shewanella piezotolerans WP3

For microorganisms, heat shock is a major stressful condition. Heat shock is characterized by sudden temperature increases that damage important protein structures and interfere with essential cellular functions. In this study, global gene expression patterns of the deep-sea bacterium Shewanella piezotolerans WP3 in response to heat shock were studied by DNA microarray analysis. Overall, 438, 573, and 627 genes were found to be differentially expressed after heat shock for 30, 60, and 90min, respectively. Functional classification of differentially transcribed genes was performed using the Clusters of Orthologous Groups of Proteins database. Additionally, 361 genes were identified as common differentially expressed genes. These genes may comprise the core genes responsible for coping with heat shock stress of WP3. Moreover, comparative analysis of gene expression pattern in WP3 and other bacteria indicated the presence of different adaptive strategies. These data represent the first transcriptome resource for the response of this deep-sea bacterium to high-temperature stress. This study contributes to the understanding of the global adaptation mechanisms of benthic bacteria toward environmental stresses.

Filamentous phage SW1 is active and influences the transcriptome of the host at high-pressure and low-temperature.

As the most abundant biological entities on the planet, viruses are involved in global biogeochemical cycles, and they have been shown to play an important role in the overall functioning of the deep-sea ecosystem. Nevertheless, little is known about whether and how deep-sea viruses affect the physiology of their bacterial hosts. Previously, the filamentous phage SW1 was identified in the bathypelagic bacterium Shewanella piezotolerans WP3, which was isolated from the upper sediment of West Pacific ocean. In this study, phage SW1 was shown to be active under in situ environmental conditions (20MPaand 4°C) by transmission electron microscopy and reverse-transcription quantitative polymerase chain reaction. Further comparative analysis showed that SW1 had a significant influence on the growth and transcriptome of its host. The transcription of genes responsible for basic cellular activities, including the transcriptional/translational apparatus, arginine synthesis, purine metabolism and the flagellar motor, were down-regulated by the phage. Our results present the firstcharacterization of a phage-host interaction under high-pressure and low-temperature conditions, which indicated that the phage adjusted the energy utilization strategy of the host for improved survival in deep-sea environments.

The Histone-Like Nucleoid Structuring Protein (H-NS) Is a Negative Regulator of the Lateral Flagellar System in the Deep-Sea Bacterium Shewanella piezotolerans WP3.

Although the histone-like nucleoid structuring protein (H-NS) is well known for its involvement in the adaptation of mesophilic bacteria, such as Escherichia coli, to cold environments and high-pressure stress, an understanding of the role of H-NS in the cold-adapted benthic microorganisms that live in the deep-sea ecosystem, which covers approximately 60% of the earth's surface, is still lacking. In this study, we characterized the function of H-NS in Shewanella piezotolerans WP3, which was isolated from West Pacific sediment at a depth of 1,914 m. Anhns gene deletion mutant (WP3Δhns) was constructed, and comparative whole-genome microarray analysis was performed. H-NS had a significant influence (fold change, >2) on the expression of a variety of WP3 genes (274 and 280 genes were upregulated and downregulated, respectively), particularly genes related to energy production and conversion. Notably, WP3Δhnsexhibited higher expression levels of lateral flagellar genes than WP3 and showed enhanced swarming motility and lateral flagellar production compared to those of WP3. The DNA gel mobility shift experiment showed that H-NS bound specifically to the promoter of lateral flagellar genes. Moreover, the high-affinity binding sequences of H-NS were identified by DNase I protection footprinting, and the results support the "binding and spreading" model for H-NS functioning. To our knowledge, this is the first attempt to characterize the function of the universal regulator H-NS in a deep-sea bacterium. Our data revealed that H-NS has a novel function as a repressor of the expression of genes related to the energy-consuming secondary flagellar system and to swarming motility.

Long 5' untranslated regions regulate the RNA stability of the deep-sea filamentous phage SW1.

Virus production in the deep-sea environment has been found to be high, and viruses have been suggested to play significant roles in the overall functioning of this ecosystem. Nevertheless, little is known about these viruses, including the mechanisms that control their production, which makes them one of the least understood biological entities on Earth. Previously, we isolated the filamentous phage SW1, whose virus production and gene transcription were found to be active at low temperatures, from a deep-sea bacterium, Shewanella piezotolerans WP3. In this study, the operon structure of phage SW1 is presented, which shows two operons with exceptionally long 5′ and 3′ untranslated regions (UTRs). In addition, the 5′UTR was confirmed to significantly influence the RNA stability of the SW1 transcripts. Our study revealed novel regulation of the operon and led us to propose a unique regulatory mechanism for Inoviruses. This type of RNA-based regulation may represent a mechanism for significant viral production in the cold deep biosphere.

The regulatory function of LexA is temperature-dependent in the deep-sea bacterium Shewanella piezotolerans WP3.

The SOS response addresses DNA lesions and is conserved in the bacterial domain. The response is governed by the DNA binding protein LexA, which has been characterized in model microorganisms such as Escherichia coli. However, our understanding of its roles in deep-sea bacteria is limited. Here, the influence of LexA on the phenotype and gene transcription of Shewanella piezotolerans WP3 (WP3) was investigated by constructing a lexA deletion strain (WP3ΔlexA), which was compared with the wild-type strain. No growth defect was observed for WP3ΔlexA. A total of 481 and 108 genes were differentially expressed at 20 and 4°C, respectively, as demonstrated by comparative whole genome microarray analysis. Furthermore, the swarming motility and dimethylsulfoxide reduction assay demonstrated that the function of LexA was related to temperature. The transcription of the lexA gene was up-regulated during cold acclimatization and after cold shock, indicating that the higher expression level of LexA at low temperatures may be responsible for its temperature-dependent functions. The deep-sea microorganism S. piezotolerans WP3 is the only bacterial species whose SOS regulator has been demonstrated to be significantly influenced by environmental temperatures to date. Our data support the hypothesis that SOS is a formidable strategy used by bacteria against various environmental stresses.