A Genome-Scale Model of \u3cem\u3eShewanella piezotolerans\u3c/em\u3e Simulates Mechanisms of Metabolic Diversity and Energy Conservation

Shewanella piezotolerans strain WP3 belongs to the group 1 branch of the Shewanella genus and is a piezotolerant and psychrotolerant species isolated from the deep sea. In this study, a genome-scale model was constructed for WP3 using a combination of genome annotation, ortholog mapping, and physiological verification. The metabolic reconstruction contained 806 genes, 653 metabolites, and 922 reactions, including central metabolic functions that represented nonhomologous replacements between the group 1 and group 2 Shewanella species. Metabolic simulations with the WP3 model demonstrated consistency with existing knowledge about the physiology of the organism. A comparison of model simulations with experimental measurements verified the predicted growth profiles under increasing concentrations of carbon sources. The WP3 model was applied to study mechanisms of anaerobic respiration through investigating energy conservation, redox balancing, and the generation of proton motive force. Despite being an obligate respiratory organism, WP3 was predicted to use substrate-level phosphorylation as the primary source of energy conservation under anaerobic conditions, a trait previously identified in other Shewanella species. Further investigation of the ATP synthase activity revealed a positive correlation between the availability of reducing equivalents in the cell and the directionality of the ATP synthase reaction flux. Comparison of the WP3 model with an existing model of a group 2 species, Shewanella oneidensis MR-1, revealed that the WP3 model demonstrated greater flexibility in ATP production under the anaerobic conditions. Such flexibility could be advantageous to WP3 for its adaptation to fluctuating availability of organic carbon sources in the deep sea

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

Environmental Adaptation: Genomic Analysis of the Piezotolerant and Psychrotolerant Deep-Sea Iron Reducing Bacterium Shewanella piezotolerans WP3

Shewanella species are widespread in various environments. Here, the genome sequence of Shewanella piezotolerans WP3, a piezotolerant and psychrotolerant iron reducing bacterium from deep-sea sediment was determined with related functional analysis to study its environmental adaptation mechanisms. The genome of WP3 consists of 5,396,476 base pairs (bp) with 4,944 open reading frames (ORFs). It possesses numerous genes or gene clusters which help it to cope with extreme living conditions such as genes for two sets of flagellum systems, structural RNA modification, eicosapentaenoic acid (EPA) biosynthesis and osmolyte transport and synthesis. And WP3 contains 55 open reading frames encoding putative c-type cytochromes which are substantial to its wide environmental adaptation ability. The mtr-omc gene cluster involved in the insoluble metal reduction in the Shewanella genus was identified and compared. The two sets of flagellum systems were found to be differentially regulated under low temperature and high pressure; the lateral flagellum system was found essential for its motility and living at low temperature

Synthetic approaches to molecular motors and conjugated oligomer libraries

Chapter 1 describes the synthesis of four caltrop-shaped molecules that might be useful as surface-bound electrical field driven molecular motors. The caltrops have a pair of electron donor-accepter arms which form a strong dipole moment. The direction and movement of the dipolar unit could be influenced by an outer electric field. The caltrops also have three benzylic thiols for bonding to a gold surface. Ellipsometry results indicate that monolayers of caltrop molecules form on a gold surface. Chapter 2 and Chapter 3 detail efforts to synthesize libraries of oligo (phenylene vinylene)s (OPVs) using combinatorial synthesis. A series of building blocks for OPVs were synthesized. Model studies were carried out to optimize the reaction strategies and to make OPV tetramers from the building blocks. Chapter 2 describes using the fluorous mixture synthesis (FMS) technology to synthesize a library of OPVs in solution phase. Chapter 3 describes the synthesis of OPVs using solid phase organic synthesis (SPOS). The NMR spectral properties of a group of intermediates frequently encountered in combinatorial synthesis, 1-aryl-3,3-dialkyltriazenes are discussed in Chapter 4

Microarray analysis of lexA gene deletion mutant of deep-sea bacterium Shewanella piezotolerans WP3 at low-temperature and high-pressure

Addressing DNA lesion, the SOS response is conserved in bacterial domain and governed by DNA binding protein LexA, which have been well characterized in model microorganism such as Escherichia coli. However, our understanding of the roles of SOS pathway in deep-sea bacteria is limited. To indentify the composition of SOS regulon and function of LexA, we performed whole genome transcriptional profiling using a custom designed microarray which contains 95% open reading frames of Shewanella piezotolerans WP3. Here we describe the experimental procedures and methods in detail to reproduce the results (available at Gene Expression Omnibus database under GSE66790) and provide resource to be employed for comparative analyses of SOS response in microorganisms which inhabited in different environments, and thus broaden our understanding of life strategy of bacteria against various environment stresses

Transcriptional profiling of CRP-regulated genes in deep-sea bacterium Shewanella piezotolerans WP3

The cAMP receptor protein (CRP) is a conserved regulator in bacteria and involved in regulation of energy metabolism, such as glucose, galactose, and citrate (Green et al., 2014 [1]). As an important catabolite activator protein, it has been well characterized in model microorganism such as Escherichia coli. However, our understanding of the roles of CRP in deep-sea bacteria is rather limited. To indentify the function of CRP, we performed whole genome transcriptional profiling using a custom designed microarray which contains 95% open reading frames of Shewanella piezotolerans WP3, which was isolated from West Pacific sediment at a depth of 1914 m (Xiao et al., 2007 [2]; Wang et al., 2008 [3]). Here we describe the experimental procedures and methods in detail to reproduce the results (available at Gene Expression Omnibus database under GSE67731 and GSE67732) and provide resource to be employed for comparative analyses of CRP regulon and the regulatory network of anaerobic respiration in microorganisms which inhabited in different environments, and thus broaden our understanding of mechanism of bacteria against various environment stresses

Microbial Community Structure of Deep-sea Hydrothermal Vents on the Ultraslow Spreading Southwest Indian Ridge

Southwest Indian Ridge (SWIR) is a typical oceanic ultraslow spreading ridge with intensive hydrothermal activities. The microbial communities in hydrothermal fields including primary producers to support the entire ecosystem by utilizing geochemical energy generated from rock-seawater interactions. Here we have examined the microbial community structures on four hydrothermal vents from SWIR, representing distinct characteristics in terms of temperature, pH and metal compositions, by using Illumina sequencing of the 16S small subunit ribosomal RNA (rRNA) genes, to correlate bacterial and archaeal populations with the nature of the vents influenced by ultraslow spreading features. Epsilon-, Gamma-, Alpha-, and Deltaproteobacteria and members of the phylum Bacteroidetes and Planctomycetes, as well as Thaumarchaeota, Woesearchaeota, and Euryarchaeota were dominant in all the samples. Both bacterial and archaeal community structures showed distinguished patterns compared to those in the fast-spreading East Pacific Ridge or the slow-spreading Mid-Atlantic Ridge as previously reported. Furthermore, within SWIR, the microbial communities are highly correlated with the local temperatures. For example, the sulfur-oxidizing bacteria were dominant within bacteria from low-temperature vents, but were not represented as the dominating group recovered from high temperature (over 300°C) venting chimneys in SWIR. Meanwhile, Thaumarchaeota, the ammonium oxidizing archaea, only showed high relative abundance of amplicons in the vents with high-temperature in SWIR. These findings provide insights on the microbial community in ultraslow spreading hydrothermal fields, and therefore assist us in the understanding of geochemical cycling therein