Vibrio natriegens genome-scale modeling reveals insights into halophilic adaptations and resource allocation.

Vibrio natriegens is a Gram-negative bacterium with an exceptional growth rate that has the potential to become a standard biotechnological host for laboratory and industrial bioproduction. Despite this burgeoning interest, the current lack of organism-specific qualitative and quantitative computational tools has hampered the community's ability to rationally engineer this bacterium. In this study, we present the first genome-scale metabolic model (GSMM) of V. natriegens. The GSMM (iLC858) was developed using an automated draft assembly and extensive manual curation and was validated by comparing predicted yields, central metabolic fluxes, viable carbon substrates, and essential genes with empirical data. Mass spectrometry-based proteomics data confirmed the translation of at least 76% of the enzyme-encoding genes predicted to be expressed by the model during aerobic growth in a minimal medium. iLC858 was subsequently used to carry out a metabolic comparison between the model organism Escherichia coli and V. natriegens, leading to an analysis of the model architecture of V. natriegens' respiratory and ATP-generating system and the discovery of a role for a sodium-dependent oxaloacetate decarboxylase pump. The proteomics data were further used to investigate additional halophilic adaptations of V. natriegens. Finally, iLC858 was utilized to create a Resource Balance Analysis model to study the allocation of carbon resources. Taken together, the models presented provide useful computational tools to guide metabolic engineering efforts in V. natriegens.

Corrigendum: Saliniramus fredricksonii gen. nov., sp. nov., a heterotrophic halophile isolated from Hot Lake, Washington, a member of a novel lineage (Salinarimonadaceae fam. nov.) within the order Rhizobiales, and reclassification of the genus Salinarimonas Liu et al. 2010 into Salinarimonadaceae.

There was an error in the proposed genus name in the published article, in that the genus 'Salinivirga' was effectively published while this article was in review. Therefore, the genus 'Salinivirga' should be replaced with 'Saliniramus'. For the convenience of future readers, we have included the complete corrected article below, in which all occurrences of the incorrect genus name have been amended: A halophilic bacterial strain, HL-109T, was isolated from the unicyanobacterial consortium UCC-O, which was obtained from the photosynthetic mat of Hot Lake (Washington, USA). A polyphasic approach using phenotypic, genotypic and chemotaxonomic data was used to classify the strain within the order Rhizobiales. The organism stained Gram-negative and was a moderate thermophile with a growth optimum of 45 °C. It was obligately aerobic, heterotrophic and halophilic, growing in both NaCl and MgSO4 brines. The novel isolate had a polymorphic cellular morphology of short rods with occasional branching, and cells were monotrichous. The major fatty acids detected were C18 : 1, C18 : 0, C16 : 0 and C18 : cyc. Phylogenetic analysis of the 16S rRNA gene placed the strain in the order Rhizobiales and it shared 94 % identity with the type strain of its nearest relative, Salinarimonas ramus. Morphological, chemotaxonomic and phylogenetic results did not affiliate the novel organism with any of the families in the Rhizobiales; therefore, HL-109T is representative of a new lineage, for which the name Saliniramus fredricksonii gen. nov., sp. nov. is proposed, with the type strain HL-109T (=JCM 31876T=DSM 102886T). In addition, examination of the phylogenetics of strain HL-109T and its nearest relatives, Salinarimonas ramus and Salinarimonasrosea, demonstrates that these halophiles form a clade distinct from the described families of the Rhizobiales. We further propose the establishment of a new family, Salinarimonadaceae fam. nov., to accommodate the genera Saliniramus and Salinarimonas (the type genus of the family).

Salinivirga fredricksonii gen. nov., sp. nov., a heterotrophic halophile isolated from a photosynthetic mat, a member of a novel lineage (Salinarimonadaceae fam. nov.) within the order Rhizobiales, and reclassification of the genus Salinarimonas Liu et al. 2010 into Salinarimonadaceae.

A halophilic bacterial strain, HL-109T, was isolated from the unicyanobacterial consortium UCC-O, which was obtained from the photosynthetic mat of Hot Lake (Washington, USA). A polyphasic approach using phenotypic, genotypic and chemotaxonomic data was used to classify the strain within the order Rhizobiales. The organism stained Gram-negative and was a moderate thermophile with a growth optimum of 45 °C. It was obligately aerobic, heterotrophic and halophilic, growing in both NaCl and MgSO4 brines. The novel isolate had a polymorphic cellular morphology of short rods with occasional branching, and cells were monotrichous. The major fatty acids detected were C18 : 1, C18 : 0, C16 : 0 and C18 : cyc. Phylogenetic analysis of the 16S rRNA gene placed the strain in the order Rhizobiales and it shared 94 % identity with the type strain of its nearest relative, Salinarimonas ramus. Morphological, chemotaxonomic and phylogenetic results did not affiliate the novel organism with any of the families in the Rhizobiales; therefore, HL-109T is representative of a new lineage, for which the name Salinivirga fredricksonii gen. nov., sp. nov. is proposed, with the type strain HL-109T (=JCM 31876T=DSM 102886T). In addition, examination of the phylogenetics of strain HL-109T and its nearest relatives, Salinarimonas ramus and Salinarimonasrosea, demonstrates that these halophiles form a clade distinct from the described families of the Rhizobiales. We further propose the establishment of a new family, Salinarimonadaceae fam. nov., to accommodate the genera Salinivirga and Salinarimonas (the type genus of the family).

VapC6, a ribonucleolytic toxin regulates thermophilicity in the crenarchaeote Sulfolobus solfataricus.

The phylum Crenarchaeota includes hyperthermophilic micro-organisms subjected to dynamic thermal conditions. Previous transcriptomic studies of Sulfolobus solfataricus identified vapBC6 as a heat-shock (HS)-inducible member of the Vap toxin-antitoxin gene family. In this study, the inactivation of the vapBC6 operon by targeted gene disruption produced two recessive phenotypes related to fitness, HS sensitivity and a heat-dependent reduction in the rate of growth. In-frame vapBC6 deletion mutants were analyzed to examine the respective roles of each protein. Since vapB6 transcript abundance was elevated in the vapC6 deletion, the VapC6 toxin appears to regulate abundance of its cognate antitoxin. In contrast, vapC6 transcript abundance was reduced in the vapB6 deletion. A putative intergenic terminator may underlie these observations by coordinating vapBC6 expression. As predicted by structural modeling, recombinant VapC6 produced using chaperone cosynthesis exhibited heat-dependent ribonucleolytic activity toward S. solfataricus total RNA. This activity could be blocked by addition of preheated recombinant VapB6. In vivo transcript targets were identified by assessing the relative expression of genes that naturally respond to thermal stress in VapBC6-deficient cells. Preferential increases were observed for dppB-1 and tetR, and preferential decreases were observed for rpoD and eIF2 gamma. Specific VapC6 ribonucleolytic action could also be demonstrated in vitro toward RNAs whose expression increased in the VapBC6-deficient strain during heat shock. These findings provide a biochemical mechanism and identify cellular targets underlying VapBC6-mediated control over microbial growth and survival at temperature extremes.

Daylight-driven carbon exchange through a vertically structured microbial community.

Interactions between autotrophs and heterotrophs are central to carbon (C) exchange across trophic levels in essentially all ecosystems and metabolite exchange is a frequent mechanism for distributing C within spatially structured ecosystems. Yet, despite the importance of C exchange, the timescales at which fixed C is transferred in microbial communities is poorly understood. We employed a stable isotope tracer combined with spatially resolved isotope analysis to quantify photoautotrophic uptake of bicarbonate and track subsequent exchanges across a vertical depth gradient in a stratified microbial mat over a light-driven diel cycle. We observed that C mobility, both across the vertical strata and between taxa, was highest during periods of active photoautotrophy. Parallel experiments with 13C-labeled organic substrates (acetate and glucose) showed comparably less exchange of C within the mat. Metabolite analysis showed rapid incorporation of 13C into molecules that can both comprise a portion of the extracellular polymeric substances in the system and serve to transport C between photoautotrophs and heterotrophs. Stable isotope proteomic analysis revealed rapid C exchange between cyanobacterial and associated heterotrophic community members during the day with decreased exchange at night. We observed strong diel control on the spatial exchange of freshly fixed C within tightly interacting mat communities suggesting a rapid redistribution, both spatially and taxonomically, primarily during daylight periods.

Metal resistance and lithoautotrophy in the extreme thermoacidophile Metallosphaera sedula.

Archaea such as Metallosphaera sedula are thermophilic lithoautotrophs that occupy unusually acidic and metal-rich environments. These traits are thought to underlie their industrial importance for bioleaching of base and precious metals. In this study, a genetic approach was taken to investigate the specific relationship between metal resistance and lithoautotrophy during biotransformation of the primary copper ore, chalcopyrite (CuFeS(2)). In this study, a genetic system was developed for M. sedula to investigate parameters that limit bioleaching of chalcopyrite. The functional role of the M. sedula copRTA operon was demonstrated by cross-species complementation of a copper-sensitive Sulfolobus solfataricus copR mutant. Inactivation of the gene encoding the M. sedula copper efflux protein, copA, using targeted recombination compromised metal resistance and eliminated chalcopyrite bioleaching. In contrast, a spontaneous M. sedula mutant (CuR1) with elevated metal resistance transformed chalcopyrite at an accelerated rate without affecting chemoheterotrophic growth. Proteomic analysis of CuR1 identified pleiotropic changes, including altered abundance of transport proteins having AAA-ATPase motifs. Addition of the insoluble carbonate mineral witherite (BaCO(3)) further stimulated chalcopyrite lithotrophy, indicating that carbon was a limiting factor. Since both mineral types were actively colonized, enhanced metal leaching may arise from the cooperative exchange of energy and carbon between surface-adhered populations. Genetic approaches provide a new means of improving the efficiency of metal bioleaching by enhancing the mechanistic understanding of thermophilic lithoautotrophy.

Carbohydrate hydrolysis and transport in the extreme thermoacidophile Sulfolobus solfataricus.

Extremely thermoacidophilic microbes, such as Sulfolobus solfataricus, are strict chemoheterotrophs despite their geologic niche. To clarify their ecophysiology, the overlapping roles of endoglucanases and carbohydrate transporters were examined during growth on soluble cellodextrins as the sole carbon and energy source. Strain-specific differences in genome structure implied a unique role for one of three endogenous endoglucanases. Plasmid-based endoglucanase expression promoted the consumption of oligosaccharides, including cellohexaose (G6) through cellonanaose (G9). Protein transporters required for cellodextrin uptake were identified through mutagenesis and complementation of an ABC transporter cassette, including a putative oligosaccharide binding protein. In addition, ablation of the binding protein compromised growth on glucose and alpha-linked oligosaccharides while inactivation of a previously described glucose transporter had no apparent impact. These data demonstrate that S. solfataricus employs a redundant mechanism for soluble cellodextrin catabolism having both substrate uptake and extracytoplasmic hydrolytic components.

Forfeiting the priority effect: turnover defines biofilm community succession.

Microbial community succession is a fundamental process that affects underlying functions of almost all ecosystems; yet the roles and fates of the most abundant colonizers are often poorly understood. Does early abundance spur long term persistence? How do deterministic and stochastic processes influence the ecological contribution of colonizers? We performed a succession experiment within a hypersaline ecosystem to investigate how different processes contributed to the turnover of founder species. Bacterial and eukaryotic colonizers were identified during primary succession and tracked through a defined, 79-day biofilm maturation period using 16S and 18S rRNA gene sequencing in combination with high resolution imaging that utilized stable isotope tracers to evaluate successional patterns of primary producers and nitrogen fixers. The majority of the founder species did not maintain high abundance throughout succession. Species replacement (versus loss) was the dominant process shaping community succession. We also asked if different ecological processes acted on bacteria versus Eukaryotes during succession and found deterministic and stochastic forces corresponded more with microeukaryote and bacterial colonization, respectively. Our results show that taxa and functions belonging to different kingdoms, which share habitat in the tight spatial confines of a biofilm, were influenced by different ecological processes and time scales of succession.

Primary and heterotrophic productivity relate to multikingdom diversity in a hypersaline mat.

Benthic microbial ecosystems are widespread yet knowledge gaps still remain on the relationships between the diversity of species across kingdoms and productivity. Here, we ask two fundamental questions: (i) How does species diversity relate to the rates of primary and heterotrophic productivity? (ii) How do diel variations in light-energy inputs influence productivity and microbiome diversity? To answer these questions, microbial mats from a magnesium sulfate hypersaline lake were used to establish microcosms. Both the number and relatedness between bacterial and eukaryotic taxa in the microbiome were assayed via amplicon-based sequencing of 16S and 18S rRNA genes over two diel cycles. These results correlated with biomass productivity obtained from substrate-specific 13C stable isotope tracers that enabled comparisons between primary and heterotrophic productivity. Both bacterial and eukaryotic species richness and evenness were related only to the rates of 13C-labeled glucose and acetate biomass incorporation. Interestingly, measures of these heterotrophic relationships changed from positive and negative correlations depending on carbon derived from glucose or acetate, respectively. The bacterial and eukaryotic diversity of this ecosystem is also controlled, in part, from energy constraints imposed by changing irradiance over a diel cycle.

Trade-offs between microbiome diversity and productivity in a stratified microbial mat.

Productivity is a major determinant of ecosystem diversity. Microbial ecosystems are the most diverse on the planet yet very few relationships between diversity and productivity have been reported as compared with macro-ecological studies. Here we evaluated the spatial relationships of productivity and microbiome diversity in a laboratory-cultivated photosynthetic mat. The goal was to determine how spatial diversification of microorganisms drives localized carbon and energy acquisition rates. We measured sub-millimeter depth profiles of net primary productivity and gross oxygenic photosynthesis in the context of the localized microenvironment and community structure, and observed negative correlations between species richness and productivity within the energy-replete, photic zone. Variations between localized community structures were associated with distinct taxa as well as environmental profiles describing a continuum of biological niches. Spatial regions in the photic zone corresponding to high primary productivity and photosynthesis rates had relatively low-species richness and high evenness. Hence, this system exhibited negative species-productivity and species-energy relationships. These negative relationships may be indicative of stratified, light-driven microbial ecosystems that are able to be the most productive with a relatively smaller, even distributions of species that specialize within photic zones.

Engineering thermoacidophilic archaea using linear DNA recombination.

Thermoacidophilic archaea comprise one of the major classes of extremophiles. Most belong to the family Sulfolobales within the phylum Crenarchaeota. They are of applied interest as sources of hyperstable enzymes, for biomining of base and precious metals, and for evolutionary studies because of their use of eukaryotic-like subcellular mechanisms. Genetic methods are available for several species particularly Sulfolobus solfataricus. This organism has a considerable number of methods available for the construction of novel cell lines with unique functions. This chapter presents recent developments in the use of homologous recombination and linear DNA for the engineering of site-specific changes in the genome of S. solfataricus.