TbsP and TrmB jointly regulate gapII to influence cell development phenotypes in the archaeon Haloferax volcanii.

Microbial cells must continually adapt their physiology in the face of changing environmental conditions. Archaea living in extreme conditions, such as saturated salinity, represent important examples of such resilience. The model salt-loving organism Haloferax volcanii exhibits remarkable plasticity in its morphology, biofilm formation, and motility in response to variations in nutrients and cell density. However, the mechanisms regulating these lifestyle transitions remain unclear. In prior research, we showed that the transcriptional regulator, TrmB, maintains the rod shape in the related species Halobacterium salinarum by activating the expression of enzyme-coding genes in the gluconeogenesis metabolic pathway. In Hbt. salinarum, TrmB-dependent production of glucose moieties is required for cell surface glycoprotein biogenesis. Here, we use a combination of genetics and quantitative phenotyping assays to demonstrate that TrmB is essential for growth under gluconeogenic conditions in Hfx. volcanii. The ∆trmB strain rapidly accumulated suppressor mutations in a gene encoding a novel transcriptional regulator, which we name trmB suppressor, or TbsP (a.k.a. "tablespoon"). TbsP is required for adhesion to abiotic surfaces (i.e., biofilm formation) and maintains wild-type cell morphology and motility. We use functional genomics and promoter fusion assays to characterize the regulons controlled by each of TrmB and TbsP, including joint regulation of the glucose-dependent transcription of gapII, which encodes an important gluconeogenic enzyme. We conclude that TrmB and TbsP coregulate gluconeogenesis, with downstream impacts on lifestyle transitions in response to nutrients in Hfx. volcanii.

Integration of VDR genome wide binding and GWAS genetic variation data reveals co-occurrence of VDR and NF-κB binding that is linked to immune phenotypes.

BACKGROUND: The nuclear hormone receptor superfamily acts as a genomic sensor of diverse signals. Their actions are often intertwined with other transcription factors. Nuclear hormone receptors are targets for many therapeutic drugs, and include the vitamin D receptor (VDR). VDR signaling is pleotropic, being implicated in calcaemic function, antibacterial actions, growth control, immunomodulation and anti-cancer actions. Specifically, we hypothesized that the biologically significant relationships between the VDR transcriptome and phenotype-associated biology could be discovered by integrating the known VDR transcription factor binding sites and all published trait- and disease-associated SNPs. By integrating VDR genome-wide binding data (ChIP-seq) with the National Human Genome Research Institute (NHGRI) GWAS catalog of SNPs we would see where and which target gene interactions and pathways are impacted by inherited genetic variation in VDR binding sites, indicating which of VDR's multiple functions are most biologically significant. RESULTS: To examine how genetic variation impacts VDR function we overlapped 23,409 VDR genomic binding peaks from six VDR ChIP-seq datasets with 191,482 SNPs, derived from GWAS-significant SNPs (Lead SNPs) and their correlated variants (r 2 > 0.8) from HapMap3 and the 1000 genomes project. In total, 574 SNPs (71 Lead and 503 SNPs in linkage disequilibrium with Lead SNPs) were present at VDR binding loci and associated with 211 phenotypes. For each phenotype a hypergeometric test was used to determine if SNPs were enriched at VDR binding sites. Bonferroni correction for multiple testing across the 211 phenotypes yielded 42 SNPs that were either disease- or phenotype-associated with seven predominately immune related including self-reported allergy; esophageal cancer was the only cancer phenotype. Motif analyses revealed that only two of these 42 SNPs reside within a canonical VDR binding site (DR3 motif), and that 1/3 of the 42 SNPs significantly impacted binding and gene regulation by other transcription factors, including NF-κB. This suggests a plausible link for the potential cross-talk between VDR and NF-κB. CONCLUSIONS: These analyses showed that VDR peaks are enriched for SNPs associated with immune phenotypes suggesting that VDR immunomodulatory functions are amongst its most important actions. The enrichment of genetic variation in non-DR3 motifs suggests a significant role for the VDR to bind in multimeric complexes containing other transcription factors that are the primary DNA binding component. Our work provides a framework for the combination of ChIP-seq and GWAS findings to provide insight into the underlying phenotype-associated biology of a given transcription factor.

Genetic engineering of Synechocystis PCC6803 for the photoautotrophic production of the sweetener erythritol.

BACKGROUND: Erythritol is a polyol that is used in the food and beverage industry. Due to its non-caloric and non-cariogenic properties, the popularity of this sweetener is increasing. Large scale production of erythritol is currently based on conversion of glucose by selected fungi. In this study, we describe a biotechnological process to produce erythritol from light and CO2, using engineered Synechocystis sp. PCC6803. METHODS: By functionally expressing codon-optimized genes encoding the erythrose-4-phosphate phosphatase TM1254 and the erythrose reductase Gcy1p, or GLD1, this cyanobacterium can directly convert the Calvin cycle intermediate erythrose-4-phosphate into erythritol via a two-step process and release the polyol sugar in the extracellular medium. Further modifications targeted enzyme expression and pathway intermediates. CONCLUSIONS: After several optimization steps, the best strain, SEP024, produced up to 2.1 mM (256 mg/l) erythritol, excreted in the medium.

Nitric oxide reactivities of the two globins of the foodborne pathogen Campylobacter jejuni: roles in protection from nitrosative stress and analysis of potential reductants.

BACKGROUND: During infection and pathogenesis, Campylobacter, the leading cause of gastroenteritis, encounters NO and reactive nitrogen species (RNS) derived from the host. To combat these species, Campylobacter jejuni expresses two haemoglobins: the single domain haemoglobin (Cgb) detoxifies NO but the role of the truncated globin (Ctb) is unclear. Confirmation of Cgb activity and more extensive exploration of Ctb function(s) in vivo are restricted due to difficulties in expressing proteins in Campylobacter and our lack of understanding of how the globin haems are re-reduced after ligand reactions. METHODS: The cgb and ctb genes were cloned under the control of arabinose-inducible promoters and the globins expressed in an Escherichia coli mutant lacking the main NO detoxification mechanisms (Hmp and the Nor system comprising the transcription regulator NorR, the flavorubredoxin and its reductase (NorVW)); cellular responses under oxidative and nitrosative stress conditions were assessed. Spectroscopic changes of the Cgb and Ctb haems in soluble fractions after oxidation by NO were evaluated. Construction of E. coli nor mutants and a ubiquinone-defective strain allowed the exploration of the flavorubredoxin reductase and the aerobic respiratory chain as candidates for Cgb electron donors in E. coli mutants. RESULTS: Cgb, but not Ctb, complements the NO- and RNS-sensitive phenotype of an E. coli hmp mutant in aerobic conditions; however, Cgb fails to protect an hmp norR mutant in the absence of oxygen. Reduction of Cgb and Ctb in E. coli and C. jejuni soluble extracts and turnover after NO oxidation is demonstrated. Finally, we report a minor role for NorW as a Cgb reductase partner in E. coli but no role for respiratory electron flux in globin redox cycling. CONCLUSIONS: The NO detoxification capacity of Cgb is confirmed by heterologous expression in E. coli. The reducibility of Cgb and Ctb in E. coli and C. jejuni extracts and the lack of dependence of reduction upon flavorubredoxin reductase and the respiratory chain in E. coli argue in favor of a non-specific reductase system. GENERAL SIGNIFICANCE: We present the most persuasive evidence to date that Cgb, but not Ctb, confers tolerance to NO and RNS by reaction with NO. Since certain hypotheses for the mechanism of haem re-reduction in E. coli following the reaction with NO are not proven, the mechanisms of reduction in C. jejuni now require challenging experimental evaluation.

The Hypersaline Archaeal Histones HpyA and HstA Are DNA Binding Proteins That Defy Categorization According to Commonly Used Functional Criteria.

Histone proteins are found across diverse lineages of Archaea, many of which package DNA and form chromatin. However, previous research has led to the hypothesis that the histone-like proteins of high-salt-adapted archaea, or halophiles, function differently. The sole histone protein encoded by the model halophilic species Halobacterium salinarum, HpyA, is nonessential and expressed at levels too low to enable genome-wide DNA packaging. Instead, HpyA mediates the transcriptional response to salt stress. Here we compare the features of genome-wide binding of HpyA to those of HstA, the sole histone of another model halophile, Haloferax volcanii. hstA, like hpyA, is a nonessential gene. To better understand HpyA and HstA functions, protein-DNA binding data (chromatin immunoprecipitation sequencing [ChIP-seq]) of these halophilic histones are compared to publicly available ChIP-seq data from DNA binding proteins across all domains of life, including transcription factors (TFs), nucleoid-associated proteins (NAPs), and histones. These analyses demonstrate that HpyA and HstA bind the genome infrequently in discrete regions, which is similar to TFs but unlike NAPs, which bind a much larger genomic fraction. However, unlike TFs that typically bind in intergenic regions, HpyA and HstA binding sites are located in both coding and intergenic regions. The genome-wide dinucleotide periodicity known to facilitate histone binding was undetectable in the genomes of both species. Instead, TF-like and histone-like binding sequence preferences were detected for HstA and HpyA, respectively. Taken together, these data suggest that halophilic archaeal histones are unlikely to facilitate genome-wide chromatin formation and that their function defies categorization as a TF, NAP, or histone. IMPORTANCE Most cells in eukaryotic species-from yeast to humans-possess histone proteins that pack and unpack DNA in response to environmental cues. These essential proteins regulate genes necessary for important cellular processes, including development and stress protection. Although the histone fold domain originated in the domain of life Archaea, the function of archaeal histone-like proteins is not well understood relative to those of eukaryotes. We recently discovered that, unlike histones of eukaryotes, histones in hypersaline-adapted archaeal species do not package DNA and can act as transcription factors (TFs) to regulate stress response gene expression. However, the function of histones across species of hypersaline-adapted archaea still remains unclear. Here, we compare hypersaline histone function to a variety of DNA binding proteins across the tree of life, revealing histone-like behavior in some respects and specific transcriptional regulatory function in others.

Differentiation of function among the RsbR paralogs in the general stress response of Bacillus subtilis with regard to light perception.

The general stress response of Bacillus subtilis can be activated by a wide range of signals, including low intensities of visible light. It is regulated by a dedicated σ factor via a complex signal transduction pathway that makes use of stressosomes: hetero-oligomeric complexes that include one or more of the RsbR proteins (RsbRA, RsbRB, RsbRC, and RsbRD). The response to blue light is mediated by the photoreceptor YtvA. We show here which of the four RsbR proteins are necessary for the activation of the σ(B) response by blue light. Experiments performed with single-, double-, and triple-deletion strains in the rsbR genes show that RsbRB and RsbRA function antagonistically, with the former being a negative regulator and the latter a positive regulator of the YtvA-dependent light activation of the stress response. A strain with RsbRB as the only RsbR protein is unable to respond to light-activation of σ(B). Furthermore, RsbRC and RsbRD can replace RsbRA's function only in the absence of RsbRB. This differentiation of function is confined to light stress, since strains with RsbRA or RsbRB as the only RsbR protein behave similarly in our experimental conditions in response to physicochemical stresses. Interestingly, RsbRB's absence is sufficient to result in light activation of the general stress response at wild-type expression levels of ytvA, while it was previously reported that YtvA could only activate σ(B) when overproduced, or when cells are supplemented with an additional environmental stress.

Copy number variation is associated with gene expression change in archaea.

Genomic instability, although frequently deleterious, is also an important mechanism for microbial adaptation to environmental change. Although widely studied in bacteria, in archaea the effect of genomic instability on organism phenotypes and fitness remains unclear. Here we use DNA segmentation methods to detect and quantify genome-wide copy number variation (CNV) in large compendia of high-throughput datasets in a model archaeal species, Halobacterium salinarum. CNV hotspots were identified throughout the genome. Some hotspots were strongly associated with changes in gene expression, suggesting a mechanism for phenotypic innovation. In contrast, CNV hotspots in other genomic loci left expression unchanged, suggesting buffering of certain phenotypes. The correspondence of CNVs with gene expression was validated with strain- and condition-matched transcriptomics and DNA quantification experiments at specific loci. Significant correlation of CNV hotspot locations with the positions of known insertion sequence (IS) elements suggested a mechanism for generating genomic instability. Given the efficient recombination capabilities in H. salinarum despite stability at the single nucleotide level, these results suggest that genomic plasticity mediated by IS element activity can provide a source of phenotypic innovation in extreme environments.

Exploring metabolic engineering design principles for the photosynthetic production of lactic acid by Synechocystis sp. PCC6803.

BACKGROUND: Molecular engineering of the intermediary physiology of cyanobacteria has become important for the sustainable production of biofuels and commodity compounds from CO2 and sunlight by "designer microbes." The chemical commodity product L-lactic acid can be synthesized in one step from a key intermediary metabolite of these organisms, pyruvate, catalyzed by a lactate dehydrogenase. Synthetic biology engineering to make "designer microbes" includes the introduction and overexpression of the product-forming biochemical pathway. For further optimization of product formation, modifications in the surrounding biochemical network of intermediary metabolism have to be made. RESULTS: To improve light-driven L-lactic acid production from CO2, we explored several metabolic engineering design principles, using a previously engineered L-lactic acid producing mutant strain of Synechocystis sp. PCC6803 as the benchmark. These strategies included: (i) increasing the expression level of the relevant product-forming enzyme, lactate dehydrogenase (LDH), for example, via expression from a replicative plasmid; (ii) co-expression of a heterologous pyruvate kinase to increase the flux towards pyruvate; and (iii) knockdown of phosphoenolpyruvate carboxylase to decrease the flux through a competing pathway (from phosphoenolpyruvate to oxaloacetate). In addition, we tested selected lactate dehydrogenases, some of which were further optimized through site-directed mutagenesis to improve the enzyme's affinity for the co-factor nicotinamide adenine dinucleotide phosphate (NADPH). The carbon partitioning between biomass and lactic acid was increased from about 5% to over 50% by strain optimization. CONCLUSION: An efficient photosynthetic microbial cell factory will display a high rate and extent of conversion of substrate (CO2) into product (here: L-lactic acid). In the existing CO2-based cyanobacterial cell factories that have been described in the literature, by far most of the control over product formation resides in the genetically introduced fermentative pathway. Here we show that a strong promoter, in combination with increased gene expression, can take away a significant part of the control of this step in lactic acid production from CO2. Under these premises, modulation of the intracellular precursor, pyruvate, can significantly increase productivity. Additionally, production enhancement is achieved by protein engineering to increase co-factor specificity of the heterologously expressed LDH.