Expression Of Sigma Factors Research Articles

Tanshinones from Salvia miltiorrhiza inhibit Mycobacterium tuberculosis via disruption of the cell envelope surface and oxidative stress

The unique structure of Mycobacterium tuberculosis cell envelope provides impermeable barrier against environmental stimuli. In the situation that this barrier is disturbed Mycobacteria react at the transcriptional and translational level to redirect metabolic processes and to maintain integrity of the cell. In this work we aimed to explore the early metabolic response of M. tuberculosis to tanshinones, which are active antimycobacterial compounds of Salvia miltiorrhiza Bunge root. The investigation of the expression of sigma factors revealed the significant shifts in the general bacterial regulatory network, whereas LC-MS metabolomics evidenced the changes in the composition of bacterial cell envelope and indicated altered metabolic pathways. Tanshinones acted via the disruption of the cell envelope surface and generation of reactive oxygen species. Bacteria responded with overproduction of inner region of outer membrane, fluctuations in the production of glycerophosphoinositolglycans, as well as changes in the levels of mycobactins, accompanied by enrichment of metabolic pathways related to redox balance and repair of damages caused by tanshinones.

Optimal reference genes for real-time quantitative PCR and the expression of sigma factors in Acidithiobacillus caldus under various conditions.

Acidithiobacillus caldus is an important sulphur-oxidizing bacterium that plays crucial roles in the bioleaching industry. This study aims to analyse the optimal reference gene for real-time quantitative PCR (RT-qPCR) under different conditions and investigate the transcription levels of the sigma factor genes in the stress response. We selected six housekeeping genes and analysed them via RT-qPCR using two energy resources, under four stress conditions. Three statistical approaches BestKeeper, geNorm, and NormFinder were utilized to determine transcription stability of these reference genes. The gapdH gene was the best internal control gene using elemental sulphur as an energy resource and under heat stress, map was the best internal control gene under pH and osmotic stress, era was the best internal control gene for the K2 S4 O6 energy resource, and rpoC was the best internal control gene under Cu2+ stress. Furthermore, the expressional levels of 11 sigma factors were analysed by RT-qPCR in the stress response. Stable internal control genes for RT-qPCR analysis of A. caldus were determined, and the expression patterns of sigma factor genes of A. caldus were investigated. The identification of the optimal reference gene and analysis of transcription levels of sigma factors in A. caldus can provide clues for reference gene selection and the study of sigma factor function.

PG1659 functions as anti-sigma factor to extracytoplasmic function sigma factor RpoE in Porphyromonas gingivalis W83.

Anti-sigma factors play a critical role in regulating the expression of sigma factors in response to environmental stress signals. PG1659 is cotranscribed with an upstream gene PG1660 (rpoE), which encodes for a sigma factor that plays an important role in oxidative stress resistance and the virulence regulatory network of P. gingivalis. PG1659, which is annotated as a hypothetical gene, is evaluated in this study. PG1659, composed of 130 amino acids, is predicted to be transmembrane protein with a single calcium (Ca2+ ) binding site. In P. gingivalis FLL358 (ΔPG1659::ermF), the rpoE gene was highly upregulated compared to the wild-type W83 strain. RpoE-induced genes were also upregulated in the PG1659-defective isogenic mutant. Both protein-protein pull-down and bacterial two-hybrid assays revealed that the PG1659 protein could interact with/bind RpoE. The N-terminal domain of PG1659, representing the cytoplasmic fragment of the protein, is critical for interaction with RpoE. In the presence of PG1659, the initiation of transcription by the RpoE sigma factor was inhibited. Taken together, our data suggest that PG1659 is an anti-sigma factor which plays an important regulatory role in the modulation of the sigma factor RpoE in P. gingivalis.

Systematic identification of novel regulatory interactions controlling biofilm formation in the bacterium Escherichia coli

Here, we investigated novel interactions of three global regulators of the network that controls biofilm formation in the model bacterium Escherichia coli using computational network analysis, an in vivo reporter assay and physiological validation experiments. We were able to map critical nodes that govern planktonic to biofilm transition and identify 8 new regulatory interactions for CRP, IHF or Fis responsible for the control of the promoters of rpoS, rpoE, flhD, fliA, csgD and yeaJ. Additionally, an in vivo promoter reporter assay and motility analysis revealed a key role for IHF as a repressor of cell motility through the control of FliA sigma factor expression. This investigation of first stage and mature biofilm formation indicates that biofilm structure is strongly affected by IHF and Fis, while CRP seems to provide a fine-tuning mechanism. Taken together, the analysis presented here shows the utility of combining computational and experimental approaches to generate a deeper understanding of the biofilm formation process in bacteria.

Expression of Heterologous Sigma Factor Expands the Searchable Space for Biofuel Tolerance Mechanisms.

Microorganisms can produce hydrocarbons that can serve as replacements or additions to conventional liquid fuels for use in the transportation sector. However, a common problem in the microbial synthesis of biofuels is that these compounds often have toxic effects on the cell. In this study, we focused on mitigating the toxicity of the biojet fuel precursor pinene on Escherichia coli. We used genomic DNA from Pseudomonas putida KT2440, which has innate solvent-tolerance properties, to create transgenic libraries in an E.coli host. We exposed cells containing the library to pinene, selecting for genes that improved tolerance. Importantly, we found that expressing the sigma factor RpoD from P.putida greatly expanded the diversity of tolerance genes recovered. With low expression of rpoDP.putida, we isolated a single pinene tolerance gene; with increased expression of the sigma factor our selection experiments returned multiple distinct tolerance mechanisms, including some that have been previously documented and also new mechanisms. Interestingly, high levels of rpoDP.putida induction resulted in decreased diversity. We found that the tolerance levels provided by some genes are highly sensitive to the level of induction of rpoDP.putida, while others provide tolerance across a wide range of rpoDP.putida levels. This method for unlocking diversity in tolerance screening using heterologous sigma factor expression was applicable to both plasmid and fosmid-based transgenic libraries. These results suggest that by controlling the expression of appropriate heterologous sigma factors, we can greatly increase the searchable genomic space within transgenic libraries.

Sigma Factor Regulated Cellular Response in a Non-solvent Producing Clostridium beijerinckii Degenerated Strain: A Comparative Transcriptome Analysis.

Clostridium beijerinckii DG-8052, derived from NCIMB 8052, cannot produce solvent or form spores, a phenomenon known as degeneration. To explore the mechanisms of degeneration at the gene level, transcriptomic profiles of the wild-type 8052 and DG-8052 strains were compared. Expression of 5168 genes comprising 98.6% of the genome was assessed. Interestingly, 548 and 702 genes were significantly up-regulated in the acidogenesis and solventogenesis phases of DG-8052, respectively, and mainly responsible for the phosphotransferase system, sugar metabolic pathways, and chemotaxis; meanwhile, 699 and 797 genes were significantly down-regulated, respectively, and mainly responsible for sporulation, oxidoreduction, and solventogenesis. The functions of some altered genes, including 286 and 333 at the acidogenesis and solventogenesis phases, respectively, remain unknown. Dysregulation of the fermentation machinery was accompanied by lower transcription levels of glycolysis rate-limiting enzymes (pfk and pyk), and higher transcription of cell chemotaxis genes (cheA, cheB, cheR, cheW, and cheY), controlled mainly by σ54 at acidogenesis. Meanwhile, abnormal spore formation was associated with repressed spo0A, sigE, sigF, sigG, and sigK which are positively regulated by σ70, and correspondingly inhibited expression of CoA-transferase at the solventogenesis phase. These findings indicated that morphological and physiological changes in the degenerated Clostridium strain may be related to altered expression of sigma factors, providing valuable targets for strain development of Clostridium species.

Molecular and physiological evidence of genetic assimilation to high CO2 in the marine nitrogen fixer Trichodesmium

Most investigations of biogeochemically important microbes have focused on plastic (short-term) phenotypic responses in the absence of genetic change, whereas few have investigated adaptive (long-term) responses. However, no studies to date have investigated the molecular progression underlying the transition from plasticity to adaptation under elevated CO2 for a marine nitrogen-fixer. To address this gap, we cultured the globally important cyanobacterium Trichodesmium at both low and high CO2 for 4.5 y, followed by reciprocal transplantation experiments to test for adaptation. Intriguingly, fitness actually increased in all high-CO2 adapted cell lines in the ancestral environment upon reciprocal transplantation. By leveraging coordinated phenotypic and transcriptomic profiles, we identified expression changes and pathway enrichments that rapidly responded to elevated CO2 and were maintained upon adaptation, providing strong evidence for genetic assimilation. These candidate genes and pathways included those involved in photosystems, transcriptional regulation, cell signaling, carbon/nitrogen storage, and energy metabolism. Conversely, significant changes in specific sigma factor expression were only observed upon adaptation. These data reveal genetic assimilation as a potentially adaptive response of Trichodesmium and importantly elucidate underlying metabolic pathways paralleling the fixation of the plastic phenotype upon adaptation, thereby contributing to the few available data demonstrating genetic assimilation in microbial photoautotrophs. These molecular insights are thus critical for identifying pathways under selection as drivers in plasticity and adaptation.

The effect of Mycobacterium tuberculosis CRISPR-associated Cas2 (Rv2816c) on stress response genes expression, morphology and macrophage survival of Mycobacterium smegmatis

Clustered regularly interspaced short palindromic repeats (CRISPR) are present in the genome of 40% bacteria and 90% archaea. CRISPR and accompanying Cas proteins constitute an adaptive immune system against disruptive mobile genetic elements. Two CRISPRs and 9 genes encoding CRISPR-associated proteins have been found in the genome of Mycobacterium tuberculosis. The CRISPR-associated Cas2 is an endoribonuclease required for the acquisition of new spacers. In this study, Cas2 encoded by Rv2816c was expressed in Mycobacterium smegmatis lacking CRISPR-Cas system and its role in stress responses of M. smegmatis in vitro and within macrophages was studied. We found that Cas2 mediated M. smegmatis stress response changes were associated with the altered expression of sigma factors which involved in mycobacterial stress response and virulence. We also found that Cas2 decreased the survival of M. smegmatis within macrophages. This study provides new insights on the role of Cas2.

Interactions of anti-sigma factor antagonists of Mycobacterium tuberculosis in the yeast two-hybrid system

Anti-sigma factor antagonists (anti-anti-sigma factors) play critical roles in regulating the expression of alternative sigma factors in response to specific stress signals. The Clusters of Orthologous Groups (COG) database has identified the existence of six genes, Rv0516c, Rv1364c, Rv1365c, Rv1904, Rv2638 and Rv3687c (grouped under the cluster COG1366), encoding potential anti-sigma factor antagonists in Mycobacterium tuberculosis. These molecules are speculated to regulate the expression of sigma factor SigF of M. tuberculosis in response to stress signals. Since signaling occurs via physical interactions of proteins (protein-protein interaction), we investigated whether the anti-sigma factor antagonists of M. tuberculosis interact with anti-sigma factor RsbW (Rv3287c) or the sigma factor SigF (Rv3286c) in the yeast two-hybrid system. The results revealed that most of the anti-sigma factor antagonists interact with either RsbW or SigF or both. In addition, some anti-sigma factor antagonists also displayed limited interactions between themselves. These interactions suggest that they possibly transduce some signals to SigF and between themselves.

Increased transcription of a potential sigma factor regulatory gene Rv1364c inMycobacterium bovisBCG while residing in macrophages indicates use of alternative promoters

Alternative sigma factors are key global regulators that coordinate bacterial responses to environmental changes necessary for adaptation and survival. In turn these sigma factors are controlled by regulators such as anti-sigma and anti-anti-sigma factors. In this report, using a cDNA-total RNA subtractive hybridisation strategy that we have developed previously, we identified increased transcription of a potential sigma factor regulatory gene, Rv1364c, in Mycobacterium bovis BCG upon phagocytosis by macrophages and this was confirmed by Northern blot analysis. Primer extension analysis revealed the use of alternative promotors, P1 and P2, and that the increased expression inside macrophages coincided with promoter switching from P2 to P1. Rv1364c (653 amino acids), originally annotated as RsbU, contains structural domains homologous to the PAS redox sensor, the protein phosphatases anti-anti-sigma factor RsbU/SpoIIE, the protein kinase anti-sigma factor RsbW/SpoIIAB and the anti-anti-sigma factor RsbV/SpoIIAA found in other bacteria. These findings have important implications for understanding coordination of the expression of sigma factors under intra-macrophage conditions. Other potentially differentially expressed genes, including genes for fatty acid metabolism, membrane transportors, heat shock proteins, potential sigma factors and energy metabolic pathways are also listed and their biological significance discussed.

Increased transcription of a potential sigma factor regulatory gene Rv1364c in Mycobacterium bovis BCG while residing in macrophages indicates use of alternative promoters

Alternative sigma factors are key global regulators that coordinate bacterial responses to environmental changes necessary for adaptation and survival. In turn these sigma factors are controlled by regulators such as anti-sigma and anti-anti-sigma factors. In this report, using a cDNA–total RNA subtractive hybridisation strategy that we have developed previously, we identified increased transcription of a potential sigma factor regulatory gene, Rv1364c, in Mycobacterium bovis BCG upon phagocytosis by macrophages and this was confirmed by Northern blot analysis. Primer extension analysis revealed the use of alternative promotors, P1 and P2, and that the increased expression inside macrophages coincided with promoter switching from P2 to P1. Rv1364c (653 amino acids), originally annotated as RsbU, contains structural domains homologous to the PAS redox sensor, the protein phosphatases anti-anti-sigma factor RsbU/SpoIIE, the protein kinase anti-sigma factor RsbW/SpoIIAB and the anti-anti-sigma factor RsbV/SpoIIAA found in other bacteria. These findings have important implications for understanding coordination of the expression of sigma factors under intra-macrophage conditions. Other potentially differentially expressed genes, including genes for fatty acid metabolism, membrane transportors, heat shock proteins, potential sigma factors and energy metabolic pathways are also listed and their biological significance discussed.

Statistical analysis of Pseudomonas aeruginosa biofilm development: impact of mutations in genes involved in twitching motility, cell-to-cell signaling, and stationary-phase sigma factor expression.

Four strains of Pseudomonas aeruginosa (wild type, Delta(pil)HIJK mutant, lasI mutant, and rpoS mutant) were genetically tagged with the green fluorescent protein, and the development of flow chamber-grown biofilms by each of them was investigated by confocal laser scanning microscopy. The structural developments of the biofilms were quantified by the computer program COMSTAT (A. Heydorn, A. T. Nielsen, M. Hentzer, C. Sternberg, M. Givskov, B. K. Ersbøll, and S. Molin, Microbiology 146:2395-2407, 2000). Two structural key variables, average thickness and roughness, formed the basis for an analysis of variance model comprising the four P. aeruginosa strains, five time points (55, 98, 146, 242, and 314 h), and three independent rounds of biofilm experiments. The results showed that the wild type, the Delta(pil)HIJK mutant, and the rpoS mutant display conspicuously different types of temporal biofilm development, whereas the lasI mutant was indistinguishable from the wild type at all time points. The wild type and the lasI mutant formed uniform, densely packed biofilms. The rpoS mutant formed densely packed biofilms that were significantly thicker than those of the wild type, whereas the Delta(pil)HIJK mutant formed distinct microcolonies that were regularly spaced and almost uniform in size. The results are discussed in relation to the current model of P. aeruginosa biofilm development.

Analyzing the developmental expression of sigma factors with S1-nuclease mapping.

Streptomycetes are mycelial, Gram-positive soil bacteria which produce a variety of biologically active secondary metabolites, including the majority of known antibiotics. In addition to this physiological differentiation, they undergo a complex cycle of morphological differentiation. Spores after germination on solid-growth media form a network of vegetative “substrate mycelium” that grows into the medium. After unknown signal(s), the substrate mycelium differentiates, and white aerial mycelium is formed that grows into the air. Later it undergoes septation and, in the last step of the process, forms chains of spores. The differentiation of Streptomyces is controlled on several levels, of which heterogeneity of σ factors of RNA polymerase seems to play an important role (). Therefore, we examined the Streptomyces differentiation through the study of these key proteins in this process.

Conditional sigma factor expression, using the inducible acetamidase promoter, reveals that the Mycobacterium tuberculosis sigF gene modulates expression of the 16-kilodalton alpha-crystallin homologue.

A chemically inducible acetamidase promoter-sigF fusion gene was integrated into the chromosome of Mycobacterium bovis BCG. Two-dimensional protein gel analysis permitted the identification of a number of protein spots whose expression was SigF related. One spot upregulated by inappropriate induction of sigF expression corresponded to the 16-kDa antigen alpha-crystallin.

The effect of supercoiling on the in vitro transcription of the spoIIA operon from Bacillus subtilis

The spoIIA operon codes for an alternative sigma factor which appears in the early stages of a sigma factor expression cascade during sporulation in Bacillus subtilis. We have used a single round in vitro transcription assay to probe requirements for transcription initiation at the spoIIA promoter. Core RNA polymerase or holyenzyme containing σ A was reconstituted with σ H protein and used to transcribe the spoIIA promoter. Formation of heparin resistant transcription initiation complexes required that the spoIIA template be supercoiled. Topoisomers of the spoIIA template were created and transcribed at various temperatures. Changes in the superhelicity of template DNA had a significant influence on the amount of transcription complexes formed at the spoIIA promoter.