Transcriptional regulation by σ factor phosphorylation in bacteria.

A major form of transcriptional regulation in bacteria occurs through the exchange of the primary σ factor of RNA polymerase (RNAP) with an alternative extracytoplasmic function (ECF) σ factor1. ECF σ factors are generally intrinsically active and are retained in an inactive state via the sequestration into σ factor-anti-σ factor complexes until their action is warranted2-20. Here, we report a previously uncharacterized mechanism of transcriptional regulation that relies on intrinsically inactive ECF σ factors, the activation of which and interaction with the ß'-subunit of RNAP depends on σ factor phosphorylation. In Vibrio parahaemolyticus, the threonine kinase PknT phosphorylates the σ factor EcfP, which results in EcfP activation and expression of an essential polymyxin-resistant regulon. EcfP phosphorylation occurs at a highly conserved threonine residue, Thr63, positioned within a divergent region in the σ2.2 helix. Our data indicate that EcfP is intrinsically inactive and unable to bind the ß'-subunit of RNAP due to the absence of a negatively charged DAED motif in this region. Furthermore, our results indicate that phosphorylation at residue Thr63 mimics this negative charge and licenses EcfP to interact with the ß'-subunit in the formation of the RNAP holoenzyme, which in turn results in target gene expression. This regulatory mechanism is a previously unrecognized paradigm in bacterial signal transduction and transcriptional regulation, and our data suggest that it is widespread in bacteria.

Stress-Responsive Alternative Sigma Factor SigB Plays a Positive Role in the Antifungal Proficiency of Bacillus subtilis.

Different Bacillus species with PGPR (plant growth-promoting rhizobacterium) activity produce potent biofungicides and stimulate plant defense responses against phytopathogenic fungi. However, very little is known about how these PGPRs recognize phytopathogens and exhibit the antifungal response. Here, we report the antagonistic interaction between Bacillus subtilis and the phytopathogenic fungus Fusarium verticillioides We demonstrate that this bacterial-fungal interaction triggers the induction of the SigB transcription factor, the master regulator of B. subtilis stress adaptation. Dual-growth experiments performed with live or dead mycelia or culture supernatants of F. verticillioides showed that SigB was activated and required for the biocontrol of fungal growth. Mutations in the different regulatory pathways of SigB activation in the isogenic background revealed that only the energy-related RsbP-dependent arm of SigB activation was responsible for specific fungal detection and triggering the antagonistic response. The activation of SigB increased the expression of the operon responsible for the production of the antimicrobial cyclic lipopeptide surfactin (the srfA operon). SigB-deficient B. subtilis cultures produced decreased amounts of surfactin, and B. subtilis cultures defective in surfactin production (ΔsrfA) were unable to control the growth of F. verticillioidesIn vivo experiments of seed germination efficiency and early plant growth inhibition in the presence of F. verticillioides confirmed the physiological importance of SigB activity for plant bioprotection.IMPORTANCE Biological control using beneficial bacteria (PGPRs) represents an attractive and environment-friendly alternative to pesticides for controlling plant diseases. Different PGPR Bacillus species produce potent biofungicides and stimulate plant defense responses against phytopathogenic fungi. However, very little is known about how PGPRs recognize phytopathogens and process the antifungal response. Here, we report how B. subtilis triggers the induction of the stress-responsive sigma B transcription factor and the synthesis of the lipopeptide surfactin to fight the phytopathogen. Our findings show the participation of the stress-responsive regulon of PGPR Bacillus in the detection and biocontrol of a phytopathogenic fungus of agronomic impact.

Structural basis of ECF-σ-factor-dependent transcription initiation.

Extracytoplasmic (ECF) σ factors, the largest class of alternative σ factors, are related to primary σ factors, but have simpler structures, comprising only two of six conserved functional modules in primary σ factors: region 2 (σR2) and region 4 (σR4). Here, we report crystal structures of transcription initiation complexes containing Mycobacterium tuberculosis RNA polymerase (RNAP), M. tuberculosis ECF σ factor σL, and promoter DNA. The structures show that σR2 and σR4 of the ECF σ factor occupy the same sites on RNAP as in primary σ factors, show that the connector between σR2 and σR4 of the ECF σ factor-although shorter and unrelated in sequence-follows the same path through RNAP as in primary σ factors, and show that the ECF σ factor uses the same strategy to bind and unwind promoter DNA as primary σ factors. The results define protein-protein and protein-DNA interactions involved in ECF-σ-factor-dependent transcription initiation.

Markerless Genome Editing in Competent Streptococci.

Selective markers employed in classical mutagenesis methods using natural genetic transformation can affect gene expression, risk phenotypic effects, and accumulate as unwanted genes during successive mutagenesis cycles. In this chapter, we present a protocol for markerless genome editing in Streptococcus mutans and Streptococcus pneumoniae achieved with an efficient method for natural transformation. High yields of transformants are obtained by combining the unimodal state of competence developed after treatment of S. mutans with sigX-inducing peptide pheromone (XIP) in a chemically defined medium (CDM) or of S. pneumoniae with the competence-stimulating peptide (CSP) together with use of a donor amplicon carrying extensive flanking homology. This combination ensures efficient and precise integration of a new allele by the recombination machinery present in competent cells.

Involvement of B. pseudomallei RpoS in apoptotic cell death in mouse macrophages.

Burkholderia pseudomallei, the causative agent of melioidosis, is a facultative intracellular Gram-negative bacillus which can survive and multiply in both phagocytic and nonphagocytic cells. This bacterium could also induce apoptosis in various cell types. In the present study, we extend our finding to demonstrate the role of RpoS of B. pseudomallei in apoptosis induction. Unlike the wild-type strain, the B. pseudomallei rpoS mutant strain failed to induce cytotoxicity in mouse macrophages (RAW264.7). Furthermore, the mutant produced less extensive mitochondrial membrane potential changes and caspase-3 activation in the macrophages than did the wild-type strain. These data suggest that the RpoS of B. pseudomallei plays an essential role in the regulation of cell death in mouse macrophages.

Mechanisms of adaptation to nitrosative stress in Bacillus subtilis.

Bacteria use a number of mechanisms for coping with the toxic effects exerted by nitric oxide (NO) and its derivatives. Here we show that the flavohemoglobin encoded by the hmp gene has a vital role in an adaptive response to protect the soil bacterium Bacillus subtilis from nitrosative stress. We further show that nitrosative stress induced by the nitrosonium cation donor sodium nitroprusside (SNP) leads to deactivation of the transcriptional repressor NsrR, resulting in derepression of hmp. Nitrosative stress induces the sigma B-controlled general stress regulon. However, a sigB null mutant did not show increased sensitivity to SNP, suggesting that the sigma B-dependent stress proteins are involved in a nonspecific protection against stress whereas the Hmp flavohemoglobin plays a central role in detoxification. Mutations in the yjbIH operon, which encodes a truncated hemoglobin (YjbI) and a predicted 34-kDa cytosolic protein of unknown function (YjbH), rendered B. subtilis hypersensitive to SNP, suggesting roles in nitrosative stress management.

The major cold shock gene, cspA, is involved in the susceptibility of Staphylococcus aureus to an antimicrobial peptide of human cathepsin G.

A Tn551 insertional library of Staphylococcus aureus strain ISP479 was challenged with an antimicrobial peptide (CG 117-136) derived from human neutrophil cathepsin G (CG). After repeated selection and screening of surviving colonies, a mutant was identified that expressed increased resistance to CG 117-136. Southern hybridization analysis revealed that the Tn551 insert in this mutant (SK1) was carried on a 10.6-kb EcoRI chromosomal DNA fragment. Subsequent physical mapping of this Tn551 insert revealed that it was positioned between a putative promoter sequence and the translational start codon of the cspA gene, which encodes a protein (CspA) highly similar to the major cold shock proteins CspA and CspB of Escherichia coli and Bacillus subtilis, respectively. This Tn551 insertion as well as a separate deletion-insertion mutation in cspA decreased the capacity of S. aureus to respond to the stress of cold shock and increased resistance to CG 117-136. The results indicate for the first time that a physiologic link exists between bacterial susceptibility to an antimicrobial peptide and a stress response system.

The mechanism of transcriptional activation by the topologically DNA-linked sliding clamp of bacteriophage T4.

Three viral proteins participate directly in transcription of bacteriophage T4 late genes: the sigma-family protein gp55 provides promoter recognition, gp33 is the co-activator, and gp45 is the activator of transcription; gp33 also represses transcription in the absence of gp45. Transcriptional activation by gp45, the toroidal sliding clamp of the T4 DNA polymerase holoenzyme, requires assembly at primer-template junctions by its clamp loader. The mechanism of transcriptional activation has been analyzed by examining rates of formation of open promoter complexes. The basal gp55-RNA polymerase holoenzyme is only weakly held in its initially formed closed promoter complex, which subsequently opens very slowly. Activation ( approximately 320-fold in this work) increases affinity in the closed complex and accelerates promoter opening. Promoter opening by gp55 is also thermo-irreversible: the T4 late promoter does not open at 0 degrees C, but once opened at 30 degrees C remains open upon shift to the lower temperature. At a hybrid promoter for sigma(70) and gp55-holoenzymes, only gp55 confers thermo-irreversibility of promoter opening. Interaction of gp45 with a C-terminal epitope of gp33 is essential for the co-activator function of gp33.

The effect of alginate on the invasion of cystic fibrosis respiratory epithelial cells by clinical isolates of Pseudomonas aeruginosa.

Chronic infection in the cystic fibrosis (CF) lung is characterized by Pseudomonas aeruginosa strains that overproduce the mucoid exopolysaccharide, alginate. Previous experiments have shown that long-term survival of P. aeruginosa in the CF lung may be facilitated by increased adherence and decreased invasion of respiratory epithelial cells. Therefore, mucoid and nonmucoid clinical isolates of P. aeruginosa were assayed for their ability to associate with and invade the CF respiratory epithelial cell line, CF/T43. Association assays and gentamicin exclusion assays demonstrated that mucoid P. aeruginosa associates with and invades CF/T43 cell monolayers significantly less than nonmucoid P. aeruginosa strains (P = .004, .02). Fluorescence microscopy invasion assays confirmed these results. The differences in association and invasion by the P. aeruginosa strains were not due to differences in lipopolysaccharide phenotype or cytotoxicity for CF/T43 respiratory epithelial cells. Exogenous bacterial alginate had no effect on the invasion of CF respiratory epithelia by a nonmucoid strain. Invasion assays with the wild-type P. aeruginosa strain PAO1 and isogenic algU and mucA mutant strains failed to show differences in invasion (P = .25). We conclude that (i) mucoid P. aeruginosa isolates associate with and invade CF/T43 respiratory epithelial cells with less efficiency than nonmucoid P. aeruginosa, (ii) these differences are not due to variations in lipopolysaccharide phenotype between strains, (iii) neither exogenous nor endogenous alginate affects the ability of P. aeruginosa to invade CF/T43 respiratory epithelial cells, and (iv) invasion of CF/T43 respiratory epithelial cells by a laboratory reference strain of P. aeruginosa does not appear to be regulated by AlgU.

Regulation of the thdF gene, which is involved in thiophene oxidation by Escherichia coli K-12.

The thdF gene of Escherichia coli encodes a 48 kD protein which is involved in the oxidation of derivatives of the sulphur-containing heterocycle thiophene and which appears to be induced during stationary phase. In this work the upstream regulatory region of the thdF gene was isolated by polymerase chain reaction and inserted in front of the lacZ structural gene. Examination of the resulting thdF-lacZ operon fusions showed that expression of the thdF gene increased as E. coli entered the stationary phase. However, the expression of thdF was not dependent on RpoS (KatF), the stationary phase sigma factor. The thdF gene was subject to substantial catabolite repression by glucose and its expression was also greatly decreased in the absence of oxygen. The thdF-lacZ fusions were not significantly affected by elevated temperature or medium of high osmolarity, nor by mutations in thdA, fadR, arcA, arcB, or fnr. Both multicopy, plasmid-borne fusions and single-copy fusions gave similar results in all of the above cases except that the plasmid-borne fusions still showed substantial expression in the absence of oxygen. The heterocyclic compounds thiophene carboxylic acid, furan carboxylic acid and proline increased expression of the thdF gene by 2- to 3-fold, but only during the stationary phase. Tryptophan, indole, and several indole derivatives had no effect.

In vitro transcription system using reconstituted RNA polymerase (Esigma(70), Esigma(H), Esigma(E) and Esigma(S)) of Pseudomonas aeruginosa.

We have developed an in vitro transcription system for Pseudomonas aeruginosa genes, using RNA polymerase (RNAP) holoenzyme reconstituted with purified sigma protein and RNAP core enzyme. The RNAP core enzyme was directly purified from P. aeruginosa PAO1 cells. The sigma factors of P. aeruginosa (sigma(70), sigma(H), sigma(E) and sigma(S)) were prepared in a hexa-histidine tagged form, which were expressed in Escherichia coli and purified using a HisTrap Chelating column. The RNAP holoenzyme reconstituted from core enzyme with each sigma factor recognized correctly each of the cognate promoters. This system will be useful for the promoter analysis of many genes in P. aeruginosa.

The anti-sigma factors.

A mechanism for regulating gene expression at the level of transcription utilizes an antagonist of the sigma transcription factor known as the anti-sigma (anti-sigma) factor. The cytoplasmic class of anti-sigma factors has been well characterized. The class includes AsiA form bacteriophage T4, which inhibits Escherichia coli sigma 70; FlgM, present in both gram-positive and gram-negative bacteria, which inhibits the flagella sigma factor sigma 28; SpoIIAB, which inhibits the sporulation-specific sigma factor, sigma F and sigma G, of Bacillus subtilis; RbsW of B. subtilis, which inhibits stress response sigma factor sigma B; and DnaK, a general regulator of the heat shock response, which in bacteria inhibits the heat shock sigma factor sigma 32. In addition to this class of well-characterized cytoplasmic anti-sigma factors, a new class of homologous, inner-membrane-bound anti-sigma factors has recently been discovered in a variety of eubacteria. This new class of anti-sigma factors regulates the expression of so-called extracytoplasmic functions, and hence is known as the ECF subfamily of anti-sigma factors. The range of cell processes regulated by anti-sigma factors is highly varied and includes bacteriophage phage growth, sporulation, stress response, flagellar biosynthesis, pigment production, ion transport, and virulence.

Characterization of csgA, a new member of the forespore-expressed sigmaG-regulon from Bacillus subtilis.

A new locus, csgA, has been identified in a search for developmental genes transcribed by E sigmaG in Bacillus subtilis. csgA has the potential to encode three small proteins, CsgAA, CsgAB and CsgAC. The latter two would be encoded by overlapping ORFs. csgA is expressed in the spore chamber of the differentiating cell and is under the control of sigmaG and the transcriptional regulatory protein SpoVT. Mutation of csgA did not affect spore formation but produced a subtle defect in the ability of the germinating spore to resume vegetative growth.

Heat-shock and general stress response in Bacillus subtilis.

The induction of stress proteins is an important component of the adaptional network of a non-growing cell of Bacillus subtilis. A diverse range of stresses such as heat shock, salt stress, ethanol, starvation for oxygen or nutrients etc. induce the same set of proteins, called general stress proteins. Although the adaptive functions of these proteins are largely unknown, they are proposed to provide general and rather non-specific protection of the cell under these adverse conditions. In addition to these non-specific general stress proteins, all extracellular signals induce a set of specific stress proteins that may confer specific protection against a particular stress factor. In B. subtilis at least three different classes of heat-inducible genes can be defined by their common regulatory characteristics: Class I genes, as exemplified by the dnaK and groE operons, are most efficiently induced by heat stress. Their expression involves a sigma A-dependent promoter, an inverted repeat (called the CIRCE element) highly conserved among eubacteria, and probably a repressor interacting with the CIRCE element. The majority of general stress genes (class II, more than 40) are induced at sigma B-dependent promoters by different growth-inhibiting conditions. The activation of sigma B by stress or starvation is the crucial event in the induction of this large stress regulon. Only a few genes, including Ion, clpC, clpP, and ftsH, can respond to different stress factors independently of sigma B or CIRCE (class III). Stress induction of these genes occurs at promoters presumably recognized by sigma A and probably involves additional regulatory elements which remain to be defined.

Identification of a conditionally essential heat shock protein in Escherichia coli.

Protein D48.5 was recognized as a heat-inducible protein of Escherichia coli during the screening of a group of random, temperature-inducible Mud-Lac fusion mutants. Physiological and genetic analysis demonstrated that (i) the structural gene for this protein, designated htpI, is a member of the sigma 32-dependent heat shock regulon, (ii) at 37 degrees C the synthesis of protein D48.5 is nearly constitutive, increasing slightly with growth rate in media of different composition, and (iii) this protein is essential for growth at high temperature.

Enhancement of in vitro transcription by addition of cloned, overexpressed major sigma factor of Chlamydia psittaci 6BC.

Obligate parasitic bacteria of the genus Chlamydia possess a developmental cycle that takes place entirely within eucaryotic host cells. Because standard methods of genetic analysis are not available for chlamydiae, an in vitro transcription system has been developed to elucidate the mechanisms by which chlamydiae regulate gene expression. The in vitro system is specific for chlamydial promoters but is inefficient, presumably because the RNA polymerase is not saturated with sigma factor. Therefore, we prepared recombinant Chlamydia psittaci 6BC major sigma factor to enhance transcription in the in vitro system. The gene encoding the major sigma factor (sigA) was identified by using an rpoD box oligonucleotide and was subsequently cloned and sequenced. It was found to encode a potential 571-amino-acid protein (sigma 66) that is greater than 90% identical to the previously identified major sigma factors from the L2 and MoPn strains of Chlamydia trachomatis. sigA was recloned into a T7 RNA polymerase expression system to produce large quantities of sigma 66 in Escherichia coli. Overexpressed sigma 66 was identified by immunoblot by using monoclonal antibodies 2G10 (reactive) and 2F8 (nonreactive) generated against E. coli sigma 70. After purification by polyacrylamide gel electrophoresis, the recombinant protein was found to stimulate, by 10-fold or more, promoter-specific in vitro transcription by C. psittaci 6BC and C. trachomatis L2 RNA polymerases. Transcription was dependent on added chlamydial sigma 66, rather than on potentially contaminating E. coli sigma 70 or other fortuitous activators, since the monoclonal antibody 2G10, and not 2F8, inhibited transcription initiation. Recombinant omega(66) had no effect on transcription by E. coli core polymerase. The addition of recombinant omega(66) to the in vitro system should be useful for distinguishing omega(66)-dependent transcription of developmentally regulated chlamydial genes from omega(66)-independent transcription.

Effect of precisely identified mutations in the spoIIAC gene of Bacillus subtilis on the toxicity of the sigma-like gene product to Escherichia coli.

Yudkin (1986) has shown that the spoIIAC gene of Bacillus subtilis cannot be cloned in Escherichia coli in such an orientation that it is expressed. This toxicity of the gene product has been attributed to its close homology with the sigma subunit of the E. coli RNA polymerase. The effect of six individual mutations in spoIIAC has now been studied. All six mutant genes could be cloned in E. coli in an orientation that does not allow expression. When in the orientation that permits expression, one mutant gene could not be cloned, and a second substantially hampered growth; both mutations lie in the region that is believed to encode the DNA-binding domain of the protein. By contrast, two missense mutations in the region of the gene thought to encode the domain that binds to the core RNA polymerase rendered the protein harmless in E. coli, as did two nonsense mutations.

Transcription termination factor rho mediates simultaneous release of RNA transcripts and DNA template from complexes with Escherichia coli RNA polymerase.

Dissociation of RNA and DNA from Escherichia coli RNA polymerase in transcription complexes prepared with enzyme molecules located within and near a rho-dependent transcription termination region on bacteriophage T7 D111 DNA has been studied using a membrane filter-binding assay. Rho protein with ATP present mediated rapid (half-time approximately 27 s) simultaneous dissociation of about 50% of both RNA and DNA. RNA molecules were preferentially released from enzyme molecules located within the termination region. Rapid release of RNA and DNA depended on a nucleoside triphosphate but did not depend on sigma factor. Pretreatment of complexes with ribonuclease prevented dissociation of DNA. Nearly simultaneous dissociation of both RNA and DNA was also detected after a lag of 3 min when the isolated transcription complexes were incubated with all four ribonucleoside triphosphates in the absence of rho factor. In this case, release presumably occurred at the rho-independent termination site that is 5990 nucleotides downstream from the A1 promoter. Thus, the dissociation of DNA from RNA polymerase at rho-dependent and rho-independent transcription termination sites is coupled with or occurs spontaneously soon after the release of transcripts at both sites.