Lon protease influences antibiotic production and UV tolerance of Pseudomonas fluorescens Pf-5

Pseudomonas fluorescens Pf-5 is a soil bacterium that suppresses plant pathogens due in part to its production of the antibiotic pyoluteorin. Previous characterization of Pf-5 revealed three global regulators, including the stationary-phase sigma factor sigma(S) and the two-component regulators GacA and GacS, that influence both antibiotic production and stress response. In this report, we describe the serine protease Lon as a fourth global regulator influencing these phenotypes in Pf-5. lon mutants overproduced pyoluteorin, transcribed pyoluteorin biosynthesis genes at enhanced levels, and were more sensitive to UV exposure than Pf-5. The lon gene was preceded by sequences that resembled promoters recognized by the heat shock sigma factor sigma(32) (sigma(H)) of Escherichia coli, and Lon accumulation by Pf-5 increased after heat shock. Therefore, sigma(H) represents the third sigma factor (with sigma(S) and sigma(70)) implicated in the regulation of antibiotic production by P. fluorescens. Lon protein levels were similar in stationary-phase and exponentially growing cultures of Pf-5 and were not positively affected by the global regulator sigma(S) or GacS. The association of antibiotic production and stress response has practical implications for the success of disease suppression in the soil environment, where biological control organisms such as Pf-5 are likely to encounter environmental stresses.     

Trehalose Is Not Relevant for In Vivo Activity of ςS-Containing RNA Polymerase in Escherichia coli

The ς^S- and ς⁷⁰-associated forms of RNA polymerase core enzyme (E) of Escherichia coli have very similar promoter recognition specificities in vitro. Nevertheless, the in vivo expression of many stress response genes is strongly dependent on ς^S. Based on in vitro assays, it has recently been proposed that the disaccharide trehalose specifically stimulates the formation and activity of Eς^S and thereby contributes to promoter selectivity (S. Kusano and A. Ishihama, J. Bacteriol. 179:3649–3654, 1997). However, we demonstrate here that a trehalose-free otsA mutant exhibits growth phase-related and osmotic induction of various ς^S-dependent genes which is indistinguishable from that of an otherwise isogenic wild-type strain and that stationary-phase cells do not accumulate trehalose (even though the trehalose-synthesizing enzymes are induced). We conclude that in vivo trehalose does not play a role in the expression of ς^S-dependent genes and therefore also not in sigma factor selectivity at the promoters of these genes.     

uspB, a New ςS-Regulated Gene in Escherichia coli Which Is Required for Stationary-Phase Resistance to Ethanol

The open reading frame immediately upstream of uspA is demonstrated to encode a 14-kDa protein which we named UspB (universal stress protein B) because of its general responsiveness to different starvation and stress conditions. UspB is predicted to be an integral membrane protein with at least one and perhaps two membrane-spanning domains. Overexpression of UspB causes cell death in stationary phase, whereas mutants of uspB are sensitive to exposure to ethanol but not heat in stationary phase. In contrast to uspA, stationary-phase induction of uspB requires the sigma factor ς^S. The expression of uspB is modulated by H-NS, consistent with the role of H-NS in altering ς^S levels. Our results demonstrate that a gene of the RpoS regulon is involved in the development of stationary-phase resistance to ethanol, in addition to the regulon’s previously known role in thermotolerance, osmotolerance, and oxidative stress resistance.     

Bacterial Cell Division Regulation: Lysogenization of Conditional Cell Division lon− Mutants of Escherichia coli by Bacteriophage Lambda

The lon⁻ mutants of Escherichia coli grow apparently normally except that, after temporary periods of inhibition of deoxyribonucleic acid synthesis, septum formation is specifically inhibited. Under these conditions, long, multinucleate, nonseptate filaments result. The lon⁻ mutation also creates a defect such that wild-type bacteriophage λ fails to lysogenize lon⁻ mutants efficiently and consequently forms clear plaques on a lon⁻ host. Two lines of evidence suggest that this failure probably results from interference with expression of the λcI gene, which codes for repressor, or with repressor action:-(i) when a lon⁻ mutant was infected with a λcII, cIII, or c Y mutant, there was an additive effect between the lon⁻ mutation and the λc mutations upon reduction of lysogenization frequency; and (ii) lon⁻ mutants permitted the growth of the λcro⁻ mutant under conditions in which the repressor was active. The isolation of λ mutants (λtp) which gained the ability to form turbid plaques on lon⁻ cells is also reported.     

Inactivation of the Regulatory Gene algU or gacA Can Affect the Ability of Biocontrol Pseudomonas fluorescens CHA0 To Persist as Culturable Cells in Nonsterile Soil

Rifampin-resistant Pseudomonas fluorescens CHA0-Rif and mutants in which the regulatory gene algU (encoding sigma factor σ^E) or gacA (encoding a global regulator of secondary metabolism) was inactivated were compared for persistence in three nonsterile soils. Functional algU and (particularly) gacA were needed for CHA0-Rif to maintain cell culturability in soil.     

The yhhP Gene Encoding a Small Ubiquitous Protein Is Fundamental for Normal Cell Growth of Escherichia coli

H-NS is a major constituent of the Escherichia coli nucleoid, whereas ς^S is a stress-induced sigma factor. An hns null mutation affects the cellular content of ς^S in such a way that a remarkable accumulation of ς^S is observed in the logarithmic growth phase, which results in enhanced expression of a number of ς^S-dependent genes, including the katE gene. We isolated an extragenic mutation that affects the expression of the katE-lacZ fusion gene in the Δhns background. The relevant gene was identified as yhhP, which encodes a small polypeptide of 81 amino acids. Lesion of this gene seemed to affect the stability of ς^S. A deletion analysis of yhhP revealed that this small protein plays a fundamental role in the general physiology of E. coli. The yhhP-deficient cell is not capable of growing in standard laboratory rich medium (i.e., Luria broth), resulting in the formation of filamentous cells. Homologs of this intriguing protein occur in a wide variety of bacterial species, including archaeal species.     

The Staphylococcus aureus Alternative Sigma Factor ςB Controls the Environmental Stress Response but Not Starvation Survival or Pathogenicity in a Mouse Abscess Model

The role of ς^B, an alternative sigma factor of Staphylococcus aureus, has been characterized in response to environmental stress, starvation-survival and recovery, and pathogenicity. ς^B was mainly expressed during the stationary phase of growth and was repressed by 1 M sodium chloride. A sigB insertionally inactivated mutant was created. In stress resistance studies, ς^B was shown to be involved in recovery from heat shock at 54°C and in acid and hydrogen peroxide resistance but not in resistance to ethanol or osmotic shock. Interestingly, S. aureus acquired increased acid resistance when preincubated at a sublethal pH 4 prior to exposure to a lethal pH 2. This acid-adaptive response resulting in tolerance was mediated via sigB. However, ς^B was not vital for the starvation-survival or recovery mechanisms. ς^B does not have a major role in the expression of the global regulator of virulence determinant biosynthesis, staphylococcal accessory regulator (sarA), the production of a number of representative virulence factors, and pathogenicity in a mouse subcutaneous abscess model. However, SarA upregulates sigB expression in a growth-phase-dependent manner. Thus, ς^B expression is linked to the processes controlling virulence determinant production. The role of ς^B as a major regulator of the stress response, but not of starvation-survival, is discussed.     

Regulation of expression of the fibronectin-binding protein BBK32 in Borrelia burgdorferi

The BBK32 protein binds to host extracellular ligand fibronectin and contributes to the pathogenesis of Borrelia burgdorferi. Here we showed that expression of the BBK32 gene is influenced by multiple environmental factors and that its regulation is governed by the response regulator Rrp2 and RpoN-RpoS (σ⁵⁴-σ^S) sigma cascade in B. burgdorferi.     

A possible extended family of regulators of sigma factor activity in Streptomyces coelicolor

SCO4677 is one of a large number of similar genes in Streptomyces coelicolor that encode proteins with an HATPase_c domain resembling that of anti-sigma factors such as SpoIIAB of Bacillus subtilis. However, SCO4677 is not located close to genes likely to encode a cognate sigma or anti-anti-sigma factor. SCO4677 was found to regulate antibiotic production and morphological differentiation, both of which were significantly enhanced by the deletion of SCO4677. Through protein-protein interaction screening of candidate sigma factor partners using the yeast two-hybrid system, SCO4677 protein was found to interact with the developmentally specific σ^F, suggesting that it is an antagonistic regulator of σ^F. Two other proteins, encoded by SCO0781 and SCO0869, were found to interact with the SCO4677 anti-σ^F during a subsequent global yeast two-hybrid screen, and the SCO0869-SCO4677 protein-protein interaction was confirmed by coimmunoprecipitation. The SCO0781 and SCO0869 proteins resemble well-known anti-anti-sigma factors such as SpoIIAA of B. subtilis. It appears that streptomycetes may possess an extraordinary abundance of anti-sigma factors, some of which may influence diverse processes through interactions with multiple partners: a novel feature for such regulatory proteins.