The extracytoplasmic function sigma factor SigY is important for efficient maintenance of the Spβ prophage that encodes sublancin in Bacillus subtilis

Many strains of the soil bacterium Bacillus subtilis are capable of producing and being resistant to the antibiotic sublancin because they harbor the Spβ prophage. This 135?kb viral genome is integrated into the circular DNA chromosome of B. subtilis, and contains genes for the production of and resistance to sublancin. We investigated the role of SigY in sublancin production and resistance, finding that it is important for efficient maintenance of the Spβ prophage. We were unable to detect the prophage in mutants lacking SigY. Additionally, these mutants were no longer able to produce sublancin, were sensitive to killing by this factor, and displayed a delay in sporulation. Wild-type cells with normal SigY activity were found to partially lose the Spβ prophage during growth and early sporulation, suggesting a mechanism for the bistable outcome of sibling cells capable of killing and of being killed. The appropriate regulation of SigY appears to be essential for growth as evidenced by the inability to disrupt the gene for its putative antisigma. Our results confirm a role for SigY in antibiotic production and resistance, as has been found for other members of the extracytoplasmic function sigma factor family in B. subtilis, and shows that this role is achieved by affecting maintenance of the Spβ prophage.     

Contributions of individual σB-dependent general stress genes to oxidative stress resistance of Bacillus subtilis

The general stress regulon of Bacillus subtilis comprises approximately 200 genes and is under the control of the alternative sigma factor σ(B). The activation of σ(B) occurs in response to multiple physical stress stimuli as well as energy starvation conditions. The expression of the general stress proteins provides growing and stationary nonsporulating vegetative cells with nonspecific and broad stress resistance. A previous comprehensive phenotype screening analysis of 94 general stress gene mutants in response to severe growth-inhibiting stress stimuli, including ethanol, NaCl, heat, and cold, indicated that secondary oxidative stress may be a common component of severe physical stress. Here we tested the individual contributions of the same set of 94 mutants to the development of resistance against exposure to the superoxide-generating agent paraquat and hydrogen peroxide (H(2)O(2)). In fact, 62 mutants displayed significantly decreased survival rates in response to paraquat and/or H(2)O(2) stress compared to the wild type at a confidence level of an α value of ≤ 0.01. Thus, we were able to assign 47 general stress genes to survival against superoxide, 6 genes to protection from H(2)O(2) stress, and 9 genes to the survival against both. Furthermore, we show that a considerable overlap exists between the phenotype clusters previously assumed to be involved in oxidative stress management and the actual group of oxidative-stress-sensitive mutants. Our data provide information that many general stress proteins with still unknown functions are implicated in oxidative stress resistance and further support the notion that different severe physical stress stimuli elicit a common secondary oxidative stress.     

The Activity of σV,an Extracytoplasmic Function σ Factor of Bacillus subtilis,Is Controlled by Regulated Proteolysis of the Anti-σ Factor RsiV

During growth in the environment, bacteria encounter stresses which can delay or inhibit their growth. To defend against these stresses, bacteria induce both resistance and repair mechanisms. Many bacteria regulate these resistance mechanisms using a group of alternative σ factors called extracytoplasmic function (ECF) σ factors. ECF σ factors represent the largest and most diverse family of σ factors. Here, we demonstrate that the activation of a member of the ECF30 subfamily of ECF σ factors, σ^V in Bacillus subtilis, is controlled by the proteolytic destruction of the anti-σ factor RsiV. We will demonstrate that the degradation of RsiV and, thus, the activation of σ^V requires multiple proteolytic steps. Upon exposure to the inducer lysozyme, the extracellular domain of RsiV is removed by an unknown protease, which cleaves at site 1. This cleavage is independent of PrsW, the B. subtilis site 1 protease, which cleaves the anti-σ factor RsiW. Following cleavage by the unknown protease, the N-terminal portion of RsiV requires further processing, which requires the site 2 intramembrane protease RasP. Our data indicate that the N-terminal portion of RsiV from amino acid 1 to 60, which lacks the extracellular domain, is constitutively degraded unless RasP is absent, indicating that RasP cleavage is constitutive. This suggests that the regulatory step in RsiV degradation and, thus, σ^V activation are controlled at the level of the site 1 cleavage. Finally, we provide evidence that increased resistance to lysozyme decreases σ^V activation. Collectively, these data provide evidence that the mechanism for σ^V activation in B. subtilis is controlled by regulated intramembrane proteolysis (RIP) and requires the site 2 protease RasP.     

DNA-binding properties of the Bacillus subtilis and Aeribacillus pallidus AC6 σ(D) proteins

σ(D) proteins from Aeribacillus pallidus AC6 and Bacillus subtilis bound specifically, albeit weakly, to promoter DNA even in the absence of core RNA polymerase. Binding required a conserved CG motif within the -10 element, and this motif is known to be recognized by σ region 2.4 and critical for promoter activity.     

Analysis of the expression and function of the σB-dependent general stress regulon of Bacillus subtilis during slow growth

Glucose-limited continuous cultures were used to analyze σ^B activity at decreasing growth rates. Expression of the σ^B-dependent genes gsiB and ctc started to increase at a growth rate of 0.2 h^–1, and both genes were induced approximately fivefold at a growth rate of 0.1 h^–1 as compared to expression at the maximal growth rate. However, maximal σ^B activity was only reached when the growth stopped as a result of the exhaustion of the carbon and energy source glucose. During glucose-limited growth, increased expression of the general stress regulon at growth rates below 0.2 h^–1 did not provide wild-type cells with a growth advantage over sigB mutants. Instead, expression of the stress regulon seems to constitute a significant burden during glucose-limited growth, resulting in a selective growth advantage of the sigB mutant as compared to the wild-type at a growth rate of 0.08 h^–1. Received: 7 January 1999 / Accepted: 22 March 1999     

Evidence that Bacillus subtilis Bacteriophage SPO2 is Temperate and Heteroimmune to Bacteriophage φ105

Some characteristics of Bacillus subtilis phage SPO2 which show that it is a temperate phage are presented. Wild-type SPO2 forms turbid plaques, similar to those of other temperate phages. SPO2 lysogenic strains which are resistant to SPO2 can be isolated; these strains remain stable lysogens despite the fact that they can no longer adsorb SPO2. SPO2 lysogenic strains can be grown for many generations in SPO2 antiserum and remain lysogenic. Phage SPO2 plates on phi105 lysogens and phage phi105 plates on SPO2 lysogens; this indicates that SPO2 and phi105 are heteroimmune. Phage phi105 plates on an SPO2-resistant strain; this indicates that SPO2 and phi105 adsorb to different receptor sites on the bacterial surface.     

Conversion of Purine Bases and Their Ribosides to 5′-Inosinic Acid by Bacillus subtilis

Among various nutritional mutants with weak 5′-nucleotidase derived from Bacillus subtilis IAM 1145, the adenine-requiring mutants could convert exogenously added hypo- xanthine, guanine, xanthine and their ribosides to 5′-inosinic acid (IMP) and accumulate it in the medium. Synthesis of IMP from purine derivatives was observed predominantly in an early stage of the cultivation. The conversion was stimulated by Fe²⁺ or Mn²⁺, and markedly depressed by an excess amount of adenine in the production-medium.     

A rubber tree��s durable resistance to Microcyclus ulei is conferred by a qualitative gene and a major quantitative resistance factor

The components of genetic resistance from the Hevea brasiliensis cultivar MDF 180 against South American Leaf Blight (SALB) caused by Microcyclus ulei were investigated by QTL mapping. MDF 180 has already been described as a cultivar with a high level of partial and long-lasting resistance. The resistance of progeny individuals from a cross between a susceptible cultivar and MDF 180 was assessed both under controlled conditions of inoculation by three M. ulei isolates and under natural infection in a field trial. Genetic maps of the two parents of this progeny were mainly established based on microsatellites and AFLP markers. No resistance QTL were found in the susceptible parent. In the resistant parent, we identified a qualitative gene responsible for the resistance against isolates from French Guiana and a major quantitative resistance factor determining the resistance against isolates from the state of Bahia (Brazil). The qualitative resistance gene was denominated M15md and was located in the linkage group g15. Four minor resistance QTLs were also identified, two of which showed an epistatic interaction with M15md. The durability of the resistance of MDF 180 is discussed in light of these data.     

Analysis of the role of Bacillus subtilis σ(M) in β-lactam resistance reveals an essential role for c-di-AMP in peptidoglycan homeostasis

The Bacillus subtilis extracytoplasmic function (ECF) σ factor σ(M) is inducible by, and confers resistance to, several cell envelope-acting antibiotics. Here, we demonstrate that σ(M) is responsible for intrinsic β-lactam resistance, with σ(X) playing a secondary role. Activation of σ(M) upregulates several cell wall biosynthetic enzymes including one, PBP1, shown here to be a target for the beta-lactam cefuroxime. However, σ(M) still plays a major role in cefuroxime resistance even in cells lacking PBP1. To better define the role of σ(M) in β-lactam resistance, we characterized suppressor mutations that restore cefuroxime resistance to a sigM null mutant. The most frequent suppressors inactivated gdpP (yybT) which encodes a cyclic-di-AMP phosphodiesterase (PDE). Intriguingly, σ(M) is a known activator of disA encoding one of three paralogous diadenylate cyclases (DAC). Overproduction of the GdpP PDE greatly sensitized cells to β-lactam antibiotics. Conversely, genetic studies indicate that at least one DAC is required for growth with depletion leading to cell lysis. These findings support a model in which c-di-AMP is an essential signal molecule required for cell wall homeostasis. Other suppressors highlight the roles of ECF σ factors in counteracting the deleterious effects of autolysins and reactive oxygen species in β-lactam-treated cells.     

Coupling of σG Activation to Completion of Engulfment during Sporulation of Bacillus subtilis Survives Large Perturbations to DNA Translocation and Replication

Spore formation in Bacillus subtilis is characterized by activation of RNA polymerase sigma factors, including the late-expressed σ^G. During spore formation an asymmetric division occurs, yielding the smaller prespore and the larger mother cell. At division, only 30% of the chromosome is in the prespore, and the rest is then translocated into the prespore. Following completion of engulfment of the prespore by the mother cell, σ^G is activated in the prespore. Here we tested the link between engulfment and σ^G activation by perturbing DNA translocation and replication, which are completed before engulfment. One approach was to have large DNA insertions in the chromosome; the second was to have an impaired DNA translocase; the third was to use a strain in which the site of termination of chromosome replication was relocated. Insertion of 2.3 Mb of Synechocystis DNA into the B. subtilis genome had the largest effect, delaying engulfment by at least 90 min. Chromosome translocation was also delayed and was completed shortly before the completion of engulfment. Despite the delay, σ^G became active only after the completion of engulfment. All results are consistent with a strong link between completion of engulfment and σ^G activation. They support a link between completion of chromosome translocation and completion of engulfment.     

The mouse alpha-albumin (afamin) promoter is differentially regulated by hepatocyte nuclear factor 1α and hepatocyte nuclear factor 1β

Alpha-albumin (AFM), a member of the albumin gene family that also includes albumin, alpha-fetoprotein, and vitamin D-binding protein, is expressed predominantly in the liver and activated at birth. Here, we identify two hepatocyte nuclear factor 1 (HNF1)-binding sites in the AFM promoter that are highly conserved in different mammals. These two sites bind HNF1α and HNF1β. The distal site (centered at -132, relative to AFM exon 1) is more important than proximal site (centered at -58), based on HNF1 binding and mutational analysis in transfected cells. Our data indicate that HNF1α is a more potent activator of AFM promoter than is HNF1β. However, HNF1β can act in a dominant manner to inhibit HNF1α-dependent transactivation of the AFM promoter when both proteins are expressed together. This suggests that the differential timing with which the albumin family genes are activated in the liver may be influenced by their responsiveness to HNF1α and HNF1β. Our comparison of HNF1-binding sites in the promoters in the albumin family of genes indicates that the primordial albumin-like gene contained two HNF1 sites; one of these sites was lost from the albumin promoter, but both sites still are present in other members of this gene family.