Genetic studies of a secondary RNA polymerase sigma factor in Bacillus subtilis

Abstract

crB ((r37) is a secondary species of RNA polymerase sigma factor found in the gram-positive bacterium Bacillus subtilis. To study the function of (rB genetically, we sought mutations that block the expression of a gene (ctc) known to be transcribed by B-containing RNA polymerase in vitro. One such mutation, called crl, was found to map in or near the structural gene (sigB) for (yB. To determine directly whether mutations in sigB would prevent transcription of ctc, we replaced sigB in the B. subtilis chromosome with insertion and deletion mutations that disrupted the cJB coding sequence. Like crl, these in vitro-constructed mutations blocked expression of ctc, but had little or no effect on viability, sporulation, expression of the sporulation gene spoVG, or production of sporulation-associated alkaline protease. Using fusions of ctc to the reporter genes xylE and lacZ, we also identified mutations that enhanced ctc expression. One such mutation, called socB, was found to be located in an open reading frame immediately downstream of sigB.The DNA-dependent RNA polymerase of the grampositive bacterium Bacillus subtilis is a highly heterogenous enzyme that exists in several different holoenzyme forms (4, 13). Each form of holoenzyme consists of core RNA polymerase and one of several different species of sigma factor that confers on the core enzyme the capacity to recognize and initiate transcription from a particular class of promoters. We have been interested in a secondary species of sigma factor called aB (formerly J7; 9, 10). This 29.9-kilodalton polypeptide (1, 7), which is found in vegetative and earlystationary-phase cells, directs the transcription in vitro of certain genes that are activated at the end of the exponential phase of growth (9-12). However, little is known about the contribution of arB to the expression of these genes in vivo or its role in B. subtilis gene expression. The recent identification of the structural gene for crJB (sigB; 1, 7) and the availability of plasmid and bacteriophage vectors bearing fusions of genes transcribed in vitro by cB_containing RNA polymerase to the reporter genes cat, xylE, and lacZ (11,17,21) have made it possible to study the function of this minor sigma factor genetically. In this paper we describe the identification and characterization of mutations in and near sigB that enhance or inhibit the expression of a B. subtilis gene, ctc, which is known to be actively transcribed by aB-containing RNA polymerase in vitro. One such mutation that significantly stimulates ctc transcription was found to be located in an open reading frame immediately adjacent to and downstream from sigB. We also describe the construction of insertion and deletion mutations in sigB and report, in confirmation and extension of previous work (1, 7), that they have little or no effect on cell viability, sporulation, or sporulation-associated gene expression.

Four additional genes in the sigB operon of Bacillus subtilis that control activity of the general stress factor sigma B in response to environmental signals

“…That is, loss of rsbU function decreases ctc expression under inducing conditions and increases expression under noninducing conditions. It was originally proposed that the addition of glucose and glutamine provides a nutritional signal that increases Bdependent gene expression as cells enter the stationary growth phase (24,25). However, our results for salt-stressed cells raise the possibility that high levels of glucose and glutamine may provide an osmotic rather than a nutritional signal (30) and that RsbU is involved in the transmission of general stress signals to B .…”

“…The transcription factor B of B. subtilis controls a set of genes that are induced both upon entry into stationary phase and by a variety of physiological stresses encountered during logarithmic growth (4,6,7,9,25,29,51). Previous genetic analysis indicated that the products of the rsbV, rsbW, and rsbX genes in the sigB operon form a hierarchical regulatory pathway that controls B activity in response to stationary-phase signals as well as to signals of salt and ethanol stress (2,6,8,24,29).…”

“…We indirectly measured B activity at the well-characterized B -dependent ctc promoter (24,38), using either a translational fusion to a lacZ reporter gene carried in single copy by the SP␤ prophage of strain PB197 (constructed by Igo and Losick [25]) or a transcriptional fusion carried in single copy by the pDH32 vector integrated at the amyE locus of strain PB198 (constructed by Boylan et al [8]). We also used the pDH32 vector (41) to construct a new transcriptional fusion to the B -dependent P B promoter of the sigB operon (29).…”

“…The RsbV protein counters the effect of RsbW and thus acts as a positive regulator that is normally required for B activity to appear in stationary phase, or during logarithmic growth in response to salt or ethanol stress (2,6,8,13). The role of the RsbX negative regulator is less well understood, but genetic analysis indicates that it acts before the RsbV-RsbW pair in a regulatory hierarchy (2,8,24,29).…”

“…The RsbV protein counters the effect of RsbW and thus acts as a positive regulator that is normally required for B activity to appear in stationary phase, or during logarithmic growth in response to salt or ethanol stress (2,6,8,13). The role of the RsbX negative regulator is less well understood, but genetic analysis indicates that it acts before the RsbV-RsbW pair in a regulatory hierarchy (2,8,24,29).During characterization of the RsbV-RsbW-RsbX hierarchy, it became evident that none of the known regulators of B activity was sufficient to fully account for the enhanced stationary-phase activation of B in response to glucose, suggesting that additional regulatory elements remained to be discovered (8). We earlier presented evidence that a second, B -independent promoter must exist for the sigB operon and that this promoter must lie more than 1,100 bp upstream from the B -dependent promoter immediately preceding rsbV (29).…”