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Sequence analysis of 236 promoters recognized by the Bacillus subtilis sigma A-RNA polymerase reveals an extended promoter structure. The most highly conserved bases include the -35 and -10 hexanucleotide core elements and a TG dinucleotide at position -15, -14. In addition, several weakly conserved A and T residues are present upstream of the -35 region. Analysis of dinucleotide composition reveals A2- and T2-rich sequences in the upstream promoter region (-36 to -70) which are phased with the DNA helix: An tracts are common near -43, -54 and -65; Tn tracts predominate at the intervening positions. When compared with larger regions of the genome, upstream promoter regions have an excess of An and Tn sequences for n > 4. These data indicate that an RNA polymerase binding site affects DNA sequence as far upstream as -70. This sequence conservation is discussed in light of recent evidence that the alpha subunits of the polymerase core bind DNA and that the promoter may wrap around RNA polymerase.

Summary Gene regulation in cis by riboswitches is prevalent in bacteria. The yybP-ykoY riboswitch family is quite widespread, yet its ligand and function remained unknown. Here we characterize the Lactococcus lactis yybP-ykoY riboswitch as a Mn2+-dependent transcription-ON riboswitch, with a ~30–40 μM affinity for Mn2+. We further determined its crystal structure at 2.7 Å to elucidate the metal sensing mechanism. The riboswitch resembles a hairpin, with two coaxially stacked helices tethered by a four-way junction and a tertiary docking interface. The Mn2+-sensing region, strategically located at the highly conserved docking interface, has two metal binding sites. Whereas the one site tolerates binding of both Mg2+ and Mn2+, the other site strongly prefers Mn2+ due to a direct contact from the N7 of an invariable adenosine. Mutagenesis and a Mn2+-free E. coli yybP-ykoY structure further reveal that Mn2+ binding is coupled with stabilization of the Mn2+-sensing region and the aptamer domain.

We have cloned two metal-regulated genes (mrgA and mrgC) from BaciUus subtilis by using transposon Tn917-lacZ. Both were isolated as iron-repressible gene fusions, but the metal specificity and sensitivity of gene repression are distinct. Transcription of mrgA-lacZ is induced at the end of logarithmic-phase growth in minimal medium, and this induction is prevented by excess manganese, iron, cobalt, or copper. Limitation for metal ions is sufficient for mrgA-lacZ induction, since resuspension in medium lacking both manganese and iron rapidly induces transcription. Transcription of mrgC-lacZ is also induced by iron deprivation but is not repressed by added manganese or other metal ions. Expression of mrgC-lacZ and a 2,3-dihydroxybenzoic acid-based siderophore is repressed in parallel by iron, and in both cases, only iron effects repression. We have cloned and sequenced the promoter and regulatory regions of both mrgA and mrgC. Both genes are preceded by a predicted crA-dependent promoter element with overlapping sequences similar to the iron box consensus element for recognition by the Escherichia coli ferric uptake regulator protein (Fur). Mutation of the putative iron box for gene mrgC leads to partial derepression in iron-replete medium.The regulation of gene expression in response to changing levels of metal ions requires a sensor function, to monitor metal ion levels, and an effector function, to mediate a transcriptional or translational response (12). Two of the best-studied metalloregulatory proteins, the mercury-sensing MerR protein and the iron-sensing Fur protein, serve both as direct metal ion sensors and as transcription factors (2,33). The importance of iron as an essential nutrient and enzyme cofactor has led to sophisticated regulatory mechanisms in both bacteria and eukaryotes to control intracellular iron levels. We are interested in the mechanisms by which iron regulates gene expression in the gram-positive organism Bacillus subtilis.Iron is an important nutrient for virtually all microorganisms, yet it is largely insoluble at neutral pH under aerobic conditions. Like many other organisms, B. subtilis synthesizes and excretes a Fe(III)-specific chelator (siderophore) under conditions of iron starvation. Early studies identified 2,3-dihydroxybenzoic acid (DHBA) and 2,3-dihydroxybenzoylglycine in cell culture supernatants of B. subtilis grown under iron limitation (14). Recent studies indicate that some B. subtilis strains have inducible uptake systems for hydroxamate-type siderophores as well (30). The synthesis of siderophore and subsequent uptake of ferri-siderophore complexes are inducible processes, suggesting that B. subtilis has one or more iron-sensing metalloregulatory proteins (24,25,30,39). We report the cloning and characterization of two metalloregulated promoter regions from B. subtilis that are affected by iron availability.We have determined the growth phase and metal ion dependence of gene expression for two strains containing transcriptional fusions to lacZ. Expression of mrgC-la...

In Bacillus subtilis, hydrogen peroxide induces the synthesis of catalase (KatA), alkyl hydroperoxide reductase (AhpCF), and a DNA-binding protein of the Dps family (MrgA). KatA, AhpCF, heme biosynthesis enzymes, and MrgA are also induced upon entry into stationary phase under conditions of iron and manganese limitation. In an effort to define the peroxide regulon repressor, PerR, we used mini-Tn10 mutagenesis to identify loci affecting the regulation of mrgA. From this screen, we isolated two mini-Tn10 insertions in ahpC, the gene encoding the small subunit of AhpCF, that increase the transcription of mrgA-lacZ even in ironsupplemented minimal medium. Indeed, these ahpC::Tn10 insertions lead to elevated expression from all peroxide regulon promoters, including those for mrgA, katA, hemAXCDBL, and ahpCF. As a result, the ahpC::Tn10 mutants display an increased resistance to H 2 O 2 . The ahpCF promoter region contains three sequences similar to the peroxide regulon consensus operator (per box). We demonstrate that the ability of ahpC::Tn10 mutations to derepress mrgA requires aerobic growth. In contrast, a second distinct trans-acting regulatory mutation bypasses this requirement for aerobic growth. Since the peroxide regulon is activated in the absence of AhpCF, which degrades alkyl hydroperoxides, we propose that organic hydroperoxides may be physiologically relevant inducers in vivo.

Bacillus subtilis contains multiple forms of RNA pol,ymerase holoenzyme, distinguished by the presence of different specificiy determinants known as (r factors.. The au factor was initially purified as a unique transcriptional activity in vegetatively growing B. subtilis cells. Purification of the a8 protein has allowed tryptic peptides to be prepared and sequenced. The sequence of one tryptic peptide fragment was used to prepare an oligonucleotide probe specific for the a8 -structural gene, and the gene was isolated from a B. subtilis subgenomic library. The complete nucleotide sequence of the a8 gene was determined, and the cloned 28 (F gene was used to construct a mutant strain which does not express the ('8 protein. This strain also failed to synthesize flageilin protein and grew as long filaments. The predicted (28 gene product is a 254-amino-acid polypeptide with a calculated molecular weight of 29,500. The d8 protein sequence was similar to that of other sequenced 'r factors and to theflbB gene product of Escherichia coli. Since theflbB gene product is a positive regulator of flagellar synthesis in E. coli, it is likely that cr8 functions to regulate flagellar synthesis in B. subtilis.Bacterial RNA polymerase is a multisubunit enzyme central to the process of gene expression. Although the catalytic activity resides in the core subunits of the enzyme, the promoter specificity of a particular holoenzyme is determined by the nature of the associated a factor (31). The majority of cellular transcription is dependent on the primary r factor, which exhibits a conserved promoter recognition specificity throughout the eubacteria (43). Many bacterial species also contain alternative a factors that are specific for transcription of distinct regulons of coordinately regulated genes. These alternative a factors normally recognize promoter sequences that are differett from those recognized by the primary cr factor. Examhples of alternative of factors in the enteric bacteria include a32 (15), specific for the transcription of heat shock genes, and (94 (19, 20), specific for transcription of fnitrogen-regulated genes. For Bacillus subtilis, at least six alternative factors have been described (26,31).In B. subtilis, alterations in cellular transcription, mediated at least in part by alternative cr factors, effect the precise temporal changes in gene expression necessary for endospore formation (26). The products of the spoOH (U30) and spoIIGB (r29) genes are sporulation-specific cr factors that have been characterized both genetically and biochemically (6,22,24,26,40,41). In addition, the spoIIAC gene product is homologous to other sequenced a factors (9) and may also function as a cf factor. The cr3 and a98 factors are found in vegetatively growing cells and are dispensable for sporulation, since disruption of these genes does not impair sporulation (3, 8; see below). To define the biological function of the B. subtilis ur28 factor, we have begun a genetic and structural analysis of the Cr28 structural gene (sigD) bas...

Gene expression in Bacillus subtilis can be controlled by alternative forms of RNA polymerase programmed by distinct a factors. One such factor, SD ((r28), is expressed during vegetative growth and has been implicated in the transcription of a regulon of genes expressed during exponential growth and the early stationary phase. We have studied several functions related to flagellar synthesis and chemotaxis in B. subtilis strains in which ufD is missing or is present at reduced levels. Previous studies showed that a null mutant, which contains a disrupted copy of the JD structural gene (sigD), fails to synthesize flagellin and grows as long filaments. We now show that these defects are accompanied by the lack of synthesis of the methyl-accepting chemotaxis proteins and a substantial decrease in two autolysin activities implicated in cell separation. A strain containing an insertion upstream of the sigD gene that reduces the level of e protein grew as short chains and was flagellated but was impaired in chemotaxis and/or motility. This reduced level of cr3 expression suggests that the sigD gene may be part of an operon. A strain containing an insertion downstream of the sigD gene expressed nearly wild-type levels of crD protein but was also impaired in chemotaxis and/or motility, suggesting that genes