Spore formation inthrough the newly formed spore septum. We propose that translocation of the prespore chromosome occurs by a mechanism that is functionally related to the conjugative transfer of plasmid DNA.

The xylA and xylB genes of Bacillus subtilis BR151 encoding xylose isomerase and xylulokinase, respectively, were disrupted by gene replacement rendering the constructed mutant strain unable to grow on xylose as the sole carbon source. The Bacillus megaterium encoded xyl genes were cloned by complementation of this strain to xylose utilization. The nucleotide sequence of about 4 kbp of the insertion indicates the presence of the xylA and xylB genes on the complementing plasmid. Furthermore, a regulatory gene, xylR, is located upstream of xylA and has opposite polarity to it. The intergenic region between the divergently oriented reading frames of xylR and xylA contains palindromic sequences of 24 bp spaced by five central bp and 29 bp spaced by 11 bp, respectively, and two promoters with opposite orientation as determined by primer extension analysis. They overlap with one nucleotide of their--35 consensus boxes. Transcriptional fusions of lacZ to xylA, xylB and xylR were constructed and revealed that xylA and xylB are repressed in the absence and can be 200-fold induced in the presence of xylose. The increased level of xylAB mRNA in induced and its absence in repressed cells confirms that this regulation occurs on the level of transcription. Deletion of the xylR gene encoding the Xyl repressor results in constitutive expression of xylAB. The transcription of xylR is autoregulated and can be induced 9-fold by xylose. The mechanism of this regulation is not clear. While the apparent xyl operator palindrome is upstream of the xylR promoter, the potential recognition of another palindrome downstream of this promoter by Xyl repressor is discussed.

The Bacillus subtilis xyl operon encoding enzymes for xylose utilization is repressed in the absence of xylose and in the presence of glucose. Transcriptional fusions of spoVG-lacZ to this operon show regulation of beta-galactosidase expression by glucose, indicating that glucose repression operates at the level of transcription. A similar result is obtained when glucose is replaced by glycerol, thus defining a general catabolite repression mechanism. A deletion of xylR, which encodes the xylose-sensitive repressor of the operon, does not affect glucose repression. The cis element mediating glucose repression was identified by Bal31 deletion analysis. It is confined to a 34 bp segment located at position +125 downstream of the xyl promoter in the coding sequence for xylose isomerase. Cloning of this segment in the opposite orientation leads to reduced catabolite repression. The homology of this element to various proposed consensus sequences for catabolite repression in B. subtilis is discussed.

The genes coding for methyl coenzyme M reductase were cloned from a genomic library ofMethanobacterium thermoautotrophicum Marburg into Escherichia coli by using plasmid expression vectors. When introduced into E. coli, the reductase genes were expressed, yielding polypeptides identical in size to the three known subunits of the isolated enzyme, a, 13, and y. The polypeptides also reacted with the antibodies raised against the respective enzyme subunits. In M. thermoautotrophicum, the subunits are encoded by a gene cluster whose transcript boundaries were mapped. Sequence analysis revealed two more open reading frames of unknown function located between two of the methyl coenzyme M reductase genes.Methanogenic bacteria are archaebacteria, which gain their energy by anaerobic formation of methane (6). Independent of the methanogenic substrate, the final step of methanogenesis is the reduction of methyl coenzyme M (CoM) to methane and CoM. This reaction, which is coupled to the synthesis of ATP in vivo (7,20), is catalyzed by the methyl CoM reductase (MCR) (3,12). This enzyme amounts to about 10% of the total protein (3, 12) and has been isolated from a large number of methanogenic bacteria (3,18,21 Q359 (19) hsdR supE tonA (P2) Y1089 (42) AlacUl69 Alon araDI39 strA hflA150 chr::TnlO (pMC9) Y1090 (42) A1acUl69 Alon araD139 strA supF trpC22::TnlO (pMC9) BNN97 (41) hsdR supE thr leu thi lacYI tonA21 (Xgtll) pUC8 (26, 39) XEMBL4 (13) Agtll (37) previously (23), using formaldehyde gels. Transfer of the DNA to GeneScreen sheets essentially followed the procedure described by Thomas (37) with the modifications given in the GeneScreen manual by the supplier. The DNA probe was labeled by nick translation as described in reference 24, using a-32P-labeled deoxynucleoside triphosphates to a specific activity of 0.5 x 108 to 1 x 108 cpm/,Lg of DNA. Hybridization and further processing of the filters were performed as described in reference 15.Antisera against MCR subunits. Antisera were raised in rabbits against the subunits of purified MCR, which were eluted from an SDS-polyacrylamide gel after their electrophoretic separation. The specificities were checked in Western blot (immunoblot) experiments, using total extracts of E. coli or M. thermoautotrophicum cells or purified MCR. No reactions were observed with E. coli extracts. The antisera were found to react with one polypeptide band each in the M. thermoautotrophicum extract, which corresponded in size to the respective subunit reacting with the same antiserum.Screening of M. thermoautotrophicum genomic libraries. Two DNA libraries were constructed. First, an EcoRI total digest of M. thermoautotrophicum DNA was ligated to Agtll DNA, which was digested with EcoRI and treated with calf intestinal phosphatase (41). After in vitro packaging (42), bacteriophages were plated on E. coli Y1090 and screened with antisera against the isolated subunits after induction with isopropyl-1-D-thiogalactopyranoside as described below. Positive phages were lysogenized in E. coli Y1089 an...

A crude protein extract of Bacillus subtilis W23 contains a sequence-specific DNA binding activity for the xyl operator as detected by the gel mobility shift assay. A xylR determinant encoded on a multicopy plasmid leads to increased expression of this binding activity. In situ footprinting analysis of the protein-DNA complex in a polyacrylamide gel shows that the xyl operator is sequence-specifically bound and protected from cleavage by copper-phenanthroline at 26 phosphodiester bonds on each strand. Quantitative competition assays for repressor binding reveal that a 25 bp synthetic xyl operator cloned into a polylinker is bound with the same affinity as the operator in the wild-type xyl regulatory region. This confirms that no additional sites in the wild-type sequence contribute to repressor binding. The xyl operator consists of ten palindromic base pairs flanking five central non-palindromic base pairs. A mutational analysis shows that the sequence of the central base pairs contributes to recognition by the repressor protein and that the spacing of the palindromic elements is crucial for repressor binding. An operator half site is not bound by the repressor. In vivo and in vitro induction studies suggest that, of several structurally similar sugars, xylose is the only molecular inducer of the Xyl repressor.

The xyl operator of Bacillus subtilis W23 was identified by deletion analysis of the xyl regulatory region as a 25-base-pair (bp) sequence located 10 bp downstream from the xyl promoter. The outer 10 bp of the xyl operator exhibit perfect palindromic symmetry, while 5 central bp are nonpalindromic. It was demonstrated that the penultimate base pair near the end of this sequence is important for repressor binding. In both strains, expression of xylose-utilizing enzymes appears to be negatively regulated at the level of transcription (5, 6). In B. subtilis 168, a regulatory gene called xy/R has been identified and characterized by complementation (6). We are interested in studying repressor-operator recognition mediating regulation of the xyl operon. In this article, we report the identification by deletion analysis and nucleotide sequencing of the xylR gene and xyl operator from B.subtilis W23 and the first data characterizing a base pair of the xyl operator important for Xyl repressor recognition. MATERIALS AND METHODSBacterial strains and plasmids. The bacterial strains and plasmids used in this study are listed in Table 1. Escherichia coli RRI and HB101 and B. siubtilis 512 and BR151 were generally used for transformations as described previously (1, 2). E. coli RRI was used for transformation with M13 DNA, and E. coli HB101 was used for all other cloning experiments. E. coli JM101 was the host for M13 infection. The culture and growth conditions were as described previously (5). We constructed pWH423 (see Fig. 2) by cloning a 2.2-kilobase-pair BamHI-HindIII DNA fragment from pIWll (21) in pWH331 (5). pWH423ABc/I was constructed from pWH423 by digestion with Bc/1I followed by mung bean nuclease digestion and ligation. DNA sequence analysis revealed that 14 base pairs (bp) (TCATTCTTGATCAA) were missing. pWH419 contains the 100-bp xy/ promoteroperator SspI-NsiI fragment from pWH416 (5) in the EcoRV-PstI site of pWH341. pWH341 was constructed by inserting the polylinker from pIC20R (10) EcoRV and HindIII sites of pWH341. The deletion plasmids were constructed by digesting pWH419 with BamHI followed by BAL 31 as previously described (9), ligating either an EcoRI linker (pWH429 and pWH430) or a Sall linker (pWH428 and pWH432) to the ends, transforming E. coli HB101, preparing the bulk of plasmid DNA, digesting it with EcoRI or EcoRI and Sa/I, separating the products on a 5% polyacrylamide gel, eluting fragments of the desired length from the gel, and recloning them into the respective sites of pWH331 (pWH428 and pWH432) or pWH341 (pWH429 and pWH430). pWH439, pWH440, and pWH441 contain the respective deletions from the EcoRI site with the HindlIl linker CAAGCTTG recloned into pWH331. All deletions were verified by sequencing.General methods. Preparation of plasmid DNA from E. coli (7,8) and B. subtilis (16) and elution of DNA from polyacrylamide gels (11) were done as described before. All of the other general methods used, including BAL 31 and mung bean nuclease digestions, were done as already described (9).Enzym...

Bacillus subtilis 168 is unable to grow on xylose and galactose as sole carbon sources, owing to the lack of specific transporters. We show that they are imported into the cell by the activity of AraE, an arabinose transporter whose synthesis is induced by l-arabinose.

The NfrA protein, an oxidoreductase from the soil bacterium Bacillus subtilis, is synthesized during the stationary phase and in response to heat. Analysis of promoter mutants revealed that the nfrA gene belongs to the class III heat shock genes in B. subtilis. An approximate 10-fold induction at both the transcriptional and the translational levels was found after thermal upshock. This induction resulted from enhanced synthesis of mRNA. Genetic and Northern blot analyses revealed that nfrA and the gene downstream of nfrA are transcribed as a bicistronic transcriptional unit. The unstable full-length transcript is processed into two short transcripts encoding nfrA and ywcH. The nfrA-ywcH operon is not induced by salt stress or by ethanol. According to previously published data, the transcription of class III genes in general is activated in response to the addition of these stressors. However, this conclusion is based on experiments which lacked a valid control. Therefore, it seems possible that the transcription of all class III genes is specifically induced by heat shock.The continuous demand of Bacillus subtilis to adapt to everchanging conditions in its natural environment has forced the generation of complex regulatory mechanisms governing the transcription of stress-specific proteins. Stress-inducible genes from B. subtilis in general are subdivided into three groups (17,18). Class I genes are specifically induced by heat stress (17). The well-known chaperonins GroEL, GroES, DnaK, DnaJ, and GrpE are encoded by genes belonging to this group (30,41,48,54,55). The transcription of the respective genes is regulated by HrcA, a transcription repressor which binds to the CIRCE element (43,56,57,59). Genes transcribed in a Bdependent manner constitute class II stress-responsive genes (17, 18). B activity is triggered by different kinds of stress and by starvation (5, 7-9). Members of the last group of stressinduced genes, class III, are induced not by starvation but by several different stressful conditions. The transcription of class III genes is neither repressed by HrcA nor solely dependent on B . The regulator of the clpC operon, which encodes class III proteins, is known (11,27). This operon is transcribed by the activity of RNA polymerases containing B and A (28). Nevertheless, transcription is not induced at the onset of the stationary phase (28), likely because of the activity of this regulator (11,27).Some of the stress-responsive proteins are regulated by two transcription factors. clpC, dps, trxA, opuE, and clpP (1,4,15,29,40,46) are transcribed by RNA polymerase containing either A or B . csbB is under the additional control of X (22). The csb40 operon (50) and the yvyD gene (13) are transcribed from B and H promoters, respectively. This genetic organization enables the bacterial cell to modulate the regulation of the respective genes in response to additional challenges.In this communication, we describe the transcriptional regulation of the nfrA-ywcH operon encoding an oxidoreductase (34, 58) and a p...