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.