We have constructed a shuttle plasmid for Bacillus megaterium and Escherichia coli that contains the promoter and repressor gene of the B. megaterium-borne operon for xylose utilization. A polylinker downstream of the promoter allows versatile cloning of genes under its transcriptional control. We have placed gdhA (encoding glucose dehydrogenase) from B. megaterium, lacZ (encoding beta-galactosidase) from E. coli, mro (encoding mutarotase) from Acinetobacter calcoaceticus, and human puk (encoding single-chain urokinase-like plasminogen activator, rscuPA) under xylose control in this vector. All four genes were between 130-fold and 350-fold inducible by 0.5% xylose in the growth medium in B. megaterium. Enzymatically active glucose dehydrogenase and mutarotase accumulated to 20% and 30% of the total soluble protein, respectively. beta-Galactosidase and rscuPA were also expressed at a high level. A gel analysis of the products demonstrated their proteolytic stability in the cytoplasm, even up to 5 h after induction. The expression properties of this new host-vector system are discussed in comparison to the ones available for B. subtilis and E. coli.

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.

We characterized catabolite repression of the genes encoding xylose utilization in Bacillus megaterium. A transcriptional fusion ofhy4 encoding xylose isomerase to the spoVG-lacZ indicator gene on a plasmid with a temperature-sensitive origin of replication was constructed and efficiently used for single-copy replacement cloning in the B. megaterium chromosome starting from a single transformant. In the resulting strain, Gene regulation in gram-positive bacteria has been mainly studied in Bacillus subtilis, focusing on developmentally regulated cell differentiation processes. In particular, genetic competence (3) and sporulation (1, 21) have been studied in great detail. Much effort has also been focused on the regulation of vegetatively expressed genes for the utilization of different carbon sources in B. subtilis (6). Other members of the bacilli, e.g., B. amyloliquefaciens, B. licheniformis, and B. megaterium, play important roles in the industrial production of enzymes and for the expression of heterologous proteins with high yields (2,9,11,18,22,24,27). B. megaterium has been used for protein expression (18,26) and as a host with improved stability of plasmids compared with B. subtilis (27,32,34,35 at the level of enzymatic activities (5) was described some time ago, it has only recently been established that catabolite repression for several genes in B. subtilis is mediated at the level of transcription (6, 12). Different cis-acting catabolic control sequences have been proposed for a number of operons (7,15,20,36). Catabolite repression of xyl in B. megaterium is more efficient than that in B. subtilis W23 (8,15,16), thus facilitating the study of effects of other carbon sources repressing less effectively than glucose. MATERUILS AND METHODSBacterial strains and plasmids. The bacterial strains and plasmids used are described in Table 1. B. megaterium WH320, a derivative of strain DSM319, was constructed by ethyl methanesulfonate mutagenesis (27) and has no detectable P-galactosidase activity, whereas the wild-type strain shows low P-galactosidase activity in the media used in this study. Escherichia coli RR1 lacZAM15 was generally used for transformations as described previously (17). B. megaterium WH320 was transformed by using protoplasts as described previously (23). pWH1505 was constructed by first inserting the 3.0-kbp BglII (nucleotide positions 1 to 2131 in the xyl sequence [27]) fragment from pWH1500 into the single BglII site (27) of pWH1503, a pIC20H derivative containing a promoterless spoVG-lacZ fusion within the SmaI site of the pIC20H polylinker (27); this was followed by insertion of the BamHI (nucleotide position 2103 in the xyl sequence [27])-PstI fragment from pWH1500 into the respective sites on pWH1503. This results in axylA4-spoVGlacZ fusion flanked by DNA originating from the xyl operon of B. megaterium.Culture and growth conditions. Bacilli and E. coli were grown in LB medium (10 g of tryptone, 5 g of NaCl, 5 g of yeast extract per liter of deionized water; pH 7.3). MOPSO medium was...

The Staphylococcus xylosus xyl genes were cloned in Staphylococcus carnosus by complementation to xylose utilization. Xylose isomerase assays under inducing (xylose present) and non-inducing (xylose absent) conditions indicated the presence of a regulated xylA gene on the recombinant plasmid. The nucleotide sequence (4520 bases) revealed three open reading frames with the same polarity. They were identified by sequence homologies as xylR, encoding the Xyl repressor, xylA, encoding xylose isomerase and xylB, encoding xylulokinase. Primer extension analyses indicated constitutive transcription of xylR and xylose-inducible transcription of xylA. Promoter consensus sequences were found upstream of both transcriptional start sites. A transcriptional terminator between xylR and xylA separates the different transcriptional units. Potential regulatory elements were identified by sequence analysis and suggest a repressor-operator mechanism for the regulation of xylAB expression.

In this article we describe the cloning of the xyl regulon encoding xylose utilization from Bacillus licheniformis by complementation of a xyl mutant of B. subtilis. The xylose isomerase encoding gene, xylA, was sequenced and identified by its extensive homology to other xylose isomerases. The expression of xylA is regulated on the level of transcription by a repressor protein encoded by xylR. Its gene has the opposite orientation of xylA and the start codons are 181 bp apart. A deletion of xylR renders xylA expression constitutive. The xylR sequence was determined and is discussed with respect to its homology to other xylR structures. Primer extension analyses of the xylA and xylR transcripts under repressing and including conditions define their promoters and confirm the regulation of xylA transcription. Furthermore, some induction of the xylR transcript by xylose is also observed. The regulatory sequence of both genes consists of a bipolar promoter system and contains three palindromic sequence elements. Their potential functions with respect to xylA and xylR regulation are discussed. The primary structures of the genes, promoters and regulatory sequences are compared to the xyl regulons encoded by B. subtilis, B. megaterium, Staphylococcus xylosus and E. coli. Homology is greatest between the B. subtilis and B. megaterium encoded xyl genes while the B. licheniformis borne genes are clearly more distant. The next greater differences are found to the S. xylosus and the greatest to the E. coli encoded genes. These results are discussed with respect to the taxonomic relations of these bacteria.