Molecular cloning, structure, promoters and regulatory elements for transcription of the Bacillus megaterium encoded regulon for xylose utilization

Abstract

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.

Genetic Analysis of a Novel Pathway for d -Xylose Metabolism in Caulobacter crescentus

“…Two routes for D-xylose degradation in microorganisms have been described. Numerous bacteria, including Escherichia coli (15), Bacillus species (24,25), and Lactobacillus species (16), use xylose isomerase to convert D-xylose to xylulose, which is then phosphorylated to enter the pentose phosphate pathway. Although some fungi have recently been shown to use this "bacterial" pathway (11), fungi more commonly transform Dxylose into xylitol by using xylose reductase and xylitol dehydrogenase (13).…”

Catabolite repression of the xyl operon in Bacillus megaterium

“…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.…”

“…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).…”

“…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.…”

“…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). B. megaterium strains lack natural competence, and most of them are less efficiently transformable than B. subtilis.…”