Identification and sequence analysis of the Bacillus subtilis W23 xylR gene and xyl operator

Kreuzer, Paul E.; Gärtner, Dagmar; Allmansberger, Rudolf; Hillen, Wolfgang

doi:10.1128/jb.171.7.3840-3845.1989

Cited by 74 publications

(39 citation statements)

References 20 publications

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“…As described above, the upstream region influences promoter activity and heat shock induction. The sequence upstream of P xylA , which is necessary for the full activity of this promoter (25), had no positive effect on the nfrA promoter (Fig. 5).…”

Section: Resultsmentioning

confidence: 99%

Transcription of the nfrA-ywcH Operon from Bacillus subtilis Is Specifically Induced in Response to Heat

2000

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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...

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Section: Resultsmentioning

confidence: 99%

Transcription of the nfrA-ywcH Operon from Bacillus subtilis Is Specifically Induced in Response to Heat

2000

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“…The deduced amino acid sequence of XylR is homologous to that of XylR of Bacillus subtilis (17), Bacillus megaterium (26), Bacillus licheniformis (30), and Staphylococcus xylosus (31). The repressor function of XylR has been unambiguously demonstrated in these organisms (15,17,29,32).…”

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Regulation of expression of the Lactobacillus pentosus xylAB operon

et al. 1997

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The xylose cluster of Lactobacillus pentosus consists of five genes, two of which, xylAB, form an operon and code for the enzymes involved in the catabolism of xylose, while a third encodes a regulatory protein, XylR. By introduction of a multicopy plasmid carrying the xyl operator and by disruption of the chromosomal xylR gene, it was shown that L. pentosus xylR encodes a repressor. Constitutive expression of xylAB in the xylR mutant is repressed by glucose, indicating that glucose repression does not require XylR. The xylR mutant displayed a prolonged lag phase compared to wild-type bacteria when bacteria were shifted from glucose to xylose medium. Differences in the growth rate in xylose medium at different stages of growth are not correlated with differences in levels of xylAB transcription in L. pentosus wild-type or xylR mutant bacteria but are positively correlated in Lactobacillus casei with a plasmid containing xylAB. Glucose repression was further investigated with a ccpA mutant. An 875-bp internal fragment of the ccpA gene of L. pentosus was isolated by PCR and used to construct a ccpA knockout mutant. Transcription analysis of L. pentosus xylA showed that CcpA is involved in glucose repression. CcpA was also shown to be involved in glucose repression of the ␣-amylase promoter of Lactobacillus amylovorus by demonstrating that glucose repression of the chloramphenicol acetyltransferase gene under control of the ␣-amylase promoter is strongly reduced in the L. pentosus ccpA mutant strain.

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“…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. …”

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Catabolite repression of the xyl operon in Bacillus megaterium

Rygus

Hillen

1992

J Bacteriol

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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...

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Identification and sequence analysis of the Bacillus subtilis W23 xylR gene and xyl operator

Cited by 74 publications

References 20 publications

Transcription of the nfrA-ywcH Operon from Bacillus subtilis Is Specifically Induced in Response to Heat

Transcription of the nfrA-ywcH Operon from Bacillus subtilis Is Specifically Induced in Response to Heat

Regulation of expression of the Lactobacillus pentosus xylAB operon

Catabolite repression of the xyl operon in Bacillus megaterium

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