Integration and mapping of Bacillus megaterium genes which code for small, acid-soluble spore proteins and their protease

Sussman, Michael D.; Vary, Patricia S.; Hartman, C; Setlow, Peter

doi:10.1128/jb.170.10.4942-4945.1988

Cited by 27 publications

(14 citation statements)

References 17 publications

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“…Since the gpr fragments cloned into pJH101 were entirely within the coding sequence, integration of these recombinant plasmids into the chromosome by a Campbell-like mechanism should disrupt the gpr gene. Southern blot analysis of chromosomal DNA from these integrants showed that the gpr coding sequence was indeed disrupted (27,28). The putative gpr strains of both species grew and sporulated normally (27,28; data not shown).…”

Section: Resultsmentioning

confidence: 99%

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Properties of Bacillus megaterium and Bacillus subtilis mutants which lack the protease that degrades small, acid-soluble proteins during spore germination

et al. 1992

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During germination of spores of Bacillus species the degradation of the spore's pool of small, acid-soluble proteins (SASP) is initiated by a protease termed GPR, the product of the gpr gene. Bacillus megaterium and B. subtilis mutants with an inactivated gpr gene grew, sporulated, and triggered spore germination as did gpr+ strains. However, SASP degradation was very slow during germination ofgpr mutant spores, and in rich media the time taken for spores to return to vegetative growth (defined as outgrowth) was much longer in gpr than in gpr+ spores. Not surprisingly, gpr spores had much lower rates of RNA and protein synthesis during outgrowth than did gpr+ spores, although both types of spores had similar levels of ATP. The rapid decrease in the number of negative supertwists in plasmid DNA seen during germination of gpr+ spores was also much slower in gpr spores. Additionally, UV irradiation of gpr B. subtilis spores early in germination generated significant amounts of spore photoproduct and only small amounts of thymine dimers (TT); in contrast UV irradiation of germinated gpr+ spores generated almost no spore photoproduct and three to four times more TT. Consequently, germinated gpr spores were more UV resistant than germinated gpr+ spores. Strikingly, the slow outgrowth phenotype of B. subtilis gpr spores was suppressed by the absence of major od/,-type SASP. These data suggest that (i) a$/,-type SASP remain bound to much, although not all, of the chromosome in germinated gpr spores; (ii) the a$d-type SASP bound to the chromosome in gpr spores alter this DNA's topology and UV photochemistry; and (iii) the presence of o/,-type SASP on the chromosome is detrimental to normal spore outgrowth.Approximately 10 to 20% of the protein of dormant spores of Bacillus species is degraded to free amino acids in the first minutes of spore germination (22). These amino acids can support much of the protein synthesis early in the development of the germinating and outgrowing spore (22). The proteins degraded in this process are a group of small, acid-soluble proteins (SASP) of two types, al and y (22). There are multiple ao/-type SASP in spores of Bacillus species, and the amino acid sequences of these proteins have been highly conserved both within and across species (22). The aJ,-type SASP are associated with spore DNA and play a key role in the resistance of spores to UV light (22, 23). Bacillus spores have only one y-type SASP, and the primary sequence of this protein has not been as highly conserved in evolution as has those of a/P-type SASP. -y-Type SASP are not associated with DNA in vivo and have no known function other than generating amino acids by their degradation during spore germination.The great majority, if not all, SASP degradation during spore germination is initiated by a single protease, which has been termed GPR (8,9,22). This enzyme is an endoprotease specific for a pentapeptide sequence found once or twice in all SASP. GPR is initially synthesized during sporulation as a 46-kDa precursor (termed P...

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Section: Resultsmentioning

confidence: 99%

“…The gpr genes from B. megaterium and B. subtilis were previously mapped (27,28) (gpr). The samples were separated by electrophoresis on SDSpolyacrylamide gels, proteins were transferred to nitrocellulose, and GPR antigen was detected with antiserum against B. megaterium GPR.…”

Section: Resultsmentioning

confidence: 99%

Properties of Bacillus megaterium and Bacillus subtilis mutants which lack the protease that degrades small, acid-soluble proteins during spore germination

et al. 1992

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“…First, the gene coding for the protease (termed gpr [27]) appears to be expressed only in the forespore for a defined time in sporulation, presumably as a result of regulation at the transcriptional level, although this has not yet been proven. Second, since the protease polypeptide undegoes multiple processing reactions, at least one of which appears to activate the enzyme, there is also regulation at the posttranslational level.…”

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“…The B. megaterium, B. subtilis, and Escherichia coli strains used are described in Table 1. The sources of Agtll and various plasmids have been described previously (6,26,27). B. megaterium and B. subtilis chromosomal DNA was isolated and purified as described previously (2, 6).…”

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Cloning, nucleotide sequence, and regulation of the Bacillus subtilis gpr gene, which codes for the protease that initiates degradation of small, acid-soluble proteins during spore germination

Sussman

Setlow

1991

J Bacteriol

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The gpr gene, which codes for the protease that initiates degradation of small, acid-soluble proteins during spore germination, has been cloned from Bacillus megaterium and Bacilus subtilis, and its nucleotide sequence has been determined. Use of a translational gpr-lacZ fusion showed that the B. subtilis gpr gene was expressed primarily, if not exclusively, in the forespore compartment of the sporulating cell, with expression taking place approximately 1 h before expression of glucose dehydrogenase and ssp genes. gpr-lacZ expression was abolished in spoIlAC (sigF) and spolIlE mutants but was reduced only -50% in a spoIJIG (sigG) mutant. However, the kinetics of the initial -50% of gpr-lacZ expression were unaltered in a spolliG mutant. The in vivo transcription start site of gpr has been identified and found to be identical to the in vitro start site on this gene with either ECFF or ECG. Induction of CG synthesis in vivo turned on gpr-lacZ expression in parallel with synthesis of glucose dehydrogenase. These data are consistent with gpr transcription during sporulation first by ErF and then by EYG.During the first minutes of germination of spores of Bacillus species, 10 to 20% of the dormant spore's protein is degraded to amino acids (21). The proteins degraded in this process are a group of small, acid-soluble spore proteins (SASP), and their degradation is initiated by an endoprotease also present in the spore (20,21). This protease has been purified from Bacillus megaterium spores and is specific for cleavage within a pentapeptide sequence found in all SASP (9, 23). The protease is synthesized during sporulation within the developing forespore as a polypeptide of 46 kDa (P46) (9,10). Approximately 1 h after its synthesis, P46 is converted to a polypeptide of 41 kDa (P41), which is the form found in the dormant spore (10). Early in spore germination, the enzyme is processed again, being converted to a slightly smaller polypeptide of 40 kDa (P40) (7,10). The active form of the protease is a tetramer, and P40, P41, and P46 all form tetramers (7, 9). However, only P40 and P41 exhibit enzymatic activity in vitro (7).It is clear that the activity of this SASP-specific protease is regulated at a variety of levels. MATERIALS AND METHODSOrganisms and plasmids used and isolation of nucleic acids. The B. megaterium, B. subtilis, and Escherichia coli strains used are described in Table 1. The sources of Agtll and various plasmids have been described previously (6,26,27). B. megaterium and B. subtilis chromosomal DNA was isolated and purified as described previously (2, 6). Plasmid DNA was isolated from E. coli strains grown overnight in L broth plus 0.5% glucose with the appropriate drug (ampicillin [50 ,ug/ml], tetracycline [10 ,ug/ml], or chloramphenicol [10 ,ug/ml]) as needed and purified by CsCl gradient centrifugation if necessary. Xgtll derivatives were grown and phage DNA was isolated as described previously (6). B. subtilis strains were made competent and transformed as previously described (2,11,12).Molecular ...

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“…B. megaterium strains lack natural competence, and most of them are less efficiently transformable than B. subtilis. Therefore, integration of markers by homologous recombination with the chromosome has as yet only been demonstrated as Campbell-type integration of plasmids (4,31). In a different approach, the transducing bacteriophage MP13 has been used for marker transfer and mapping (33).…”

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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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Integration and mapping of Bacillus megaterium genes which code for small, acid-soluble spore proteins and their protease

Cited by 27 publications

References 17 publications

Properties of Bacillus megaterium and Bacillus subtilis mutants which lack the protease that degrades small, acid-soluble proteins during spore germination

Properties of Bacillus megaterium and Bacillus subtilis mutants which lack the protease that degrades small, acid-soluble proteins during spore germination

Cloning, nucleotide sequence, and regulation of the Bacillus subtilis gpr gene, which codes for the protease that initiates degradation of small, acid-soluble proteins during spore germination

Catabolite repression of the xyl operon in Bacillus megaterium

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