Development of Genetic Methods in Bacillus Megaterium

Vary, Patricia S.; Tao, Y-P.

doi:10.1016/b978-0-12-274161-6.50072-3

Cited by 44 publications

(39 citation statements)

References 14 publications

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“…However, as shown in this paper, it was not possible to gain expression of ORF3 in commonly applied E. coli systems. We assume different codon preferences to be responsible for the observed lack of expression, as the genomic G+C ratio of E. coli is very different from that of the yeast plasmids (52% vs 26%, respectively); the G+C ratio (39%) of B. megaterium (Vary, 1992) is closer to pGKL2 (Tommasino et al, 1988) and that is why we have chosen this Grampositive bacterium as the host for heterologous expression of Orf3p. Such an experimental approach allowed us to express the protein with a C-terminal His tag useful for identification and isolation of the protein, which was subsequently used to demonstrate mRNA-triphosphatase activity and formation of a stable EpG.…”

Section: Discussionmentioning

confidence: 99%

“…Since the E. coli genome has a G+C content of 50.1% (Oliver and Marin, 1996), whereas the killer plasmid pair, pGKL1 and pGKL2, exhibits a G+C content of only 26% (Tommasino et al, 1988), we used as an alternative the B. megaterium expression system (G+C=39%; Vary 1992). For this purpose, plasmid p03His (Figure 1), based on the E. coli/ Bacillus shuttle plasmid pWH1520 (Rygus and Hillen, 1991), was constructed and used for expression studies in strain B. megaterium DSM319.…”

Section: Heterologous Expression Of Orf3pmentioning

confidence: 99%

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Kluyveromyces lactis cytoplasmic plasmid pGKL2: heterologous expression of Orf3p and proof of guanylyltransferase and mRNA–triphosphatase activities

Tiggemann

Jeske

Larsen

et al. 2001

Yeast

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The predicted ORF3 polypeptide (Orf3p) of the linear genetic element pGKL2 from Kluyveromyces lactis was expressed in Bacillus megaterium as a fusion protein with a His(6X)-tag at the C-terminus for isolation by Ni-affinity chromatography. This is the first time that a yeast cytoplasmic gene product has been expressed heterologously as a functional protein in a bacterial system. The purified protein was found to display both RNA 5k-triphosphatase and guanylyltransferase activities. When the lysine residue present at position 177 of the protein within the sequence motif (KXDG), highly conserved in capping enzymes and other nucleotidyl transferases, was substituted by alanine, the guanylyltransferase activity was lost, thereby proving an important role for the transfer of GMP from GTP to the 5k-diphosphate end of the mRNA. Our in vitro data provides the first direct evidence that the polypeptide encoded by ORF3 of the cytoplasmic yeast plasmid pGKL2 functions as a plasmid-specific capping enzyme. Since genes equivalent to ORF3 of pGKL2 have been identified in all autonomous cytoplasmic yeast DNA elements investigated so far, our findings are of general significance for these widely distributed yeast extranuclear genetic elements.

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Section: Discussionmentioning

confidence: 99%

Section: Heterologous Expression Of Orf3pmentioning

confidence: 99%

Kluyveromyces lactis cytoplasmic plasmid pGKL2: heterologous expression of Orf3p and proof of guanylyltransferase and mRNA–triphosphatase activities

Tiggemann

Jeske

Larsen

et al. 2001

Yeast

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“…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.…”

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“…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).…”

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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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“…Southern blot analysis of DNA from B. megaterium PV361, a derivative of B. megaterium QMB1551 that has lost all seven plasmids (15), showed no detectable 4.7-kb EcoRI fragment hybridizing to the sspG probe (Fig. 3, lanes C and D).…”

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Cloning and nucleotide sequence of a plasmid-carried gene coding for a minor small, acid-soluble protein from Bacillus megaterium spores

Carrillo-Martinez

Setlow

1993

J Bacteriol

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The gene (termed sspG) coding for a small, acid-soluble protein (SASP) from spores of Bacillus megaterium QMB1551, termed SASP-G, has been cloned, and its nucleotide sequence has been determined. SASP-G is a 42-residue protein containing 2 tryptophan and 11 lysine residues, including a hexalysine sequence, and is not homologous to any previously described SASP. The sspG gene appears to be an additional member of the sigma G regulon. No gene homologous to sspG is present in B. cereus T or B. subtilis 168. The reason for the absence of sspG from other Bacillus species appears to be that in B. megaterium, sspG is present only on a 111-kb plasmid; this plasmid is not present in B. cereus T or B. subtilis 168. The sspG gene is the first forespore-expressed gene found to be on a plasmid.

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Development of Genetic Methods in Bacillus Megaterium

Cited by 44 publications

References 14 publications

Kluyveromyces lactis cytoplasmic plasmid pGKL2: heterologous expression of Orf3p and proof of guanylyltransferase and mRNA–triphosphatase activities

Kluyveromyces lactis cytoplasmic plasmid pGKL2: heterologous expression of Orf3p and proof of guanylyltransferase and mRNA–triphosphatase activities

Catabolite repression of the xyl operon in Bacillus megaterium

Cloning and nucleotide sequence of a plasmid-carried gene coding for a minor small, acid-soluble protein from Bacillus megaterium spores

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