Highly efficient expression of homologous and heterologous genes in Bacillus megaterium

Meinhardt, Friedhelm; Ståhl, Ulf; Ebeling, Wolfgang

doi:10.1007/bf00296622

Cited by 40 publications

(19 citation statements)

References 24 publications

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“…Although it was shown previously that plasmids are preferentially located near the cell poles and are grouped in clusters of foci (25,35), it is generally assumed that high-copy-number plasmids without a partitioning system segregate by random diffusion. In contrast, low-copy-number plasmids segregated by a partitioning system were found localized near the center or quarter position of the cell and behaved like minichromosomes (42).…”

Section: Discussionmentioning

confidence: 89%

Polar Fixation of Plasmids during Recombinant Protein Production in Bacillus megaterium Results in Population Heterogeneity

Münch

Müller

Wienecke

et al. 2015

Appl Environ Microbiol

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dDuring the past 2 decades, Bacillus megaterium has been systematically developed for the gram-per-liter scale production of recombinant proteins. The plasmid-based expression systems employed use a xylose-controlled promoter. Protein production analyses at the single-cell level using green fluorescent protein as a model product revealed cell culture heterogeneity characterized by a significant proportion of less productive bacteria. Due to the enormous size of B. megaterium, such bistable behavior seen in subpopulations was readily analyzed by time lapse microscopy and flow cytometry. Cell culture heterogeneity was not caused simply by plasmid loss: instead, an asymmetric distribution of plasmids during cell division was detected during the exponential-growth phase. Multicopy plasmids are generally randomly distributed between daughter cells. However, in vivo and in vitro experiments demonstrated that under conditions of strong protein production, plasmids are retained at one of the cell poles. Furthermore, it was found that cells with accumulated plasmids and high protein production ceased cell division. As a consequence, the overall protein production of the culture was achieved mainly by the subpopulation with a sufficient plasmid copy number. Based on our experimental data, we propose a model whereby the distribution of multicopy plasmids is controlled by polar fixation under protein production conditions. Thereby, cell lines with fluctuating plasmid abundance arise, which results in population heterogeneity. Our results provide initial insights into the mechanism of cellular heterogeneity during plasmid-based recombinant protein production in a Bacillus species.

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Section: Discussionmentioning

confidence: 89%

Polar Fixation of Plasmids during Recombinant Protein Production in Bacillus megaterium Results in Population Heterogeneity

Münch

Müller

Wienecke

et al. 2015

Appl Environ Microbiol

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“…coli strains listed in Table 1 were transformed with pQE60-3b and cultivated in LB medium containing 100 mg/ml ampicillin. For expression of Orf3p in B. megaterium we used shuttle plasmid p03His, which was transformed into B. megaterium DSM319 according to a previously described method (Meinhardt et al, 1989).…”

Section: Expression and Purification 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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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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“…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).…”

mentioning

confidence: 99%

“…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.…”

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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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Highly efficient expression of homologous and heterologous genes in Bacillus megaterium

Cited by 40 publications

References 24 publications

Polar Fixation of Plasmids during Recombinant Protein Production in Bacillus megaterium Results in Population Heterogeneity

Polar Fixation of Plasmids during Recombinant Protein Production in Bacillus megaterium Results in Population Heterogeneity

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

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