Regulation of expression, genetic organization and substrate specificity of xylose uptake in <i>Bacillus megaterium</i>

Schmiedel, Dagmar; Kintrup, Martin; Küster, Elke; Hillen, Wolfgang

doi:10.1046/j.1365-2958.1997.2881654.x

Cited by 37 publications

(35 citation statements)

References 26 publications

(36 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Although xylT is the last gene of the xylABT operon, it seems to be uncoupled from the XylR-mediated regulation of xylA. This finding can be explained by the existence of an internal transcriptional terminator within the operon upstream of xylT (31). In agreement with this, xylT mutants of B. megaterium continued to show the observed culture heterogeneity (unpublished data).…”

Section: Resultssupporting

confidence: 73%

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

Münch

Müller

Wienecke

et al. 2015

Appl Environ Microbiol

View full text Add to dashboard Cite

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.

show abstract

Section: Resultssupporting

confidence: 73%

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

Münch

Müller

Wienecke

et al. 2015

Appl Environ Microbiol

View full text Add to dashboard Cite

show abstract

“…Although this finding is quite puzzling, one possible explanation is that the altered protein might impair the Pkc1-dependent cell wall integrity pathway, rendering this pathway less responsive to environmental changing conditions. The C-terminus of the Cwh43p protein shows similarities with a xylose permease from B. megaterium (Schmiedel et al, 1997), and with putative sugar transporters from A. thaliana and D. melanogaster. Interestingly, the hydropathy analysis of the Cwh43p C-terminus revealed the presence of 12 transmembrane segments that are typical of most of the yeast sugar transporters (Boles and Hollenberg, 1997).…”

Section: Discussionmentioning

confidence: 99%

Saccharomyces cerevisiae YCRO17c/CWH43 encodes a putative sensor/transporter protein upstream of the BCK2 branch of the PKC1‐dependent cell wall integrity pathway

Martin-Yken

Dagkessamanskaia

Groot

et al. 2001

Yeast

View full text Add to dashboard Cite

The Saccharomyces cerevisiae cwh43-2 mutant, originally isolated for its Calcofluor white hypersensitivity, displays several cell wall defects similar to mutants in the PKC1-MPK1 pathway, including a growth defect and increased release of b-1,6-glucan and bglucosylated proteins into the growth medium at increased temperatures. The cloning of CWH43 showed that it corresponds to YCR017c and encodes a protein with 14-16 transmembrane segments containing several putative phosphorylation and glycosylation sites. The N-terminal part of the amino acid sequence of Cwh43p shows 40% similarity with the mammalian FRAG1, a membrane protein that activates the fibroblast growth factor receptor of rat osteosarcoma (FGFR2-ROS) and with protein sequences of four uncharacterized ORFs from Caenorhabditis elegans and one from Drosophila melanogaster. The C-terminus of Cwh43p shows low similarities with a xylose permease of Bacillus megaterium and with putative sugar transporter from D. melanogaster, and has 52% similarity with a protein sequence from a Schizosaccharomyces pombe cDNA. A Cwh43-GFP fusion protein suggested a plasma membrane localization, although localization to the internal structure of the cells could not be excluded, and it concentrates to the bud tip of small budded cells and to the neck of dividing cells. Deletion of CWH43 resulted in cell wall defects less pronounced than those of the cwh43-2 mutant. This allelespecific phenotype appears to be due to a G-R substitution at position 57 in a highly conserved region of the protein. Genetic analysis places CWH43 upstream of the BCK2 branch of the PKC1 signalling pathway, since cwh43 mutations were synthetic lethal with pkc1 deletion, whereas the cwh43 defects could be rescued by overexpression of BCK2 and not by high-copy-number expression of genes encoding downstream proteins of the PKC1 pathway. However, unlike BCK2, whose disruption in a cln3 mutant resulted in growth arrest in G 1 , no growth defect was observed in a double cwh43 cln3 mutants. Taken together, it is proposed that CWH43 encodes a protein with putative sensor and transporter domains acting in parallel to the main PKC1-dependent cell wall integrity pathway, and that this gene has evolved into two distinct genes in higher eukaryotes.

show abstract

“…(16,33,38), and Staphylococcus xylosus (44). Free xylose can be transported via low-affinity symporters (XylE or XylT) or high-affinity binding-protein dependent systems (XylFGH) (1,6,9,41). Xylose isomerase (xylA gene) then converts the aldose sugar to xylulose, which is phosphorylated by xylulokinase (xylB gene).…”

mentioning

confidence: 99%

Dissolution of Xylose Metabolism in Lactococcus lactis

Erlandson

Park

Wissam

et al. 2000

Appl Environ Microbiol

View full text Add to dashboard Cite

Xylose metabolism, a variable phenotype in strains of Lactococcus lactis, was studied and evidence was obtained for the accumulation of mutations that inactivate the xyl operon. The xylose metabolism operon (xylRAB) was sequenced from three strains of lactococci. Fragments of 4.2, 4.2, and 5.4 kb that included the xyl locus were sequenced from L. lactis subsp. lactis B-4449 (formerly Lactobacillus xylosus), L. lactis subsp. lactis IO-1, and L. lactis subsp. lactis 210, respectively. The two environmental isolates, L. lactis B-4449 and L. lactis IO-1, produce active xylose isomerases and xylulokinases and can metabolize xylose. L. lactis 210, a dairy starter culture strain, has neither xylose isomerase nor xylulokinase activity and is Xyl ؊ . Xylose isomerase and xylulokinase activities are induced by xylose and repressed by glucose in the two Xyl ؉ strains. Sequence comparisons revealed a number of point mutations in the xylA, xylB, and xylR genes in L. lactis 210, IO-1, and B-4449. None of these mutations, with the exception of a premature stop codon in xylB, are obviously lethal, since they lie outside of regions recognized as critical for activity. Nevertheless, either cumulatively or because of indirect affects on the structures of catalytic sites, these mutations render some strains of L. lactis unable to metabolize xylose. Xylose metabolism has been described for a wide array of microorganisms. Extensively characterized xylose-metabolizing (Xyl ϩ ) bacteria include Escherichia coli (24, 46), Lactobacillus spp. (3,4,(26)(27)(28), Bacillus spp. (16,33,38), and Staphylococcus xylosus (44). Free xylose can be transported via low-affinity symporters (XylE or XylT) or high-affinity binding-protein dependent systems (XylFGH) (1, 6, 9, 41). Xylose isomerase (xylA gene) then converts the aldose sugar to xylulose, which is phosphorylated by xylulokinase (xylB gene). Further metabolism of xylulose-5-phosphate occurs via the pentose-phosphate or phosphoketolase pathways. Xylose metabolism is induced by xylose, mediated via XylR. In Salmonella and E. coli strains, XylR is an activator when xylose is present (42,46). In grampositive organisms, XylR is a repressor which is inactivated when xylose binds (13,26,27,37,44,45). The catabolite repression of xylose metabolism by glucose is mediated primarily through the CcpA protein (40).Lactococcus lactis subsp. cremoris and almost all L. lactis subsp. lactis strains cannot metabolize xylose. These organisms have undergone intense selection for use in dairy fermentations, but plants are thought to be their original ecological niche because L. lactis subsp. lactis isolates have been recovered from many different plants (22,34,35). Also, the plant isolate Lactobacillus xylosus has been reclassified as L. lactis subsp. lactis (39). However, L. lactis subsp. cremoris strains are almost exclusively dairy associated (22).We discovered xylose metabolic genes from both plant (Xyl ϩ ) and dairy (Xyl Ϫ ) isolates of L. lactis. The sequencing of xylRAB from L. lactis strains IO-1, 210, a...

show abstract

Regulation of expression, genetic organization and substrate specificity of xylose uptake in Bacillus megaterium

Cited by 37 publications

References 26 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

Saccharomyces cerevisiae YCRO17c/CWH43 encodes a putative sensor/transporter protein upstream of the BCK2 branch of the PKC1‐dependent cell wall integrity pathway

Dissolution of Xylose Metabolism in Lactococcus lactis

Contact Info

Product

Resources

About