Characterization of a sucrase gene from Staphylococcus xylosus.
Ontology highlight
ABSTRACT:
SUBMITTER: Bruckner R
PROVIDER: S-EPMC196229 | biostudies-other | 1993 Feb
REPOSITORIES: biostudies-other
Publications
Characterization of a sucrase gene from Staphylococcus xylosus.
Brückner R R Wagner E E Götz F F
Journal of bacteriology 19930201 3
The Staphylococcus xylosus gene scrB, encoding a sucrase, has been isolated from a genomic library of S. xylosus constructed in Escherichia coli. The gene was detected by its ability to confer utilization of the glucose and fructose residues of raffinose in an E. coli strain that is not able to metabolize galactose. It was found to reside within a 1.8-kb DNA fragment, the nucleotide sequence of which was determined. One large open reading frame, which is preceded by a ribosome binding site, is e ...[more]
Similar Datasets
Project description:In Staphylococcus xylosus, scrB is one of two genes necessary for sucrose utilization. It encodes a sucrase that hydrolyzes intracellular sucrose-6-phosphate generated by the uptake of sucrose via the sucrose-specific enzyme II of the phosphotransferase system, the gene product of scrA. ScrB sucrase activity is inducible by the presence of sucrose in the culture medium. Primer extension experiments demonstrated that the observed regulation is achieved at the level of scrB transcription initiation. The protein mediating sucrose-specific regulation of scrB was found to be encoded immediately upstream of the sucrase gene. The nucleotide sequence of the regulatory gene scrR comprises an open reading frame that specifies a protein of 35.8 kDa. This protein exhibits similarity to transcriptional regulators of the GalR-LacI family. Inactivation of the scrR reading frame in the genome of S. xylosus led to the constitutive expression of scrB at a high level, identifying ScrR as a repressor of transcription. Sucrose-specific regulation of scrB was also lost upon deletion of 4 bp of a palindromic sequence (OB) covering positions +6 to +21 downstream of the scrB transcriptional start site. These results suggested a direct interaction of the ScrR repressor and the operator OB. Accordingly, a fusion protein consisting of the maltose-binding protein of Escherichia coli and the ScrR protein was able to interact with an scrB promoter fragment in gel mobility shift experiments but failed to bind an scrB fragment carrying the 4-bp deletion derivative of OB. An scrR promoter fragment, which dose not contain a sequence resembling OB, was not shifted by the fusion protein. This result corroborates scrR primer extension analyses showing that transcription of the repressor gene itself is not regulated. Therefore, the sucrase gene operator OB is the target sequence through which the ScrR protein exerts its negative effect on transcription initiation. In the promoter region of scrA, the gene essential for sucrose transport, two palindromic sequences that are similar to the scrB operator are found. Their presence in scrA suggests that ScrR controls a sucrose-specific regulon in S. xylosus.
| S-EPMC177679 | biostudies-other
Project description:The Staphylococcus xylosus gene hprK, encoding HPr kinase (HPrK), has been isolated from a genomic library. The HPrK enzyme, purified as a His(6) fusion protein, phosphorylated HPr, the phosphocarrier protein of the bacterial phosphotransferase system, at a serine residue in an ATP-dependent manner, and it also catalyzed the reverse reaction. Therefore, the enzyme constitutes a bifunctional HPr kinase/phosphatase. Insertional inactivation of the gene in the genome of S. xylosus resulted in the concomitant loss of both HPr kinase and His serine-phosphorylated-HPr phosphatase activities in cell extracts, strongly indicating that the HPrK enzyme is also responsible for both reactions in vivo. HPrK deficiency had a profound pleiotropic effect on the physiology of S. xylosus. The hprK mutant strain showed a severe growth defect in complex medium upon addition of glucose. Glucose uptake in glucose-grown cells was strongly enhanced compared with the wild type. Carbon catabolite repression of three tested enzyme activities by glucose, sucrose, and fructose was abolished. These results clearly demonstrate the prominent role of HPr kinase in global control to adjust catabolic capacities of S. xylosus according to the availability of preferred carbon sources.
| S-EPMC101872 | biostudies-literature
Project description:Staphylococcus xylosus is a facultative anaerobic bacterium used as a starter culture for fermented meat products. In an attempt to analyze the antioxidant capacities of this organism, the superoxide dismutase (SOD) was characterized. S. xylosus contains a single cytoplasmic SOD, which was not inhibited by H2O2. The SOD activity in crude extracts was completely lost upon metal depletion, but it could be recovered by manganese and very weakly by iron. It is therefore suggested that the S. xylosus SOD is a manganese-preferring enzyme. The corresponding gene, sod, was isolated from a genomic library of S. xylosus DNA and complemented the growth defect of an Escherichia coli SOD-deficient mutant. As deduced from the nucleotide sequence, sod encodes a protein of 199 amino acids with a molecular mass of 22.5 kDa. Two transcriptional start sites 25 and 120 bp upstream of the sod start codon were identified. A terminator-like structure downstream of the gene suggested a monocistronic sod mRNA. Regulation of sod expression was studied using fusions of the sod promoters to a genomic promoterless beta-galactosidase gene. The sod expression was not affected by manganese and increased slightly with paraquat. It was induced during stationary phase in a complex medium but not in a chemically defined medium. To investigate the physiological role of SOD, a mutant devoid of SOD activity was constructed. Growth experiments showed that sod is not essential for aerobic growth in complex medium. However, in chemically defined medium without leucine, isoleucine, and valine, the sod mutant hardly grew, in contrast to the wild-type strain. In addition, the mutant was sensitive to hyperbaric oxygen and to paraquat. Therefore, sod plays an important role in the protection of S. xylosus from oxidative stress.
| S-EPMC93135 | biostudies-literature
Project description:Staphylococcus xylosus is a commensal of the skin of humans and animals and a ubiquitous bacterium naturally present in food. It is one of the major starter cultures used for meat fermentation, but a few strains could potentially be hazardous and are related to animal opportunistic infections. To better understand the genetic diversity of S. xylosus intraspecies, suppressive and subtractive hybridization (SSH) was carried out with the S. xylosus C2a strain, a commensal of human skin, used as the driver for three tester strains, S04002 used as a starter culture, S04009 isolated from cow mastitis, and 00-1747, responsible for mouse dermatitis. SSH revealed 122 tester-specific fragments corresponding to 149 open reading frames (ORFs). A large proportion of these ORFs resembled genes involved in specific metabolisms. Analysis of the distribution of the tester-specific fragments in 20 S. xylosus strains of various origins showed that the S. xylosus species could be divided into two clusters with one composed only of potentially hazardous strains. The genetic content diversity of this species is colocalized in a region near the origin of replication of the chromosome. This region of speciation previously observed in the Staphylococcus genus corresponded in S. xylosus species to a strain-specific region potentially implicated in ecological fitness.
| S-EPMC2168225 | biostudies-literature
Project description:A Staphylococcus xylosus C2A gene cluster, which encodes enzymes in the pathway for choline uptake and dehydrogenation (cud), to form the osmoprotectant glycine betaine, was identified. The cud locus comprises four genes, three of which encode proteins with significant similarities to those known to be involved in choline transport and conversion in other organisms. The physiological role of the gene products was confirmed by analysis of cud deletion mutants. The fourth gene possibly codes for a regulator protein. Part of the gene cluster was shown to be transcriptionally regulated by choline and elevated NaCl concentrations as inducers.
| S-EPMC93644 | biostudies-literature
Project description:The presence of determinants of resistance to antibiotics can constitute a possible safety hazard in coagulase-negative staphylococci (CNS), which are widely present in food of animal origin. Among CNS, S. xylosus is a species frequently isolated from fermented meat products. Resistance to tetracycline was found to be one of the most distributed resistances occurring in S. xylosus strains isolated from fermented sausages. We evaluated the transfer of tetracycline resistance in vitro and in situ between S. xylosus strains. We selected three strains isolated from dry fermented sausages, resistant to tetracycline but not to minocycline, their resistance occurring by a mechanism of active efflux encoded by the tetK gene. Only one strain was able to transfer its tetracycline resistance to a recipient strain initially susceptible and plasmid-free using a filter mating procedure. Transfer of tetracycline resistance was observed at very low frequencies of 3.4 × 10-9 per recipient. To further investigate the transferability of this tetracycline resistance, the donor and recipient strains were tested in pilot-scale fermented sausage production. This transfer was possible but at a low rate, 1.4 × 10-7, and only under conditions of a high inoculation level of 108 CFU/g of meat. The tetK gene is located on a small mobilizable plasmid close to Staphylococcus aureus pT181 plasmid. In conclusion, the transfer of tetracycline resistance between strains of S. xylosus is possible, but at a really low frequency in vitro and in situ in fermented sausages. Even if this represents a very moderate risk, it should be taken into account as required by the European approach of Qualified Presumption of Safety (QPS) and AFSSA safety recommendations, advising that strains used as starter cultures should not carry any transferable antibiotic resistance.
| S-EPMC6414713 | biostudies-literature
Project description:The biofilm associated protein (Bap) is recognised as the essential component for biofilm formation in Staphylococcus aureus V329 and has been predicted as important for other species as well. Although Bap orthologs are also present in most S. xylosus strains, their contribution to biofilm formation has not yet been demonstrated. In this study, different experimental approaches were used to elucidate the effect of Bap on biofilm formation in S. xylosus and the motif structure of two biofilm-forming S. xylosus strains TMW 2.1023 and TMW 2.1523 was compared to Bap of S. aureus V329. We found that despite an identical structural arrangement into four regions, Bap from S. xylosus differs in key factors to Bap of S. aureus, i.e., isoelectric point of aggregation prone Region B, protein homology and type of repeats. Disruption of bap had no effect on aggregation behavior of selected S. xylosus strains and biofilm formation was unaffected (TMW 2.1023) or at best slightly reduced under neutral conditions (TMW 2.1523). Further, we could not observe any typical characteristics of a S. aureus Bap-positive phenotype such as functional impairment by calcium addition and rough colony morphology on congo red agar (CRA). A dominating role of Bap in cell aggregation and biofilm formation as reported mainly for S. aureus V329 was not observed. In contrast, this work demonstrates that functions of S. aureus Bap cannot easily be extrapolated to S. xylosus Bap, which appears as non-essential for biofilm formation in this species. We therefore suggest that biofilm formation in S. xylosus follows different and multifactorial mechanisms.
| S-EPMC8708073 | biostudies-literature
Project description:By transposon Tn917 mutagenesis, two mutants of Staphylococcus xylosus were isolated that showed higher levels of beta-galactosidase activity in the presence of glucose than the wild type. Both transposons integrated in a gene, designated glcU, encoding a protein involved in glucose uptake in S. xylosus, which is followed by a glucose dehydrogenase gene (gdh). Glucose-mediated repression of beta-galactosidase, alpha-glucosidase, and beta-glucuronidase activities was partially relieved in the mutant strains, while repression by sucrose or fructose remained as strong as in the wild type. In addition to the pleiotropic regulatory effect, integration of the transposons into glcU reduced glucose dehydrogenase activity, suggesting cotranscription of glcU and gdh. Insertional inactivation of the gdh gene and deletion of the glcU gene without affecting gdh expression showed that loss of GlcU function is exclusively responsible for the regulatory defect. Reduced glucose repression is most likely the consequence of impaired glucose uptake in the glcU mutant strains. With cloned glcU, an Escherichia coli mutant deficient in glucose transport could grow with glucose as sole carbon source, provided a functional glucose kinase was present. Therefore, glucose is internalized by glcU in nonphosphorylated form. A gene from Bacillus subtilis, ycxE, that is homologous to glcU, could substitute for glcU in the E. coli glucose growth experiments and restored glucose repression in the S. xylosus glcU mutants. Three more proteins with high levels of similarity to GlcU and YcxE are currently in the databases. It appears that these proteins constitute a novel family whose members are involved in bacterial transport processes. GlcU and YcxE are the first examples whose specificity, glucose, has been determined.
| S-EPMC93981 | biostudies-literature
Project description:The lactose utilization genes of Staphylococcus xylosus have been isolated and characterized. The system is comprised of two structural genes, lacP and lacH, encoding the lactose permease and the beta-galactosidase proteins, respectively, and a regulatory gene, lacR, coding for an activator of the AraC/XylS family. The lactose utilization genes are divergently arranged, the lacPH genes being opposite to lacR. The lacPH genes are cotranscribed from one promoter in front of lacP, whereas lacR is transcribed from two promoters of different strengths. Lactose transport as well as beta-galactosidase activity are inducible by the addition of lactose to the growth medium. Primer extension experiments demonstrated that regulation is achieved at the level of lacPH transcription initiation. Inducibility and efficient lacPH transcription are dependent on a functional lacR gene. Inactivation of lacR resulted in low and constitutive lacPH expression. Expression of lacR itself is practically constitutive, since transcription initiated at the major lacR promoter does not respond to the availability of lactose. Only the minor lacR promoter is lactose inducible. Apart from lactose-specific, LacR-dependent control, the lacPH promoter is also subject to carbon catabolite repression mediated by the catabolite control protein CcpA. When glucose is present in the growth medium, lacPH transcription initiation is reduced. Upon ccpA inactivation, repression at the lacPH promoter is relieved. Despite this loss of transcriptional regulation in the ccpA mutant strain, beta-galactosidase activity is still reduced by glucose, suggesting another level of control.
| S-EPMC107164 | biostudies-literature
Project description:The biofilm associated protein (Bap) is recognised as the essential component for biofilm formation in Staphylococcus aureus V329 and other species. Although Bap orthologs are also present in most S. xylosus strains, their contribution to biofilm formation has not yet been determined. In this study, different experimental approaches were used to elucidate the effect of Bap on biofilm formation in S. xylosus and the motif structure of two biofilm-forming S. xylosus strains TMW 2.1023 and TMW 2.1523 was compared to Bap of S. aureus V329. We found that despite an identical structural arrangement into four regions, Bap from S. xylosus differs in key factors to Bap of S. aureus i.e. isoelectric point of aggregation prone Region B, protein homology and type of repeats. Disruption of bap had no effect on aggregation behavior of selected S. xylosus strains and a significant reduction in biofilm was only observed for one strain (TMW 2.1523) under neutral conditions. Further we could not observe any typical characteristics of a S. aureus Bap positive phenotype such as functional impairment by calcium addition and rough colony morphology on CRA. A predominant role of Bap in cell aggregation and biofilm formation as reported for S. aureus V329 was not observed. We therefore suggest that biofilm formation follows different, multifactorial mechanisms, and cannot be referred to as the primary function of Bap in S. xylosus.
2022-01-20 | PXD029728 | Pride