scholarly journals The Functional ccpA Gene Is Required for Carbon Catabolite Repression in Lactobacillus plantarum

2001 ◽  
Vol 67 (7) ◽  
pp. 2903-2907 ◽  
Author(s):  
Lidia Muscariello ◽  
Rosangela Marasco ◽  
Maurilio De Felice ◽  
Margherita Sacco
Keyword(s):  
Lactobacillus Plantarum ◽  
Mutant Strain ◽  
Catabolite Repression ◽  
Carbon Catabolite Repression ◽  
Protein A ◽  
Regulatory Region ◽  
Gene Encoding ◽  
Upstream Promoter ◽  
In Vivo Footprinting

ABSTRACT We report the characterization of the ccpA gene ofLactobacillus plantarum, coding for catabolite control protein A. The gene is linked to the pepQ gene, encoding a proline peptidase, in the order ccpA-pepQ, with the two genes transcribed in tandem from the same strand as distinct transcriptional units. Two ccpA transcription start sites corresponding to two functional promoters were found, expression from the upstream promoter being autogenously regulated through a catabolite-responsive element (cre) sequence overlapping the upstream +1 site. During growth on ribose, the upstream promoter showed maximal expression, while growth on glucose led to transcription from the downstream promoter. In a ccpA mutant strain, the gene was transcribed mainly from the upstream promoter in both repressing and non repressing conditions. Expression of two enzyme activities, β-glucosidase and β-galactosidase, was relieved from carbon catabolite repression in the ccpA mutant strain. In vivo footprinting analysis of the catabolite-controlledbglH gene regulatory region in the ccpA mutant strain showed loss of protection of the cre under repressing conditions.

scholarly journals Overproduction of SecA Suppresses the Export Defect Caused by a Mutation in the Gene Encoding the Escherichia coli Export Chaperone SecB

1999 ◽  
Vol 181 (10) ◽  
pp. 3010-3017 ◽  
Author(s):  
Heather A. Cook ◽  
Carol A. Kumamoto
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane Proteins ◽  
Protein A ◽  
In Vivo Studies ◽  
Wild Type ◽  
Gene Encoding ◽  
Precursor Proteins ◽  
Additional Support ◽  
Insight Into

ABSTRACT SecB is a cytosolic protein required for rapid and efficient export of particular periplasmic and outer membrane proteins inEscherichia coli. SecB promotes export by stabilizing newly synthesized precursor proteins in a nonnative conformation and by targeting the precursors to the inner membrane. Biochemical studies suggest that SecB facilitates precursor targeting by binding to the SecA protein, a component of the membrane-embedded translocation apparatus. To gain more insight into the functional interaction of SecB and SecA, in vivo, mutations in the secA locus that compensate for the export defect caused by the secBmissense mutation secBL75Q were isolated. Two suppressors were isolated, both of which led to the overproduction of wild-type SecA protein. In vivo studies demonstrated that the SecBL75Q mutant protein releases precursor proteins at a lower rate than does wild-type SecB. Increasing the level of SecA protein in the cell was found to reverse this slow-release defect, indicating that overproduction of SecA stimulates the turnover of SecBL75Q-precursor complexes. These findings lend additional support to the proposed pathway for precursor targeting in which SecB promotes targeting to the translocation apparatus by binding to the SecA protein.

Disruption of regulatory gene GAL80 in Saccharomyces cerevisiae: effects on carbon-controlled regulation of the galactose/melibiose pathway genes

1984 ◽  
Vol 4 (8) ◽  
pp. 1521-1527
Author(s):  
T E Torchia ◽  
R W Hamilton ◽  
C L Cano ◽  
J E Hopper
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Catabolite Repression ◽  
Carbon Catabolite Repression ◽  
Regulatory Gene ◽  
Negative Regulator ◽  
Null Mutation ◽  
Null Mutant ◽  
Wild Type

In Saccharomyces cerevisiae, the transcriptional expression of the galactose-melibiose catabolic pathway genes is under the control of at least three regulatory genes, GAL4, GAL80, and GAL3. We have isolated the GAL80 gene and have studied the effect of a null mutation on the carbon-controlled regulation of the MEL1 and GAL cluster genes. The null mutation was achieved in vivo by replacing the chromosomal wild-type GAL80 allele with an in vitro-created GAL80 deletion-disruption mutation. Enzyme activities and RNA levels for the GAL cluster and MEL1 genes were constitutively expressed in the null mutant strain grown on glycerol-lactate and were higher than in the isogenic wild-type yeast strain when compared after growth on galactose. Carbon catabolite repression of the GAL cluster and MEL1 genes, which occurs at the level of transcription, is retained in the null mutant. Deletion of the GAL80 gene in a gal4 cell does not restore GAL cluster and MEL1 gene expression. The data demonstrate that (i) the GAL80 protein is a purely negative regulator, (ii) the GAL80 protein does not mediate carbon catabolite repression, and (iii) the GAL4 protein is not simply an antagonizer of GAL80-mediated repression.

scholarly journals Use of the mCherry Fluorescent Protein To Study Intestinal Colonization by Enterococcus mundtii ST4SA and Lactobacillus plantarum 423 in Mice

2015 ◽  
Vol 81 (17) ◽  
pp. 5993-6002 ◽  
Author(s):  
Winschau F. van Zyl ◽  
Shelly M. Deane ◽  
Leon M. T. Dicks
Keyword(s):  
Lactobacillus Plantarum ◽  
Fluorescent Protein ◽  
Intestinal Colonization ◽  
Red Fluorescent Protein ◽  
Gene Encoding ◽  
Content Type ◽  
Enterococcus Mundtii ◽  
Probiotic Strains ◽  
Reporter Systems

ABSTRACTLactic acid bacteria (LAB) are natural inhabitants of the gastrointestinal tract (GIT) of humans and animals, and some LAB species receive considerable attention due to their health benefits. Although many papers have been published on probiotic LAB, only a few reports have been published on the migration and colonization of the cells in the GIT. This is due mostly to the lack of efficient reporter systems. In this study, we report on the application of the fluorescent mCherry protein in thein vivotagging of the probiotic strainsEnterococcus mundtiiST4SA andLactobacillus plantarum423. ThemCherrygene, encoding a red fluorescent protein (RFP), was integrated into a nonfunctional region on the genome ofL. plantarum423 by homologous recombination. In the case ofE. mundtiiST4SA, themCherrygene was cloned into the pGKV223D LAB/Escherichia coliexpression vector. Expression of themCherrygene did not alter the growth rate of the two strains and had no effect on bacteriocin production. Both strains colonized the cecum and colon of mice.

scholarly journals Disruption of regulatory gene GAL80 in Saccharomyces cerevisiae: effects on carbon-controlled regulation of the galactose/melibiose pathway genes.

1984 ◽  
Vol 4 (8) ◽  
pp. 1521-1527 ◽  
Author(s):  
T E Torchia ◽  
R W Hamilton ◽  
C L Cano ◽  
J E Hopper
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Catabolite Repression ◽  
Carbon Catabolite Repression ◽  
Regulatory Gene ◽  
Negative Regulator ◽  
Null Mutation ◽  
Null Mutant ◽  
Wild Type

In Saccharomyces cerevisiae, the transcriptional expression of the galactose-melibiose catabolic pathway genes is under the control of at least three regulatory genes, GAL4, GAL80, and GAL3. We have isolated the GAL80 gene and have studied the effect of a null mutation on the carbon-controlled regulation of the MEL1 and GAL cluster genes. The null mutation was achieved in vivo by replacing the chromosomal wild-type GAL80 allele with an in vitro-created GAL80 deletion-disruption mutation. Enzyme activities and RNA levels for the GAL cluster and MEL1 genes were constitutively expressed in the null mutant strain grown on glycerol-lactate and were higher than in the isogenic wild-type yeast strain when compared after growth on galactose. Carbon catabolite repression of the GAL cluster and MEL1 genes, which occurs at the level of transcription, is retained in the null mutant. Deletion of the GAL80 gene in a gal4 cell does not restore GAL cluster and MEL1 gene expression. The data demonstrate that (i) the GAL80 protein is a purely negative regulator, (ii) the GAL80 protein does not mediate carbon catabolite repression, and (iii) the GAL4 protein is not simply an antagonizer of GAL80-mediated repression.

scholarly journals Inactivation of the gbpA Gene of Streptococcus mutans Increases Virulence and Promotes In Vivo Accumulation of Recombinations between the Glucosyltransferase B and C Genes

1998 ◽  
Vol 66 (5) ◽  
pp. 2180-2185 ◽  
Author(s):  
Karsten R. O. Hazlett ◽  
Suzanne M. Michalek ◽  
Jeffrey A. Banas
Keyword(s):  
Streptococcus Mutans ◽  
Mutant Strain ◽  
Rat Model ◽  
Binding Protein ◽  
Protein A ◽  
Wild Type ◽  
Mutant Strains

ABSTRACT Glucan-binding protein A (GbpA) of Streptococcus mutanshas been hypothesized to promote sucrose-dependent adherence and the cohesiveness of plaque and therefore to contribute to caries formation. We have analyzed the adherence properties and virulence of isogenicgbpA mutants relative to those of wild-type S. mutans. Contrary to expectations, the gbpA mutant strains displayed enhanced sucrose-dependent adherence in vitro and enhanced cariogenicity in vivo. In vitro, S. mutanswas grown in the presence of [3H]thymidine and sucrose within glass vials. When grown with constant rotation, significantly higher levels of gbpA mutant organisms than of wild type remained adherent to the vial walls. Postgrowth vortexing of rotated cultures significantly decreased adherence of wild-type organisms, whereas the adherence of gbpA mutant organisms was unaffected. In the gnotobiotic rat model, the gbpA mutant strain was hypercariogenic though the colonization levels were not significantly different from those of the wild type. ThegbpA mutant strain became enriched in vivo with organisms that had undergone a recombination involving the gtfB andgtfC genes. The incidence of gtfBC recombinant organisms increased as a function of dietary sucrose availability and was inversely correlated with caries development. We propose that the absence of GbpA elevates the cariogenic potential of S. mutans by altering the structure of plaque. However, the hypercariogenic plaque generated by gbpA mutant organisms may be suboptimal for S. mutans, leading to the accumulation of gtfBC recombinants whose reduced glucosyltransferase activity restores a less cariogenic plaque structure.

scholarly journals The local transcriptional regulators SacR1 and SacR2 act as repressors of fructooligosaccharides metabolism in Lactobacillus plantarum

2020 ◽  
Author(s):  
Chen Chen ◽  
Linlin Wang ◽  
Haiyan Yu ◽  
huaixiang tian
Keyword(s):  
Lactobacillus Plantarum ◽  
Consensus Sequence ◽  
Protein A ◽  
Pcr Analysis ◽  
Promoter Regions ◽  
Mobility Shift ◽  
Global And Local ◽  
Electrophoretic Mobility Shift Assays

Abstract Background: In Lactobacillus plantarum , fructooligosaccharides (FOS) metabolism is controlled by both global and local regulatory mechanisms. Although catabolite control protein A has been identified as a global regulator of FOS metabolism, the functions of local regulators remain unclear. This study aimed to elucidate the roles of two local regulators, SacR1 and SacR2, in the regulation of FOS metabolism in L. plantarum both in vitro and in vivo . Results: The inactivation of sacR1 and sacR2 affected the growth and production of metabolites for strains grown on FOS or glucose, respectively. A reverse transcription-quantitative PCR analysis of one wild-type and two mutant strains ( ΔsacR1 and ΔsacR2 ) of L. plantarum identified SacR1 and SacR2 as repressors of genes relevant to FOS metabolism in the absence of FOS, and these genes could be induced or derepressed by the addition of FOS. The analysis predicted four potential transcription factor binding sites (TFBSs) in the putative promoter regions of two FOS-related clusters. The binding of SacR1 and SacR2 to these TFBSs both in vitro and in vivo was verified using electrophoretic mobility shift assays and chromatin immunoprecipitation, respectively. A consensus sequence of WNNNNNAACGNNTTNNNNNW was deduced for the TFBSs of SacR1 and SacR2. Conclusion: Our results identified SacR1 and SacR2 as local repressors for FOS metabolism in L. plantarum . The regulation is achieved by the binding of SacR1 and SacR2 to TFBSs in the promoter regions of FOS-related clusters. The results provide new insights into the complex network regulating oligosaccharide metabolism by lactic acid bacteria .

scholarly journals AfadDmutant ofVibrio choleraeIs Impaired in the Production of Virulence Factors and Membrane Localization of the Virulence Regulatory Protein TcpP

2010 ◽  
Vol 79 (1) ◽  
pp. 258-266 ◽  
Author(s):  
Sreejana Ray ◽  
Epshita Chatterjee ◽  
Arpita Chatterjee ◽  
Kalidas Paul ◽  
Rukhsana Chowdhury
Keyword(s):  
Mutant Strain ◽  
Virulence Factors ◽  
Regulatory Protein ◽  
Inducible Promoter ◽  
Fatty Acyl ◽  
Membrane Localization ◽  
Logarithmic Phase ◽  
Gene Encoding ◽  
Reverse Transcription Pcr

ABSTRACTIn the enteric pathogenVibrio cholerae, expression of the major virulence factors is controlled by the hierarchical expression of several regulatory proteins comprising the ToxR regulon. In this study, we demonstrate that disruption of thefadDgene encoding a long-chain fatty acyl coenzyme A ligase has marked effects on expression of the ToxR virulence regulon, motility, andin vivolethality ofV. cholerae. In theV. cholerae fadDmutant, expression of the major virulence genesctxABandtcpA, encoding cholera toxin (CT), and the major subunit of the toxin-coregulated pilus (TCP) was drastically repressed and a growth-phase-dependent reduction in the expression oftoxT, encoding the transcriptional activator ofctxABandtcpA, was observed. Expression oftoxTfrom an inducible promoter completely restored CT to wild-type levels in theV. cholerae fadDmutant, suggesting that FadD probably acts upstream oftoxTexpression. Expression oftoxTis activated by the synergistic effect of two transcriptional regulators, TcpP and ToxR. Reverse transcription-PCR and Western blot analysis indicated that although gene expression and production of both TcpP and ToxR are unaffected in thefadDmutant strain, membrane localization of TcpP, but not ToxR, is severely impaired in thefadDmutant strain from the mid-logarithmic phase of growth. Since the decrease intoxTexpression occurred concomitantly with the reduction in membrane localization of TcpP, a direct correlation between the defect in membrane localization of TcpP and reducedtoxTexpression in thefadDmutant strain is suggested.

scholarly journals Involvement of Pyruvate Oxidase Activity and Acetate Production in the Survival of Lactobacillus plantarum during the Stationary Phase of Aerobic Growth

2006 ◽  
Vol 72 (12) ◽  
pp. 7933-7940 ◽  
Author(s):  
Philippe Goffin ◽  
Lidia Muscariello ◽  
Frederique Lorquet ◽  
Aline Stukkens ◽  
Deborah Prozzi ◽  
...  
Keyword(s):  
Stationary Phase ◽  
Lactobacillus Plantarum ◽  
Catabolite Repression ◽  
Oxidase Activity ◽  
Carbon Catabolite Repression ◽  
Aerobic Growth ◽  
Early Stationary Phase ◽  
Acetate Production ◽  
Pyruvate Oxidase ◽  
Oxidase Gene

ABSTRACT In addition to the previously characterized pyruvate oxidase PoxB, the Lactobacillus plantarum genome encodes four predicted pyruvate oxidases (PoxC, PoxD, PoxE, and PoxF). Each pyruvate oxidase gene was individually inactivated, and only the knockout of poxF resulted in a decrease in pyruvate oxidase activity under the tested conditions. We show here that L. plantarum has two major pyruvate oxidases: PoxB and PoxF. Both are involved in lactate-to-acetate conversion in the early stationary phase of aerobic growth and are regulated by carbon catabolite repression. A strain devoid of pyruvate oxidase activity was constructed by knocking out the poxB and poxF genes. In this mutant, acetate production was strongly affected, with lactate remaining the major end product of either glucose or maltose fermentation. Notably, survival during the stationary phase appeared to be dramatically improved in the poxB poxF double mutant.

scholarly journals Loss of function of the carbon catabolite repressor CreA leads to low but inducer‐independent expression from the feruloyl esterase B promoter in Aspergillus niger

2021 ◽  
Author(s):  
Jos Reijngoud ◽  
Mark Arentshorst ◽  
Claudine Ruijmbeek ◽  
Ian Reid ◽  
Ebru Demirci Alazi ◽  
...  
Keyword(s):  
Catabolite Repression ◽  
Allyl Alcohol ◽  
Carbon Catabolite Repression ◽  
Constitutive Expression ◽  
Genetic Screen ◽  
Feruloyl Esterase ◽  
Loss Of Function ◽  
Gene Encoding ◽  
Targeted Deletion ◽  
Reporter Strains

Abstract Objective With the aim to decipher the mechanisms involved in the transcriptional regulation of feruloyl esterase encoded by faeB, a genetic screen was performed to isolate A. niger mutants displaying inducer-independent expression from the faeB promoter. Result PfaeB-amdS and PfaeB-lux dual reporter strains were constructed and used to isolate trans-acting mutants in which the expression of both reporters was increased, based on the ability to grow on acetamide plates and higher luciferase activity, respectively. The genetic screen on the non-inducing carbon source D-fructose yielded in total 111 trans-acting mutants. The genome of one of the mutants was sequenced and revealed several SNPs, including a point mutation in the creA gene encoding a transcription factor known to be involved in carbon catabolite repression. Subsequently, all mutants were analyzed for defects in carbon catabolite repression by determining sensitivity towards allyl alcohol. All except four of the 111 mutants were sensitive to allyl alcohol, indicating that the vast majority of the mutants are defective in carbon catabolite repression. The creA gene of 32 allyl alcohol sensitive mutants was sequenced and 27 of them indeed contained a mutation in the creA gene. Targeted deletion of creA in the reporter strain confirmed that the loss of CreA results in constitutive expression from the faeB promoter. Conclusion Loss of function of CreA leads to low but inducer-independent expression from the faeB promoter in A. niger.

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