scholarly journals Identification of a Gene Cluster Enabling Lactobacillus casei BL23 To Utilize myo-Inositol

2007 ◽  
Vol 73 (12) ◽  
pp. 3850-3858 ◽  
Author(s):  
Mar�a Jes�s Yebra ◽  
Manuel Z��iga ◽  
Sophie Beaufils ◽  
Gaspar P�rez-Mart�nez ◽  
Josef Deutscher ◽  
...  
Keyword(s):  
Lactobacillus Casei ◽  
Protein A ◽  
Transcriptional Repressor ◽  
Catabolic Pathway ◽  
Gene Encoding ◽  
Carbohydrate Utilization ◽  
Alternative Pathways ◽  
Semialdehyde Dehydrogenase ◽  
Catabolite Control Protein A ◽  
Dna Insertion

ABSTRACT Genome analysis of Lactobacillus casei BL23 revealed that, compared to L. casei ATCC 334, it carries a 12.8-kb DNA insertion containing genes involved in the catabolism of the cyclic polyol myo-inositol (MI). Indeed, L. casei ATCC 334 does not ferment MI, whereas strain BL23 is able to utilize this carbon source. The inserted DNA consists of an iolR gene encoding a DeoR family transcriptional repressor and a divergently transcribed iolTABCDG1G2EJK operon, encoding a complete MI catabolic pathway, in which the iolK gene probably codes for a malonate semialdehyde decarboxylase. The presence of iolK suggests that L. casei has two alternative pathways for the metabolism of malonic semialdehyde: (i) the classical MI catabolic pathway in which IolA (malonate semialdehyde dehydrogenase) catalyzes the formation of acetyl-coenzyme A from malonic semialdehyde and (ii) the conversion of malonic semialdehyde to acetaldehyde catalyzed by the product of iolK. The function of the iol genes was verified by the disruption of iolA, iolT, and iolD, which provided MI-negative strains. By contrast, the disruption of iolK resulted in a strain with no obvious defect in MI utilization. Transcriptional analyses conducted with different mutant strains showed that the iolTABCDG1G2EJK cluster is regulated by substrate-specific induction mediated by the inactivation of the transcriptional repressor IolR and by carbon catabolite repression mediated by the catabolite control protein A (CcpA). This is the first example of an operon for MI utilization in lactic acid bacteria and illustrates the versatility of carbohydrate utilization in L. casei BL23.

scholarly journals Transcriptional Activation of the Bacillus subtilis ackA Gene Requires Sequences Upstream of the Promoter

1998 ◽  
Vol 180 (22) ◽  
pp. 5961-5967 ◽  
Author(s):  
Andrew J. Turinsky ◽  
Frank J. Grundy ◽  
Jeong-Ho Kim ◽  
Glenn H. Chambliss ◽  
Tina M. Henkin
Keyword(s):  
Bacillus Subtilis ◽  
Carbon Source ◽  
Transcriptional Activation ◽  
Consensus Sequence ◽  
Protein A ◽  
Acetate Kinase ◽  
Gene Encoding ◽  
Gram Positive Bacteria ◽  
Catabolite Control Protein A

ABSTRACT Transcriptional activation of the Bacillus subtilis ackA gene, encoding acetate kinase, was previously shown to require catabolite control protein A (CcpA) and sequences upstream of the ackA promoter. CcpA, which is responsible for catabolite repression of a number of secondary carbon source utilization genes in B. subtilis and other gram-positive bacteria, recognizes a cis-acting consensus sequence, designated cre (catabolite response element), generally located within or downstream of the promoter of the repressed gene. Two sites resembling this sequence are centered at positions −116.5 and −56.5 of the ackA promoter and have been termedcre1 and cre2, respectively. Synthesis of acetate kinase, which is involved in the conversion of acetyl coenzyme A to acetate, is induced when cells are grown in the presence of an easily metabolized carbon source such as glucose. In this study,cre2, the site closer to the promoter, and the region upstream of cre2 were shown to be indispensable for CcpA-dependent transcriptional activation of ackA, whereascre1 was not required. In addition, insertion of 5 bp between cre2 and the promoter disrupted activation, while 10 bp was tolerated, suggesting face-of-the-helix dependence of the position of cre2 and/or upstream sequences. DNase footprinting experiments demonstrated binding of CcpA in vitro tocre2 but not cre1, consistent with the genetic data. Activation of ackA transcription was blocked in aptsH1/crh double mutant, suggesting involvement of this pathway in CcpA-mediated transcriptional activation.

scholarly journals The Carbohydrate Metabolism of Lactiplantibacillus plantarum

2021 ◽  
Vol 22 (24) ◽  
pp. 13452
Author(s):  
Yanhua Cui ◽  
Meihong Wang ◽  
Yankun Zheng ◽  
Kai Miao ◽  
Xiaojun Qu
Keyword(s):  
Carbohydrate Metabolism ◽  
Protein A ◽  
Sugar Metabolism ◽  
Regulatory Genes ◽  
Strain Specificity ◽  
Carbohydrate Utilization ◽  
Catabolite Control Protein A ◽  
Carbohydrate Fermentation ◽  
Two Component Systems ◽  
Carbohydrate Metabolism Genes

Lactiplantibacillus plantarum has a strong carbohydrate utilization ability. This characteristic plays an important role in its gastrointestinal tract colonization and probiotic effects. L. plantarum LP-F1 presents a high carbohydrate utilization capacity. The genome analysis of 165 L. plantarum strains indicated the species has a plenty of carbohydrate metabolism genes, presenting a strain specificity. Furthermore, two-component systems (TCSs) analysis revealed that the species has more TCSs than other lactic acid bacteria, and the distribution of TCS also shows the strain specificity. In order to clarify the sugar metabolism mechanism under different carbohydrate fermentation conditions, the expressions of 27 carbohydrate metabolism genes, catabolite control protein A (CcpA) gene ccpA, and TCSs genes were analyzed by quantitative real-time PCR technology. The correlation analysis between the expressions of regulatory genes and sugar metabolism genes showed that some regulatory genes were correlated with most of the sugar metabolism genes, suggesting that some TCSs might be involved in the regulation of sugar metabolism.

Molecular characterization of three mutations in katG affecting the activity of hydroperoxidase I of Escherichia coli

1990 ◽  
Vol 68 (7-8) ◽  
pp. 1037-1044 ◽  
Author(s):  
Peter C. Loewen ◽  
Jacek Switala ◽  
Mark Smolenski ◽  
Barbara L. Triggs-Raine
Keyword(s):  
Escherichia Coli ◽  
Specific Activity ◽  
Polymerase Chain Reaction Amplification ◽  
Protein A ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Gene Encoding ◽  
Reaction Amplification ◽  
Mutant Genes

Hydroperoxidase I (HPI) of Escherichia coli is a bifunctional enzyme exhibiting both catalase and peroxidase activities. Mutants lacking appreciable HPI have been generated using nitrosoguanidine and the gene encoding HPI, katG, has been cloned from three of these mutants using either classical probing methods or polymerase chain reaction amplification. The mutant genes were sequenced and the changes from wild-type sequence identified. Two mutants contained G to A changes in the coding strand, resulting in glycine to aspartate changes at residues 119 (katG15) and 314 (katG16) in the deduced amino acid sequence of the protein. A third mutant contained a C to T change resulting in a leucine to phenylalanine change at residue 139 (katG14). The Phe139-, Asp119-, and Asp314-containing mutants exhibited 13, < 1, and 18%, respectively, of the wild-type catalase specific activity and 43, 4, and 45% of the wild-type peroxidase specific activity. All mutant enzymes bound less protoheme IX than the wild-type enzyme. The sensitivities of the mutant enzymes to the inhibitors hydroxylamine, azide, and cyanide and the activators imidazole and Tris were similar to those of the wild-type enzyme. The mutant enzymes were more sensitive to high temperature and to β-mercaptoethanol than the wild-type enzyme. The pH profiles of the mutant catalases were unchanged from the wild-type enzyme.Key words: catalase, hydroperoxidase I, mutants, sequence analysis.

Carboxymethylhydroxymuconic semialdehyde dehydrogenase in the 4-hydroxyphenylacetate catabolic pathway of Pseudomonas putida

1986 ◽  
Vol 64 (12) ◽  
pp. 1288-1293 ◽  
Author(s):  
Josefa M. Alonso ◽  
Amando Garrido-Pertierra
Keyword(s):  
Pseudomonas Putida ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Competitive Inhibitor ◽  
Catabolic Pathway ◽  
Ph Optimum ◽  
Semialdehyde Dehydrogenase ◽  
Standard Assay ◽  
Assay Conditions

5-Carboxymethyl-2-hydroxymuconic semialdehyde (CHMSA) dehydrogenase in the 4-hydroxyphenylacetate meta-cleavage pathway was purified from Pseudomonas putida by gel filtration, anion-exchange, and affinity chromatographies. Sodium dodecyl sulfate – polyacrylamide gel electrophoresis analysis suggested an approximate tetrameric molecular weight of 200 000. The purified enzyme showed a pH optimum at 7.8. The temperature–activity relationship for the enzyme from 27 to 45 °C showed broken Arrhenius plots with an inflexion at 36–37 °C. Under standard assay conditions, the enzyme acted preferentially with NAD. It could also catalyze the reduction with NADP (which had a higher Km), at 18% of the rate observed for NAD. The following kinetic parameters were found: Km(NAD) = 20.0 ± 3.6 μM, Km(CHMSA) = 8.5 ± 1.8 μM, and Kd(enzyme–NAD complex) = 7.8 ± 2.0 μM. The product NADH acted as a competitive inhibitor against NAD.

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.

scholarly journals Expression of cbsA Encoding the Collagen-Binding S-Protein of Lactobacillus crispatusJCM5810 in Lactobacillus casei ATCC 393T

2000 ◽  
Vol 182 (23) ◽  
pp. 6857-6861 ◽  
Author(s):  
Beatriz Martı́nez ◽  
Jouko Sillanpää ◽  
Egbert Smit ◽  
Timo K. Korhonen ◽  
Peter H. Pouwels
Keyword(s):  
Cell Wall ◽  
Lactobacillus Casei ◽  
Surface Rendering ◽  
Recombinant Bacteria ◽  
Gene Encoding ◽  
Collagen Binding ◽  
S Protein ◽  
Translational Fusion ◽  
Sorting Signal ◽  
Lactobacillus Crispatus

The cbsA gene encoding the collagen-binding S-layer protein of Lactobacillus crispatus JCM5810 was expressed inL. casei ATCC 393T. The S-protein was not retained on the surface of the recombinant bacteria but was secreted into the medium. By translational fusion of CbsA to the cell wall sorting signal of the proteinase, PrtP, of L. casei, CbsA was presented at the surface, rendering the transformants able to bind to immobilized collagens.

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