scholarly journals Absence of Malolactic Activity Is a Characteristic of H+-ATPase-Deficient Mutants of the Lactic Acid Bacterium Oenococcus oeni

2003 ◽  
Vol 69 (4) ◽  
pp. 1973-1979 ◽  
Author(s):  
Delphine Galland ◽  
Raphaëlle Tourdot-Maréchal ◽  
Maud Abraham ◽  
Ky Son Chu ◽  
Jean Guzzo
Keyword(s):  
Lactic Acid ◽  
Regulatory Protein ◽  
Oenococcus Oeni ◽  
Growth Conditions ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Regulation Of Expression ◽  
Gene Encoding ◽  
Malolactic Enzyme ◽  
Malate Metabolism

ABSTRACT The lack of malolactic activity in H+-ATPase-deficient mutants of Oenococcus oeni selected previously was analyzed at the molecular level. Western blot experiments revealed a spot at 60 kDa corresponding to the malolactic enzyme only in the parental strain. Moreover, the mleA transcript encoding the malolactic enzyme was not detected by reverse transcription (RT)-PCR analysis of mutants. These results suggest that the malolactic operon was not transcribed in ATPase-deficient mutants. The mleR gene encoding a LysR-type regulatory protein which should be involved in expression of the malolactic genes was described previously for O. oeni. Results obtained in this study show that the mleR transcript was not detected in the mutants by RT-PCR. No mutation in the nucleotide sequences of the mleR gene and the malolactic operon was found. The effect of a reduction in H+-ATPase activity on l-malate metabolism was then investigated by using other malolactic bacteria. Spontaneous H+-ATPase-deficient mutant strains of Lactococcus lactis and Leuconostoc mesenteroides were isolated by using neomycin resistance. Two mutants were selected. These mutants exhibited ATPase activities that were reduced to 54 and 70% of the activities obtained for the L. lactis and L. mesenteroides parental strains, respectively. These mutants were also acid sensitive. However, in contrast to the ATPase-deficient mutants of O. oeni, activation of l-malate metabolism was observed with the L. lactis and L. mesenteroides mutants under optimal or acidic growth conditions. These data support the suggestion that expression of the genes encoding malolactic enzymes in O. oeni is regulated by the mleR product, as it is in L. lactis. Nevertheless, our results strongly suggest that there is a difference between the regulation of expression of the malolactic locus in O. oeni and the regulation of expression of this locus in less acidophilic lactic acid bacteria.

scholarly journals The fluorene catabolic linear plasmid in Terrabacter sp. strain DBF63 carries the β-ketoadipate pathway genes, pcaRHGBDCFIJ, also found in proteobacteria

Microbiology ◽  
2005 ◽  
Vol 151 (11) ◽  
pp. 3713-3722 ◽  
Author(s):  
Hiroshi Habe ◽  
Jin-Sung Chung ◽  
Ayako Ishida ◽  
Kano Kasuga ◽  
Kazuki Ide ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Tricarboxylic Acid Cycle ◽  
Bacterial Species ◽  
Linear Plasmid ◽  
Pcr Analysis ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Gene Encoding ◽  
Positive Trait ◽  
Tricarboxylic Acid Cycle Intermediates

Terrabacter sp. strain DBF63 is capable of degrading fluorene (FN) to tricarboxylic acid cycle intermediates via phthalate and protocatechuate. Genes were identified for the protocatechuate branch of the β-ketoadipate pathway (pcaR, pcaHGBDCFIJ) by sequence analysis of a 70 kb DNA region of the FN-catabolic linear plasmid pDBF1. RT-PCR analysis of RNA from DBF63 cells grown with FN, dibenzofuran, and protocatechuate indicated that the pcaHGBDCFIJ operon was expressed during both FN and protocatechuate degradation in strain DBF63. The gene encoding β-ketoadipate enol-lactone hydrolase (pcaD) was not fused to the next gene, which encodes γ-carboxymuconolactone decarboxylase (pcaC), in strain DBF63, even though the presence of the pcaL gene (the fusion of pcaD and pcaC) within a pca gene cluster has been thought to be a Gram-positive trait. Quantitative RT-PCR analysis revealed that pcaD mRNA levels increased sharply in response to protocatechuate, and a biotransformation experiment with cis,cis-muconate using Escherichia coli carrying both catBC and pcaD indicated that PcaD exhibited β-ketoadipate enol-lactone hydrolase activity. The location of the pca gene cluster on the linear plasmid, and the insertion sequences around the pca gene cluster suggest that the ecologically important β-ketoadipate pathway genes, usually located chromosomally, may be spread widely among bacterial species via horizontal transfer or transposition events.

scholarly journals Modulation of transcription and characterization of the promoter organization of the autotransporter adhesin heptosyltransferase and the autotransporter adhesin AIDA-I

Microbiology ◽  
2010 ◽  
Vol 156 (4) ◽  
pp. 1155-1166 ◽  
Author(s):  
Inga Benz ◽  
Tessa van Alen ◽  
Julia Bolte ◽  
Mirka E. Wörmann ◽  
M. Alexander Schmidt
Keyword(s):  
Protein Translocation ◽  
Growth Conditions ◽  
Limited Attention ◽  
Gram Negative Bacteria ◽  
Regulation Of Expression ◽  
Gene Encoding ◽  
E Coli ◽  
K 12 ◽  
Transcriptional Fusions

In Gram-negative bacteria, autotransporter proteins constitute the largest family of secreted proteins, and exhibit many different functions. In recent years, research has largely focused on mechanisms of autotransporter protein translocation, where several alternative models are still being discussed. In contrast, the biogenesis of only a few autotransporters has been studied and, likewise, regulation of expression has received only very limited attention. The glycosylated autotransporter adhesin involved in diffuse adherence (AIDA)-I system consists of the aah gene, encoding a specific autotransporter adhesin heptosyltransferase (AAH), and the aidA gene, encoding the autotransporter protein (AIDA-I). In this study, we investigated the promoter organization and transcription of these two genes using reporter plasmids carrying lacZ transcriptional fusions. The two genes, aah and aidA, are transcribed as a bicistronic message. However, aidA is additionally transcribed from its own promoter. There are two distinct start sites for each of the two genes. Interestingly, transcription of both genes is enhanced in hns and rfaH mutant backgrounds. Furthermore, we addressed the influence of environmental factors and different genetic backgrounds of Escherichia coli K-12 strains on transcription activity. We found that transcription varied considerably in different E. coli K-12 laboratory strains and under different growth conditions.

Molecular characterization and expression analysis of a gene encoding mannose-binding lectin from bulb of Zephyranthes grandiflora

Biologia ◽  
2006 ◽  
Vol 61 (6) ◽  
Author(s):  
Guoyin Kai ◽  
Yang Lu ◽  
Zhongying Qian ◽  
Yongting Luo ◽  
Genyu Zhou ◽  
...  
Keyword(s):  
Mannose Binding Lectin ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Gene Encoding ◽  
Acid Protein ◽  
Mannose Binding ◽  
Lectin Family ◽  
Binding Lectin ◽  
Amino Acid Protein ◽  
Chinese Medicinal Plant

AbstractIn this paper we report on the molecular cloning, sequencing and partially characterisation of a lectin from bulb of the Chinese medicinal plant Zephyranthes grandiflora. The full-length cDNA of Z. grandiflora bulb lectin (ZGBL) consisted of 986 bp and contained a 576 bp ORF encoding a 191 amino acid protein. Bioinformatics analysis results clearly indicate that ZGBL belongs to the monocot mannose-binding lectin family, which contains 3 putative mannose-binding sites per subunit. RT-PCR analysis results indicate that ZGBL is constitutively expressed in all the tested tissue types including root, bulb, leaf and flower. Interestingly, ZGBL is more closely related to the Orchidaceae rather than the Amaryllidaceae family on molecular evolution.

scholarly journals Liver receptor homologue-1 is expressed in the adrenal and can regulate transcription of 11 beta-hydroxylase

2001 ◽  
Vol 27 (2) ◽  
pp. 255-258 ◽  
Author(s):  
ZN Wang ◽  
M Bassett ◽  
WE Rainey
Keyword(s):  
Adrenal Glands ◽  
Pcr Analysis ◽  
Orphan Nuclear Receptor ◽  
Rt Pcr ◽  
Hormone Production ◽  
Mobility Shift ◽  
Gene Encoding ◽  
Liver And Pancreas ◽  
Genes Encoding ◽  
Mobility Shift Assay

Liver receptor homologue-1 (LRH-1, designated NR5A2) is a mammalian homologue of Drosophila fushi tarazu factor (dFTZ-F1) and structurally belongs to the orphan nuclear receptor superfamily. LRH-1 can recognize the DNA sequence 5'-AAGGTCA-3', the canonical recognition motif for steroidogenic factor 1 (SF-1). Herein, we hypothesized that LRH-1 might play a role in the regulation of human adrenal expression of steroidogenic enzymes. To test this hypothesis, LRH-1 expression in human adult and fetal adrenal glands was examined by RT-PCR analysis. The fetal and adult adrenal glands, as well as liver and pancreas, were observed to express LRH-1 mRNA using RT-PCR. The ability of LRH-1 to enhance transcription of the gene encoding human 11 beta- hydroxylase (hCYP11B1) was then examined using the H295R adrenal cell line. LRH-1 co-transfection with hCYP11B1 luciferase promoter constructs caused a 25-fold induction of luciferase activity. Furthermore, co-transfection of a hCYP11B1 reporter construct containing a mutation in the SF-1 binding cis-element abolished the stimulatory effect of both SF-1 and LRH-1. Electrophoretic mobility shift assay (EMSA) demonstrated that LRH-1 could bind to the SF-1 response element. Taken together, our data suggested that LRH-1 is expressed in the adrenal, and can substitute for SF-1 to enhance transcription of genes encoding certain of the steroid-metabolizing enzymes. A role for LRH-1 in the regulation of adrenal or gonadal steroid hormone production should be further studied.

Pediococcus parvulus gtf Gene Encoding the GTF Glycosyltransferase and Its Application for Specific PCR Detection of β-d-Glucan–Producing Bacteria in Foods and Beverages

2006 ◽  
Vol 69 (1) ◽  
pp. 161-169 ◽  
Author(s):  
MARIA LAURA WERNING ◽  
IDOIA IBARBURU ◽  
MARIA TERESA DUEÑAS ◽  
ANA IRASTORZA ◽  
JESÚS NAVAS ◽  
...  
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Blot Hybridization ◽  
Pcr Amplification ◽  
Southern Blot Hybridization ◽  
Oenococcus Oeni ◽  
Alcoholic Beverages ◽  
Gene Encoding ◽  
Specific Pcr ◽  
Genomic Locations

Exopolysaccharide production by lactic acid bacteria is beneficial in the dairy and oat-based food industries and is used to improve the texture of the fermented products. However, β-D-glucan–producing bacteria are considered spoilage microorganisms in alcoholic beverages because their secreted exopolysaccharides alter the viscosity of cider, wine, and beer, rendering them unpalatable. The plasmidic glycosyltransferase (gtf) gene of the Pediococcus parvulus 2.6 strain isolated from ropy cider has been cloned and sequenced, and its GTF product was functionally expressed in Streptococcus pneumoniae. The GTF protein, which has glycosyltransferase activity, belongs to the COG1215 membrane-bound glycosyltransferase family, and agglutination tests revealed that the enzyme enables S. pneumoniae to synthesize β-D-glucan. PCR amplification and Southern blot hybridization showed that the gtf gene is also present at different genomic locations in the β-d-glucan producers Lacto-bacillus diolivorans G77 and Oenococcus oeni I4 strains, also isolated from ropy cider. A PCR assay has been developed for the detection of exopolysaccharide-producing bacteria. Forward and reverse primers, included respectively in the coding sequences of the putative glycosyltransferase domain and the fifth trans-membrane segment of the GTF, were designed. Analysis of 76 ropy and nonropy lactic acid bacteria validated the method for specific detection of β-d-glucan homopolysaccharide producer Pediococcus, Lactobacillus, and Oenococcus strains. The limit of the assay in cider was 3 × 102 CFU/ml. This molecular method can be useful for the detection of ropy bacteria in cider before spoilage occurs, as well as for isolation of new exopolysaccharide-producing strains of industrial interest.

scholarly journals Expression of the Gonococcal Global Regulatory Protein Fur and Genes Encompassing the Fur and Iron Regulon during In Vitro and In Vivo Infection in Women

2008 ◽  
Vol 190 (9) ◽  
pp. 3129-3139 ◽  
Author(s):  
Sarika Agarwal ◽  
Shite Sebastian ◽  
Borys Szmigielski ◽  
Peter A. Rice ◽  
Caroline A. Genco
Keyword(s):  
Epithelial Cells ◽  
Microarray Analysis ◽  
Regulatory Protein ◽  
Growth Conditions ◽  
Pcr Analysis ◽  
Gonococcal Infection ◽  
Cervical Epithelial Cells ◽  
Female Subjects

ABSTRACT The ferric uptake regulatory protein, Fur, functions as a global regulatory protein of gene transcription in the mucosal pathogen Neisseria gonorrhoeae. We have shown previously that several N. gonorrhoeae Fur-repressed genes are expressed in vivo during mucosal gonococcal infection in men, which suggests that this organism infects in an iron-limited environment and that Fur is expressed under these conditions. In this study we have demonstrated expression of the gonococcal fur gene in vitro, in human cervical epithelial cells, and in specimens from female subjects with uncomplicated gonococcal infection. In vitro studies confirmed that the expression of the gonococcal fur gene was repressed during growth under iron-replete growth conditions but that a basal level of the protein was maintained. Using GFP transcriptional fusions constructed from specific Fur binding sequences within the fur promoter/operator region, we determined that this operator region was functional during N. gonorrhoeae infection of cervical epithelial cells. Furthermore, reverse transcription-PCR analysis, as well as microarray analysis, using a custom Neisseria Fur and iron regulon microarray revealed that several Fur- and iron-regulated genes were expressed during N. gonorrhoeae infection of cervical epithelial cells. Microarray analysis of specimens obtained from female subjects with uncomplicated gonococcal infection corroborated our in vitro findings and point toward a key role of gonococcal Fur- and iron-regulated genes in gonococcal disease.

scholarly journals Evidence for Multiple Levels of Regulation of Oenococcus oeni clpP-clpL Locus Expression in Response to Stress

2004 ◽  
Vol 186 (7) ◽  
pp. 2200-2205 ◽  
Author(s):  
Charlotte Beltramo ◽  
Cosette Grandvalet ◽  
Fabrice Pierre ◽  
Jean Guzzo
Keyword(s):  
Lactic Acid ◽  
Reverse Transcription ◽  
Mrna Stability ◽  
Oenococcus Oeni ◽  
Pcr Analysis ◽  
Reverse Transcription Pcr ◽  
Atpase Gene ◽  
Response To Stress ◽  
Multiple Levels ◽  
Posttranscriptional Level

ABSTRACT A locus containing the clpP and clpL genes in the lactic acid bacterium Oenococcus oeni was studied. Real-time reverse transcription-PCR analysis revealed different induction factors involved in expression of these genes during stress. According to the conditions, clpP and clpL genes could be transcripted as two distinct transcripts or cotranscripted. The clpP promoter depended on the CtsR regulator, but surprisingly the clpL promoter did not. The amount of the clpL transcript depended on mRNA stability. This clp ATPase gene is at least controlled at the posttranscriptional level.

scholarly journals Phenotypical and Functional Characteristics of Human Regulatory T Cells during Ex Vivo Maturation from CD4+ T Lymphocytes

2021 ◽  
Vol 11 (13) ◽  
pp. 5776
Author(s):  
Varvara G. Blinova ◽  
Natalia S. Novachly ◽  
Sofya N. Gippius ◽  
Abdullah Hilal ◽  
Yulia A. Gladilina ◽  
...  
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Ex Vivo ◽  
Pcr Analysis ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Inflammatory Reactions ◽  
Effector Cells ◽  
Cycle Regulation ◽  
Further Development

Regulatory T cells (Tregs) participate in the negative regulation of inflammatory reactions by suppressing effector cells. In a number of autoimmune disorders, the suppressive function and/or the number of Tregs is compromised. The lack of active functioning Tregs can be restored with adoptive transfer of expanded ex vivo autologous Tregs. In our study, we traced the differentiation and maturation of Tregs CD4+CD25+FoxP3+CD127low over 7 days of cultivation from initial CD4+ T cells under ex vivo conditions. The resulting ex vivo expanded cell population (eTregs) demonstrated the immune profile of Tregs with an increased capacity to suppress the proliferation of target effector cells. The expression of the FoxP3 gene was upregulated within the time of expansion and was associated with gradual demethylation in the promotor region of the T cell-specific demethylation region. Real-time RT-PCR analysis revealed changes in the expression profile of genes involved in cell cycle regulation. In addition to FOXP3, the cells displayed elevated mRNA levels of Ikaros zinc finger transcription factors and the main telomerase catalytic subunit hTERT. Alternative splicing of FoxP3, hTERT and IKZF family members was demonstrated to be involved in eTreg maturation. Our data indicate that expanded ex vivo eTregs develop a Treg-specific phenotype and functional suppressive activity. We suggest that eTregs are not just expanded but transformed cells with enhanced capacities of immune suppression. Our findings may influence further development of cell immunosuppressive therapy based on regulatory T cells.

scholarly journals Lactobacillus reuteri BM53-1 Produces a Compound That Inhibits Sticky Glucan Synthesis by Streptococcus mutans

2021 ◽  
Vol 9 (7) ◽  
pp. 1390
Author(s):  
Masafumi Noda ◽  
Naho Sugihara ◽  
Yoshimi Sugimoto ◽  
Ikue Hayashi ◽  
Sachiko Sugimoto ◽  
...  
Keyword(s):  
Antibacterial Activity ◽  
Lactic Acid ◽  
Dental Caries ◽  
Lactobacillus Reuteri ◽  
Early Stage ◽  
Pcr Analysis ◽  
Cariogenic Bacteria ◽  
Qrt Pcr ◽  
Actinidia Polygama ◽  
Glucan Synthesis

Cariogenic bacteria, such as Streptococcus (S.) mutans and S. sobrinus, produce insoluble and sticky glucans as a biofilm material. The present study demonstrates that a lactic acid bacterium (LAB) named BM53-1 produces a substance that inhibits the sticky glucan synthesis. The BM53-1 strain was isolated from a flower of Actinidia polygama and identified as Lactobacillus reuteri. The substance that inhibits sticky glucan synthesis does not exhibit antibacterial activity against S. mutans. The cariogenic S. mutans produces glucans under the control of three glucosyltransferase (GTF) enzymes, named GtfB, GtfC, and GtfD. Although GtfB and GtfC produce insoluble glucans, GtfD forms soluble glucans. Through quantitative reverse-transcriptional (qRT)-PCR analysis, it was revealed that the BM53-1-derived glucan-production inhibitor (GI) enhances the transcriptions of gtfB and gtfC genes 2- to 7-fold at the early stage of cultivation. However, that of gtfD was not enhanced in the presence of the GI, indicating that the glucan stickiness produced by S. mutans was significantly weaker in the presence of the GI. Our result demonstrates that Lb. reuteri BM53-1 is useful to prevent dental caries.

scholarly journals Native Yeasts and Lactic Acid Bacteria Isolated from Spontaneous Fermentation of Seven Grape Cultivars from the Maule Region (Chile)

Foods ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 1737
Author(s):  
Wendy Franco ◽  
Sergio Benavides ◽  
Pedro Valencia ◽  
Cristian Ramírez ◽  
Alejandra Urtubia
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Regional Growth ◽  
Starter Culture ◽  
Growth Conditions ◽  
Microbial Evolution ◽  
Spontaneous Fermentation ◽  
Wine Production ◽  
Grape Juices ◽  
Grape Cultivars

Grapes are a source of native yeasts and lactic acid bacteria (LAB); however, the microbial make up is dependent on the grape cultivar and the regional growth conditions. Therefore, the aim of this study was to characterize the yeast and LAB in seven grape cultivars cultivated in Chile. Grape juices were fermented at 25 °C for 7 days. Samples were collected to analyze sugar, organic acids, and ethanol. Microbial evolution was measured with culture-dependent and molecular approaches. Then, a native isolated Candida oleophila was selected for further sequential fermentations with Saccharomyces cerevisiae. The grape cultivars in the Maule showed a diversity of non-Saccharomyces yeasts, with a greater diversity observed at the beginning of the fermentation. However, species from the Hansenasporia, Metschnikowia, Torulaspora, Lachancea, and Candida genera were detected after 7 days, suggesting tolerance to environments rich in ethanol, capability may be associated to the terroir studied, which is characterized by torrid weather and antique and traditional vineyards. The alcoholic fermentation negatively impacted the LAB population, and after 7 days only Leuconostoc mesenteroides was isolated. In the sequential fermentations, C. oleophila was able to produce fermented grape juices with <1.5 g/L glucose, 12.5% (v/v) alcohol, and low concentrations of malic (<1.00 g/L) and succinic (2.05 g/L) acids, while acetic acid reached values >0.3 (g/L). To our knowledge this is the first time C. oleophila has been reported as a potential starter culture for wine production. However, more studies are necessary to fully characterize the potential of C. oleophila on wine attributes.

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