Transcriptional Pattern of Genes Coding for the Proteolytic System of Lactococcus lactis and Evidence for Coordinated Regulation of Key Enzymes by Peptide Supply

ABSTRACT The transcription of 16 genes encoding 12 peptidases (pepC, pepN, pepX, pepP, pepA, pepF2, pepDA1, pepDA2, pepQ, pepT, pepM, and pepO1), PI and PIII proteinases (prtP1 and prtP3), and three transport systems (dtpT, dtpP, and opp-pepO1) ofLactococcus lactis MG1363 was analyzed in response to different environmental factors. Promoter fusions with luciferase reporter genes and/or mRNA analysis were used to study the effects of sugar sources, growth at 37°C, and peptide supply on the transcription of these genes. Only transcription of thepepP gene is modulated by the source of sugar. The presence of potential catabolite-responsive element (CRE) boxes in its promoter region suggests that expression of this gene is directly controlled by catabolic repression. Elevated temperature had no significant effect on the level of transcription of these genes. prtP1, prtP3, pepC, pepN, pepX, and the opp-pepO1 operon are the most highly expressed genes in chemically defined medium, and their expression is repressed 5- to 150-fold by addition of peptide sources such as Casitone in the medium. Moreover, the transcription ofprtP1, prtP3, pepC, pepN, and the opp-pepO1operon is repressed two- to eight-fold by the dipeptides leucylproline and prolylleucine. The transcription of pepDA2 might also be repressed by the peptide sources, but this effect is not observed on the regulation of dtpT, pepP, pepA, pepF2, pepDA1, pepQ, pepT, pepM, and the dtpP operon. The significance of these results with respect to the functions of different components of the proteolytic system in L. lactis are discussed.

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A General Method for Selection of α-Acetolactate Decarboxylase-Deficient Lactococcus lactis Mutants To Improve Diacetyl Formation

Applied and Environmental Microbiology ◽

10.1128/aem.65.3.1202-1206.1999 ◽

1999 ◽

Vol 65 (3) ◽

pp. 1202-1206 ◽

Cited By ~ 33

Author(s):

Mirjana Curic ◽

Birgitte Stuer-Lauridsen ◽

Pierre Renault ◽

Dan Nilsson

Keyword(s):

Amino Acids ◽

Lactococcus Lactis ◽

Genetically Modified Organisms ◽

Defined Medium ◽

Isoleucine Leucine ◽

Genes Encoding ◽

Resistant Mutants ◽

Branched Chain ◽

Selection Of

ABSTRACT The enzyme acetolactate decarboxylase (Ald) plays a key role in the regulation of the α-acetolactate pool in both pyruvate catabolism and the biosynthesis of the branched-chain amino acids, isoleucine, leucine, and valine (ILV). This dual role of Ald, due to allosteric activation by leucine, was used as a strategy for the isolation of Ald-deficient mutants of Lactococcus lactis subsp.lactis biovar diacetylactis. Such mutants can be selected as leucine-resistant mutants in ILV- or IV-prototrophic strains. Most dairy lactococcus strains are auxotrophic for the three amino acids. Therefore, the plasmid pMC004 containing the ilv genes (encoding the enzymes involved in the biosynthesis of IV) of L. lactis NCDO2118 was constructed. Introduction of pMC004 into ILV-auxotrophic dairy strains resulted in an isoleucine-prototrophic phenotype. By plating the strains on a chemically defined medium supplemented with leucine but not valine and isoleucine, spontaneous leucine-resistant mutants were obtained. These mutants were screened by Western blotting with Ald-specific antibodies for the presence of Ald. Selected mutants lacking Ald were subsequently cured of pMC004. Except for a defect in the expression of Ald, the resulting strain, MC010, was identical to the wild-type strain, as shown by Southern blotting and DNA fingerprinting. The mutation resulting in the lack of Ald in MC010 occurred spontaneously, and the strain does not contain foreign DNA; thus, it can be regarded as food grade. Nevertheless, its application in dairy products depends on the regulation of genetically modified organisms. These results establish a strategy to select spontaneous Ald-deficient mutants from transformable L. lactis strains.

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Two Nucleoside Uptake Systems in Lactococcus lactis: Competition between Purine Nucleosides and Cytidine Allows for Modulation of Intracellular Nucleotide Pools

Journal of Bacteriology ◽

10.1128/jb.185.5.1503-1508.2003 ◽

2003 ◽

Vol 185 (5) ◽

pp. 1503-1508 ◽

Cited By ~ 25

Author(s):

Jan Martinussen ◽

Steen L. L. Wadskov-Hansen ◽

Karin Hammer

Keyword(s):

Growth Rate ◽

Lactococcus Lactis ◽

Laboratory Strain ◽

Defined Medium ◽

Nucleoside Transport ◽

Transport Systems ◽

Nucleoside Triphosphate ◽

Buffer System ◽

Uridine Uptake ◽

Nucleotide Pools

ABSTRACT A method for measuring internal nucleoside triphosphate pools of lactococci was optimized and validated. This method is based on extraction of 33P-labeled nucleotides with formic acid and evaluation by two-dimensional chromatography with a phosphate buffer system for the first dimension and with an H3BO3-LiOH buffer for separation in the second dimension. We report here the sizes of the ribo- and deoxyribonucleotide pools in laboratory strain MG1363 during growth in a defined medium. We found that purine- and pyrimidine-requiring strains may be used to establish physiological conditions in batch fermentations with altered nucleotide pools and growth rates by addition of nucleosides in different combinations. Addition of cytidine together with inosine to a purine-requiring strain leads to a reduction in the internal purine nucleotide pools and a decreased growth rate. This effect was not seen if cytidine was replaced by uridine. A similar effect was observed if cytidine and inosine were added to a pyrimidine-requiring strain; the UTP pool size was significantly decreased, and the growth rate was reduced. To explain the observed inhibition, the nucleoside transport systems in Lactococcus lactis were investigated by measuring the uptake of radioactively labeled nucleosides. The Km for for inosine, cytidine, and uridine was determined to be in the micromolar range. Furthermore, it was found that cytidine and inosine are competitive inhibitors of each other, whereas no competition was found between uridine and either cytidine or inosine. These findings suggest that there are two different high-affinity nucleoside transporters, one system responsible for uridine uptake and another system responsible for the uptake of all purine nucleosides and cytidine.

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Characterization of the Role of para-Aminobenzoic Acid Biosynthesis in Folate Production by Lactococcus lactis

Applied and Environmental Microbiology ◽

10.1128/aem.02174-06 ◽

2007 ◽

Vol 73 (8) ◽

pp. 2673-2681 ◽

Cited By ~ 77

Author(s):

Arno Wegkamp ◽

Wietske van Oorschot ◽

Willem M. de Vos ◽

Eddy J. Smid

Keyword(s):

Gene Cluster ◽

Lactococcus Lactis ◽

Gene Clusters ◽

Defined Medium ◽

Biosynthesis Pathway ◽

Chemically Defined Medium ◽

Aminobenzoic Acid ◽

Biosynthesis Gene Cluster ◽

Gene Coding ◽

Aba Biosynthesis

ABSTRACT The pab genes for para-aminobenzoic acid (pABA) biosynthesis in Lactococcus lactis were identified and characterized. In L. lactis NZ9000, only two of the three genes needed for pABA production were initially found. No gene coding for 4-amino-4-deoxychorismate lyase (pabC) was initially annotated, but detailed analysis revealed that pabC was fused with the 3′ end of the gene coding for chorismate synthetase component II (pabB). Therefore, we hypothesize that all three enzyme activities needed for pABA production are present in L. lactis, allowing for the production of pABA. Indeed, the overexpression of the pABA gene cluster in L. lactis resulted in elevated pABA pools, demonstrating that the genes are involved in the biosynthesis of pABA. Moreover, a pABA knockout (KO) strain lacking pabA and pabB C was constructed and shown to be unable to produce folate when cultivated in the absence of pABA. This KO strain was unable to grow in chemically defined medium lacking glycine, serine, nucleobases/nucleosides, and pABA. The addition of the purine guanine, adenine, xanthine, or inosine restored growth but not the production of folate. This suggests that, in the presence of purines, folate is not essential for the growth of L. lactis. It also shows that folate is not strictly required for the pyrimidine biosynthesis pathway. L. lactis strain NZ7024, overexpressing both the folate and pABA gene clusters, was found to produce 2.7 mg of folate/liter per optical density unit at 600 nm when the strain was grown on chemically defined medium without pABA. This is in sharp contrast to L. lactis strains overexpressing only one of the two gene clusters. Therefore, we conclude that elevated folate levels can be obtained only by the overexpression of folate combined with the overexpression of the pABA biosynthesis gene cluster, suggesting the need for a balanced carbon flux through the folate and pABA biosynthesis pathway in the wild-type strain.

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Potency of isothiocyanates to induce luciferase reporter gene expression via the electrophile-responsive element from murine glutathione S-transferase Ya

Toxicology in Vitro ◽

10.1016/j.tiv.2009.02.005 ◽

2009 ◽

Vol 23 (4) ◽

pp. 617-621 ◽

Cited By ~ 8

Author(s):

Martijn Vermeulen ◽

Anne-Marie M.J.F. Boerboom ◽

Barry M.G. Blankvoort ◽

Jac M.M.J.G. Aarts ◽

Ivonne M.C.M. Rietjens ◽

...

Keyword(s):

Gene Expression ◽

Reporter Gene ◽

Reporter Gene Expression ◽

Luciferase Reporter ◽

Glutathione S Transferase ◽

Luciferase Reporter Gene ◽

Responsive Element

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Effect of serum proteins on sodium-linked transport processes in cultured kidney cells.

Journal of the American Society of Nephrology ◽

10.1681/asn.v5111964 ◽

1995 ◽

Vol 5 (11) ◽

pp. 1964-1970

Author(s):

S S Blumenthal ◽

D L Lewand ◽

P A Tipnis ◽

J G Kleinman

Keyword(s):

Amino Acid ◽

Dextran Sulfate ◽

Cultured Cells ◽

Serum Proteins ◽

Fluid Phase ◽

Defined Medium ◽

Transport Processes ◽

Transport Systems ◽

Heat Treated ◽

Fluid Phase Endocytosis

The mechanism for increased Na+ retention in the nephrotic syndrome is unknown. To determine if Na+ transport systems in the proximal tubule might be affected by filtered proteins, mouse cortical tubule cells grown in defined medium were exposed to concentrations of bovine serum albumin (BSA) ranging from 0.01 to 0.5%. Activity of the Na(+)-glucose cotransporter, measured as Na(+)-dependent uptake of alpha-methylglucoside, increased progressively to a maximum of 2.3-fold above baseline (P < 0.001; N = 10). The increase in transporter activity was due to an increased Vmax, and the magnitude of the increase was inversely related to the basal cotransporter activity of the cultures. Increased cotransporter activity was detectable 6 h after exposure, was sustained for 24 h after cells were removed from an albumin-free medium, and was prevented by cycloheximide. Heat-treated BSA, fatty-acid and globulin-free BSA, and gamma-globulins were as effective at increasing Na(+)-glucose cotransporter activity as untreated Fraction V BSA. Dextran, dextran-sulfate, and amino acid supplements were ineffective. Neither protease inhibitors nor chloroquine added to an albumin-containing medium prevented increased alpha-methylglucoside uptake. Albumin did not change the rate of fluid-phase endocytosis in the cultured cells. Na(+)-amino acid cotransport and Na(+)-H+ exchange were either decreased or unchanged after BSA exposure. Exposing apical surfaces of cells grown on permeable membranes to BSA led to a greater increase in activity of the Na(+)-glucose cotransporter relative to controls than did exposing the basolateral surface (145 versus 89%; P < 0.05; N = 5).(ABSTRACT TRUNCATED AT 250 WORDS)

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Amino acid uptake systems in Bacteroides ruminicola

Canadian Journal of Microbiology ◽

10.1139/m79-180 ◽

1979 ◽

Vol 25 (10) ◽

pp. 1161-1168 ◽

Cited By ~ 15

Author(s):

Roselynn M. W. Stevenson

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Nitrogen Sources ◽

Amino Acid Uptake ◽

Defined Medium ◽

High Rate ◽

Transport Systems ◽

Acid Uptake ◽

Chemical Structures ◽

Kinetic Inhibition

Uptake of amino acids by Bacteroides ruminicola was observed in cells grown in a complete defined medium, containing ammonia as the nitrogen source. A high rate of uptake occurred only in fresh medium, as an inhibitory substance, possibly acetate, apparently accumulated during growth. All amino acids except proline were taken up and incorporated into cold trichloroacetic acid precipitable material. Different patterns of incorporation and different responses to 2,4-dinitrophenol and potassium ferricyanide indicated multiple uptake systems were involved. Kinetic inhibition patterns suggested six distinct systems were present for amino acid uptake, with specificities related to the chemical structures of the amino acids. Thus, the failure of free amino acids to act as sole nitrogen sources for growth of B. ruminicola is not due to the absence of transport systems for these compounds.

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microRNA and mRNA profiles in the amygdala are relevant to fear memory induced by physical or psychological stress

Journal of Neurophysiology ◽

10.1152/jn.00215.2019 ◽

2019 ◽

Vol 122 (3) ◽

pp. 1002-1022 ◽

Cited By ~ 9

Author(s):

Yan Sun ◽

Wei Lu ◽

Kaixin Du ◽

Jin-Hui Wang

Keyword(s):

Psychological Stress ◽

High Throughput Sequencing ◽

Fear Memory ◽

Physical Stress ◽

Luciferase Reporter ◽

Pcr Analysis ◽

Rt Pcr ◽

Genes Encoding ◽

Reporter Assays ◽

Molecular Profiles

Anxiety is presumably driven by fear memory. Molecular profiles in the amygdala of mice with fear memory induced by psychological and physical stresses remain to be elucidated. Fear memory in mice was induced by a paradigm of social defeat. Physical and psychological stresses (PPS) to an intruder were given by attacks from an aggressive resident. Psychological stress (PS) to an observer was given by the witnessing of aggressor attacks. Amygdala tissues from these mice showing fear memory and anxiety vs. tissues from control mice were harvested to analyze mRNA and microRNA profiles by high-throughput sequencing. In the amygdala of intruders and observers with fear memory, the genes encoding 5-HTR1b, 5-HTR2a, DAR2, AChRM3, and IP3R1 are upregulated, whereas genes encoding GPγ11, GPγ13, GPγT2, RasC3, and P450 are downregulated, indicating that these molecules are involved in fear memory induced by physical/psychological stresses. In the comparison of intruders with observers, the upregulation of genes encoding 5-HTR6, GPγ8, P2R7, NFκ2, CREB3/1, and Itgα9 as well as the downregulation of genes encoding DAR5, 5-HTR1a, and HSP1a are involved in fear memory induced by physical stress. The upregulation of genes encoding DAR1, 5-HTR5a and SSR2/3 as well as the downregulation of AdRα1, CREB3/1, GPγ13 and GPγ8 are involved in fear memory induced by psychological stress. Results obtained by sequencing mRNA and microRNA profiles are consistent with results of quantitative RT-PCR analysis and dual-luciferase reporter assays performed for validation. In conclusion, fear memories and anxiety induced by PPS vs. PS are caused by the imbalanced regulation of different synapses and signaling pathways in the amygdala. NEW & NOTEWORTHY The current study identifies the molecular mechanism underlying fear memory and anxiety induced by psychological stress vs. physical stress, in which the imbalanced expression of microRNA-regulated mRNAs relevant to dopaminergic, adrenergic, and serotonergic synapses in the amygdala plays an important role. This result reveals different molecular profiles for psychological and physical stresses.

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The Oligopeptide Transport System Is Essential for the Development of Natural Competence in Streptococcus thermophilus Strain LMD-9

Journal of Bacteriology ◽

10.1128/jb.00257-09 ◽

2009 ◽

Vol 191 (14) ◽

pp. 4647-4655 ◽

Cited By ~ 97

Author(s):

Rozenn Gardan ◽

Colette Besset ◽

Alain Guillot ◽

Christophe Gitton ◽

Véronique Monnet

Keyword(s):

Quorum Sensing ◽

Streptococcus Thermophilus ◽

Defined Medium ◽

Mass Spectrometry Analysis ◽

Transport Systems ◽

Label Free ◽

Spectrometry Analysis ◽

Proteomic Approach ◽

Liquid Chromatography Tandem Mass ◽

Oligopeptide Transport

ABSTRACT In gram-positive bacteria, oligopeptide transport systems, called Opp or Ami, play a role in nutrition but are also involved in the internalization of signaling peptides that take part in the functioning of quorum-sensing pathways. Our objective was to reveal functions that are controlled by Ami via quorum-sensing mechanisms in Streptococcus thermophilus, a nonpathogenic bacterium widely used in dairy technology in association with other bacteria. Using a label-free proteomic approach combining one-dimensional electrophoresis with liquid chromatography-tandem mass spectrometry analysis, we compared the proteome of the S. thermophilus LMD-9 to that of a mutant deleted for the subunits C, D, and E of the ami operon. Both strains were grown in a chemically defined medium (CDM) without peptides. We focused our attention on proteins that were no more detected in the ami deletion mutant. In addition to the three subunits of the Ami transporter, 17 proteins fulfilled this criterion and, among them, 7 were similar to proteins that have been identified as essential for transformation in S. pneumoniae. These results led us to find a condition of growth, the early exponential state in CDM, that allows natural transformation in S. thermophilus LMD-9 to turn on spontaneously. Cells were not competent in M17 rich medium. Furthermore, we demonstrated that the Ami transporter controls the triggering of the competence state through the control of the transcription of comX, itself controlling the transcription of late competence genes. We also showed that one of the two oligopeptide-binding proteins of strain LMD-9 plays the predominant role in the control of competence.

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Transcriptional Modulation of Resistance against Xanthomonas oryzae pv. oryzae Korean Race K2 in japonica Rice

Agronomy ◽

10.3390/agronomy10070960 ◽

2020 ◽

Vol 10 (7) ◽

pp. 960

Author(s):

Marjohn C. Niño ◽

Yong-Gu Cho

Keyword(s):

Bacterial Blight ◽

Cellular Reprogramming ◽

Xanthomonas Oryzae ◽

Common Disease ◽

Post Inoculation ◽

Japonica Rice ◽

Defense Compounds ◽

Twin Arginine Translocation ◽

Genes Encoding ◽

Highly Expressed Genes

Bacterial blight is a common disease found in the rice-growing regions in the Korean peninsula. Identification of the gene network involved against Xanthomonas oryzae pv. oryzae Korean race K2 in popular japonica cultivars is essential in underpinning the molecular mechanism of resistance. A microarray of two popular Korean japonica rice cultivars, a bacterial blight susceptible Dongjin and resistant Jinbaek, was performed to investigate the transcripts of inducible genes at 48 h post-inoculation. A total of 771 differentially expressed genes were identified in Jinbaek, whereas 298 were found in Dongjin. The resistance observed in Jinbaek is likely participated by genes with predicted functions in transmembrane perception, intracellular signal transduction, and transcription activity. Moreover, the remarkable involvement of numerous WRKY proteins signifies orchestration of defense signals via robust cellular reprogramming, which leads to resistance. To discover genes essential to bacterial blight resistance in Jinbaek, 13 highly expressed genes encoding different protein classes were cloned and overexpressed in rice. Although none of the overexpression plants exhibited resistance comparable to Jinbaek, four candidate genes, including one twin-arginine translocation pathway signal (LOC_Os01g45640.1), one cytochrome p450 (LOC_Os09g10340.1), and two uncharacterized expressed protein (LOC_Os08g26230.4, LOC_Os09g04310.1) conferred partial resistance. However, of these four genes, only p450s have been reported to play an important role in the synthesis of plant defense compounds. These findings revealed the complexity of key immune signaling conduits critical to mounting a full defense against Xanthomonas. oryzae pv. oryzae race K2 in japonica rice.

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Iron Regulation and Pathogenicity in Erwinia chrysanthemi 3937: Role of the Fur Repressor Protein

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.1999.12.2.119 ◽

1999 ◽

Vol 12 (2) ◽

pp. 119-128 ◽

Cited By ~ 60

Author(s):

Thierry Franza ◽

Christele Sauvage ◽

Dominique Expert

Keyword(s):

Iron Transport ◽

Reverse Genetics ◽

Erwinia Chrysanthemi ◽

Transport Systems ◽

Iron Regulation ◽

Iron Availability ◽

Reading Frame ◽

Genes Encoding ◽

Coordinated Expression ◽

Main Determinants

Low iron availability is a triggering signal for coordinated expression of the genes encoding pectate lyases PelB, PelC, PelD, and PelE, and chrysobactin iron transport functions, which are two main determinants of phytopathogenicity of the Erwinia chrysanthemi strain 3937. The possible implication of the ferric uptake regulation (Fur) protein in this process was investigated. The E. chrysanthemi fur gene was cloned by functional complementation of an Escherichia coli fur mutant and sequenced. The 444-bp open reading frame identified was found to code for a protein highly similar to the E. coli Fur regulator. An E. chrysanthemi fur null mutant was constructed by reverse genetics. This mutant showed altered growth capacity and reduced pathogenicity on African violets. In a fur background, transcriptional lacZ fusions to genes belonging to the E. chrysanthemi high affinity iron transport systems were constitutively expressed. Transcription of the pelA, pelD, and pelE genes was analyzed, using fusions to the uidA reporter gene. Iron availability and a fur mutation did not influence the expression of pelA. In the presence of iron, pelD and pelE transcription levels were higher in the fur mutant than in the parental strain. Furthermore, iron deficiency stimulated the expression of both fusions in the fur mutant. These findings indicate that, in E. chrysanthemi 3937, (i) Fur negatively controls iron transport and genes encoding PelD and PelE, and (ii) additional factor(s) mediate iron regulation of the pel genes.

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