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Characterization of the CtsR stress response regulon in Lactobacillus plantarum.

ABSTRACT: Lactobacillus plantarum ctsR was characterized. ctsR was found to be cotranscribed with clpC and induced in response to various abiotic stresses. ctsR deletion conferred a heat-sensitive phenotype with peculiar cell morphological features. The transcriptional pattern of putative CtsR regulon genes was examined in the Delta ctsR mutant. Direct CtsR-dependent regulation was demonstrated by DNA-binding assays using recombinant CtsR and the promoters of the ctsR-clpC operon and hsp1.

SUBMITTER: Fiocco D

PROVIDER: S-EPMC2812460 | biostudies-literature | 2010 Feb

REPOSITORIES: biostudies-literature

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Characterization of the CtsR stress response regulon in Lactobacillus plantarum.

Fiocco Daniela D Capozzi Vittorio V Collins Michael M Gallone Anna A Hols Pascal P Guzzo Jean J Weidmann Stephanie S Rieu Aurélie A Msadek Tarek T Spano Giuseppe G

Journal of bacteriology 20091120 3

Lactobacillus plantarum ctsR was characterized. ctsR was found to be cotranscribed with clpC and induced in response to various abiotic stresses. ctsR deletion conferred a heat-sensitive phenotype with peculiar cell morphological features. The transcriptional pattern of putative CtsR regulon genes was examined in the Delta ctsR mutant. Direct CtsR-dependent regulation was demonstrated by DNA-binding assays using recombinant CtsR and the promoters of the ctsR-clpC operon and hsp1. ...[more]

PMID: 19933364

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Project description:BackgroundBacteriocin production in the lactic acid bacterium Lactobacillus plantarum C11 is regulated through a quorum sensing based pathway involving two highly homologous response regulators (59% identity and 76% similarity), PlnC as a transcriptional activator and PlnD as a repressor. Previous in vitro studies have shown that both regulators bind, as homodimers, to the same DNA regulatory repeats to exert their regulatory functions. As the genes for these two proteins are located on the same auto-regulatory operon, hence being co-expressed upon gene activation, it is plausible that their opposite functions must somehow be differentially regulated, either in terms of timing and/or binding kinetics, so that their activities do not impair each other in an uncontrolled manner. To understand the nature behind this potential differentiation, we have studied the binding kinetics of the two regulators on five target promoters (PplnA, PplnM, PplnJ, PplnE and PplnG) from the bacteriocin regulon of L. plantarum C11.ResultsBy using surface plasmon resonance spectroscopy we obtained parameters such as association rates, dissociation rates and dissociation constants, showing that the two regulators indeed differ greatly from each other in terms of cooperative binding and binding strength to the different promoters. For instance, cooperativity is very strong for PlnC binding to the promoter of the regulatory operon (PplnA), but not to the promoter of the transport operon (PplnG), while the opposite is seen for PlnD binding to these two promoters. The estimated affinity constants indicate that PlnC can bind to PplnA to activate transcription of the key regulatory operon plnABCD without much interference from PlnD, and that the repressive function of PlnD might act through a different mechanism than repression of the regulatory operon.ConclusionWe have characterised the DNA binding kinetics of the two regulators PlnC and PlnD from the bacteriocin locus in L. plantarum C11. Our data show that PlnC and PlnD, despite their strong homology to each other, differ greatly from each other in terms of binding affinity and cooperativity to the different promoters of the pln regulon.

Project description:Heat stress resistance and response were studied in strains of Lactobacillus plantarum. Stationary-phase cells of L. plantarum DPC2739 had decimal reduction times (D values) (D value was the time that it took to reduce the number of cells by 1 log cycle) in sterile milk of 32.9, 14.7, and 7.14 s at 60, 72, and 75 degrees C, respectively. When mid-exponential-phase cells were used, the D values decreased. The temperature increases which caused a 10-fold reduction in the D value ranged from 9 to 20 degrees C, depending on the strain. Part of the cell population treated at 72 degrees C for 90 s recovered viability during incubation at 7 degrees C in sterile milk for 20 days. When mid-exponential- or stationary-phase cells of L. plantarum DPC2739 were adapted to 42 degrees C for 1 h, the heat resistance at 72 degrees C for 90 s increased ca. 3 and 2 log cycles, respectively. Heat-adapted cells also showed increased growth at pH 5 and in the presence of 6% NaCl. Two-dimensional gel electrophoresis of proteins expressed by control and heat-adapted cells revealed changes in the levels of expression of 31 and 18 proteins in mid-exponential- and stationary-phase cells, respectively. Twelve proteins were commonly induced. Nine proteins induced in the heat-adapted mid-exponential- and/or stationary-phase cells of L. plantarum DPC2739 were subjected to N-terminal sequencing. These proteins were identified as DnaK, GroEL, trigger factor, ribosomal proteins L1, L11, L31, and S6, DNA-binding protein II HlbA, and CspC. All of these proteins have been found to play a role in the mechanisms of stress adaptation in other bacteria. Antibodies against GroES detected a protein which was induced moderately, while antibodies against DnaJ and GrpE reacted with proteins whose level of expression did not vary after heat adaptation. This study showed that the heat resistance of L. plantarum is a complex process involving proteins with various roles in cell physiology, including chaperone activity, ribosome stability, stringent response mediation, temperature sensing, and control of ribosomal function. The physiological mechanisms of response to pasteurization in L. plantarum are fundamental for survival in cheese during manufacture.

Project description:Two independent chemostat cultivations were performed for both wild type and trxB1 over-expression strain, NZ7602. Three hybridization experiments, all with the same hybridization scheme (see overall design), were performed with samples obtained from these chemostats before and after treatment with hydrogen peroxide (30 minutes 2.5 mM peroxide). Two of these experiments used samples obtained from the same fermentation. In each experiment three arrays were used. Per array two cDNA labeled targets were hybridized on custom designed L. plantarum WCFS1 11K Agilent oligo microarrays (GEO Acc. Nr. GPL4318) using the Agilent 60-mer oligo microarray processing protocol version 4.1. These microarrays contained an average of 3 probes per gene. Dried slides were scanned in the Scan Array Express (PerkinElmer Life Sciences; Packard Bioscience) at 10 microns. Spot intensity data was quantified (average intensity) in ImaGene version 5.0 (BioDiscovery, Inc., El Segundo, CA). Signal intensities of all probes were corrected against background and normalized by fitting a plot of M (= 2log [cy5 intensity/cy3 intensity]) against A (= 0.5 • 2log [cy5 intensity • cy3 intensity]) using the lowess algorithm in BASE [32]. The fold change (FC) is defined as 2M. For the statistical analysis we used microarray analysis of variation (R/maanova) [33] In this maanova test we used three variables: fermentation, treatment, and genotype. We tested the model taking into consideration the interaction between genotype and treatment. Significantly regulated genes were defined as genes whose nominal adjusted pvalues were less than 10% and a FC>1.5. The maanova test resulted only in two sets of interesting data because the interaction effect did not reveal significant changes in transcript levels. One dataset representing the transcripts affected as a result of the overproduction of TR and another set representing the transcripts affected due to oxidative stress. These two datasets were denominated genotype and hydrogen peroxide datasets respectively. Keywords: hydrogen peroxide stress response

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Characterization of the CtsR stress response regulon in Lactobacillus plantarum.

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Characterization of the CtsR stress response regulon in Lactobacillus plantarum.

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