Dataset Information

Lactobacillus plantarum ldhL gene: overexpression and deletion.

ABSTRACT: Lactobacillus plantarum is a lactic acid bacterium that converts pyruvate to L-(+)- and D-(-)-lactate with stereospecific enzymes designated L-(+)- and D-(-)-lactate dehydrogenase (LDH), respectively. A gene (designated ldhL) that encodes L-(+)-lactate dehydrogenase from L. plantarum DG301 was cloned by complementation in Escherichia coli. The nucleotide sequence of the ldhL gene predicted a protein of 320 amino acids closely related to that of Lactobacillus pentosus. A multicopy plasmid bearing the ldhL gene without modification of its expression signals was introduced in L. plantarum. L-LDH activity was increased up to 13-fold through this gene dosage effect. However, this change had hardly any effect on the production of L-(+)- and D-(-)-lactate. A stable chromosomal deletion in the ldhL gene was then constructed in L. plantarum by a two-step homologous recombination process. Inactivation of the gene resulted in the absence of L-LDH activity and in exclusive production of the D isomer of lactate. However, the global concentration of lactate in the culture supernatant remained unchanged.

SUBMITTER: Ferain T

PROVIDER: S-EPMC205095 | biostudies-other | 1994 Feb

REPOSITORIES: biostudies-other

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Lactobacillus plantarum ldhL gene: overexpression and deletion.

Ferain T T Garmyn D D Bernard N N Hols P P Delcour J J

Journal of bacteriology 19940201 3

Lactobacillus plantarum is a lactic acid bacterium that converts pyruvate to L-(+)- and D-(-)-lactate with stereospecific enzymes designated L-(+)- and D-(-)-lactate dehydrogenase (LDH), respectively. A gene (designated ldhL) that encodes L-(+)-lactate dehydrogenase from L. plantarum DG301 was cloned by complementation in Escherichia coli. The nucleotide sequence of the ldhL gene predicted a protein of 320 amino acids closely related to that of Lactobacillus pentosus. A multicopy plasmid bearing ...[more]

PMID: 8300514

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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

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.

Dataset Information

Lactobacillus plantarum ldhL gene: overexpression and deletion.

Publications

Lactobacillus plantarum ldhL gene: overexpression and deletion.

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