Cloning, Nucleotide Sequence, and Transcriptional Analysis of the Pediococcus acidilactici L-(1)Lactate Dehydrogenase Gene (original) (raw)
Related papers
Applied and …, 1995
Recombinant plasmids containing the Pediococcus acidilactici L-(؉)-lactate dehydrogenase gene (ldhL) were isolated by complementing for growth under anaerobiosis of an Escherichia coli lactate dehydrogenase-pyruvate formate lyase double mutant. The nucleotide sequence of the ldhL gene predicted a protein of 323 amino acids showing significant similarity with other bacterial L-(؉)-lactate dehydrogenases and especially with that of Lactobacillus plantarum. The ldhL transcription start points in P. acidilactici were defined by primer extension, and the promoter sequence was identified as TCAACT-(17 bp)-TATAAT. This sequence is closely related to the consensus sequence of vegetative promoters from gram-positive bacteria as well as from E. coli. Northern analysis of P. acidilactici RNA showed a 1.1-kb ldhL transcript whose abundance is growth rate regulated. These data, together with the presence of a putative rho-independent transcriptional terminator, suggest that ldhL is expressed as a monocistronic transcript in P. acidilactici.
Pediococcus acidilactici ldhD gene: cloning, nucleotide sequence, and transcriptional analysis
Journal of Bacteriology, 1995
The gene encoding D-lactate dehydrogenase was isolated on a 2.9-kb insert from a library of Pediococcus acidilactici DNA by complementation for growth under anaerobiosis of an Escherichia coli lactate dehydrogenase and pyruvate-formate lyase double mutant. The nucleotide sequence of ldhD encodes a protein of 331 amino acids (predicted molecular mass of 37,210 Da) which shows similarity to the family of D-2-hydroxyacid dehydrogenases. The enzyme encoded by the cloned fragment is equally active on pyruvate and hydroxypyruvate, indicating that the enzyme has both D-lactate and D-glycerate dehydrogenase activities. Three other open reading frames were found in the 2.9-kb insert, one of which (rpsB) is highly similar to bacterial genes coding for ribosomal protein S2. Northern (RNA) blotting analyses indicated the presence of a 2-kb dicistronic transcript of ldhD (a metabolic gene) and rpsB (a putative ribosomal protein gene) together with a 1-kb monocistronic rpsB mRNA. These transcripts are abundant in the early phase of exponential growth but steadily fade away to disappear in the stationary phase. Primer extension analysis identified two distinct promoters driving either cotranscription of ldhD and rpsB or transcription of rpsB alone.
Pediococcus acidilactici IdhD gene: cloning, nucleotide sequence, and transcriptional analysis
Journal of Bacteriology
The gene encoding D-lactate dehydrogenase was isolated on a 2.9-kb insert from a library of Pediococcus acidilactici DNA by complementation for growth under anaerobiosis of an Escherichia coli lactate dehydrogenase and pyruvate-formate lyase double mutant. The nucleotide sequence of ldhD encodes a protein of 331 amino acids (predicted molecular mass of 37,210 Da) which shows similarity to the family of D-2-hydroxyacid dehydrogenases. The enzyme encoded by the cloned fragment is equally active on pyruvate and hydroxypyruvate, indicating that the enzyme has both D-lactate and D-glycerate dehydrogenase activities. Three other open reading frames were found in the 2.9-kb insert, one of which (rpsB) is highly similar to bacterial genes coding for ribosomal protein S2. Northern (RNA) blotting analyses indicated the presence of a 2-kb dicistronic transcript of ldhD (a metabolic gene) and rpsB (a putative ribosomal protein gene) together with a 1-kb monocistronic rpsB mRNA. These transcripts are abundant in the early phase of exponential growth but steadily fade away to disappear in the stationary phase. Primer extension analysis identified two distinct promoters driving either cotranscription of ldhD and rpsB or transcription of rpsB alone.
Alexandria Journal of Food Science and Technology, 2006
As a cloning vector for transformation in Propionibacterium sp., pPK705 shuttle vector was experimented, where it consists of the indigenous plasmid of Propionibacterium acidipropionici E214 (pRG01), plasmid pUC18 and the hygromycin resistance gene (hyg B). A 1.2 Kb BamHI-EcoRI DNA fragment encoding ldh L gene of Pediococcus acidilactici DG302 was cut from plasmid pGID150 and further subcloned in pPK705. Transformation and expression of the ldh L gene of pediococci in NAD +-independent LDH Propionibacterium freudenreichii var. shermanii LAG16424T was confirmed. The absolute L (+) LDH enzyme activity was found to be (25.67 to 42.79 IU/ml) for Propionibacterium transformants cell free extract. Twenty Propionibacterium transformants were further inoculated in skimmed milk, where curdling was detected after 48 h with one of the prominent transformants. After 144 h, all transformants coagulated skimmed milk as compared to parental bacterial strain. Maximum % of lactic acid produced by Propionibacterium transformants was 0.79 % as lactic acid after 96 h. Further proof for the expression of NAD +-dependent L (+) LDH through the transformed ldh L gene was confirmed by polyacrylamide gel electrophoresis.
Journal of Bioscience and Bioengineering, 2017
The present study revealed the effect of nitrogen sources on lactic acid production and stimulation of D-and Llactate dehydrogenases (LDH) of parent Lactobacillus lactis NCIM 2368 and its mutant RM2-24 generated after UV mutagenesis. Both the parent and mutant strains were evaluated for D-lactic acid production in control and modified media. The modified media did not show remarkable effect on lactic acid production in case of parent whereas mutant exhibited significant enhancement in D-lactic acid production along with the appearance of L-lactic acid in the broth. Both LDH activities and specific activities were found to be higher in mutant than the parent strain. These results suggested that the diammonium hydrogen phosphate in modified media triggered the expression of LDH genes leading to enhanced lactic acid production. This observation has been proved by studying the expression levels of Dand L-LDH genes of parent and mutant in control and modified media using quantitative RT-PCR technique. In case of mutant, the transcriptional levels of D-LDH and L-LDH increased w17 fold and w1.38 fold respectively in modified medium compared to the values obtained with control medium. In case of parent, no significant change in transcriptional levels of D-and L-LDH was found when the cells were grown in either control medium or modified medium. This study suggested that the mutant, RM2-24 has L-LDH gene which is expressed in presence of (NH 4) 2 HPO 4 resulting in L-lactic acid production. Co-production of L-lactic acid in D-lactic acid fermentation may be detrimental in the PLA production.
FEBS Letters, 1991
A straiti of Escherichiu coli (FMJ144) deficient for pyruvate formate lyase and lactate dehydrogenase (LWH) was complemented with a genomic DNA iibc\lry from Laciobacilius delbrueckii subsp. Lirlgaricus. One positive clone showed LWH activity and production of W(-)lactate was demonstrated,. The nucleotide sequence of the W-LDH gene (/&A) revealed the spontaneous inseetion of an E. coli insertion sequence IS2 upstream OF the gene cod&g region. The open reading frame encoded a 333-amino acid protein, showing no similarity with known L-LWH sequences but closely related to L. cosei W-hydroxyisocaproate dehydrogenase (W-HicDW). W-Lactate dehydtogenase; Ins&on sequence 1st; Lactobacillus delbrueckii subsp. bulgaricus; W-Hydroxyisocapeoate dehydrogenase Published by Elsevier Science Publishers B. V.
Analysis of ldh genes in Lactobacillus casei BL23: role on lactic acid production
Journal of Industrial Microbiology & Biotechnology, 2008
Lactobacillus casei is a lactic acid bacterium that produces L-lactate as the main product of sugar fermentation via L-lactate dehydrogenase (Ldh1) activity. In addition, small amounts of the D-lactate isomer are produced by the activity of a D-hydroxycaproate dehydrogenase (HicD). Ldh1 is the main L-lactate producing enzyme, but mutation of its gene does not eliminate L-lactate synthesis. A survey of the L. casei BL23 draft genome sequence revealed the presence of three additional genes encoding Ldh paralogs. In order to study the contribution of these genes to the global lactate production in this organism, individual, as well as double mutants (ldh1 ldh2, ldh1 ldh3, ldh1 ldh4 and ldh1 hicD) were constructed and lactic acid production was assessed in culture supernatants. ldh2, ldh3 and ldh4 genes play a minor role in lactate production, as their single mutation or a mutation in combination with an ldh1 deletion had a low impact on L-lactate synthesis. A ldh1 mutant displayed an increased production of D-lactate, which was probably synthesized via the activity of HicD, as it was abolished in a ldh1 hicD double mutant. Contrarily to HicD, no Ldh1, Ldh2, Ldh3 or Ldh4 activities could be detected by zymogram assays. In addition, these assays revealed the presence of extra bands exhibiting D-/L-lactate dehydrogenase activity, which could not be attributed to any of the described genes. These results suggest that L. casei BL23 possesses a complex enzymatic system able to reduce pyruvic to lactic acid.
Lactobacillus plantarum ldhL gene: overexpression and deletion
Journal of bacteriology, 1994
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 i...
1997
The Lactococcus lactis pfl gene, encoding pyruvate formate-lyase (PFL), has been cloned and characterized. The deduced amino acid sequence of the L. lactis PFL protein showed high similarity to those of other bacterial PFL proteins and included the conserved glycine residue involved in posttranslational activation of PFL. The genetic organization of the chromosomal pfl region in L. lactis showed differences from other characterized pfl loci, with an upstream open reading frame independently transcribed in the same orientation as the pfl gene. The gene coding for PFL-activase (act), normally found downstream of pfl, was not identified in L. lactis. Analysis of pfl expression showed a strong induction under anaerobiosis at the transcriptional level independent of the growth medium used. During growth with galactose, pfl showed the highest levels of expression. Constructed L. lactis pfl strains were unable to produce formate under anaerobic growth. Higher levels of diacetyl and acetoin were produced anaerobically in the constructed Lactococcus lactis subsp. lactis biovar diacetylactis pfl strain.
Applied and Environmental Microbiology, 2011
Several lactic acid bacteria use homolactic acid fermentation for generation of ATP. Here we studied the role of the lactate dehydrogenase enzyme on the general physiology of the three homolactic acid bacteria Lactococcus lactis, Enterococcus faecalis, and Streptococcus pyogenes. Of note, deletion of the ldh genes hardly affected the growth rate in chemically defined medium under microaerophilic conditions. However, the growth rate was affected in rich medium. Furthermore, deletion of ldh affected the ability for utilization of various substrates as a carbon source. A switch to mixed acid fermentation was observed during glucose-limited continuous growth and was dependent on the growth rate for S. pyogenes and on the pH for E. faecalis. In S. pyogenes and L. lactis, a change in pH resulted in a clear change in Y ATP (cell mass produced per mole of ATP). The pH that showed the highest Y ATP corresponded to the pH of the natural habitat of the organisms.