Knockout of the two ldh genes has a major impact on peptidoglycan precursor synthesis in Lactobacillus plantarum

Most bacteria synthesize muramyl-pentapeptide peptidoglycan precursors ending with a D-alanyl residue (e.g., UDP-N-acetylmuramyl-L-Ala-gamma-D-Glu-L-Lys-D-Ala-D-Ala). However, it was recently demonstrated that other types of precursors, notably D-lactate-ending molecules, could be synthesized by several lactic acid bacteria. This particular feature leads to vancomycin resistance. Vancomycin is a glycopeptide antibiotic that blocks cell wall synthesis by the formation of a complex with the extremity of peptidoglycan precursors. Substitution of the terminal D-alanine by D-lactate reduces the affinity of the antibiotic for its target. Lactobacillus plantarum is a lactic acid bacterium naturally resistant to vancomycin. It converts most of the glycolytic pyruvate to L- and D-lactate by using stereospecific enzymes designated L- and D-lactate dehydrogenases, respectively. In the present study, we show that L. plantarum actually synthesizes D-lactate-ending peptidoglycan precursors. We also report the construction of a strain which is deficient for both D- and L-lactate dehydrogenase activities and which produces only trace amounts of D- and L-lactate. As a consequence, the peptidoglycan synthesis pathway is drastically affected. The wild-type precursor is still present, but a new type of D-alanine-ending precursor is also synthesized in large quantities, which results in a highly enhanced sensitivity to vancomycin.

[1]  M. Arthur,et al.  Characterization of Tn1546, a Tn3-related transposon conferring glycopeptide resistance by synthesis of depsipeptide peptidoglycan precursors in Enterococcus faecium BM4147 , 1993, Journal of bacteriology.

[2]  H. Blöcker,et al.  Cloning, sequencing and expression in Escherichia coli of the D-2-hydroxyisocaproate dehydrogenase gene of Lactobacillus casei. , 1989, Gene.

[3]  Y. Burstein,et al.  Purification of D-alanine carboxypeptidase from Escherichia coli B on a penicillin-Sepharose column. , 1975, The Biochemical journal.

[4]  M. M. Bradford A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[5]  E. Guittet,et al.  Modification of peptidoglycan precursors is a common feature of the low-level vancomycin-resistant VANB-type Enterococcus D366 and of the naturally glycopeptide-resistant species Lactobacillus casei, Pediococcus pentosaceus, Leuconostoc mesenteroides, and Enterococcus gallinarum , 1994, Journal of bacteriology.

[6]  G. Venema,et al.  Construction of plasmid cloning vectors for lactic streptococci which also replicate in Bacillus subtilis and Escherichia coli , 1984, Applied and environmental microbiology.

[7]  W. J. Dower,et al.  High efficiency transformation of E. coli by high voltage electroporation , 1988, Nucleic Acids Res..

[8]  M. Arthur,et al.  Structural relationship between the vancomycin resistance protein VanH and 2-hydroxycarboxylic acid dehydrogenases. , 1991, Gene.

[9]  L. Morelli,et al.  Drug resistance plasmids in Lactobacillus acidophilus and Lactobacillus reuteri , 1982, Applied and environmental microbiology.

[10]  W. Sandine,et al.  Common occurrence of plasmid DNA and vancomycin resistance in Leuconostoc spp , 1984, Applied and environmental microbiology.

[11]  S. Mizushima,et al.  PURIFICATION AND PROPERTIES OF LACTIC DEHYDROGENASE OF LACTOBACILLUS CASEI , 1962 .

[12]  S. Fukui,et al.  Purification and properties of lactate racemase from Lactobacillus sake. , 1968, Journal of biochemistry.

[13]  W. Alborn,et al.  Inhibition of peptidoglycan biosynthesis in gram-positive bacteria by LY146032 , 1987, Antimicrobial Agents and Chemotherapy.

[14]  J. Heijenoort Chapter 3 Biosynthesis of the bacterial peptidoglycan unit , 1994 .

[15]  A. Efstratiou,et al.  Vancomycin-resistant leuconostocs, lactobacilli and now pediococci. , 1987, Journal of Hospital Infection.

[16]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[17]  S. Handwerger,et al.  The cytoplasmic peptidoglycan precursor of vancomycin-resistant Enterococcus faecalis terminates in lactate , 1992, Journal of bacteriology.

[18]  F. Radler,et al.  Malolactic enzyme of Lactobacillus plantarum. Purification, properties, and distribution among bacteria. , 1983, The Journal of biological chemistry.

[19]  M. Arthur,et al.  Sequence of the vanC gene of Enterococcus gallinarum BM4174 encoding a D-alanine:D-alanine ligase-related protein necessary for vancomycin resistance. , 1992, Gene.

[20]  P. Courvalin,et al.  Analysis of genes encoding D-alanine:D-alanine ligase-related enzymes in Leuconostoc mesenteroides and Lactobacillus spp. , 1995, Gene.

[21]  M. Kula,et al.  Dehydrogenases for the synthesis of chiral compounds. , 1989, European journal of biochemistry.

[22]  J. Collins,et al.  Cloning, sequencing and expression of the L-2-hydroxyisocaproate dehydrogenase-encoding gene of Lactobacillus confusus in Escherichia coli. , 1989, Gene.

[23]  C. Walsh,et al.  Evidence for in vivo incorporation of D-lactate into peptidoglycan precursors of vancomycin-resistant enterococci , 1992, Antimicrobial Agents and Chemotherapy.

[24]  R. Leer,et al.  Incompatibility of Lactobacillus Vectors with Replicons Derived from Small Cryptic Lactobacillus Plasmids and Segregational Instability of the Introduced Vectors , 1991, Applied and environmental microbiology.

[25]  P. Reynolds,et al.  Modified peptidoglycan precursors produced by glycopeptide-resistant enterococci. , 1992, FEMS microbiology letters.

[26]  M. Collins,et al.  Lactobacillus pentosus (Fred, Peterson, and Anderson) sp. nov., nom. rev. , 1987 .

[27]  R. Facklam,et al.  Antimicrobial susceptibility of vancomycin-resistant Leuconostoc, Pediococcus, and Lactobacillus species , 1990, Antimicrobial Agents and Chemotherapy.

[28]  J. Frère,et al.  Streptomyces K15 DD-peptidase-catalysed reactions with ester and amide carbonyl donors. , 1986, The Biochemical journal.

[29]  P. Stanssens,et al.  Characterization of a gram-positive broad-host-range plasmid isolated from Lactobacillus hilgardii. , 1989, Plasmid.

[30]  J. Duval,et al.  Vancomycin resistance gene vanC is specific to Enterococcus gallinarum , 1992, Antimicrobial Agents and Chemotherapy.

[31]  C. Walsh,et al.  Molecular basis for vancomycin resistance in Enterococcus faecium BM4147: biosynthesis of a depsipeptide peptidoglycan precursor by vancomycin resistance proteins VanH and VanA. , 1991, Biochemistry.

[32]  Jinping,et al.  Vancomycin-resistant Leuconostoc mesenteroides and Lactobacillus casei synthesize cytoplasmic peptidoglycan precursors that terminate in lactate , 1994, Journal of bacteriology.

[33]  D. Morrison,et al.  Construction and properties of a new insertion vector, pJDC9, that is protected by transcriptional terminators and useful for cloning of DNA from Streptococcus pneumoniae. , 1988, Gene.

[34]  M. Arthur,et al.  Genetics and mechanisms of glycopeptide resistance in enterococci , 1993, Antimicrobial Agents and Chemotherapy.

[35]  C. Walsh,et al.  Identification of vancomycin resistance protein VanA as a D-alanine:D-alanine ligase of altered substrate specificity. , 1991, Biochemistry.

[36]  M. Arthur,et al.  Glycopeptide resistance mediated by enterococcal transposon Tn 1546 requires production of VanX for hydrolysis of D‐alanyl‐D‐alanine , 1994, Molecular microbiology.

[37]  J. Richardson,et al.  Biosynthesis of modified peptidoglycan precursors by vancomycin-resistant Enterococcus faecium. , 1992, FEMS microbiology letters.

[38]  T. Ohta,et al.  D-lactate dehydrogenase is a member of the D-isomer-specific 2-hydroxyacid dehydrogenase family. Cloning, sequencing, and expression in Escherichia coli of the D-lactate dehydrogenase gene of Lactobacillus plantarum. , 1991, The Journal of biological chemistry.

[39]  D. Williams,et al.  The structure and mode of action of glycopeptide antibiotics of the vancomycin group. , 1984, Annual review of microbiology.

[40]  D. Garmyn,et al.  Lactobacillus plantarum ldhL gene: overexpression and deletion , 1994, Journal of bacteriology.

[41]  C. Kaneuchi,et al.  Production of Succinic Acid from Citric Acid and Related Acids by Lactobacillus Strains , 1988, Applied and environmental microbiology.

[42]  M. Collins,et al.  Identification of gram-positive coccal and coccobacillary vancomycin-resistant bacteria , 1989, Journal of clinical microbiology.

[43]  J. Holbrook,et al.  NAD(+)-dependent D-2-hydroxyisocaproate dehydrogenase of Lactobacillus delbrueckii subsp. bulgaricus. Gene cloning and enzyme characterization. , 1994, European journal of biochemistry.

[44]  K. Schleifer,et al.  Peptidoglycan Types of Bacterial Cell Walls and Their Taxonomic Implications , 1973, Bacteriological reviews.