Dataset Information

Role of PdxR in the activation of vitamin B6 biosynthesis in Listeria monocytogenes.

ABSTRACT: Listeria monocytogenes?PdxR is a member of the poorly characterized but widespread group of MocR/GabR-type chimeric bacterial proteins that have DNA-binding and aminotransferase-like domains. Using mutational analysis, real-time RT-PCR, transcriptional fusions, gel-shift assays, DNase I footprinting, and in vitro transcription, it was shown that PdxR is a direct activator of the pdxST operon, transcribed divergently from pdxR and responsible for the de novo synthesis of pyridoxal 5'-phosphate (PLP), the major active form of vitamin B6 . PLP acts as an anti-activator of PdxR and is the only effector required to reduce the activity of PdxR. PdxR is also a negative autoregulator, and its ability to repress is increased by PLP. A dyad-symmetry sequence, which overlaps the -35 region of the pdxS promoter and lies downstream of the pdxR transcription start point, serves as an important element of the PdxR binding site. Unexpectedly, some mutations in this activator binding site, disrupting the dyad-symmetry element, caused constitutive, B6 -independent expression from the pdxS promoter. The data suggest that PdxR-like proteins, for which PLP plays just a signalling role, form a separate functional group among the MocR/GabR-type proteins.

SUBMITTER: Belitsky BR

PROVIDER: S-EPMC4299700 | biostudies-literature | 2014 Jun

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Role of PdxR in the activation of vitamin B6 biosynthesis in Listeria monocytogenes.

Belitsky Boris R BR

Molecular microbiology 20140505 5

Listeria monocytogenes PdxR is a member of the poorly characterized but widespread group of MocR/GabR-type chimeric bacterial proteins that have DNA-binding and aminotransferase-like domains. Using mutational analysis, real-time RT-PCR, transcriptional fusions, gel-shift assays, DNase I footprinting, and in vitro transcription, it was shown that PdxR is a direct activator of the pdxST operon, transcribed divergently from pdxR and responsible for the de novo synthesis of pyridoxal 5'-phosphate (P ...[more]

PMID: 24730374

Similar Datasets

Project description:Lysozyme is an important component of the innate immune system. It functions by hydrolyzing the peptidoglycan (PG) layer of bacteria. The human pathogen Listeria monocytogenes is intrinsically lysozyme resistant. The peptidoglycan N-deacetylase PgdA and O-acetyltransferase OatA are two known factors contributing to its lysozyme resistance. Furthermore, it was shown that the absence of components of an ABC transporter, referred to here as EslABC, leads to reduced lysozyme resistance. How its activity is linked to lysozyme resistance is still unknown. To investigate this further, a strain with a deletion in eslB, coding for a membrane component of the ABC transporter, was constructed in L. monocytogenes strain 10403S. The eslB mutant showed a 40-fold reduction in the MIC to lysozyme. Analysis of the PG structure revealed that the eslB mutant produced PG with reduced levels of O-acetylation. Using growth and autolysis assays, we showed that the absence of EslB manifests in a growth defect in media containing high concentrations of sugars and increased endogenous cell lysis. A thinner PG layer produced by the eslB mutant under these growth conditions might explain these phenotypes. Furthermore, the eslB mutant had a noticeable cell division defect and formed elongated cells. Microscopy analysis revealed that an early cell division protein still localized in the eslB mutant, indicating that a downstream process is perturbed. Based on our results, we hypothesize that EslB affects the biosynthesis and modification of the cell wall in L. monocytogenes and is thus important for the maintenance of cell wall integrity.IMPORTANCE The ABC transporter EslABC is associated with the intrinsic lysozyme resistance of Listeria monocytogenes However, the exact role of the transporter in this process and in the physiology of L. monocytogenes is unknown. Using different assays to characterize an eslB deletion strain, we found that the absence of EslB affects not only lysozyme resistance but also endogenous cell lysis, cell wall biosynthesis, cell division, and the ability of the bacterium to grow in media containing high concentrations of sugars. Our results indicate that EslB is, by means of a yet-unknown mechanism, an important determinant for cell wall integrity in L. monocytogenes.

Project description:UnlabelledPhotorhabdus luminescens is a Gram-negative entomopathogenic bacterium which symbiotically associates with the entomopathogenic nematode Heterorhabditis bacteriophora P. luminescens is highly virulent to many insects and nonsymbiotic nematodes, including Caenorhabditis elegans To understand the virulence mechanisms of P. luminescens, we obtained virulence-deficient and -attenuated mutants against C. elegans through a transposon-mutagenized library. From the genetic screening, we identified the pdxB gene, encoding erythronate-4-phosphate dehydrogenase, as required for de novo vitamin B6 biosynthesis. Mutation in pdxB caused growth deficiency of P. luminescens in nutrient-poor medium, which was restored under nutrient-rich conditions or by supplementation with pyridoxal 5'-phosphate (PLP), an active form of vitamin B6 Supplementation with three other B6 vitamers (pyridoxal, pyridoxine, and pyridoxamine) also restored the growth of the pdxB mutant, suggesting the existence of a salvage pathway for vitamin B6 biosynthesis in P. luminescens Moreover, supplementation with PLP restored the virulence-deficient phenotype against C. elegans Combining these results with the fact that pdxB mutation also caused attenuation of insecticidal activity, we concluded that the production of appropriate amounts of vitamin B6 is critical for P. luminescens pathogenicity.ImportanceThe Gram-negative entomopathogenic bacterium Photorhabdus luminescens symbiotically associates with the entomopathogenic nematode Heterorhabditis bacteriophora P. luminescens is highly virulent to many insects and nonsymbiotic nematodes, including Caenorhabditis elegans We have obtained several virulence-deficient and -attenuated P. luminescens mutants against C. elegans through genetic screening. From the genetic analysis, we present the vitamin B6 biosynthetic pathways in P. luminescens that are important for its insecticidal activity. Mutation in pdxB, encoding erythronate-4-phosphate dehydrogenase and required for the de novo vitamin B6 biosynthesis pathway, caused virulence deficiency against C. elegans and growth deficiency of P. luminescens in nutrient-poor medium. Because such phenotypes were restored under nutrient-rich conditions or by supplementation with B6 vitamers, we showed the presence of the two vitamin B6 synthetic pathways (de novo and salvage) in P. luminescens and also showed that the ability to produce an appropriate amount of vitamin B6 is critical for P. luminescens pathogenicity.

Dataset Information

Role of PdxR in the activation of vitamin B6 biosynthesis in Listeria monocytogenes.

Publications

Role of PdxR in the activation of vitamin B6 biosynthesis in Listeria monocytogenes.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets