The Lactobacillus casei ptsHI47T mutation causes overexpression of a LevR-regulated but RpoN-independent operon encoding a mannose class phosphotransferase system.
A proteome analysis of Lactobacillus casei mutants that are affected in carbon catabolite repression revealed that a 15-kDa protein was strongly overproduced in a ptsHI47T mutant. This protein was identified as EIIA of a mannose class phosphotransferase system (PTS). A 7.1-kb DNA fragment containing the EIIA-encoding open reading frame and five other genes was sequenced. The first gene encodes a protein resembling the RpoN (sigma54)-dependent Bacillus subtilis transcription activator LevR. The following pentacistronic operon is oriented in the opposite direction and encodes four proteins with strong similarity to the proteins of the B. subtilis Lev-PTS and one protein of unknown function. The genes present on the 7.1-kb DNA fragment were therefore called levR and levABCDX. The levABCDX operon was induced by fructose and mannose. No "-12, -24" promoter typical of RpoN-dependent genes precedes the L. casei lev operon, and its expression was therefore RpoN independent but required LevR. Phosphorylation of LevR by P approximately His-HPr stimulates its activity, while phosphorylation by P approximately EIIBLev inhibits it. Disruption of the EIIBLev-encoding levB gene therefore led to strong constitutive expression of the lev operon, which was weaker in a strain carrying a ptsI mutation preventing phosphorylation by both P approximately EIIBLev and P approximately His-HPr. Expression of the L. casei lev operon is also subject to P-Ser-HPr-mediated catabolite repression. The observed slow phosphoenolpyruvate- and ATP-dependent phosphorylation of HPrI47T as well as the slow phosphoryl group transfer from the mutant P approximately His-HPr to EIIALev are assumed to be responsible for the elevated expression of the lev operon in the ptsHI47T mutant.
Mazé
Alain
A
Laboratoire de Microbiologie et Génétique Moléculaire, INRA-INAPG-CNRS, Thiverval-Grignon, France.
Boël
Grégory
G
Poncet
Sandrine
S
Mijakovic
Ivan
I
Le Breton
Yoann
Y
Benachour
Abdellah
A
Monedero
Vicente
V
Deutscher
Josef
J
Hartke
Axel
A
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D004269:DNA, Bacterial / Q000737:chemistry / Q000302:isolation & purification; D004268:DNA-Binding Proteins; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D005632:Fructose / Q000378:metabolism / Q000494:pharmacology; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D007780:Lacticaseibacillus casei / Q000235:genetics* / Q000378:metabolism; D008358:Mannose / Q000378:metabolism / Q000494:pharmacology; D008969:Molecular Sequence Data; D009154:Mutation; D009876:Operon; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D051877:RNA Polymerase Sigma 54; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D012808:Sigma Factor / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D012808:Sigma Factor; D014157:Transcription Factors; C067962:LevR protein, Bacillus subtilis; D005632:Fructose; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C030182:phosphocarrier protein HPr; D012321:DNA-Directed RNA Polymerases; D051877:RNA Polymerase Sigma 54; D008358:Mannose