The Lactobacillus casei ptsHI47T mutation causes overexpression of a LevR-regulated but RpoN-independent operon encoding a mannose class phosphotransferase system

Abstract

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.

FIG. 1.

Analytical 2-D gel electrophoresis carried out with crude extracts of L. casei wild-type BL23 (WT) and five CCR-relieved mutants. Shown is the region around the migration position of HPr (encircled), which was determined on preparative gels by carrying out Western blots with a rabbit polyclonal antibody. The arrows indicate the position of EIIA^Lev, which is overproduced in the ptsHI47T strain.

FIG. 2.

(A) Schematic representation of the lev operon and the preceding levR gene in B. subtilis and in L. casei (L.c.) strains ATCC 334 and BL23. Hairpin loops indicate transcription terminators. (B) Domain organization of the L. casei and B. subtilis transcription activator LevR. DNA-B indicates the DNA binding domain with the helix-turn-helix motif. C-LevR indicates a region located between the NtrC central domain and PRD1 in B. subtilis LevR which differs from the corresponding region in L. casei LevR. An alignment of the sequence around the RpoN binding motif GAFTGA (RpoN-B) of several proteins containing an NtrC central domain is also shown. PspF, NtrC, and TyrR are proteins from E. coli, and BkdR and RocR are from B. subtilis. The GAFTGA sequence motif is absent from TyrR and L. casei LevR, which are both RpoN independent, but is present in B. subtilis LevR, which is RpoN (SigL) dependent, and in the other transcription activators included in the alignment.

FIG. 3.

Northern blots carried out with 5 μg of RNA isolated from either the L. casei wild-type strain BL23 (WT) or one of the five CCR-relieved mutants grown in MRS fermentation medium complemented with glucose. A levA-specific probe was used to detect the lev operon transcript of 3.4 kb in the various samples. The positions of 16S and 23S rRNAs are indicated with arrows.

FIG. 4.

Schematic representation of the 283-bp intergenic region between levR and levA of L. casei. This sequence contains the transcription start sites (+1, underlined capital letters), the start codons (letters in boldface type), and presumed −10 and −35 promoter regions (arrows) for both the levR gene and the levRABCDX operon as well as a potential cre site (boxed area) which overlaps the transcription start site of the lev operon and the −10 promoter sequence of levR.

FIG. 5.

Northern blots carried out with 5 μg of RNA isolated from wild-type strain BL23 (WT), the ptsHI47T or levR mutant, or the ptsHI47T levR double mutant (A) and wild-type strain BL23 (WT), the ptsHI47T or rpoN mutant, or the ptsHI47T rpoN double mutant (B). The strains were grown in MRS fermentation medium complemented with glucose. The levA-specific probe was used to detect the lev operon transcript of 3.4 kb. The positions of the 16S and 23S rRNAs are indicated with arrows.

FIG. 6.

Phosphorylation of MBP-LevR by [³²P]P∼His-HPr (A) and [³²P]P∼EIIB^Lev (B). (A) Samples containing [³²P]PEP and the indicated proteins were incubated at 37°C before they were separated on a 0.1% SDS-15% polyacrylamide gel which was dried and exposed to autoradiography. The sample loaded on lane 3 contained MBP-LevR, which had been treated with factor Xa before the phosphorylation reaction was carried out. The migration positions of HPr, EI, LevR, and MBP-LevR are indicated by arrows. The slowest-migrating radioactive band (arrow with the question mark) probably corresponds to MBP-LevR dimers. (B) Samples containing [³²P]PEP, EI, HPr, and the indicated proteins were incubated at 37°C before they were separated on a 0.1% SDS-8% polyacrylamide gel, which was dried and exposed to autoradiography. In LevR-H1, His-488 in the EIIA^Man domain was replaced with an alanine, while in LevR-H12, His-776 in PRD2 was also replaced with an alanine. The migration positions of EI and wild-type and mutant LevRs are indicated by arrows.

FIG. 7.

ATP-dependent HprK/P-catalyzed phosphorylation at Ser-46 of HPr (A) and HPrI47T (B). Samples were prepared as described in Materials and Methods, incubated for different time periods at 37°C, and separated on nondenaturing 12.5% polyacrylamide gels, which allowed us to separate HPr and P-Ser-HPr. The gels were stained with Coomassie blue.

FIG. 8.

[³²P]PEP-dependent EI-catalyzed phosphorylation of HPr (A) and HPrI47T (B). Samples were prepared as described in Materials and Methods, incubated for different time periods at 37°C, and separated on 0.1% SDS-15% polyacrylamide gels which were dried and exposed to autoradiography. The migration positions of EI and HPr are indicated by arrows.

FIG. 9.

Phosphoryl group transfer from [³²P]P∼His-HPr (A) and [³²P]P∼His-HPrI47T (B) to EIIA^Lev. [³²P]PEP, EI, and HPr or HPrI47T were preincubated to allow their exhaustive phosphorylation before EIIA^Lev was added to the assay mixtures, which were further incubated at 37°C. Aliquots were withdrawn after different time periods and separated on 0.1% SDS-15% polyacrylamide gels which were dried and exposed to autoradiography. The migration positions of EI, HPr, HPrI47T, EIIA^Lev, and its dimer are indicated by arrows.

FIG. 10.

Northern blots with 5 μg of RNA isolated from either the L. casei wild-type strain BL23 (WT); the ptsHI47T, ptsI, or levB mutant; or the ptsHI47T levB double mutant, which were grown in MRS fermentation medium complemented with ribose. The levA-specific probe was used to detect the lev operon transcript of 3.4 kb. The positions of the 16S and 23S rRNAs are indicated with arrows.