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Molecular characterization of the Lactococcus lactis ptsHI operon and analysis of the regulatory role of HPr.

ABSTRACT: The Lactococcus lactis ptsH and ptsI genes, encoding the general proteins of the phosphoenolpyruvate-dependent phosphotransferase system, HPr and enzyme I, respectively, were cloned, and the regulatory role of HPr was studied by mutational analysis of its gene. A promoter sequence was identified upstream of the ptsHI operon, and the transcription start site was mapped by primer extension. The results of Northern analyses showed the presence of two glucose-inducible transcripts, one of 0.3 kb containing ptsH and a second of 2.0 kb containing both ptsH and ptsI. Disruption of the ptsH and ptsI genes in strain NZ9800 resulted in a reduced growth rate at the expense of glucose, but no growth at the expense of sucrose and fructose, confirming the dominant role of the phosphotransferase system in the uptake of these sugars in L. lactis. Complementation of the ptsH and ptsI mutants with the intact genes under the control of a regulated promoter resulted in the restoration of the wild-type phenotype. The role of HPr(Ser-P) in the recently established CcpA-mediated control of galactose metabolism as well as glycolysis was analyzed by producing an HPr mutant carrying an aspartic acid on residue 46 which mimicks a phosphorylated serine. The results of these experiments demonstrated the role of HPr(Ser-P) as corepressor in the catabolite repression of the gal operon. Furthermore, we show for the first time that HPr(Ser-P) functions as a coactivator in the CcpA-mediated catabolite activation of the pyruvate kinase and L-lactate dehydrogenase genes.

SUBMITTER: Luesink EJ

PROVIDER: S-EPMC93441 | biostudies-literature | 1999 Feb

REPOSITORIES: biostudies-literature

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Molecular characterization of the Lactococcus lactis ptsHI operon and analysis of the regulatory role of HPr.

Luesink E J EJ Beumer C M CM Kuipers O P OP De Vos W M WM

Journal of bacteriology 19990201 3

The Lactococcus lactis ptsH and ptsI genes, encoding the general proteins of the phosphoenolpyruvate-dependent phosphotransferase system, HPr and enzyme I, respectively, were cloned, and the regulatory role of HPr was studied by mutational analysis of its gene. A promoter sequence was identified upstream of the ptsHI operon, and the transcription start site was mapped by primer extension. The results of Northern analyses showed the presence of two glucose-inducible transcripts, one of 0.3 kb con ...[more]

PMID: 9922238

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Project description:In Lactococcus lactis, the his operon contains all the genes necessary for histidine biosynthesis. It is transcribed from a unique promoter, localized 300 bp upstream of the first gene. The region corresponding to the untranslated 5' end of the transcript, named the his leader region, displays the typical features of the T box transcriptional attenuation mechanism which is involved in the regulation of many amino acid biosynthetic operons and tRNA synthetase genes in gram-positive bacteria. Here we describe the regulation of transcription of the his operon by the level of histidine in the growth medium. In the absence of histidine, two transcripts are present. One covers the entire operon, while the other stops at a terminator situated about 250 bp downstream of the transcription start point. DNA sequences implicated in regulation of the his operon were identified by transcriptional fusion with luciferase genes and site-directed mutagenesis. In addition to the previously defined sequences necessary for effective T-box-mediated regulation, new essential regions were identified. Eighteen percent of the positions of the his leader region were found to differ in seven distantly related strains of L. lactis. Analysis of the variable positions supports the folding model of the central part of the his leader region. Lastly, in addition to the T-box-mediated regulation, the operon is regulated at the level of initiation of transcription, which is repressed in the presence of histidine. An operator site, necessary for full repression, overlaps the terminator involved in the T box attenuation mechanism. The functionality of the operator is altered on plasmids with low and high copy numbers, suggesting that supercoiling may play a role in the expression of the his operon. The extents of regulation at the levels of initiation and attenuation of transcription are 6- to 8-fold and 14-fold, respectively. Together, the two levels of control allow a 120-fold range of regulation of the L. lactis operon by histidine.

Project description:A noticeable genomic feature of many piliated Gram-positive bacterial species is the presence of more than one pilus-encoding operon. Paradigmatically, the gut-adapted Lactobacillus rhamnosus GG strain contains two different fimbrial operons in its genome. However, whereas one of these operons (called spaCBA) is encoding for the functionally mucus-/collagen-binding SpaCBA pilus, for the other operon (called spaFED) any native expression of the SpaFED-called pili is still the subject of some uncertainty. Irrespective of such considerations, we decided it would be of relevance or interest to decipher the gross structure of this pilus type, and as well assess its functional capabilities for cellular adhesion and immunostimulation. For this, and by following the approach we had used previously to explicate the immuno-properties of SpaCBA pili, we constructed nisin-inducible expression clones producing either wild-type or SpaF pilin-deleted surface-assembled L. rhamnosus GG SpaFED pili on Lactococcus lactis cells. Using these piliated lactococcal constructs, we found that the pilin-polymerized architecture of a recombinant-produced SpaFED pilus coincides with sequence-based functional predictions of the related pilins, and in fact is prototypical of those other sortase-dependent pilus-like structures thus far characterized for piliated Gram-positive bacteria. Moreover, we confirmed that among the different pilin subunits encompassing spaFED operon-encoded pili, the SpaF pilin is a main adhesion determinant, and when present in the assembled structure can mediate pilus binding to mucus, certain extracellular matrix proteins, and different gut epithelial cell lines. However, somewhat unexpectedly, when recombinant SpaFED pili are surface-attached, we found that they could not potentiate the existing lactococcal cell-induced immune responses so elicited from intestinal- and immune-related cells, but rather instead, they could dampen them. Accordingly, we have now provided the first phenotypical description of a spaFED pilus operon, and with that furthered the functional understanding of surface piliation for a particular gut-commensalic genre of piliated Gram-positive bacteria.

Dataset Information

Molecular characterization of the Lactococcus lactis ptsHI operon and analysis of the regulatory role of HPr.

Publications

Molecular characterization of the Lactococcus lactis ptsHI operon and analysis of the regulatory role of HPr.

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