Cloning and expression of the Listeria monocytogenes scott A ptsH and ptsI genes, coding for HPr and enzyme I, respectively, of the phosphotransferase system

Abstract

The phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) utilizes high-energy phosphate present in PEP to drive the uptake of several different carbohydrates in bacteria. In order to examine the role of the PTS in the physiology of Listeria monocytogenes, we identified the ptsH and ptsI genes encoding the HPr and enzyme I proteins, respectively, of the PTS. Nucleotide sequence analysis indicated that the predicted proteins are nearly 70% similar to HPr and enzyme I proteins from other organisms. Purified L. monocytogenes HPr overexpressed in Escherichia coli was also capable of complementing an HPr defect in heterologous extracts of Staphylococcus aureus ptsH mutants. Additional studies of the transcriptional organization and control indicated that the ptsH and ptsI genes are organized into a transcription unit that is under the control of a consensus-like promoter and that expression of these genes is mediated by glucose availability and pH or by by-products of glucose metabolism.

FIG. 1

Nucleotide and deduced amino acid sequences of the ptsH and ptsI genes of L. monocytogenes Scott A. The putative −35 and −10 promoter sequences and ribosome binding sites (RBS) are underlined. The transcriptional start site is indicated by +1. The ptsH stop codon is indicated by an asterisk.

FIG. 2

Amino acid alignment comparing the HPr protein of L. monocytogenes to the HPr proteins of other gram-positive bacteria. Areas of 100% conservation are enclosed in boxes. The GenBank accession numbers for the nucleotide sequences encoding the proteins are as follows: S. carnosus, X60766; S. mutans, L15191; S. salivarius, Z17217; E. faecalis, Z19137; and B. subtilis, X12832.

FIG. 3

Northern blot analysis of L. monocytogenes Scott A RNA performed with probes specific for ptsH (A) and ptsI (B). (A) The lanes contained total RNA from cells harvested under the following conditions: lane 1, pH 5.4, 1.2 mM glucose; lane 2, pH 5.3, 0.4 mM glucose; lane 4, pH 6.2, 70.6 mM glucose; lane 5, pH 5.34, 64.2 mM glucose; lane 6, pH 4.4, 54.9 mM glucose. Lane 3 contained no RNA. (B) The lanes contained total RNA from cells harvested under the following conditions: lane 1, pH 6.6, 7.4 mM glucose; lane 2, pH 5.4, 1.2 mM glucose; lane 3, pH 5.3, 0.4 mM glucose; lane 4, pH 6.6, 72.9 mM glucose; lane 5, pH 6.2, 70.6 mM glucose; lane 6, pH 4.4, 54.9 mM glucose.

FIG. 4

Primer extension of the ptsHI transcriptional start site. The position of the cDNA primer extension product is indicated by an arrow.

FIG. 5

SDS-PAGE. Lane 1, protein molecular weight standard; lane 2, E. coli E509 containing pRSET; lane 3, uninduced E. coli E509 containing pRSET-ptsH gene construct; lane 4, IPTG-induced E. coli E509 containing pRSET-ptsH gene construct; lane 5, purified L. monocytogenes HPr. kD, kilodaltons.