ABSTRACT
Mutational analysis of a 5.5 kb fragment of the genome Streptococcus pneumoniae led to the identification of a putative new virulence gene, designated orfD. Insertion mutagenesis of flanking genes on the fragment suggested that the corresponding gene products were required for in vitro growth. In contrast, insertion mutation of orfD did not alter in vitro growth or the transformability pattern of the mutated strain. However, it did reduce bacterial growth in mice and attenuated virulence in an intraperitoneal model of infection. orfD is flanked by orfC (63 codons) and ftsL (105 codons) and all three genes are upstream of pbpx. orfC showed no similarity with other known proteins. ftsL of S. pneumoniae exhibits minimal sequence similarity with ftsL of E. coli, but shares 16% identical residues with the ftsL homologue encoded by ylld of B. subtilis. Also, ftsL of S. pneumoniae has a predicted topology similar to that described for ftsL of E. coli. Putative promoters with an extended -10 box could be identified upstream of both orfC or orfD. The four open reading frames (including pbpx) are orientated in the same direction, and polycistronic transcription could theoretically start at either promoter. Interestingly, this region shows organizational and sequence homologies with genes controlling division and cell wall biosynthesis (DCW) in other bacteria. The attenuation of virulence in the orfD insertion mutant might be due to the loss of function of the orfD gene product or to an altered level of expression of downstream genes.
Subject(s)
Cell Cycle Proteins , Escherichia coli Proteins , Genes, Bacterial , Mutagenesis, Insertional , Open Reading Frames/genetics , Streptococcus pneumoniae/genetics , Streptococcus pneumoniae/pathogenicity , Amino Acid Sequence , Animals , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Base Sequence , Cell Division/genetics , Cell Wall/genetics , Cell Wall/metabolism , Male , Membrane Proteins/genetics , Membrane Proteins/metabolism , Methyltransferases/genetics , Methyltransferases/metabolism , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Pneumococcal Infections/microbiology , Sequence Alignment , Sequence Analysis, DNA , Streptococcus pneumoniae/growth & development , Virulence/geneticsABSTRACT
Expression of the PrfA-controlled virulence gene hly (encoding the pore-forming cytolysin listeriolysin) is down-regulated by readily metabolized carbon sources in Listeria monocytogenes. We isolated a Tn917-insertional mutant of L. monocytogenes (strain LO28), which expressed a hemolytic phenotype in the presence of cellobiose. Using hly fusions to luxAluxB genes, we show that hly expression was derepressed in the presence of cellobiose at the transcriptional level. Surprisingly, hly expression was still repressed by glucose, as observed for the parental strain. Genetic analysis of the Tn917-flanking regions indicated that the transposon had inserted in a non-coding region located between two genes in opposite orientations. These two newly identified genes were designated orfA and mdrL. The insertion occurred immediately upstream of orfA, likely into its promoter region. Transcriptional analysis of orfA and mdrL revealed that Tn917 had abolished orfA expression whereas it had activated expression of mdrL. orfA encodes a putative protein of 176 amino acids homologous to YfiO of Bacillus subtilis (28% identity), a protein of unknown function. mdrL codes for a putative protein of 398 amino acids homologous to Bmr and Blt of B. subtilis (21-24% identity), two members of the multidrug resistance efflux pump family. Our results indicate that we have identified a new locus which plays a crucial role in the cellobiose-dependent repression of hly expression.
