Identification, cloning, and characterization of the Ima operon, whose gene products are unique to Listeria monocytogenes

Abstract

The lmaA gene of Listeria monocytogenes encodes a protein capable of inducing delayed-type hypersensitivity reactions in L. monocytogenes-immune mice (S. Göhmann, M. Leimeister-Wächter, E. Schiltz, W. Goebel, and T. Chakraborty, M. Microbiol. 4:1091-1099, 1990). Here we show that it is the last gene of the lma operon, which now comprises four genes, lmaDCBA. Maxicell analysis of peptides encoded by the lma operon identified four polypeptides of 16.7, 16.4, 14.9, and 21 kDa which correspond to the gene products encoded by the lmaD, -C, -B, and -A genes, respectively. Northern blot analysis of the lma operon showed that lmaA is expressed by two transcripts: the longer lmaDCBA transcript of 2,100 nucleotides, which was observed at growth temperatures of 37 and 20؇C, and a shorter transcript consisting of lmaBA, which is detected only at low temperatures (20؇C). Two promoters, one preceding the lmaD gene and another located upstream of the lmaB gene, were detected. An extended stem-loop structure resembling box elements found in other gram-positive pathogens was also present in the lmaC-lmaB intergenic region. By immunoblot analysis, we found that although LmaA was produced at both temperatures (20 and 37؇C), it was secreted into culture supernatants only at 20؇C. However, LmaA lacks a bona fide signal peptide sequence and could, like flagellin, be secreted by a type III transport system. DNA hybridization studies indicate that the lma operon is species specific and restricted to pathogenic strains of L. monocytogenes.The gram-positive, facultative intracellular bacterium Listeria monocytogenes is a foodborne pathogen responsible for infections in immunocompromised persons and pregnant women. Listerial infections can result in meningitis, meningoencephalitis, septicemia, and death (10, 12, 16). The consumption of contaminated food has been shown to be the major route of infection. In addition to causing sporadic listeriosis, L. monocytogenes has been implicated in several epidemics in Europe and North America (2, 37). The ability of L. monocytogenes to grow at 4ЊC, a temperature at which many off-theshelf foodstuffs are stored, opens up a unique environment for listerial enrichment (16,43). Listerial products that are expressed at low growth temperatures are therefore a relevant area of study.Virulence factors necessary for entry, intracellular survival, multiplication, and motility have been previously characterized. The genes encoding these factors are organized in two clusters on the listerial chromosome. The main cluster contains the genes coding for hemolysin (hly), phospholipases (plcA, plcB), metalloprotease (mpl), and actin nucleation factor (actA) and includes the virulence regulator protein PrfA (6, 18-21, 27, 28). The internalin genes inlA and inlB, which are required for uptake of the bacteria into the eukaryotic cell, are arranged as an operon close to but separate from the other virulence locus (29). The expression of the inlA and inlB genes is regulated by both PrfA-dependent and -independent mechani...

Natural Atypical Listeria innocua Strains with Listeria monocytogenes Pathogenicity Island 1 Genes

“…L. innocua can be serotype 4f (30), but it has some undefined serotypes, and so the 4f designation is somewhat tentative. Also, L. grayi and L. seeligeri have some undefined serotypes, (27). The presence of hly ϩ , in an apparently non-L. monocytogenes genotypic background, was suggestively similar to the genotype of PRL/ NW 15B95, so these strains were further studied.…”

“…To more clearly show the evolutionary relationships occurring among members of the Listeria species, the species subbranches of the phylogenetic tree were clustered. (4,27). The virulence gene cluster may have been acquired by phage-mediated transfer or via transformation, the possibility of which has been suggested following identification of DNA uptake genes in the genome of both L. monocytogenes and L. innocua (11).…”

“…Since these methods are generic for Listeria spp., a crucial step in contamination analysis is the identification of Listeria isolates to species level. Conveniently, speciation of a bona fide Listeria isolate involves only a few simple tests (14,27,32). These are production, or not, of acid (without gas) from L-rhamnose, D-xylose, or mannitol and the lysis of sheep or horse red blood cells.…”

Genetic and phenotypic characterization of Listeria monocytogenes lineage III

“…Virulence gene alleles for actA and hly were determined by PCR-RFLP as described previously (Wiedmann et al, 1997). Detection of the putative virulence gene lmaA was carried out by PCR using primers lmaA-F (59-TTC TGC TGG TGC TAC AGG TG-39and lmaA-R (59-CCA ACA AGG TCT AAC TGT AAA CCG-39), which amplify an approximately 420 bp fragment of the lmaA ORF (Schaferkordt & Chakraborty, 1997). Each reaction contained 1 unit Taq polymerase (Promega), 1?5 mM MgCl 2 , 16 PCR buffer, 50 mM of each dNTP and 0?5 mM of each primer.…”

“…Subgroups IIIA, IIIB and IIIC contained predominantly hly PCR-RFLP type 4b (28 %, 80 % and 57 %, respectively). Interestingly, 19 lineage III isolates did not yield a PCR product for lmaA, indicating the absence of this putative virulence gene (Schaferkordt & Chakraborty, 1997) in some lineage III isolates. While the majority (88 %) of IIIB and IIIC isolates carried lmaA, only 58 % of lineage IIIA isolates were confirmed to carry this gene, and this difference was statistically significant (P= 0?002).…”