A monoclonal antibody obtained by immunization of mice with heat-killed cells of Listeria monocytogenes serotype 4d showed reactivity towards a protein (P45) from L. monocytogenes with an apparent molecular mass of 45 kDa. This protein was detected in the culture supernatant and at the cell surface of L. monocytogenes. Proteins cross-reacting with the monoclonal antibody were present in all Listeria strains investigated, except L. grayi. The structural gene was cloned in Escherichia coli and sequenced. Translation of the gene starts at a TTG initiation codon. The gene was found to code for a protein of 402 amino acid residues with a predicted molecular mass of 42.7 kDa. It has a signal peptide of 27 amino acid residues, resulting in a molecular mass for the mature polypeptide of 39.9 kDa. Protein database searches showed that this protein has 55% similarity and 38% identity to protein p60 of L. monocytogenes and exhibits significant sequence similarities to p54 from Enterococcus faecium and Usp45 from Lactococcus lactis. P45 was shown to have peptidoglycan lytic activity and the encoding gene was named spl (secreted protein with lytic property).

The distribution of distinct sequences in pathogenic and commensal Neisseria species was investigated systematically by dot blot analysis. Probes representing the genes of Rmp, pilin and IgA1 protease were found to hybridize exclusively to the chromosomal DNA of the pathogenic species, Neisseria gonorrhoeae and/or Neisseria meningitidis. In contrast, specific sequences for the genes of the porin protein Por and the opacity protein (Opa) were also detected in a panel of commensal Neisseria species such as N. lactamica, N. subflava, N. flava, N. mucosa and N. sicca. Using opa-specific oligonucleotides as probes in chromosomal blots, the genomes of the commensal Neisseria species show a totally reduced repertoire of cross-hybridizing loci compared to the complex opa gene family of N. gonorrhoeae. DNA sequence analysis of one opa-related gene derived from N. flava and N. sicca, respectively, revealed a large degree of homology with previously described gonococcal and meningococcal genes, e.g., a typical repetitive sequence in the leader peptide and the distribution of the hypervariable and conserved regions. This observation, together with the finding, that the gene is constitutively transcribed, leads to the assumption that some of the commensal Neisseria species may have the potential for the expression of a protein harboring similar functions as the Opa proteins in pathogenic Neisseriae.

The distribution of distinct sequences in pathogenic and commensal Neisseria species was investigated systematically by dot blot analysis. Probes representing the genes of Rmp, pilin and IgA1 protease were found to hybridize exclusively to the chromosomal DNA of the pathogenic species, Neisseria gonorrhoeae and/or Neisseria meningitidis. In contrast, specific sequences for the genes of the porin protein Por and the opacity protein (Opa) were also detected in a panel of commensal Neisseria species such as N. lactamica, N. subflava, N. flava, N. mucosa and N. sicca. Using opa-specific oligonucleotides as probes in chromosomal blots, the genomes of the commensal Neisseria species show a totally reduced repertoire of cross-hybridizing loci compared to the complex opa gene family of N. gonorrhoeae. DNA sequence analysis of one opa-related gene derived from N. flava and N. sicca, respectively, revealed a large degree of homology with previously described gonococcal and meningococcal genes, e.g., a typical repetitive sequence in the leader peptide and the distribution of the hypervariable and conserved regions. This observation, together with the finding, that the gene is constitutively transcribed, leads to the assumption that some of the commensal Neisseria species may have the potential for the expression of a protein harboring similar functions as the Opa proteins in pathogenic Neisseriae.

The genus Neisseria includes two important human pathogens, Neisseria gonorrhoeae and Neisseria meningitidis, causing gonorrhea and meningitidis, respectively. The very close relationship between these two species has been shown by a variety of approaches, including DNA-DNA hybridization, interspecies transformation and DNA restriction endonuclease pattern (1, 2, 3). Based on 16S rRNA sequence comparisons Neisseria gonorrhoeae was grouped to the (-proteobacteria (4). By Southern hybridization we identified four distinct rRNA loci in the genome of Neisseria gonorrhoeae and Neisseria meningitidis, respectively (data not shown). This confirms the results previously found for Neisseria gonorrhoeae (5). The polymerase chain reaction (PCR) was used to amplify one 23S rRNA gene from the chromosome of Neisseria gonorrhoeae and Neisseria meningitidis and their nucleotide sequence was determined. The DNA sequence of the 23S rRNA genes from the two Neisseria species is 2890 bp in length. The G+C content of the 23S rRNA gene is 52.1 mol% and 51.6 mol% for Neisseria gonorrhoeae and Neisseria meningitidis, respectively, which is in accordance with the overall base composition of the genomic DNA (50-53 mol%). The high sequence similarity (>98%) (Table 1) confirms the close relationship of these organisms. The sequence differences are mainly restricted to the known variable regions of the 23S rRNA gene (6). In Table 1 sequence similarities with two other proteobacteria, Pseudomonas cepacia (6) representative for the f-subgroup and E. coli belonging to the 'y-subgroup, are shown. A phylogenetic tree based on the Kn." values (7) is outlined in Figure 1. The analysis of the secondary structure reveals that both Neisseria 23S rRNAs harbour a typical helix 16 found in all proteobacteria rRNAs (6). In spite of the great homology between the two investigated neisserial rRNAs the differences may be useful for the design of species-specific probes for rapid identification of these pathogens.

The class 3 protein (PorB) is an important component of the meningococcal outer membrane. The structural gene (porB) encoding the class 3 protein has been cloned using primers suitable for the amplification of the corresponding chromosomal fragment by the polymerase chain reaction (PCR). The complete nucleotide sequence was determined and predicts a mature protein of 310 amino acids, preceded by a signal peptide of 19 residues. The predicted protein sequence of the class 3 protein exhibits essential structural homology to the gonococcal porin PIA. The class 3 protein encoding gene was expressed in Escherichia coli under the control of an inducible promotor.