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Universal primers targeting conserved sequences flanking the 3' end of the 16S and the 5' end of the 23S rRNA genes (rDNAs) were used to amplify the 16S-23S rDNA internal transcribed spacers (ITS) from eight species of pseudomonads which have been associated with human infections. Amplicons from reference strains of Pseudomonas aeruginosa, Pseudomonas cepacia, Pseudomonas gladioli, Pseudomonas mallei, Pseudomonas mendocina, Pseudomonas pickettii, Pseudomonas pseudomallei, and Xanthomonas maltophilia were cloned from each species, and sequence analysis revealed a total of 19 distinct ITS regions, each defining a unique sequevar with ITS sizes ranging from 394 (P. cepacia) to 641 (P. pseudomallei) bp. Five distinct ITS sequevars in P. cepacia, four in P. mendocina, three in P. aeruginosa, two each in P. gladioli and P. pseudomallei, and one each in P. mallei, P. pickettii, and X. maltophilia were identified. With the exception of one P. cepacia ITS, all ITS regions contained potential tRNA sequences for isoleucine and/or alanine. On the basis of these ITS sequence data, species-specific oligonucleotide primers were designed to differentiate P. aeruginosa, P. cepacia, and P. pickettii. The specificities of these primers were investigated by testing 220 clinical isolates, including 101 strains of P. aeruginosa, 103 strains of P. cepacia, and 16 strains of P. pickettii, in addition to 24 American Type Culture Collection (ATCC) Pseudomonas strains. The results showed that single primer pairs directed at particular ITSs were capable of specifically identifying the ATCC reference strains and all of the clinical isolates of P. aeruginosa and P. pickettii, but this was not the case with several ITS-based primer pairs tested for P. cepacia. This pathogen, on the other hand, could be specifically identified by primer pairs directed against the 23S rDNA.

The structural gene of the Pasteurella haemolytica leukotoxin determinant is highly homologous to that of the Escherichia coli hemolysin determinant, which also encodes a specialized set of genes involved in the secretion of the hemolysin. In this report, we describe the cloning and nucleotide sequence of the analogous secretion genes from P. haemolytica which make up the remainder of the leukotoxin determinant. The secretion genes were cloned directly from the P. haemolytica chromosome to form the recombinant plasmid pPH5B. By subcloning the secretion genes together with the leukotoxin structural gene, the cloned leukotoxin determinant was reconstructed on a single plasmid, pLKT52, which directs the synthesis of active leukotoxin to the culture supernatant when expressed in E. coli. DNA sequence analysis showed the presence of two secretion genes, designated lktB and lktD in order of their genetic organization, which code for proteins of 79.7 and 54.7 kilodaltons, both of which were detected when pLKT52 was expressed in E. coli minicells. The lktB and lktD genes were found to be highly homologous to the hlyB and hlyD secretion genes of the hemolysin determinant, and the predicted LktB-HlyB and LktD-HlyD proteins were 90.5 and 75.6% homologous. Nucleotide sequence homology between the leukotoxin and hemolysin determinants was limited to the C, A, B, and D coding regions, although the presence of similar transcriptional terminators in the A-B intercistronic region is suggestive of a similar transcriptional organization. On the basis of these data, we hypothesize that the two determinants share a common evolutionary history and are prototypes for a widely disseminated family of virulence factors, the RTX cytotoxins.

The Pasteurella haemolytica leukotoxin determinant is composed of four contiguous genes encoded on the same DNA strand and denoted lktCABD, in the order of their genetic organization. To gain a better understanding of the expression and regulation of the leukotoxin, the transcripts and promoters of the lkt determinant were mapped. Northern (RNA) blot analysis revealed two sets of transcripts. One set was 3.7 and 3.4 kilobases long, encoded lktCA, and comprised approximately 90% of the transcripts, whereas the other set was 7.4 and 7.1 kilobases long and encoded lktCABD. Two promoters were present, and each had features similar to the Escherichia coli consensus promoter sequences. Both promoters were located upstream from lktC; they were separated by 258 base pairs, as mapped by primer extension analysis. These results suggest a mechanism of expression similar to that of the related E. coli hemolysin. Transcription initiated upstream from lktC at either promoter and continued through lktC and lktA to a rho-independent transcriptional termination signal in the lktA-lktB intercistronic region. This signal attenuated expression by terminating 90% of transcription to generate the 3.7- and 3.4-kilobase lktCA transcripts. The remaining readthrough transcription generated full-length 7.4- and 7.1-kilobase lktCABD transcripts. Expression of the leukotoxin was greatly reduced by growth at 30 degrees C, pH 6.5, and Fe2+ limitation. These conditions also modulated the expression of a number of other secreted proteins, which suggests that all of these secreted proteins are controlled by the same regulatory mechanism.

A 4.4-kilobase-pair DNA fragment coding for the leukotoxin of Pasteurella haemolytica A1 has been isolated, and its nucleotide sequence has been determined. Two open reading frames, designated lktC and lktA, coding for proteins of 19.8 and 101.9 kilodaltons, respectively, were identified. Expression of the two genes in minicell-labeling experiments resulted in the production of the predicted proteins LKTC and LKTA. By using an antiserum against the soluble antigens of P. haemolytica A1 in Western blot (immunoblot) analysis of total cellular proteins from the Escherichia coli clones, LKTA was identified as an additional antigenic protein. Results from subcloning of the DNA fragment suggested that expression from both lktC and lktA is required for leukotoxin activity, indicating that the leukotoxin of P. haemolytica A1 is encoded by two genes. A comparison of the organization and the DNA sequence of the leukotoxin genes with those of the E. coli alpha-hemolysin genes showed a significant degree of homology between the two loci. This analysis suggested that the leukotoxin genes of P. haemolytica A1 and the E. coli alpha-hemolysin genes may have evolved from a common ancestor and that the two toxins may share similar activities or functional domains or both.