No abstract

Plasmid analysis was used in the investigation of an outbreak of nosocomial Legionnaires' disease in four patients. Serogroup 1 strains were isolated from two patients, the air-conditioning cooling tower, and two hot-water tanks. All serogroup 1 strains contained two plasmids with approximate molecular masses of 21 and 48 megadaltons (Mdal). The serogroup 1 strain found in the cooling-tower isolate also contained an additional 1.9 Mdal-plasmid. Restriction-endonuclease analysis of the 21-Mdal plasmid that was present in patient and hot water-tank isolates revealed identical EcoRI and HaeIII fragment patterns. Digestion of the similarly sized plasmid in the cooling-tower isolate resulted in a unique fragment pattern. The data provide direct bacteriologic evidence implicating the hot-water tanks rather than the cooling tower as the source of the infecting strain.

The Salmonella typhimurium pepE gene, encoding an N-terminal-Asp-specific dipeptidase, has been cloned on pBR328 by complementation of the Asp-Pro growth defect conferred by a pepE mutation. Strains carrying the complementing plasmids greatly overproduce peptidase E. The enzyme has been purified from an extract of such a strain, its N-terminal amino acid sequence has been determined, and crystals suitable for X-ray diffraction have been grown. A new assay using L-aspartic acid p-nitroanilide as a substrate has been used to determine the pH optimum (-7.5) and to test the elfect of potential inhibitors. Insertions of transposon y8 (TnlOOO) into one of the plasmids have been used to localize the gene and as sites for priming sequencing reactions. The nucleotide sequence of a 1,088-bp region of one of these plasmids has been determined. This sequence contains an open reading frame that predicts a 24.8-kDa protein with an N-terminal sequence that agrees with that determined for peptidase E. The predicted peptidase E amino acid sequence is not similar to that of any other known protein. The nucleotide sequence of the region upstream from pepE contains a promoter with a cyclic AMP receptor protein (CRP) site, and the effects of growth medium and of a crp mutation on expression of a pepE-lacZ fusion indicate that pepE is a member of the CRP regulon. The unique specificity of peptidase E and its lack of sequence similarity to any other peptidase suggest that this enzyme may be the prototype of a new class of peptidases. Its regulation by CRP and its specificity suggest that the enzyme may play a role in allowing the cell to use peptide aspartate to spare carbon otherwise required for the synthesis of the aspartate family of amino acids.

Mutations at the Escherichia coli prlC locus suppress the export defect of certain lamB signal sequence mutations. The SalmoneUla typhimurium opdA gene encodes an endoprotease that can participate in the catabolism of certain peptides and is required for normal development of phage P22. Plasmids carrying either the wild-type (pTC100 prlC+) or suppressor alleles ofpriC complemented all phenotypes associated with an S. typhimurium opdA mutation. A plasmid carrying an amber mutation in priC (prlC31(Am)] was unable to complement except in an amber suppressor background. TnlO00 insertions which eliminated the ability of pTC100 (prlC+) to complement opdA4 mapped to the region of the plasmid shown by deletion analysis and subcloning to contain priC. The nucleotide sequence of a 2.7-kb fragment including this region was determined, revealing an open reading frame encoding a 77-kDa protein. The sequences of this open reading frame and its putative promoter region were very similar (84% nucleotide sequence identity and 95% amino acid identity) to those of S. typhimurium opdA, showing that these genes are homologs. The nucleotide sequence of the priCi suppressor allele was determined and predicts an in-frame duplication of seven amino acids, providing further confirmation that the priC suppressor phenotype results from changes in the endopeptidase OpdA.

Lipopolysaccharide (LPS) is a major constituent of the outer membrane of gram-negative bacteria. We used sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteinase-K-digested cell lysates to provide preliminary data on the LPS chemotypes for 20 strains of LegioneUa pneumophila (serogroups 1 to 8). The profiles of all strains except Chicago 2 (serogroup 6) were similar in the number, spacing, and size distribution of the bands visualized on silver-stained gels and were indicative of smooth LPS. However, compared with the bands from Salmonella minnesota smooth LPS, their banding pattern was much tighter, with three to four legionella bands for every salmonella band. The proteinase K digest of Chicago 2 was unique in that only two widely separated silver-stained bands were seen. LPS profiles of 10 serogroup 1 strains were identical, and the profile of Knoxville 1 was not altered by extensive in vitro passage. We used immunoblotting to investigate the serological specificities of the LPSs. When a rabbit antiserum prepared against a serogroup 1 strain was used to probe nitrocellulose sheets that bound LPS from strains belonging to eight different serogroups, it recognized only the LPS from the homologous serogroup. Similar results were observed with serogroup 2, 4, and 6 antisera. Our data indicate that L. pneumophila has a smooth-type LPS with an unusual banding pattern and that it is a serogroup-specific antigen.