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Two different hemolysins, HpmA and HlyA, have been reported in Proteus spp. To study the distribution of these hemolysins among Proteus strains, isolates from various infections and normal feces were screened for hemolysin production. All 63 Proteus mirabilis strains and 23 of the 24 Proteus vulgaris strains produced a calcium-independent hemolytic activity detectable in cell-free supernatants. The calcium-independent activity was due to HpmA; this activity correlated with the presence of hpmA sequences and the production of an extracellular 166-kilodalton (kDa) protein that reacted with anti-HpmA antiserum. HpmA- mutants, constructed by deletion of the central portion of the hpmA gene, did not produce the 166-kDa protein and were no longer hemolytic when compared with their respective parent strains. Among the 87 P. mirabilis and P. vulgaris isolates examined, calcium-dependent hemolytic activity was produced by only two P. vulgaris strains. These strains produced a 110-kDa protein which comigrated with the Escherichia coli hemolysin (HlyA) antibodies in immunoblots. These studies show that Proteus spp. produce two distinct extracellular hemolysins, with nearly all strains producing the calcium-independent hemolysin, HpmA, but only an occasional P. vulgaris isolate producing HlyA.

The lipopolysaccharides (LPS) extracted from Proteus strains OX2, OX19, and OXK used as antigens in the Weil-Felix test, were characterized by chemical analysis and SDS-polyacrylamide gel electrophoresis (SDS-PAGE). To separate the O-polysaccharide, core-oligosaccharide, and lipid A moieties, each LPS was treated with 2% acetic acid, centrifuged, and applied to Sephadex G-50 column. The core-oligosaccharides contained L-glycero-D-mannoheptose, D-glycero-D-mannoheptose, glucose (Glc), galactose, 3-deoxy-D-mannooctulosonic acid, uronic acid, phosphate, glucosamine (GlcN), and galactosamine (GalN). The lipid A preparations contained GlcN, GlcN-phosphate, and three fatty acids (myristic, plamitic, and beta-hydroxymyristic acids). However, the O-polysaccharides of OX2- and OXK-LPS had different chemical compositions which consisted of Glc, GlcN, and quinovosamine, and Glc, uronic acid, and GalN, respectively, while OX19-LPS seemed to lack O-polysaccharide.

The bacteria harvested in the early log phase lyse when they are submitted to a pH above 10. The peptidoglycan is not degraded in these conditions. Thus, the authors used these properties to extract the peptidoglycan from several gram negative and gram positive bacteria.

Morganella ("Proteus") morganii is the only species in the recently proposed genus Morganella, but we suspect there are yet undescribed species in the genus. Two candidates for new species were recently investigated. Nineteen strains isolated from clinical specimens resembled M. morganii but were lysine positive and nonmotile and fermented glycerol within 24 h. Typical M. morganii are lysine negative and motile and ferment glycerol slowly or not at all. The three-test variance from typical M. morganii suggested that this new group might be a new Morganella species; however, strains of the group were closely related to typical M. morganii by deoxyribonucleic acid (DNA)-DNA hybridization. A second group of 14 strains isolated from clinical specimens was ornithine negative but was otherwise very similar to M. morganii. This group was also closely related to M. morganii by DNA hybridization. Both sets of strains clearly belong in the species Morganella morganii as distinct biogroups; they are not separate species as originally suspected. Initially both biogroups posed a problem in identification until their true relationship to M. morganii was determined.

Enteric bacteria having a high content of cyclopropane fatty acids steeply increase their synthesis when grown on insufficiently propitious culture media (meat-peptone agar or modified Drobot'ko synthetic medium) as compared with bacteria grown under more favourable conditions (meat-peptone broth). Simultaneously, a decrease in monounsaturated fatty acids and increase in palmitic acid are observed. One of the main factors underlying the change in the proportion of fatty acids in bacteria grown on synthetic medium is an increase in medium pH in the process of their growth. Enteric bacteria containing minute amounts/or not containing cyclopropane fatty acids at all (under the experimental conditions used) change their fatty-acid profile little if the culture medium is changed. When grown under insufficiently favourable conditions, these bacteria mainly display an enhanced content of palmitic acid and a lowered content of octadacenoic acid as compared with bacteria grown under more favourable conditions. Of the culture media used, meat-peptone broth, which affords the most favourable conditions for eneteric bacteria growth, is the most suitable medium for obtaining data of taxonomic value.

Yersinia pestis has been characterized in terms of fingerprints of digests (pancreatic and/or T1 ribonuclease) of its 16S and 5S ribosomal RNAs. These show clearly that Y. pestis is a member of the Enterobacteriaceae and suggest that within the Family it is most closely related to Serratia and/or Proteus.

Proteocines derived from twelve previously described bacteriocinogenic strains of Proteus mirabilis and Proteus vulgaris were investigated. All proteocine preparations were particulate, unaffected by trypsin, and destroyed by freezing and thawing or by heating at 60 degrees C for 30 minutes. Proteocine activity was removed by adsorption with the appropriate sensitive organisms. The active principles of all preparations were partially purified by precipitation with 70% (w/v NH4(SO4)2 followed by ultra-centrifugation. Column chromatography showed that proteocine activity was associated with only one of the peaks of material which absorbed strongly at 257 mu. All twelve proteocine preparations were revealed by electron microscopy as a phage-tail-like structure and each particle had a sheath, a core, and a base-plate from which spine-like fibres extend. Adsorption of these particles to the cell wall of sensitive strains did not disrupt the bacterial cell wall, but the cytoplasmic membrane and the cell contents shrank, with consequent death of the "infected" cell.

The haemagglutinins and fimbriae produced by 18 strains of Proteus penneri were studied and compared with those formed by representative strains of other species of Proteeae. After repeated subcultures at 30 degrees C, 12 P. penneri strains formed only MR/K haemagglutinins which were associated with thin, non-channelled, type-3 fimbriae. Two strains formed simultaneously both MS and MR/K haemagglutinins associated with thick, channelled, type-1 fimbriae and type-3 fimbriae, respectively. Four strains formed simultaneously both MR/K and MR/P haemagglutinins. No P. penneri strain formed either MS or MR/P haemagglutinins alone under these conditions. The type-3 fimbriae from P. penneri strain E180 were isolated, purified and found to be protein of 19 Kda. Immunoelectronmicroscopy studies with antibody to the type-3 fimbriae of strain E180 showed that P. penneri strains at least two antigenic types of type-3 fimbriae. The type-3 fimbrial antigen of the vaccine strain E180 was shared by another eight strains of P. penneri. A further eight P. penneri strains and the strains representative of other genera within Proteeae had type-3 fimbriae of a different antigenic type. The formation of these haemagglutinins and fimbriae suggests that this organism is well endowed to be a urinary-tract pathogen.

Twenty strains of Providencia rustigianii (including the type strain of Prov. friedericiana) have been characterized by one-dimensional SDS-PAGE of cellular proteins. They comprised 12 strains (almost exclusively associated with the intestinal tract) from humans, plus eight largely from the intestinal tract of pig, penguin and environmental sources. The protein patterns, which contained 45-50 discrete bands, were highly reproducible and were used as the basis for two numerical analyses. In the first, which included all the protein bands, the 20 Prov. rustigianii strains formed six clusters at the 88% S level. One of these clusters included the type strains of both Prov. friedericiana and Prov. rustigianii, thereby confirming the synonymy of these two species. In the second analysis, the principal protein bands were excluded. At the 86% S level the 20 Prov. rustigianii strains formed a single cluster, whilst a field strain of Morganella morganii and the respective type strains of three other Providencia species remained unclustered. The total protein pattern of the type strain of Prov. alcalifaciens was very similar to that of Prov. rustigianii phenon 3 and the M. morganii field strain, which indicates that careful biochemical characterization may be necessary to ascribe strains to a species before typing by the PAGE technique. Alternatively, a selective analysis of the protein bands may be used to confirm the identity of the strains, as shown in this study.

The fatty acid composition of total cellular lipids and lipid A of lipopolysaccharides was studied in Providencia alcalifaciens and P. stuartii. Significant differences were found in the fatty acid composition of total cellular lipids from the above species; these are caused mainly by the presence of cyclopropane fatty acids (methylenehexadecanoic and methyleneoctadecanoic acids) in P. stuartii and their absence in P. alcalifaciens. Differences were also established in the content of fatty acids in lipid A of lipopolysaccharides from P. alcalifaciens and P. stuartii. The data are consistent with the idea that P. alcalifaciens and P. stuartii are separate taxons though, apparently, closely related.

Proteus mirabilis, Proteus vulgaris, Morganella morganii, Providencia rettgeri, and Providencia alcalifaciens, which were once classified into the same genus, Proteus, were studied. Cefoxitin-resistant mutants from these species were isolated, and it was confirmed that the resistance was attributed to the lack of an outer membrane protein, resulting in a significant decrease in the penetration of hydrophilic cephalosporins through the outer membrane. Comparison of the mutant strains with their parental strains in the diffusion rates of six monoanionic cephalosporins, a zwitterionic cephalosporin (cephaloridine), and a divalent anionic cephalosporin (cephalosporin C) suggested that each species had only one kind of porin protein, with molecular weights of 40,000 (Proteus mirabilis) or 37,000 (the other four species) and that the porins formed channels with cation selectivity, except for Proteus vulgaris. Porin proteins were purified from all the bacterial species except Providencia alcalifaciens, and the radius of the pores formed by the purified porins was estimated by the use of the liposome swelling assay. The pore radii were estimated to be approximately 0.59 nm (Proteus mirabilis), 0.63 nm (Proteus vulgaris), 0.58 nm (Providencia rettgeri), and 0.60 nm (M. morganii), similar to the size of the pore radius of Escherichia coli porins.

Penicillin-binding proteins in three species of Proteus, Proteus mirabilis, P. morganii, and P. rettgeri, were investigated by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. Penicillin-binding proteins in these Proteus species were compared with those in Escherichia coli K-12. An approximate correlation between penicillin-binding proteins in E. coli and those in Proteus species was shown by several criteria: electrophoretic mobilities; affinities of several beta-lactam antibiotics which show characteristic patterns of binding to penicillin-binding proteins in E. coli; relation between affinities of antibiotics to the proteins and effects on morphological changes in Proteus species; location of beta-lactamase activity among penicillin-binding proteins; and thermostability. The electrophoretic mobilities and several other characteristics of penicillin-binding proteins among the Proteus species examined were found to be similar from species to species and differed only slightly from those of E. coli.

The fatty acid compositions of stationary-phase cultures of Arizona arizonae, Citrobacter freundii, Providencia alcalifaciens, Providencia stuartii, and Providencia sp. were studied. The major fatty acids of A. arizonae, C. freundii, and Providencia were 16:0, 16:1, 17:cyclopropane, and 19:cyclopropane. The fatty acid compositions of the two strains of A. arizonae examined were similar to each other, but the three strains of C. freundii differed from one another in their fatty acid compositions. In both A. arizonae and C. freundii, the relative quantities of saturated, unsaturated, and cyclopropane fatty acids were similar to those which have been found in stationary-phase cultures of other members of the Enterobacteriaceae. The three strains of Providencia also differed from one another in their fatty acid compositions. In all three strains, the total quantity of unsaturated fatty acids was larger and that of the cyclopropane fatty acids was smaller than those found in stationary-phase cultures of other enteric bacteria.

A covalently-closed circular DNA species, banding at a buoyant density of rho = 1.709 g/cm(3) in CsCl, has been identified in antibiotic-sensitive colonies of E. coli strain AB2463 (rec A(-)) after mating with a Proteus mirabilis strain that carries the R-factor, R1. This plasmid, which represents a stable segregant of R1 that has lost all of the drug resistance determinants present on the parent R-factor but which has retained its ability to be transferred by conjugation, fulfills the functional definition of the R-factor transfer unit (RTF).

Eighty-seven different enterobacteria and pseudomonas strains were examined for the presence of extrachromosomal deoxyribonucleic acid (DNA). Thirty-four strains contained closed circular DNA by the ethidium bromide CsCl density technique. Extrachromosomal DNA was most frequent in Escherichia and Klebsiella strains. The extrachromosomal DNA was isolated and characterized by analytical ultracentrifugation and electron microscopy. All the extrachromosomal DNA-containing bacteria contained circular DNA molecules of small size (0.5-4 mum). Most of these bacteria also contained larger circles (20-40 mum). The number of different size classes of circular DNA in each strain varied from one to five. The buoyant density of the extrachromosomal DNA ranged from 1.692 to 1.721 g/cm(3). Many bacteria contained extrachromosomal DNA of more than one density.

Analysis of the deoxyribonucleic acid prepared from Proteus mirabilis, Escherichia coli, and Serratia marcescens in an alkaline CsCl gradient has shown that there is an asymmetric distribution of guanine plus thymine residues between the complementary strands of the deoxyribonucleic acid.

The buoyant density of deoxyribonucleic acid (DNA) from the temperature-sensitive R factor, Rts1, was determined by CsCl density-gradient centrifugation. Rts1 was found to consist of a single species of DNA of density 1.705 g/cm(3), which corresponds to a base composition of 45% guanine plus cytosine. This value is distinct from the densities previously reported for other R factors, suggesting that Rts1 represents a new molecular class of R factors.

Polyacrylamide gel electrophoretic patterns of acidified phenol extracts prepared from whole cells can be used for the identification of bacterial L-forms and Mycoplasma species of human origin. Ten human Mycoplasma serotypes and eight L-forms belonging to five different genera were studied. The gel patterns were sufficiently distinct and reproducible that it was possible not only to identify L-forms at the genus level (group with streptococci) and different Mycoplasma serotypes but also to differentiate between the two of them. The parentage of L-forms of Streptobacillus moniliformis L1, Listeria monocytogenes, Streptococcus MG, and Staphylococcus aureus Smith strain was established by relating their gel patterns directly to parent bacteria. It was found that an L-form designated S. moniliformis An (ATCC 14220) was actually an L-form of Proteus. In addition, it was shown electrophoretically that no relationship existed between the Streptococcus MG L-form and M. pneumoniae. The applicability of this method as a diagnostic and taxonomic tool for the differentiation of L-forms and mycoplasmas is discussed.

The transfer unit, RTF, derived from an R factor has been transferred to Proteus mirabilis and characterized physiochemically.