Antigenic Determinants of Salmonella for Production of “Clearance” Factor in Mouse Typhoid

The “clearance” factor produced in the peritoneal cavity of mice immunized with killed vaccines prepared from Salmonella typhimurium or S. enteritidis was identified as the specific antibodies elicited by the O side chain of the cell wall polysaccharides in the organisms used as immunogens. After immunization of mice with vaccines prepared from virulent Salmonella strains, complement-dependent antibacterial antibodies in the serum and “clearance” factors in the peritoneal cavity were found to appear coincidentally, to last for more than one year, and to have the same specificity against the virulent bacterial strains. The relationship between the complement-dependent antibacterial antibodies and “clearance” factor, and the mechanisms of bactericidal action of these antibacterial agents in experimental typhoid were discussed.     

Development,replication, and reversion of mecillinam-induced ovoid form ofSalmonella typhimurium

Concentrations of mecillinam ranging from 0.8 to 0.0125 g/ml in Trypticase soy broth causedSalmonella typhimurium to assume an ovoid morphology. The presence of chloramphenicol or the absence of nutrients or nonphysiological temperatures prevented the change to ovoid morphology, whereas the presence of nalidixic acid modified the effect of mecillinam. Ovoid forms replicated as ovoids. No intermediate rod morphology was evident during eight daily subculturings of ovoids in mecillinam. Reversion of ovoids to normal morphology occurred within 3 h in drug-free medium, or within 15 min after the addition of penicillinase. Inhibition of either DNA or protein synthesis, or the absence of nutrients, or incubation in nonphysiological temperatures prevented reversion of ovoids to normal morphology.     

Inhibition of cell division inMyxococcus xanthus by mecillinam

Increase in cell numbers is inhibited by adding mecillinam to vegetative cultures ofMyxococcus xanthus. However, both cell length and volume continue to increase in the presence of the antibiotic. Different size classes of cells increase in proportion to their initial size. Incorporation ofmeso-diamino[¹⁴C]pimelic acid into peptidoglycan and [³H]uridine into RNA is not immediately affected by mecillinam. It is suggested that mecillinam inhibits the formation of new sites of wall synthesis, and these are necessary for cell division to occur.     

Radiation resistance ofSalmonella

Summary The ionizing radiation resistances of sixSalmonella species were examined. The experimental variables were the suspending medium, the presence or absence of air, and the temperature during the irradiation process.S. typhimurium ATCC 14028,S. enteritidis ATCC 9186,S. newport ATCC 6962,S. dublin ATCC 15480,S. anatum ATCC 9270, andS. arizonae ATCC 29933 were suspended in phosphate buffer (0.1 M, pH 7.0), brain heart infusion broth (BHI) or mechanically deboned chicken and exposed to gamma radiation from cesium-137 at 0.12 kGy per min. The radiation resistance of theSalmonella increased approximately two-fold when assayed in sterile mechanically deboned chicken rather than in buffer or BHI. The average radiation (0.30 to 1.20 kGy) D-value for all sixSalmonella strains was 0.56 kGy in mechanically deboned chicken.S. enteritidis was significantly more resistant to ionizing radiation than the other five strains ofSalmonella tested on mechanically deboned chicken. The temperature of irradiation but not the presence or absence of air significantly influenced the survival ofS. typhimurium andS. enteritidis in mechanically deboned chicken. Treatment of chicken meat with ionizing radiation would be an effective means for control ofSalmonella contamination.     

Analysis of chimeric UmuC proteins: identification of regions inSalmonella typhimurium UmuC important for mutagenic activity

UnlikeEscherichia coli, the closely related bacteriumSalmonella typhimurium is relatively unresponsive to the mutagenic effects of DNA-damaging agents. Previous experiments have suggested that these phenotypic differences might result from reduced activity of theS. typhimurium UmuC protein. To investigate this possibility, we have taken advantage of the high degree of homology between the UmuC proteins ofE. coli andS. typhimurium and have constructed a series of plasmid-encoded chimeric proteins. The possibility that the phenotypic differences might be due to differential expression of the respective UmuC proteins was eliminated by constructing chimeric proteins that retained the first 25 N-terminal amino acids of either of the UmuC proteins (and presumably the same translational signals), but substituting the remaining 397 C-terminal amino acids with the corresponding segments from the reciprocal operon. Constructs expressing mostlyE. coli UmuC were moderately proficient for mutagenesis whereas those expressing mostlyS. typhimurium UmuC exhibited much lower frequencies of mutation, indicating that the activity of the UmuC protein ofS. typhimurium is indeed curtailed. The regions responsible for this phenotype were more precisely localized by introducing smaller segments of theS. typhimurium UmuC protein into the UmuC protein ofE. coli. While some regions could be interchanged with few or no phenotypic effects, substitution of residues 212–395 and 396–422 ofE. coli UmuC with those fromS. typhimurium resulted in reduced mutability, while substitution of residues 26–59 caused a dramatic loss of activity. We suggest, therefore, that the primary cause for the poor mutability ofS. typhimurium can be attributed to mutations located within residues 26–59 of theS. typhimurium UmuC protein.     

Incidence of resistance to chloramphenicol and tetracyclines among 13502Salmonella strains isolated in 1961

Summary The rate of resistance to tetracycline and chloramphenicol amongSalmonella strains isolated in the Netherlands in 1961 was found to be 3.96%, the corresponding figures for 1958/1959 and 1961 being 2.08 and 1.29 respectively. In this country the total number ofSalmonella types found to develop resistance to either tetracycline or chloramphenicol now amounts to 38. Almost 77% of all resistant strains isolated in 1961 were found among the human pathogenS.typhimurium. The relative frequency of resistance in this organism was 8.18%, as compared with 2.50% in 1958/1959 and 1.80% in 1960. In 1961 some cross infections caused by tetracycline resistant strains ofS.typhimurium were observed in man and on one occasion also in a herd of calves. A similar outbreak due to a tetracycline resistant strain ofS.bovis morbificans was seen in a hospital. As almost 87% of all antibiotic resistant strains found in 1961 originated from human patients, the resistance must be largely attributed to the therapeutic use of the drugs in question.     

Protein Engineering of Uridine Phosphorylase from Escherichia coliK-12. II. A Comparative Study of the Properties of Hybrid and Mutant Forms of Uridine Phosphorylases

Genes for hybrid uridine phosphorylases (UPases) consisting of fragments of amino acid sequences of UPases from Escherichia coliand Salmonella typhimuriumwere constructed. Producing strains of the corresponding proteins were genetically engineered. Mutant forms of the E. coliK-12 UPase were produced by site-directed mutagenesis. A comparative study of the enzyme properties of the mutant and hybrid forms of bacterial UPases was performed. It was shown that Asp27 rather than Asp5 and Asp29 residues of the E coliUPase forms part of the active site of the protein. A scheme of the involvement of Asp27 in the binding of inorganic phosphate is proposed.     

Resistance to mecillinam produced by the co-operative action of mutations affecting iipopolysaccharide, spoT, and cya or crp genes of Salmonella typhimurium

Lipopolysaccharide (LPS), spoT, and cya or crp mutations individually do not affect the minimum inhibitory concentration of mecillinam on Salmonella typhimurium. However, when mutations of two of these types were combined in the same strain, high-level resistance appeared, and increased even further when all three types of mutations were present. Most mutations affecting LPS (rfa, rfb, rfc) showed this behaviour, although to different degrees. The highest resistance to mecillinam was caused by galE and rfc mutations whereas almost no effect was noticed with rfaB or rfaK mutations. This phenomenon appears to be specific for mecillinam since none of several other antibiotics elicited it. Reduction of guanosine tetraphosphate (ppGpp) levels by introduction of a relA mutation did not significantly affect the MIC of mecillinam on strains carrying different combinations of SpoT, galE, and cya or crp mutations. All the strains produced spherical cells in medium with a low concentration (0.05 μg ml^?1) of the antibiotic. These results suggest that the antibacterial action of mecillinam on S. typhimurium is somehow dependent on the interaction of LPS, cyclic AMP/cyclic AMP receptor protein (cAMP/CRP), and SpoT. The reported resistance to mecillinam of cya and crp mutants of Escherichia coli K-12 is probably due to the natural LPS defectiveness of this strain.     

Preliminary characterization of the material released to the culture medium byCandida albicans yeast and mycelial cells

Culture filtrate concentrates were obtained fromCandida albicans yeast and mycelial cells grown in the presence of¹⁴C-protein hydrolysate for radioactive labeling of cellular polypeptides. Both growth forms released to the medium minor but significant amounts of proteinaceous materials. The analysis of culture filtrate concentrates by means of SDS-polyacrylamide gel electrophoresis and fluorography revealed a similar and complex electrophoretic pattern, though some qualitative and quantitative differences between samples obtained from yeast and mycelial cells were observed. Materials released, mostly composed of mannoproteins as shown by their affinity towards concanavalin A, presented (i) cross-reactivity (by Western immunoblotting and ELISA) against polyclonal antisera to genuine cell wall components (among them the 58-kilodalton fibrinogen-binding mannoprotein) and (ii) high affinity for polystyrene-latex microbeads. Results presented suggest a possible common identity for the molecules shed to the medium and the cell wall protein and mannoprotein constituents.Abbreviations SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electroporesis - PAS periodic acid/Schiff method     

Characterization and infectivity of a spontaneous variant isolated fromFrankia sp. WEY 0131391

Summary A spontaneous variant, obtained from aFrankia isolate fromAlnus rubra nodules, was compared with the parent strain with regard to infectivity, nitrogenase activity, and electrophoretic and immunological profiles. Both the parent and the variant strain were equally effective in inducing nodulation in seedlings ofA. rubra. All inoculated plants had an active nitrogenase system as measured by the acetylene reduction assay. Electrophoresis of whole cell homogenates on SDS-polyacrylamide slab gels showed similar electrophoretic profiles; however, the variant strain also exhibited striking differences in protein patterns that distinguish it from the parent strain. Immunological analysis of the originalFrankia strain and its variant revealed shared antigens as well as immunologically distinct antigenic determinants in the two strains. The variant strain exhibits a distinct morphology and growth patterns which remain stable after many passages through culture.     

envB mutations confer UV-sensitivity to Salmonella typhimurium and UV-resistance to Escherichia coli

Summary An envB mutation isolated in Salmonella typhimurium LT2 was transferred by conjugation to Escherichia coli K-12. The mutation produced the same alterations in E. coli as in S. typhimurium concerning cell shape, sensitivity to drugs, autolysis, and fermentation of carbohydrates. However, although the mutation conferred sensitivity to UV irradiation in Salmonella, in E. coli it behaved as a genuine envB mutation producing resistance to UV inactivation. The fact that the mutation produced opposite effects in the survival of UV-irradiated S. typhimurium and E. coli discloses an intriguing difference between these closely related species.Career Investigator of the Consejo Nacional de Investigaciones Cientificas y Técnicas, Argentina     

Surface display of domain III of Japanese encephalitis virus E protein on <Emphasis Type="Italic">Salmonella typhimurium</Emphasis> by using an ice nucleation protein

A bacterial cell surface display technique based on an ice nucleation protein has been employed for the development of live vaccine against viral infection. Due to its ubiquitous ability to invade host cells, Salmonella typhimurium might be a good candidate for displaying viral antigens. We demonstrated the surface display of domain III of Japanese encephalitis virus E protein and the enhanced green fluorescent protein on S. typhimurium BRD509 using the ice nucleation protein. The effects of the motif in the ice nucleation protein on the effective display of integral protein were also investigated. The results showed that display motifs in the protein can target integral foreign protein on the surface of S. typhimurium BRD509. Moreover, recombinant strains with surface displayed viral proteins retained their invasiveness, suggesting that the recombinant S. typhimurium can be used as live vaccine vector for eliciting complete immunogenicity. The data may yield better understanding of the mechanism by which ice nucleation protein displays foreign proteins in the Salmonella strain.     

An ultrastructural comparison of the cell envelopes of selected strains of Nocardia asteroides and Nocardia brasiliensis

Growth curves were determined for three strains each ofNocardia asteroides andNocardia brasiliensis. Two strains ofN. brasiliensis and one strain ofN. asteroides had longer lag periods of growth than the remaining three strains. All strains had generation times of approximately 5.5 hours.The ultrastructure of the cell envelope of eachNocardia strain in early stationary phase growth was also examined. All the strains had typical trilaminar cell walls and cell membranes. The thickness of the cell wall layers, especially the inner peptidoglycan layer, varied from strain to strain. The inner layer of two strains ofN. brasiliensis and one strain ofN. asteroides was 12 nm or more in thickness, while that of the remaining three strains was 7 nm thick. These observed differences in growth patterns and/or thickness of the cell wall layers could be correlated to the varying degress of virulence as well as the divergent pathologies exhibited by these organisms.     

SlyA, a regulatory protein from Salmonella typhimurium, induces a haemolytic and pore-forming protein in Escherichia coli

A chromosomal fragment from Salmonella typhimurium, when cloned in Escherichia coli, generates a haemolytic phenotype. This fragment carries two genes, termed slyA and slyB. The expression of slyA is sufficient for the haemolytic phenotype. The haemolytic activity of E. coli carrying multiple copies of slyA is found mainly in the cytoplasm, with some in the periplasm of cells grown to stationary phase, but overexpression of SlyB, a 15 kDa lipoprotein probably located in the outer membrane, may lead to enhanced, albeit unspecific, release of the haemolytic activity into the medium. Polyclonal antibodies raised against a purified SlyA-HlyA fusion protein identified the over-expressed monomeric 17 kDa SlyA protein mainly in the cytoplasm of E. coli grown to stationary phase, although smaller amounts were also found in the periplasm and even in the culture supernatant. However, the anti-SlyA antibodies reacted with the SlyA protein in a periplasmic fraction that did not contain the haemolytic activity. Conversely, the periplasmic fraction exhibiting haemolytic activity did not contain the 17 kDa SlyA protein. Furthermore, S. typhimurium transformed with multiple copies of the slyA gene did not show a haemolytic phenotype when grown in rich culture media, although the SlyA protein was expressed in amounts similar to those in the recombinant E. coli strain. These results indicate that SlyA is not itself a cytolysin but rather induces in E. coli (but not in S. typhimurium) the synthesis of an uncharacterised, haemolytically active protein which forms pores with a diameter of about 2.6 nm in an artificial lipid bilayer. The SlyA protein thus seems to represent a regulation factor in Salmonella, as is also suggested by the similarity of the SlyA protein to some other bacterial regulatory proteins. slyA- and slyB-related genes were also obtained by PCR from E. coli, Shigella sp. and Citrobacter diversus but not from several other gram-negative bacteria tested.     

Bdellovibrio possesses a prey-derived OmpF protein in its outer membrane

Bdellovibrio bacteriovorus 109D andBdellovibrio stolpii derive one of their major outer membrane proteins from the outer membrane of their prey. This prey-derived protein corresponds to the OmpF protein ofEscherichia coli. Bdellovibrios cultivated onSalmonella typhimurium prey acquire theSalmonella OmpF protein; this protein is distinguishable electrophoretically from the OmpF protein ofE. coli. Bdellovibrios containing the prey-derived OmpF protein are sensitive to killing by colicin A but not colicin E1, whereas bdellovibrios without this protein are completely resistant to colicin killing.     

Electrophoretical studies of proteins of the hypopharyngeal glands and of the larval food ofMelipona quadrifasciata anthidioides Lep. (Hymenoptera,Meliponinae)

Summary The electrophoretical protein patterns of hypopharyngeal glands, larval food ofMelipona, and royal jelly ofApis were compared.Since protein patterns of hypopharyngeal glands from newly emerged workers, brood cell provisioners and foragers are similar to freshly deposited larval food, the identical protein bands probably represent actual gland secretion. This suggests that, as inApis, the glands secrete proteins to the larval food, and maintain this ability throughout life, although at slightly different intensities, according to the activity of the bees.The similarity on the electrophoretic profiles of the major larval food protein inApis andMelipona is an interesting finding because of its probable evolutionary significance.     

A New Fungal Metabolite and Root Growth-stimulating Activity of Its Methyl Ester

Escherichia coli rodA mutant AOS151 grows as round cells at 30 and 42°C (H. Matsuzawa, K. Hayakawa, T. Sato, and K. Imahori, J. Bacteriol., 115, 436–442 (1973)). The mutant was found to be resistant to mecillinam at both temperatures. lip⁺ transductants were prepared by Pl phage transduction via strain AOS151, the cotransduction frequency of round morphology (Rod^?) at 42°C with the lip gene being about 90%. At 42°C all 54 Rod^? transductants tested were resistant to mecillinam. At 30°C all but two of these Rod^? (at 42°C)-type transductants were rod-shaped, and all were sensitive to mecillinam; the two strains grew as ovoid cells. The original rodA mutant AOS151 probably involves an additional mutation(s), that expresses the round cell shape at lower temperature, whereas the rodA51 mutation alone seems to result in temperature-sensitive expression of round cell morphology and mecillinam resistance. rodA mutant cells cultured at either 30 or 42°C had wild-type penicillin-binding protein 2, judging from penicillin-binding activity, electrophoretic mobility, and thermosensitivity.     

A sub-division ofSalmonella typhimurium into phage types based on the method of craigie and yen; Phages adaptable to species of the B and D group ofSalmonella; Phase adsorption as diagnostic aid

Summary and conclusions Phages were isolated which were adsorbed bySalmonella strains of groups B and D. These phages were adaptable to strains of species from these groups. The behaviour of some adaptations of these phages on strains ofS. paratyphi B has been described. A typing scheme forS. typhimurium has been established. So far, this consists of 32 types which have all been demonstrated with adaptations of a single phage.     

Analysis of chimeric UmuC proteins: identification of regions inSalmonella typhimurium UmuC important for mutagenic activity

UnlikeEscherichia coli, the closely related bacteriumSalmonella typhimurium is relatively unresponsive to the mutagenic effects of DNA-damaging agents. Previous experiments have suggested that these phenotypic differences might result from reduced activity of theS. typhimurium UmuC protein. To investigate this possibility, we have taken advantage of the high degree of homology between the UmuC proteins ofE. coli andS. typhimurium and have constructed a series of plasmid-encoded chimeric proteins. The possibility that the phenotypic differences might be due to differential expression of the respective UmuC proteins was eliminated by constructing chimeric proteins that retained the first 25 N-terminal amino acids of either of the UmuC proteins (and presumably the same translational signals), but substituting the remaining 397 C-terminal amino acids with the corresponding segments from the reciprocal operon. Constructs expressing mostlyE. coli UmuC were moderately proficient for mutagenesis whereas those expressing mostlyS. typhimurium UmuC exhibited much lower frequencies of mutation, indicating that the activity of the UmuC protein ofS. typhimurium is indeed curtailed. The regions responsible for this phenotype were more precisely localized by introducing smaller segments of theS. typhimurium UmuC protein into the UmuC protein ofE. coli. While some regions could be interchanged with few or no phenotypic effects, substitution of residues 212–395 and 396–422 ofE. coli UmuC with those fromS. typhimurium resulted in reduced mutability, while substitution of residues 26–59 caused a dramatic loss of activity. We suggest, therefore, that the primary cause for the poor mutability ofS. typhimurium can be attributed to mutations located within residues 26–59 of theS. typhimurium UmuC protein.     

Occurrence of Salmonella spp in estuarine and coastal waters of Portugal

The presence of Salmonella and its relationship with indicator organisms of fecal pollution, such as total coliforms, fecal coliforms and fecal streptococci, was studied at two marine zones in Portugal. Seventeen different Salmonella serotypes were isolated and identified, S. virchow was the most frequently isolated (21.6%). In addition, a high percentage (35.1%) was recorded for some Salmonella serotypes of clinical significance, namely S. enteritidis, S. infantis, S. typhimurium and S. virchow. In any of the samples from the two zones Salmonella was not detected in the absence of any of the indicator organisms. However, the incidence of Salmonella as a function of indicator concentration intervals established by the EEC standards was 0, 10 and 19.3% at guide values of total coliforms, fecal coliforms and fecal streptococci, respectively in the Faro samples (south of Portugal). In contrast, Salmonella incidence rates of 37.5, 36.4 and 33.3% were recorded at the corresponding guide values the Caminha samples (north of Portugal). No significant correlations (p>0.005) were obtained between Salmonella and the indicators at the sampling stations; however, total coliforms and fecal streptococci were the indicators most closely related to Salmonella in Caminha and Faro samples, respectively. Survival experiments in Escherichia coli, Enterococcus faecalis and S. typhimurium, using diffusion chambers, were performed to verify whether the lack of correlation between indicators and Salmonella was due to different inactivation rates in seawater. The results indicate that survival percentages of the three microorganisms tested were similar after 48 h of exposure to seawater.