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Protective effects of a supernatant factor from Salmonella typhimurium on Salmonella typhimurium infection of inbred mice.

A supernatant factor prepared from 48-h cultures of Salmonella typhimurium has been used to immunize mice against subsequent challenge with normally lethal doses of S. typhimurium. The mouse strains used, C57BL and BALB/c, were sensitive to S. typhimurium with 50% lethal doses of less than 50 organisms. Two doses of supernatant factor, given intraperitoneally 20 days apart, protected mice against a subcutaneous challenge dose 10 days later of 100 50% lethal doses of S. typhimurium, resulting in 50 to 80% survival. The viable counts were reduced initially in organs of immunized mice compared with controls, and the multiplication of bacteria was delayed, although the final levels found in the organs would normally have been lethal. Protection obtained was specific for S. typhimurium in that no increased survival was shown after Salmonella enteritidis challenge of immunized mice. Although lipopolysaccharide was demonstrated in the supernatant factor, lipopolysaccharide alone did not protect challenged mice. Supernatant factor produced delayed-type hypersensitivity reactions in mice sensitized with nonlethal doses of Salmonella. The nature of the active factor, found to be partially protein, has yet to be elucidated.

Animals

Nematospiroides dubius as a vector for Salmonella typhimurium.

Salmonella typhimurium within the 3rd stage larvae of Nematospiroides dubius was shown to infect mice, evidenced by prolonged shedding of salmonellae in the feces. Numbers of S typhimurium needed to infect mice were approximately 1,000- fold less if incorporated within the 3rd-stage larvae of N dubius. Results of these experiments demonstrate that nematode parasites may act as a vector for pathogenic species of enteric bacteria.

Ancylostomatoidea

A transport system for phosphoenolpyruvate, 2-phosphoglycerate, and 3-phosphoglycerate in Salmonella typhimurium.

Salmonella typhimurium strain LT-2 was found to utilize phosphoenolpyruvate, 2-phosphoglycerate, and 3-phosphoglycerate as sole sources of carbon and energy for growth, but Escherichia coli strains did not. The following evidence suggests that this growth difference was due to the presence in Salmonella cells of an inducible phosphoglycerate permease distinct from previously studied transport systems: (a) The ability of cells to take up 3-phospho[14-C]glycerate was induced by growth in the presence of phosphoenolpyruvate, 2-phosphoglycerate, or 3-phosphoglycerate, but not glycerate, alpha-glycerophosphate, or other carbon sources tested. (b) Uptake of 3-phospho[14-C]glycerate was strongly inhibited by the three nonradioactive inducers of 3-phosphoglycerate uptake, but not by glycerate or alpha-glycerophosphate. (c) Mutants which lost the ability to utilize and take up 3-phosphoglycerate simultaneously lost the ability to utilize 2-phosphoglycerate and phosphoenolpyruvate, but not other compounds tested. (d) Mutant strains which constitutively synthesized the phosphoglycerate transport system could use both phosphoglycerates and phosphoenolpyruvate as sole sources of phosphate at low substrate concentrations. (e) A strain lacking alkaline and acid phosphatases could still grow with 3-phosphoglycerate as sole carbon source. Maximal rates of 3-phospho[14-C]glycerate uptake occurred at pH 6 in the presence of an exogenous energy source. The apparent Km for 3-phosphoglycerate uptake under these conditions was about 10-minus 4 M. The maximal uptake rate (but not the Km) was dependent on potassium ions. Although synthesis of the phosphoglycerate transport system appeared to be under adenosine 3:5-monophosphate control, glucose repressed induction only slightly. The genes controlling synthesis of the phosphoglycerate transport system (pgt genes) appeared to map at about 74 min on the Salmonella chromosome.

Biological Transport

Redox potential-dependent nitrite metabolism by Salmonella typhimurium.

Salmonellae are generally resistant to the inhibitory effects of NaNO2. Removal of the lipopolysaccharide of Salmonella typhimurium by ethylenediaminetetraacetic acid pretreatment did not result in subsequent inhibtion of growth by NaNO2, indicating that lipopolysaccharide does not function to exclude NaNO2 from the cell. NaNO2 disappeared from the medium while the cells were growing, but, after stationary phase was reached, no further losses were observed unless the pH was maintained above 7.0. Similar losses were observed in a cell-free system if the redox potential of the medium was between -250 and -175 mV. If the disrupted cell suspension was first heated in a boiling water bath for 15 to 18 min, no NaNO2 loss was observed regardless of the redox potential. S. typhimurium is capable of metabolizing NaNO2, possibly by means of a nitrite-reducing enzyme function which is redox controlled.

Drug Resistance, Microbial

The metabolism of N4-hydroxycytidine-a mutagen for Salmonella typhimurium.

Salmonella typhimurium cells were grown in the presence of (14C)-N4-hydroxycytidine (N4OHcyd), a mutagenic nucleoside, and labelling in DNA and RNA digest was traced. The results show that this analogue is incorporated into RNA at a level of 40 mug/100 mg, and into DNA with a yield at least 100 times smaller. Some N-minus4OHcyd was rapidly metabolized and labelling was found in all ribo- and deoxyribo-nucleosides.

Centrifugation, Density Gradient

In vivo conversion of sodium azide to a stable mutagenic metabolite in Salmonella typhimurium.

Salmonella typhimurium TA1530 and G46 strains growing in minimal medium supplemented with sodium azide produce a stable mutagenic metabolite which is not azide. The production of this metabolite is restricted to the log phase of bacteria grown in the presence of azide. The metabolite is highly mutagenic in DNA-repair defective base-substitution strains TA1530 and TA1535, but ineffective in frameshift strains TA1538 and TA1537. The metabolite induces mutations in resting cells of the TA1530 strain.

Azides

Genetic modification of substrate specificity of hypoxanthine phosphoribosyltransferase in Salmonella typhimurium.

Salmonella typhimurium strain GP660 (proAB-gpt deletion, purE) lacks guanine phosphoribosyltransferase and hence cannot utilize guanine as a purine source and is resistant to inhibition by 8-azaguanine. Strain GP660 was mutagenized and a derivative strain (GP36) was isolated for utilization of guanine and hypoxanthine, but not xanthine, as purine sources. This alteration was designated sug. The strain was then sensitive to inhibition by 8-azaguanine. Column chromatographic analysis revealed the altered phosphoribosyltransferase peaks for both hypoxanthine and guanine to be located together, in the same position as hypoxanthine phosphoribosyltransferase (hpt gene product) of the wild-type strain. Genetic analysis showed the sug mutation to be allelic with hpt. Therefore sug represented a modification of the substrate specificity of the hpt gene product.

Alleles

Aflatoxin B1 mutagenesis, DNA binding, and adduct formation in Salmonella typhimurium.

Salmonella typhimurium strain TM677 was mutagenized with aflatoxin B1 (AFB1) in liquid suspension culture in the presence of a rat liver postmitochondrial supernatant. Forward mutation to 8-azaguanine resistance was measured in the treated cultures and was found to increase linearly with AFB1 concentration. DNA purified from mutagenized cells was analyzed for AFB1 adduct formation by high-pressure liquid chromatography after adduct liberation. AFB1 exposures at 0.16 and 0.32 micrometer for 35 min produced 15 and 22 AFB1--DNA adducts per genome, respectively, and induced 8-azaguanine-resistant fractions of 4.9 X 10(-4) and 9.6 X 10(-4). Approximately 70% of the AFB1 bound to DNA was chromatographically identical to 2,3-dihydro-2-(N7-guanyl)-3-hydroxyaflatoxin B1 at the two AFB1 levels used.

Aflatoxins

Selection for a large genetic duplication in Salmonella typhimurium.

Salmonella typhimurium strains containing a duplication of nearly a third of the genome have been isolated by a simple procedure involving selection for improved utilization of L-malate as sole carbon source. The duplication occurs at a very high spontaneous frequency. Strains containing the duplication can be isolated selectively on malate medium, or by a non-selective procedure involving Hfr conjugation. When strains containing the duplication are maintained on non-selective medium, the duplication is readily lost. Genetic evidence suggests that the duplication is chromosomal and tandem. The fact that the recA gene is included in the duplication has been used to obtain evidence that the recA1 marker is recessive to its wild-type allele. Unlike tandem duplications previously described in I. coli, the duplication described in this report appears to have unique endpoints.

Acetates

Acylaminoacid esterase mutants of Salmonella typhimurium.

Salmonella typhimurium contains three electrophoretically separable enzyme activities that hydrolyze N-acetyl phenylalanine beta-naphthyl ester (NAPNE). One of these enzymes is an endoprotease, protease I. Mutations at a locus apeA near purE lead to loss of this enzyme. We have found that N-acetyl leucine alpha-naphthyl ester (NALNE) is not hydrolyzed by protease I but is a good substrate for the other two activities. Using NALNE as a chromogenic substrate to screen colonies growing on agar, we have isolated mutants (apeB) that simultaneously lose both of the two other esterase activities. The chromosomal positions of apeB and nearby markers in the proC-purE region have been determined using both phage P1 and phage P22 mediated transduction. The observed order is proC thiC apeB apt apeA purE. Strains lacking all three activities (apeA apeB double mutants) have been constructed and have growth rates similar to wild-type strains.

Amino Acids

An alteration in outer membrane permeability associated with a division lesion in a strain of Salmonella typhimurium.

Salmonella typhimurium strain 4a is a temperature sensitive mutant with defects in both septation and separation. The separation lesion was reversed by phenethylalcohol but this agent failed to allow septation or growth at restrictive temperature. Organisms of strain 4a grown at 42 degrees C were, unlike the parental strain, resistant to lysis by lysozyme plus EDTA and lipopolysaccharide was poorly extracted by EDTA from cultures of strain 4a grown at 42 degrees C. Such cultures may, therefore, be resistant to lysis with lysozyme plus EDTA not because the murein is altered but because the EDTA fails to permeabilize the outer membrane to lysozyme. In confirmation of this, murein isolated from strain 4a after growth at 42 degrees C showed the same sensitivity to lysozyme as murein from the parental strain. In spite of the altered envelope properties of strain 4a after growth at 42 degrees C, no major changes in protein or phospholipid composition have so far been demonstrated.

Cell Division

Purification and properties of a citrate-binding transport component, the C protein of Salmonella typhimurium.

Salmonella typhimurium was shown to contain a citrate-binding protein (C protein) which was purified to homogeneity from the periplasmic fraction released by cold osmotic shock. The protein is dimeric, has an apparent molecular weight of 28 000 and an isoelectric point of 6.1. Sodium ions were required for optimum substrate binding, however, the divalent cations Zn2+, Mg2+, and Co2+ were inhibitory. The C protein was relatively stable but sensitive to various detergents and chaotropic agents. Approximately one citrate molecule was bound per molecule of protein and citrate binding (Kd = 1-2.6 microM) was strongly competitively inhibited by DL-isocitrate and DL-fluorocitrate but not by other carboxylates. Neither succinate, glutamate, nor acetate were bound to the C protein. No apparent enzyme activity was associated with this protein. A concomitant reduction in the level of binding protein and in citrate transport activity occurred in osmotically shocked cells as well as with L-malate- or succinate-grown cells. Fluorocitrate-resistant mutants were simultaneously defective in citrate transport, citrate binding, and production of cross-reacting material. One transport-defective mutant did produce citrate binding protein.

Biological Transport, Active

Interaction of complement components with a serum-resistant strain of Salmonella typhimurium.

Salmonella typhimurium C5 is under normal conditions (physiological saline containing 0.002 M Mg2+) resistant to the action of antibody and complement (C). It becomes sensitive, however, when suspended in tris(hydroxymethyl)-aminomethane buffer (Reynolds and Pruul, 1971; Reynolds and Rowley, 1969). The interaction of complement components with this strain sensitized with specific antibody has been studied to identify the intermediate step at which inhibition occurs. The components C1 yields C2 react normally, as has been shown by lysis of complement-treated cells incubated with complement in ethylenediaminetetraacetic acid. Also, the reaction of C3 can be demonstrated by positive immune adherence and agglutination with anti-C3. The complement-treated cells do not, however, react with rabbit C6 to 9 or rabbit serum lacking C6 in tris(hydroxymethyl)aminomethan buffer. We conclude from these date that C5 can not react effectively under normal conditions. In contrast, if bacteria-antibody complexes are pretreated with rabbit serum lacking C6 in tris(hydroxymethyl)aminomethane buffer, they are readily lysed by incubation with C6 to 9. Thus, C5 can react with the bacterial surface in tris(hydroxymethyl)-aminomethane buffer.

Animals

Outer membrane of gram-negative bacteria. XVIII. Electron microscopic studies on porin insertion sites and growth of cell surface of Salmonella typhimurium.

Salmonella typhimurium contains three "major proteins" or "porins" (34K, 35K, and 36K) in the outer membrane. A mutant strain producing only the 35K porin was first grown in media containing high concentrations of NaCl to "repress" the porin synthesis and then was shifted into a medium without NaCl. The newly made porin molecules were then labeled with the ferritin-coupled antibody at various times after the shift, and the samples were examined by whole-mount, freeze-etching, and thin-section electron microscopy. These experiments showed that newly inserted porins appeared as discrete patches uniformly distributed over the surface of the cell and, furthermore, that the sites of adhesion between the inner and outer membrane were most probably the pathway by which the newly made porin molecules appeared on cell surface. The 34K and 36K porins were also inserted in the same manner, since the appearance of new porins at discrete sites all over the cell surface was also observed when cells with wild-type porin phenotype were treated with unlabeled antibody to block existing antigenic sites, subsequently regrown, and labeled with the ferritin-coupled antibody. Since porins comprise a major portion of the densely packed, relatively immobile, "protein framework" of the outer membrane, these results lead us to conclude that the outer membrane grows predominantly by diffuse intercalation rather than by the zonal growth mechanism.

Antibodies, Bacterial

Mutants defective in the 33K outer membrane protein of Salmonella typhimurium.

Salmonella typhimurium LT2 lines, if phenotypically rough, are fully sensitive to bacteriocin 4-59, produced by Salmonella canastel strain SL1712. Bacteriocin-resistant mutants fell into three classes. Those resistant to phage ES18 and to albomycin proved to be mutants of class chr (equivalent to tonB of Escherichia coli); these mutants still adsorb the bacteriocin and so are classified as tolerant. Another class of (incompletely) tolerant mutants was resistant to phage PH51; their envelope fractions lacked the band corresponding to outer membrane protein 34K, known to serve for adsorption of phage PH51. A third class of mutants, which did not adsorb the bacteriocin, was unaltered in sensitivity to phages. Their envelopes lacked the 33K band, indicating absence of the outer membrane protein 33K, considered to correspond to outer membrane protein II* of E. coli, which in that species is determined at locus ompA (formerly tolG or con). Phage P22 HT105/1 cotransduced the 33K S. typhimurium gene (to be called ompA, to accord with E. coli usage) with pyrD+ at about 30% frequency when the donor allele was ompA+ or one ompA, but at only 3 to 11% when the donor allele was another ompA. When the donor carried either of two long deletions of the put (proline utilization) operon, phage P22 HT105/1 cotransduced put (and ompA+) with pyrD+ at low frequency. The cotransduction data indicate that ompA of S. typhimurium is located between pyrD and put, nearer the former. This corresponds to the map position of ompA in E. coli K-12.

Bacterial Proteins

Deoxyribonucleic acid adenine and cytosine methylation in Salmonella typhimurium and Salmonella typhi.

The methylations of adenine in the sequence -GATC- and of the second cytosine in the sequence - [Formula: see text] - were studied in Salmonella typhimurium and in Salmonella typhi. The study was carried out by using endonucleases which restrict the plasmid pBR322 by cleavage at the sequences -GATC- (DpnI and MboI) and - [Formula: see text] - (EcoRII). The restriction patterns obtained for this plasmid isolated from transformed S. typhimurium and S. typhi were compared with those of pBR322 isolated from Escherichia coli K-12. In E. coli K-12, adenines at the sequence -GATC- and the second cytosines at - [Formula: see text] - are met hylated by enzymes coded for by the genes dam and dem, respectively. From comparison of the restriction patterns obtained, it is concluded that S. typhimurium and S. typhi contain genes responsible for deoxyribonucleic acid methylation equivalent to E. coli K-12 genes dam and dcm.

Adenine

The effect on the virulence and infectivity of Salmonella typhimurium and Salmonella gallinarum of acquiring antibiotic resistance plasmids from organisms that had caused serious outbreaks of disease.

Antibiotic resistance plasmids from organisms that had caused serious epidemics, including those responsible for epidemics of chloramphenicol-resistant typhoid fever and dysentery in Central America, were transferred to a strain of Salmonella typhimurium and of Salmonella gallinarum. The virulence and infectivity of these R(+) forms were then compared with the R(-) parent forms in orally inoculated chickens.None of the R(+) forms were more virulent than their R(-) parent forms. The mortality rates they produced were either the same as or less than that of their R(-) parent forms. The mortality rates were not increased by feeding the chickens on diets containing antibiotics against which the plasmids provided resistance.The removal of the plasmids from some R(+) forms of decreased virulence was not accompanied by any alteration in virulence, indicating that they were less virulent mutants of the parent strain that had conjugated preferentially. In other cases their virulence was increased, indicating that the very possession of the plasmid was involved in their decreased virulence. Of four forms of the S. gallinarum strain harbouring the plasmid that had been incriminated in the Central American dysentery outbreak, one was as virulent as the parent R(-) form and the other three were less virulent. Preferential conjugation by an avirulent mutant was responsible for the lack of virulence of one of them but the very possession of the plasmid appeared responsible for the decreased virulence of the other two. The decreased virulence of de-repressed F(+) and I(+) forms of the S. typhimurium strain was increased to that of repressed F(+) form and of the parent form by plasmid removal.Organisms of the R(+) forms of the S. typhimurium strain were not excreted in larger amounts or for longer periods of time by infected chickens than organisms of the R(-) parent form were. Neither did organisms of the R(+) forms of this strain or the S. gallinarum strain spread more rapidly or more extensively from infected chickens to in-contact chickens than organisms of the R(-) parent forms did. When antibiotics against which the infecting R(+) organisms provided resistance were included in the diet of these chickens the R(+) organisms were usually excreted in greater amounts, for longer periods of time and spread more rapidly and more extensively from the infected chickens to the in-contact chickens.

Ampicillin

Influence of temperature on growth of lipopolysaccharide-deficient (rough) mutants of Salmonella typhimurium and Salmonella minnesota.

Smooth strains of Salmonella typhimurium and S. minnesota, and chemotypes Ra, Rb, and Rc, which are deficient in lipopolysaccharide components of the somatic side chains and outer core region, grow normally on nutrient agar and nutrient broth up to 45 degrees C. However, most mutants with defects in the heptose region of the LPS (chemotypes Rd2 and Re) do not grow on this medium at 42 degrees C or above; a few grow at 42 degrees C but not at 45 degrees C. In liquid medium (nutrient broth, or phosphate minimal medium), growth, measured as turbidity or as colony-forming units, stops 60 to 90 min after shift from 30 to 42 degrees C; DNA and protein synthesis cease at the same time. Growth does not reoccur at 42 degrees C; protein synthesis and growth reinitiate upon shift to 30 or 37 degrees C. Growth cessation does not alter cell morphology in the phase-contrast microscope. Growth of heptose-deficient strains at 42 degrees C in nutrient broth is restored by MgCl2 (0.5 mM), NaCl (50 mM), or sucrose (100 mM). Sensitivity to smooth-specific and rough-specific phages, and analysis of LPS composition, indicate that heptose-deficient mutants grown at temperatures from 30 to 45 degrees C, and in the presence or absence of high salt, do not contain heptose or O-specific sugars in their LPS.

Bacterial Proteins