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Salmonella typhimurium ST39 exhibits reduced virulence in mice and decreased survival in mouse macrophages compared with the parent strain SL3201. Strain ST39 is nonmotile, carries an indeterminate deletion in and near the flgB operon, and is defective in the mviS (mouse virulence Salmonella) locus. In flagellum-defective strains, the flgM gene product of S. typhimurium negatively regulates flagellar genes by inhibiting the activity of FliA, the flagellin-specific sigma factor. In this study, flgM of wild-type S. typhimurium LT2 was found to complement the mviS defect in ST39 for virulence in mice and for enhanced survival in macrophages. Transduction of flgM::Tn10dCm into the parent strain SL3201 resulted in attenuation of mouse virulence and decreased survival in macrophages. However, a flgM-fliA double mutant was fully virulent in mice and survived in macrophages at wild-type levels. Thus, the absolute level of FliA activity appears to affect the virulence of S. typhimurium SL3201 in mice. DNA hybridization studies showed that flgM-related sequences were present in species other than Salmonella typhimurium and that sequences related to that of fliA were common among members of the family Enterobacteriaceae. Our results demonstrate that flgM and fliA, two genes previously shown to regulate flagellar operons, are also involved in the regulation of expression of virulence of S. typhimurium and that this system may not be unique to the genus Salmonella.

The generation of aromatic-dependent ( aro − ) Salmonella havana (Group G2, 01, 13, 23) from a smooth wild-type parent strain by transduction with phage P1 is reported. Mice immunized with this live aro − S. havana strain (CS234) by the intraperioneal (i.p.) route were protected against challenge with wild-type S. havana , whereas those immunized by the oral route were not. Mice immunized with two doses of formalin-killed aro − S. havana by the i.p. route were also unprotected, in spite of high antibody titers. However, only those mice immunized with live aro − S. havana by the i.p. route developed significant delayed-type hypersensitivity. Following i.p. inoculation in mice, the aro − S. havana strain CS234 was detected in the liver, spleen and mesenteric lymph nodes on day 9 but not on day 15 post-inoculation (p.i.). On the other hand, when mice were inoculated with the parent wild-type strain (CS4) or the aro − derivative strain CS234 by the oral route, the organisms were recovered from the mesenteric lymph nodes and intestine only on day 3 but onot on day 6 post-inoculation. In sheep inoculated with the aro − strain CS234 in the gastroc muscle, organisms were recovered from the muscle, and popliteal and medial iliac lymph nodes for up to 21 but not 28 days p.i. However, no mutant organisms were recovered from liver, spleen, mesenteric lymph nodes or faeces. In orally-inoculated sheep, the mutant organisms were recovered from the mesenteric lymph nodes. rumen, intestinal contents, and faeces up to 14–21 days post-inoculation but not at 28 days. When sheep immunised with the aro − S. havana strain CS234 by the intramuscular or oral route were challenged with the parent wild-type S. havana strain CS4 by the oral route, the latter strain was detectable in the mesenteric lymph nodes and faeces of immune sheep up to 14 days post-challenge in contrast with the non-immune sheep, where the challenge strain was detectable even at 28 days post-challenge. Only sheep immunized by the intramuscular route developed high antibody levels and delayed-type hypersensitivity.

The generation of smooth aromatic-dependent Salmonella serotype Bovismorbificans (Group C2, O6, 8) from a smooth wild-type parent strain by transduction with phage P1, and conjugation with Salmonella serotype Typhimurium carrying F'-8gal is described. The smooth aromatic-dependent S. serotype Bovismorbificans was non-lethal for mice at an oral challenge dose of 2 x 10(9) cfu (equivalent to 200 LD50 of the parent, wild-type strain). The safety of the auxotrophic mutant was further substantiated by comparing its multiplication kinetics in vivo with that of its virulent parent organisms. Mice immunised with live, smooth aromatic-dependent S. serotype Bovismorbificans by either the oral or intraperitoneal (i.p.) route were protected against oral challenge with virulent S. serotype Bovismorbificans; the degree of protection was significantly better (p less than 0.05) at a challenge dose of 100 or 200 LD50 in mice receiving two rather than one vaccination. In contrast, mice immunised with three doses of the formalin-killed virulent, parent organisms by the i.p. route were not protected, in spite of high antibody titres. Only those mice immunised with the live, smooth aromatic-dependent S. serotype Bovismorbificans i.p. developed significant (p less than 0.01-0.05) delayed-type hypersensitivity.

We have reported that motile Escherichia coli K-12 placed in an electric field swims toward the anode but that motile Salmonella typhimurium strains swim toward the cathode, a phenomenon called galvanotaxis (J. Adler and W. Shi, Cold Spring Harbor Symp. Quant. Biol. 53:23-25, 1988). In the present study, we isolated mutants with an altered direction of galvanotaxis. By further analyses of these mutants and by examination of E. coli and Salmonella strains with altered cell surface structure, we have now established a correlation between the direction of galvanotaxis and the surface structure of the cell: motile rough bacteria (that is, those without O polysaccharide; for example, E. coli K-12 and S. typhimurium mutants of classes galE and rfa) swam toward the anode, whereas motile smooth bacteria (that is, those with O polysaccharide; for example, wild-type S. typhimurium LT2) swam toward the cathode. However, smooth bacteria with acidic polysaccharide capsules (K1 in E. coli and Vi in Salmonella typhi) swam toward the anode. Measurements of passive electrophoretic mobility of strains representative of each set were made. We propose that the different directions of galvanotaxis of rough (or capsulate) bacteria and of smooth bacteria are explicable if the negative electrophoretic mobility of flagellar filaments is less than that of rough bodies but greater than that of smooth bodies.

An auxotrophic Salmonella dublin (O9,12) strain, SL5631, with a deletion affecting gene aroA, was made into a partial diploid expressing the rfb (O-antigen-repeat-unit-specifying) gene cluster of Salmonella typhimurium (O4,12). By use of O4- and O9-specific antisera in indirect immunofluorescence assays, the resulting hybrid SL7103 was shown to express both the O4- and O9-antigen epitopes in the same bacterium. Qualitative and quantitative sugar analyses by gas-liquid chromatography on peralditol acetates of phenol-water-extracted lipopolysaccharides showed that the S. dublin and S. typhimurium repeating units (estimated on the basis of their tyvelose and abequose contents, respectively) were present in approximately equimolar amounts. The SL7103 hybrid auxotroph was avirulent when given intraperitoneally to NMRI mice in a dose of 10(8) CFU and elicited a protective immunity against intraperitoneal challenge with either virulent S. dublin (50% lethal dose of ca. 1.5 x 10(4) CFU versus < 1 x 10(1) CFU in nonimmunized mice) or virulent S. typhimurium (50% lethal dose of ca. 1 x 10(5) versus < 1 x 10(1) CFU in nonimmunized mice). Compared with the protection elicited in homologous systems (S. dublin SL5631 against S. dublin and S. typhimurium SL1479 against S. typhimurium), the protective efficacy of the hybrid was reduced approximately 70-fold against S. dublin challenge and 100-fold against S. typhimurium challenge. Vaccination with S. typhimurium SL1479 conferred no protection against S. dublin challenge, and vaccination with S. dublin SL5631 conferred no protection against S. typhimurium challenge. The protection elicited by the hybrid strain SL7103 is supposed to be mainly a consequence of serum antibodies directed against the immunodominant O4 and O9 epitopes.

Two Salmonella hybrid strains, SL5313 (Salmonella typhimurium with a D.rfb+ gene cluster) and SL5396 (S. enteritidis with a B.rfb+ gene cluster), each expressing both O-antigen 4 (of serogroup B) and O-antigen 9 (of serogroup D) were studied by immunofluorescence using a mixture of O4-specific mouse monoclonal and O9-specific rabbit polyclonal antibodies. Bound antibodies, detected by anti-mouse antibody labelled with fluorescein isothiocyanate and anti-rabbit antibody labelled with tetramethylrhodamine isothiocyanate showed that more than 98% of the bacteria expressed both the O4 and O9 epitopes. Phenol-water-extracted lipopolysaccharide from batch-grown cultures subjected to sugar and methylation analyses by gas-liquid chromatography and mass spectrometry were shown to contain abequose (of the O4 epitope) and tyvelose (of the O9 epitope) in ratios of 1:1.5 and 1:2.5 for SL5313 and SL5396, respectively. Isolated polysaccharide chains, obtained by weak-acid hydrolysis of the lipopolysaccharides, were found to contain both O4 and O9 specificities in the same molecule, since polysaccharide bound to O4 antibody attached to a solid-phase-adsorbed O9-specific antibody and vice versa. This demonstrates that in strains SL5313 and SL5396 O chains containing both O4 repeating units (from S. typhimurium) and O9 units (from S. enteritidis) are present.

Salmonella typhimurium SL1479, an auxotrophic mutant strain having a complete block in the aromatic biosynthetic pathway and therefore requiring p-aminobenzoic acid and 2,3-dihydroxybenzoate not available in mammalian tissues, was given orally in a dose of 10(10) live bacteria to 4- to 5-week-old calves. Only a mild transient fever response was seen. Strain SL1479 was unable to colonize and persist in the calves for more than 2 weeks. In a vaccination experiment, groups of six calves were (i) orally vaccinated with the live S. typhimurium SL1479 strain, (ii) subcutaneously vaccinated with a heat-inactivated S. typhimurium SVA1232 strain with aluminum hydroxide as adjuvant, or (iii) not vaccinated, to serve as controls. Calves were orally challenged with the live, calf-virulent S. typhimurium SVA44 strain: either 10(6) bacteria (equivalent to 100 25% lethal doses [LD25S]) or 10(9) bacteria (100,000 LD25S doses). The live oral vaccine gave significantly better protection than the heat-vaccinated, subcutaneously injected vaccine since (i) only control calves and calves given the killed vaccine developed profuse diarrhea, (ii) clinically, the mild fever responses seen after challenge in calves given the live vaccine were significantly lower (P less than 0.0005), (iii) autopsies performed 21 days after the challenge infection revealed normal findings in calves given the live vaccine, whereas calves given the killed vaccine showed signs of acute enteritis and chronic salmonellosis, (iv) all 12 calves given either 100 X or 100,000 X the LD25 survived the 21-day observation period; the mean survival time in nonvaccinated calves was 8.0 days; in calves given heat-inactivated vaccine and 100 X the LD25 it was 21.0 days, and in calves given 100,000 X the LD25 it was 11.5 days, (v) the fecal bacterial counts of the challenge S. typhimurium SVA44 strain were significantly lower (P less than 0.0005) in both groups given the live vaccine, and (vi) upon autopsy followed by culture, the qualitative recovery of the challenge strain from the alimentary canals and tissues of calves given the live vaccine was significantly lower (P less than 0.005).

Summary: Transductional mapping, with phage ES18 or ES18.h1, showed that several mutations causing the RfaH− phenotype (defective formation of galactose I and also of more distal units of the lipopolysaccharide core) were located between metE and pepQ in the Salmonella typhimurium linkage map; the affected locus is designated rfaH. The mutation of one strain of RfaH− phenotype was located elsewhere, at an unidentified rfa locus. Introduction of an F” plasmid containing the metE segment of the Escherichia coli chromosome into several rfaH mutants restored the “smooth” (Rfa+) phenotype. Several rfaH mutations, and that of the phenotypically similar rfa mutant, caused increased sensitivity to bacitracin, polymyxin, novobiocin, nafcillin and oxacillin, as expected if the mutations have no effect on the formation of the part of the lipopolysaccharide core proximal to the galactose units.

The biochemical defect in a class of Salmonella typhimurium mutants (rfaB) defective in biosynthesis of the lipopolysaccharide core is described. Structural, immunochemical and enzymologic studies showed that: (i) the core polysaccharide completely lacked the branch alpha 1,6-D-galactosyl residue of the normal lipopolysaccharide as shown by methylation analysis and 1H nmr spectroscopy; (ii) the mutant lipopolysaccharides acted as acceptors for transfer of D-galactose from UDP-D-galactose into alpha 1,6 linkage to the proximal D-glucosyl residue of the core in a reaction catalyzed by an enzyme activity present in extracts from rfaB+ cells; (iii) the UDP-D-galactose:(glucosyl)lipopolysaccharide alpha 1,6-D-galactosyltransferase activity was absent from extracts of rfaB cells.

Genetic analysis of aroA554::Tn10 derivatives of two mouse-virulent Salmonella typhimurium strains, "FIRN" and "WRAY," and of a nonreverting derivative of each constructed for use as a live vaccine, showed the site of the insertion among mapped aroA point mutants. The WRAY live-vaccine strain gave no aro+ recombinants in crosses with aroA point mutations to one side of the insertion, indicating a deletion from Tn10 through the sites of these point mutations. The FIRN live-vaccine strain gave wild-type recombinants with all tested point mutants; it probably has a deletion or inversion extending from Tn10 into aroA but not as far as the nearest point mutation. Some tetracycline-sensitive mutants of aroA554::Tn10 strains required serine and pyridoxine, indicating loss of serC function, and some that were found to be SerC- did not produce gas from glucose, indicating a loss of pfl function. These results show the gene order pfl-serC-aroA, as in Escherichia coli. Ampicillin enrichment applied to pools of tetracycline-sensitive mutants of strains with Tn10 insertions near aroA (i.e., zbj::Tn10 strains) yielded Aro- SerC- Pfl-, Aro- SerC+ Pfl+, and Aro- SerC- Pfl+ mutants but none which were Aro+ SerC-. All of the mutants are explicable by deletions or inversions extending clockwise from zbj::Tn10 into or through an operon comprising serC (promoter-proximal) and aroA. Such an operon was also shown by the identification of two Tn10 insertions causing phenotype Aro- SerC-, each able to revert to Aro+ SerC+ by precise excision. serC corresponds to the open reading frame promoter-proximal to aroA that was identified elsewhere by base sequencing of a cloned aroA segment of S. typhimurium (Comai et al., Science 221:370-371, 1983). Both serine and chorismate are precursors of enterochelin; this may be why serC and aroA are in a single operon.

Plasmid R46 (an R factor conferring resistance to ampicillin, sulfonamides, streptomycin and tetracycline) reduces the bactericidal effect of UV irradiation but increases its mutagenic effect (reversion of hisG46), and raises the frequency of spontaneous reversion (mutator effect). Putative deletion mutants of R46 were obtained by transduction of the plasmid, then two successive conjugal transfers. Plasmids of five of six deletion classes, each with a different combination of drug resistance traits, retained conjugative ability and the UV-protecting, mutagenesis-enhancing and mutator effects of R46. (pKM101, used in the Ames system to enhance responsiveness to chemical mutagens, is one such mutant of R46.) Plasmids of a sixth class, represented by pKM115, conferred resistance only to streptomycin and were non-conjugative. All of several such plasmids (of independent origin) had a much stronger mutator effect than did R46, but lacked UV-protecting ability and did not enhance the mutagenic effect of UV irradiation. We infer that R46 possesses: (i) a gene, uvp, which increases capacity for error-prone repair of UV-damaged DNA, and thus causes both UV protection and enhancement of UV mutagenesis; (ii) gene(s) whose action in the absence of gene uvp greatly increases the frequency of spontaneous reversion of hisG46. A plasmid of another incompatibility group, pLS51, has UV-protecting and mutagenesis-enhancing effect but lacks the mutator property; introduction of pLS51 into a clone of hisG46 carrying a pKM115-type plasmid greatly reduced its spontaneous reversion rate, as expected if pLS51 also has a uvp gene able to modulate the mutator effect of R46-derived gene(s) in the pKM115-type plasmid.

Isogenic pairs of strains of Salmonella typhimurium which differed only in whether or not they were flagellate were found to be equally virulent in C57BL/6J mice infected orally, intravenously, or intraperitoneally. Therefore, we investigated the genetic basis for our previous observation that in this mouse model, nonflagellate delta flagABCDE25 strains were reduced in virulence compared with isogenic wild-type flagellate strains. The recombinant plasmid pMH6, which contains several flg+ genes and a segment of the S. typhimurium chromosome adjacent to the flg genes, was introduced into a delta flgABCDE25 mutant. This restored virulence in mice challenged intraperitoneally, which suggested that a virulence gene occurs adjacent to the flg genes. When plasmid pMH64, which lacks the chromosomal segment adjacent to the flg genes, was introduced into the same delta flgABCDE25 mutant, virulence was not restored. In contrast, the introduction of pMH71, a plasmid which retains the chromosomal segment adjacent to the flg genes, restored virulence. We concluded that a hitherto unknown virulence gene, which we have named mviS, occurs adjacent to the flg genes and that its absence in delta flgABCDE25 mutants, rather than the nonflagellate phenotype of the delta flgABCDE25 mutants, caused the previously reported attenuation of such mutants.

SUMMARY: E colicins are those inactive on btuB mutants, which lack the outer membrane protein that adsorbs vitamin B12, E colicins and phage BF23; types E1, E2, E3 and E7 have been defined by the specific immunity of E-colicinogenic strains to the type of E colicin they produce. Further immunity types - E4, E5 and E6 - are now described. Shigella sonnei of colicin type 9 makes colicin E4, and Shigella sonnei of types 9A, 12 and 14 make colicin E6 (not colicin E3, as previously supposed). Many local Shigella sonnei isolates make E colicin of a new type - E5. The action of colicins E1 to E7 was (incompletely) blocked by vitamin B12, which also reduced the effect of A colicins (which are weakly active on Escherichia coli K12 btuB indicators). Escherichia coli K12 given plasmid ColA-23 by transformation was immune to A colicins but sensitive to colicins E1 to E7. A purported ‘colicin E4’ was shown to be of class colicin A. Escherichia coli K12(CloDF13) transformants were immune to colicin E6. A ‘cap’ of colicin-sensitive indicator bacteria developed over, but not around, killed colonies of btuB + E-colicinogenic strains, because of adsorption of colicin by non-induced bacteria of the colony. Killed colonies of btuB + E-colicinogenic strains gave partly discrete lobes of colicin action on indicator lawns, instead of circular zones, apparently as a result of unstable variation in colicin production. The presence of a colicin E4, E5 or E6 plasmid in K12 btuB + strains made them more sensitive to colicins E2 and E7, as shown by zones of partial translucency surrounding the ordinary colicin zones and by increased titre of colicin E2 and E7 preparations on such indicators.

Various features of salmonellosis were examined in a burned-mouse model. In this model, which uses an outbred mouse strain, a challenge dose of ca. 100 CFU with any of several strains of Salmonella typhimurium caused a fatal infection. A variety of mutated strains attenuated for virulence in Salmonella-susceptible parenterally infected mice were also attenuated in the burned-mouse model. When administered as live vaccines injected intraperitoneally the same attenuated strains provided between slight and complete protection against subsequent lethal challenge subcutaneously at the site of a burn. The correspondence of results obtained in the burned-mouse model with those seen in other mouse models coupled with the unique advantages of the burned-mouse model argue for the usefulness of the model in studies of salmonellosis and in testing of strains constructed for use as live vaccines.

SUMMARY: The presence of rare motile bacteria in 22 of 48 non-motile Salmonella strains tested has been inferred from the production of satellite micro-colonies in semi-solid medium. Such rare motile bacteria have been isolated by micromanipulation; they are not motile mutants, since their progeny are nearly all non-motile. They transmitted motility to only a few of their descendants, as if their motility were due to the presence of non-multiplying motility-conferring particles which were transmitted to their progeny at cell division. It is inferred that small numbers of such particles arise during an intracellular ‘event’; this event probably consists of a transient ability to synthesize new flagella. The frequency of events in one Salmonella O strain was found to be c. 4 x 10-5/bacterium/generation.

The dynamics of bacterial and fungal incidence in two sharply different soil samples after the addition of water and glucose was studied using direct microscopic techniques and a technique of inoculation in growth media. The ratio of the population density indices established by means of microscopy to those found from the data of inoculation characterizes the stage of microbial succession. The techniques of microscopy and inoculation are equitable, furnish different information, and must be used in parallel while examining various problems pertinent to the dynamics of the soil microflora incidence.

SUMMARY: R factors from Enterobacter strains isolated outside Japan were transferred to Salmonella typhimurium LT 2 and their resistance traits transduced by phage P 22. Of five fi + factors conferring resistance to tetracycline (Tc), streptomycin (Sm), sulphonamides (Su) and chloramphenicol (Cm), three, one of which conferred also resistance to kanamycin (Km), behaved like previously reported factors in that: Tc was transduced by itself but never with any other trait; all other traits were usually co-transduced; no transductants could transmit resistance by conjugation, even after infection with F'-13 lac. The other two fi + factors, which conferred also resistance to benzylpenicillin (Pn), behaved similarly, except that: Pn was transduced only at a low rate, and was never co-transduced with any other trait; and a few transductants when given F'-13 lac became able to transmit. Four fi - factors, two of type Tc, Sm, Su, Pn and two of type Tc, Su, Pn differed in that: nearly all Tc transductants acquired also all the other traits; Pn was usually co-transduced with the other traits; and some transductants could transmit by conjugation, even without F'-13 lac. The various transductant classes obtained by treatment with P 22 grown on LT 2 carrying both an fi + (Tc, Sm, Su, Cm, Km) and fi - (Tc, Sm, Su, Pn) factor could be accounted for by transduction of fragments of either one or the other of these factors. The sorts of transductants produced by lysates of a strain possessing Tc, Sm, Su, Cm, Km determinants obtained by growing a strain carrying an fi + (Tc, Sm, Su, Cm) factor with one carrying an fi + (Km) factor suggested that the strain carried two fi + factors, not a recombinant factor. Both fi - and fi + factors conferring resistance to benzylpenicillin conferred also resistance to cephalothin and cephaloridine and caused the constitutive production of a β-lactamase, active on both benzylpenicillin and on cephalothin. All four fi - and one of the five fi + factors resembled coll factors in that they protected strain LT 2 against the bactericidal effect of ultraviolet irradiation.

Many nonflagellate ( fla − ) mutants of Salmonella typhimurium strain LT2 yield 3–50% of fla + transductants with the phase-1 flagellar antigen determined by the H 1 allele of the donor. However, for some fla − and for all mot − (flagellar nonfunction) mutants tested, the frequency of cotransduction of H 1 with fla + or mot + was only 0.01–1%. The results suggest either that incomplete pairing of transduced fragments of uniform composition takes place or, more plausibly, that only a few of the fragments bearing the relevant fla + and mot + genes also carry the donor H 1 allele. The phase-1 flagellar antigens (i.e., H 1 phenotype) of abortive fla + transductants was inferred from the inhibition by antisera of the production of “trails” in semisolid medium by recipients in (latent) phase-1. All abortive fla + transductants expressed the recipient H 1 allele; with fla − recipients with high H 1 cotransduction rates all except 0.1–5% expressed also the donor H 1 allele, but for recipients with low cotransduction rates a higher proportion (25->50%) failed to express the donor H 1 allele. It is inferred that H 1 is absent from a few transduced fragments carrying fla + sites closely linked to H 1 and from many fragments carrying less closely linked fla sites. Heterogeneity was greater in phage A4 lysates than in P22 lysates, and seemed to vary also according to donor.

SUMMARY: In 18 hr. incubation of broth inoculated with a majority of bacteria of Salmonella typhimurium LT2 col -, i.e. non-colicinogenic, and a minority of strain LT2 (colI), i.e. carrying colicine factor colI, 30--70% of the col - bacteria acquired colI; this increased to 50--90% after 2 hr. of secondary incubation after tenfold dilution with broth. These cultures, containing a high proportion of bacteria newly infected by colI, transmitted colI to about 50% of the bacteria of a col - strain in 1 hr. (whereas pure cultures of LT2 (colI) transmitted to only 0.01%); they are termed HFC (high-frequency colicinogeny-transferring). An inoculum containing 1--3 recently infected bacteria from an HFC preparation of a streptomycin-sensitive strain sufficed to transmit colI to a resistant strain in streptomycin broth. We infer that 30--100% of bacteria newly infected by colI are ‘competent donors’, able to transmit colI. By the same test the proportion of competent donors in LT2 (colI) strains was only 0.02%. Inoculum size, ratio of inoculum components, motility, aeration and secondary incubation affected the HFC property of mixed cultures in a way explicable by the need for the epidemic spread of colI in the col - component to reach a peak at the time of testing. The rate of loss of the HFC property on further growth indicated that for 3--7 generations the progeny of newly infected bacteria are competent donors. Transmission was associated with clumping and pairing. Bacteria of an HFC preparation pair with 10% of an acceptor population within 2 min. of mixing; completion of transfer requires 2 1/2--30 min. Non-availability of nutrients and some growth inhibitors interfere with transfer. We conclude that colI multiplies autonomously in newly infected bacteria and their immediate progeny and enables them to conjugate, but does not confer ability to conjugate in established colicinogenic strains; perhaps it is then integrated into the host chromosome.

Change of flagellar antigenic phase in the Salmonellas is considered as resulting from the mutation and back mutation.A rapid method of determining the phase of numerous colonies ofSalmonella typhi-muriumis described; melted sloppy agar containing antibody for one phase is layered on tplates on which colonies have grown. On brief incubation, colonies of the agglutinated phase produce narrow dense sharp edged zones of opacity, while colonies of the other phase wide diffuse zones of swarming organisms.A theoretical analysis shows that if the growth rates of the phases are equal the proportion of cells of ‘mutant’ phase in a culture initially all in one phase and maintained in continuous logarithmic growth will (at first) increase linearly with the number of generations at a rate proportional to the mutation rate. In one strain ofSalm, typhi-muriumexperiments showed the rates of growth of the phases to be equal; the proportion of cells of mutant phase in cultures of this strain during long periods of continuous growth has been measured; linear rates of increase were found, and the mutation rates are calculated.The variance in proportion of cells of mutant phase in replicate cultures has been measured; the findings are correlated with theoretical considerations.Certain anomalous decrease in proportion of cells of mutant phase during prolonged growth are described, and a possible explanation is proposed.Eight further strains have been investigated by the same methods; in the nine strains, the rates of mutation from group to specific phase range from 10-4to 4.7 X 10-3per bacterial generation cycle, and the rates of the reverse mutation from 10-5to 8.6 X 10-4. In all the strains, including two of varietybinns, the rate of mutation from group to specific is higher than the rate of the reverse change; it is shown theoretically that such strains will tend towards an equilibrium composition in which the specific phase will predominate; in one rapidly mutating strain such an equilibrium has been demonstrated.In a limited experiment, alteration of medium and of temperature of incubation caused to detectable alteration of rate of mutation per bacterial generation.Stable O mutants, and poorly flagellated mutants which back-mutate to the fully motile form, are described.The experimental findings are compared with reports in the literature on the phase ofSalm. typhi muriumas isolated. The behavior of strains of varietybinnsis discussed, and it is suggested that this description should not now be applied to strains in which a specific phase can be demonstrated. The biological funtion of mutablility of phase is discussed.

SUMMARY: Cultures of non-motile Salmonella strains treated with phage lysates of motile strains produce in semi-solid agar: (i) swarms, each consisting of a clone of motile bacteria, attributed to complete transduction of motility; (ii) trails, i.e. unbranched linear groups of microcolonies stretching some millimetres from the site of inoculation, attributed to abortive transduction of motility; the latter was thought to result from the importation by phage of a gene conferring motility, which was not replicated but was transmitted, as a supernumerary gene, down one line of descent (unilinearly) to but one of the descendants of the organism which received it, the trail marking the path of the gene-bearing descendants of successive generations. The present work has led to a modified hypothesis: that the phage-imported non-replicated gene confers on the organism the ability to synthesize motility conferring (MC) particles, which are distributed amongst its non-gene-bearing progeny, which cannot make new ones; that each particle may be unilinearly transmitted for many generations; and that while one MC particle confers motility in broth, several are needed to enable a bacterium to travel in a semi-solid medium. The main evidence for this hypothesis is provided by pedigrees of bacteria made motile by abortive transduction, isolated by micromanipulation from treated suspensions. All such bacteria produce mainly non-motile offspring. A minority (exceptional or E bacteria) produce clones which after 10-15 generations include 20 to 100 motile descendants; the rest give clones containing only a few (0-12) motile organisms. E bacteria are identified as ones containing the supernumerary gene, the others as ones motile through possession of a few MC particles only. In each of six extensive pedigrees a single E bacterium was isolated from amongst the 9th-21st generation progeny of the original E parent. None of a large number of other motile bacteria isolated in collateral sublines (and so ex hypothesi not gene-bearing) produced more than 12 motile descendants. These data show that the E character (ability to produce 15 or more motile descendants) is unilinearly transmitted. Any motile bacteria found amongst the progeny of a non-E bacterium after six or more generations were inferred to be motile through possession of one MC particle each; of many such which were isolated none produced more than one motile descendant; when this in turn was isolated, a single motile organism was sometimes again detected in the clone produced. This shows that there is unilinear transmission of motility (in broth). Samples of populations of motile bacteria isolated by micro-manipulation from treated suspensions were transferred to semisolid agar, or to individual droplets; the proportion of bacteria which generated trails was about equal to the proportion found to be E. This was to be expected if both characters reflect the presence of the non-replicated gene. The MC particle is probably a flagellum, or a granule which determines the production of one. The particle presumed to account for the unilinear transmission of the E and trail-forming characters is most economically interpreted as a phage-imported fragment of genetic material which has failed to replace its homologue in the genetic organelle of the recipient organism.

SUMMARY: The flagellation and motility of Salmonella typhimurium were unaffected by proteolytic enzymes; but when washed stationary-phase bacteria were briefly exposed to acid (e.g. pH 2·6 for 30 sec. at 43°) the proportion of motile, and of flagellated, bacteria was decreased from more than 50% to less than 1%, with little or no decrease in viable count. When S. typhimurium or Vibrio metchnikovi grown on stiff agar were rubbed for several minutes on the agar, the proportion of motile and of flagellated bacteria was decreased from c. 90% (found in control bacteria, soaked off without rubbing) to c. 1%, without decrease in viable count. High-speed stirring in a blendor also deflagellated and destroyed the motility of S. typhimurium in suspension, without killing the bacteria or retarding their growth. Blendor treatment sufficient to deflagellate S. typhimurium had no effect on the movements of certain non-flagellated motile organisms (Leptospira canicola, Treponema pallidum and T. duttoni), except when it caused gross morphological changes, attributed to cell damage. When log-phase broth cultures of S. typhimurium deflagellated in the blendor were incubated, bacteria with flagella (at first all short) and motile bacteria soon reappeared. At first many bacteria rotated; this suggests that flagella shorter than c. 0·3 μ cause rotational, but not translational, motility. The rate of increase of mean summed flagellar length/bacterium was compatible with the hypothesis that flagella are not (spontaneously) shed in broth cultures of S. typhimurium and that the rate of growth of flagella is unaffected by deflagellation. Up to one generation time after deflagellation the mean number of flagella/bacterium was, however, less than before deflagellation. It is inferred that in a log-phase culture about half the flagella present at any one moment are ‘fully-grown’, incapable of being regenerated when broken off. All the results obtained are compatible with the hypothesis that the motility of flagellated bacteria is due to active movement of their flagella.

A Gene Determining Presence or Absence of ɛ-N-Methyl-Lysine in Salmonella Flagellar Protein

“Virulence factor” or “virulence determinant” is often used to refer to bacterial traits, such as production of a surface component hindering phagocytosis, whose presence is noted to be correlated with virulence and whose loss, by mutation, etc., causes loss or great reduction in virulence in an experimental system. Logically any bacterial property indispensable for bacterial growth in the relevant compartment of the host, such as ability to grow at the temperature there prevalent, should be considered a virulence factor; we shall discuss such properties, as well as those commonly called virulence factors.

Lysogenization of Salmonella typhimurium with either of the bacteriophages A3 and A4 results in O-acetylation of the L-rhamnose residues of the O-polysaccharide chain of the lipopolysaccharide of the bacterial cell envelope. The O-acetyl group is found on both O-2 and O-3 of the L-rhamnosyl residues. This lysogenic conversion prevents the adsorption of the A3 and A4 phages and also greatly reduces the rate of adsorption of phage P22 to the O-polysaccharide chain as measured by binding studies with whole bacteria. Isolated lipopolysaccharide from A3- and A4-lysogenized bacteria was also inefficient in inactivating these phages: the concentration required for 50% inactivation was 10,000-fold higher than that for lipopolysaccharide from S. typhimurium not lysogenized by any A phage. Binding of phages A3 and A4 is accompanied by hydrolysis of the alpha-1,3 linkage between rhamnose and galactose in the tetrasaccharide repeating unit of the O-polysaccharide. Phage hydrolysis generates saccharides of various lengths, the majority being dodecasaccharides, i.e., equivalent to three repeating units. It is surmised that O-acetylation of the rhamnosyl residue interferes with phage A3, A4, and P22 infection by preventing binding to and hydrolysis of the O-polysaccharide chain, the initial step in the phage infection cycle. The new O-acetyl-rhamnose entities did not elicit specific antibodies in rabbits in accordance with earlier experiences. The O-acetylation of O-2 and O-3 of rhamnose is a new, hitherto unknown, modification of the O-polysaccharide chain of S. typhimurium.

SUMMARY: FOR mutants of Salmonella typhimurium are resistant to Felix O phage, whose receptor includes the N-acetylglucosamine branch of the lipopolysaccharide (LPS) core, but smooth in cultural properties, antigenic character and phage sensitivity pattern (MacPhee et al., 1975). The rfa(FOR) genes determining the FOR character of nine mutants were transduced into a smooth cysEpyrE recipient: the nine FOR transductants (and a tenth FOR mutant) were then made rfb (i.e. unable to make O chains) by transduction or Hfr crosses. The rfb FOR strains were sensitive to FO phage but nearly all of them showed a somewhat reduced efficiency of plating and diminished rate of adsorption of the phage. This observation and the Ra (complete core) serological activity of their LPS (tested by haemagglutination inhibition) indicate the presence of some, but less than the normal number of, completed core chains in FOR rfb LPS. On the basis of the sensitivities of the FOR transductants and their rfb derivatives to various “rough-specific” phages, their increased sensitivities to some antibiotics and to deoxycholate and the serological activity of the rfb FOR LPS in various incomplete core systems, the mutants were divided into three groups: (i) five mutants with probable defects in previously undetected rfa gene(s) concerned with formation of both the galactose I and the galactose II units of the LPS core; (ii) two mutants with defects inferred to affect the structure of the inner part of the core and also interfere with addition of the N-acetylglucosamine branch; (iii) three mutants in which no type of incomplete core could be detected, probably affected in formation of the inner part of the core chain. The mutation of one mutant of the last class, unlike those of the other nine mutants tested, lay outside the cysE-pyrE segment, in the 90 to 116 min region of the linkage map.

Mice immunised by the oral or intraperitoneal route with a live aromatic-dependent strain of Salmonella typhimurium exhibited significantly less protection against oral challenge with 50 LD50 of an ovine isolate of S. typhimurium (12313) than when a bovine isolate with the same O antigens and phage-type as strain 12313 was used as the challenge organism. When challenged with 10 LD50, however, protection against both strains was significantly better than that obtained when mice were vaccinated with killed vaccines (heat-killed, acetone-killed or irradiated) even when the antigenic mass of the killed vaccine was increased by up to 500-fold in an attempt to compensate for the expected limited multiplication of the mutant organism. Sheep immunised with the live mutant strain by either the intramuscular or oral route were protected against oral challenge with the virulent ovine isolate of 5. typhimurium; unimmunised sheep died of acute enteritis within 7 days, although there was no evidence of systemic invasion by the challenge organism. After challenge, immunised animals ate more food than the unimmunised controls and suffered only transient, mild diarrhoea. Serum antibody titres against O and H antigens measured by direct or antiglobulin tests were significantly higher in sheep immunised by the intramuscular route than in those immunised orally. Sheep in both immunised groups developed skin swellings within 30 min after intradermal inoculation with purified homologous lipopolysaccharide indicating development of immediate-type hypersensitivity, but only those immunised by the intramuscular route showed significant indurated skin swellings characteristic of delayed-type hypersensitivity 48 and 72 h post-inoculation. Thus it appears that protection against salmonellosis in sheep immunised by the intramuscular or the oral route may involve different mechanisms.

We made delta aroA, delta purA, and delta aroA delta purA derivatives of a strain of Salmonella dublin and isolated a nalidixate-resistant mutant of each construct. An inoculum of each of the nearly isogenic nalidixate-resistant auxotrophs was administered to BALB/c mice by gavage. The ability of each strain to colonize, invade, persist in tissues, and evoke serum and mucosal antibody responses to the lipopolysaccharide of the parent strain was examined. Only the delta aroA strain colonized, invaded, persisted, and (more importantly) evoked sustained significant serum and mucosal antibody responses. Neither the delta purA nor the delta aroA delta purA strain showed any of these abilities. These observations demonstrate that the purA defect, which causes a requirement for adenine, reduces the live-vaccine efficacy of attenuated Salmonella strains and may limit the effectiveness of Salmonella strains as carriers of heterologous antigens. These findings may be important in the selection of attenuated S. typhi strains for use in humans either as antityphoid live vaccines or as vectors for antigens of other pathogens.

Mutants resistant to phage P22 by virtue of roughness, due to mutation at rouB, were obtained in P22-lysogenic and nonlysogenic forms of two genetically marked lines of Salmonella typhimurium strain LT2. Crosses permuted in respect of the lysogeny or nonlysogeny of the rough metA tryB donor and of the rough adeC proA ile str-r acceptor line were then made, using the colicine factors colI and colE1 to obtain conjugation and recombination. When the donor was P22-lysogenic and the acceptor was nonlysogenic there was a great reduction in the yield of recombinants with the linked azi, pro, and gal alleles of the donor. In the cross of the nonlysogenic donor to the lysogenic acceptor the yield of recombinants with the pro+ donor allele was normal. In this cross all the recombinant clones with the pro+ allele from the donor were either nonlysogenic or mixed in respect of lysogeny, though homogeneous in respect of all other segregating characters. By contrast only one of 54 recombinants with the ile+, but not the pro+, donor allele was nonlysogenic. The results suggest that in lysogenic S. typhimurium strain LT2 prophage P22 is always or nearly always located on or in the chromosome very close to proA.

Metabolic and Genetic Investigations of Nutritionally Exacting Strains of Staphylococcus pyogenes

IN bacteria certain genetic determinants which may be gained or lost can multiply without incorporation in the bacterial chromosome. Some such plasmids1, the class of episomes2, may become reversibly integrated into the chromosome; for others, including the transmissible drug-resistance (R) factors3, integration has not been unequivocally demonstrated. We report observations on the transmission of an R factor and of colicine factors by conjugation and transduction in Salmonella typhimurium, and show that R factor genetic material may become attached to the bacterial chromosome—but only by becoming associated with an episome, prophage P22.

An LT2 Hfr strain,his metC gal, was crossed to a multiply marked LT2 F−line. Analysis of recombinant yields, segregation of unselected markers and interrupted matings indicated injection of the Hfr chromosome in the sequenceThe introduction into the Hfr of the colicine factorscolI, colE1andcolE2and the R factorR2had little or no effect on its fertility. All four factors were transmitted at low frequency to the F−population, and to recombinants at higher frequencies (colI5–30%,colE130–80%,colE25–30%,R20–9%). Transfer ofcolE1occurred before 20 min., that ofcolE2andcolIlater than 100 min. Segregation data did not reveal close linkage of any factor to any chromosomal locus, but recombinants with a long stretch of donor chromosome were more likely than others to have acquiredcolE2andcolI. Nearly all recombinants andF−cells which acquiredcolIorcolE2acquired both, andcolE1also. Most cells which acquiredR2acquired one or more colicine factors. These plasmid associations can be formally represented by transfer of plasmids, independently of the chromosome, in the sequencecolE1—(colI, colE2)—R2. Phage P22 grown on the Hfr carrying the four plasmids transduced thetet-rtrait ofR2at very low frequency, and thesul-r str-rcharacters, together, at low frequency. Some of each sort of drug-resistance transductant, but no transductants in respect of chromosomal characters, acquiredcolEl or colE2by co-transduction.

SUMMARY: Phage lysates of a salmonella strain can transduce flagellar characters from that strain to a second strain which absorbs the phage. Non-motile strains treated with lysates o other strains produced stable motile forms detected as spreading swarms in semi-solid agar. O strains of known species produced swarms with the H antigens characteristic of theirown species, and the species of previously untypable O isolates could be inferred from the H antigens of their induced swarms. The production of swarms by O strains when treated with lysates of other O strains divided nine Salmonella typhimurium O strains into six interacting groups; there are therefore at least six non-homologous genetic factors, mutation of any of which may cause absence of flagella, thus masking other genes which control H antigenic specificity. Two flagellated but non-motile strains produced motile forms when treated with lysates of other strains, or of each other. At least two genes concerned with locomotor function are thus recognized. All the motile derivatives of one O strain spread very slowly through semi-solid agar; the ability to spread rapidly could be transduced to them by a second exposure to a lysate. In exceptional instances two flagellar characters were simultaneously transduced: three O strains sometimes acquired a flagellar antigen from the donor strain at the same time as the H character; another O strain sometimes acquired simultaneously flagellation and the slow-spreading character. There are therefore many distinct genes controlling the presence, antigenic character and function of the flagella. They may form part of an ordered gene array in which pairs of genes which are sometimes simultaneously transduced by a single phage particle are presumably closely linked.

SUMMARY: In stock cultures of Salmonella typhimurium strain lt2 carrying the factor determining the production of colicine I, colI, together with other colicine factors, colE1 or colE2, most of the small minority of bacteria competent to act as donors of colI also transmit their E factor. Most noncolicinogenic bacteria which acquire an E factor at the same time as colI become competent to transmit both factors. Similarly, bacteria carrying factors colE1 and/or colE2 accept colI normally and then usually become competent to transmit both colI and the E factor(s) they already carry. In experiments on the kinetics of colicine factor transmission by lt2 (colE1) (colE2) newly infected with colI, most acceptor bacteria that acquire any colicine factor acquire all three factors, within 5–20 min. for most pairs, but within 1 min. for a very few. When conjugation is interrupted within 5 min. of mixing, fewer acceptor bacteria acquire all three factors; all single-factor and two-factor classes are then represented, so the order of transfer of the three factors must differ in different pairs. Bacteria carrying colE2 do not transfer this factor to donor bacteria from whom they acquire colI; but they become able to transmit colE2 (and presumably colI also) within 15–20 min. of acquiring colI. Other observations support the hypothesis that in most competent donors carrying colE1 and/or colE2, these factors multiply autonomously, as does colI.

SUMMARY: Some hypotheses are considered which describe the aetiology of a fatal infection in a partially resistant host; i.e. a host which does not invariably die after inoculation with one bacterium. The hypothesis of independent action postulates that the mean probability per inoculated bacterium of multiplying to cause (or help to cause) a fatal infection is independent of the number of bacteria inoculated and, for a partially resistant host, is less than unity (1>p>0). It predicts: (1) that the slope, b, of the probit-mortality/log-dose curve will be 2·0 or less at the LD 50 point; (2) that, while hosts dying after inoculation with many LD50 die as a result of the multiplication of many of the inoculated bacteria, most of those dying from 1 LD50 or less do so following the multiplication of only one of the inoculated bacteria, regardless of the total number of bacteria inoculated. When a mixture of several equally virulent, distinguishable variants of a given pathogen are inoculated, fatal infections caused by the growth of one bacterium should result in the predominance of only one variant at post mortem. The hypotheses of maximum and of partial synergism postulate that inoculated bacteria co-operate so that the value of p increases as the size of the dose increases. They predict: (1) that the slope of the dose-response curve may be more than 2·0 at the LD50 point; (2) that all observed fatal infections will be initiated by more than one bacterium and that consequently the inoculation of a mixed inoculum will lead to the predominance of several variants at post mortem. Variants of Salmonella paratyphi-B which carried one of the three flagellar antigens, b, i, or e, h, were prepared by transduction. Variants of S. typhimurium which either fermented or did not ferment xylose were also prepared. In each case, the variants did not differ detectably in growth rate in vivo, or in virulence. The value of b was 1·8 for mice inoculated with S. paratyphi-B by intraperitoneal injection (LD50 = 7·7 × 106 organisms). As predicted by the hypothesis of independent action, the relative frequencies of the variants in samples of heart blood obtained post mortem varied greatly from mouse to mouse when the dose was 1 LD50 or less, and became progressively more uniform (and similar to the inoculum) as the size of the dose increased. The value of b was 0·66 for mice challenged by S. typhimurium by subcutaneous injection (LD50 = 2 × 103 organisms); and challenge with a mixed inoculum gave similar results. Despite this general conformity with prediction, far fewer mice than expected yielded only one variant at post mortem. This discrepancy possibly resulted from a terminal breakdown in resistance, which was demonstrated by experiment. It is concluded that these results are most economically explained by the hypothesis of independent action and that this hypothesis is probably applicable to many other infective systems.

An electron microscopic examination of eight non-flagellate strains of S. typhimurium revealed that an H1 (amber) mutant produced flagellar hooks and basal structures indistinguishable from those associated with the proximal end of normal flagella. No flagella-related structures were seen in strains with mutations in fla genes A, B, C, D, F, K or M. A mutant of a new fla complementation group, flaR, produced abnormal structures, termed “superhooks”, which resemble the hooks of normal flagella assembled end-to-end. The mutant locus, flaR, maps between flaM and flaAIII.

SUMMARY: Bacteria in cultures of Salmonella typhimurium LT2 were peritrichously flagellated when grown in nutrient broth at 370; but most were non-flagellated when grown for 6 mean-generation times or more at 440. When a culture growing exponentially at 370 was transferred to 440, growth continued at about the same rate; but the synthesis of new flagella was largely curtailed. The fate of the parental flagella was studied by staining and counting flagella on bacteria from samples taken during growth at 440 of cultures first grown at 370. After 3 mean-generation-times the average number of flagella/flagellated bacterium had fallen from about 8 to about 2 and the proportion of flagellated bacteria from about 100 % to about 60 %. The distribution of numbers of flagella/bacterium was at all times unimodal, with the mode decreasing from about 8 to 0. In non-growing cultures at 440 there was little or no change in the average number of flagella/bacterium, in the proportion of flagellated bacteria, or in the distribution of numbers of flagella/bacterium. It is inferred that parental flagella are neither rapidly shed at 440 nor retained entirely by one daughter cell at each division but are distributed about equally between the two daughter cells.

SUMMARYA total of 1537 strains ofSalmonella typhimuriumbelonging to seven prevalent phage types were examined on solid media for their ability to ferment rhamnose, xylose and inositol, for colicine production and for nutritional requirements. Most of the strains in each phage type were almost completely homogeneous, especially in their sugar fermentation reactions. However, strains of phage type 1a/2 were not homogeneous, but could be assigned to one of four subgroups on the basis of ability to ferment inositol, inhibition of growth bymeso-tartrate and auxotrophy for nicotinic acid. The subdivision proved to have epidemiological value. The inhibition of growth bymeso-tartrate was observed on a denned medium containing citrate as the energy source. Inhibition did not occur if glucose, casein hydrolysate or aspartic acid were added to the medium.

Rough Mutants of Salmonella Typhimurium (3) Enzymatic Synthesis of Nucleotide-Sugar Compounds

The Genetics of Bacteria and their Viruses Studies in Basic Genetics and Molecular Biology. By Dr. William Hayes. Pp. xii+740+25 plates. (Oxford: Blackwell Scientific Publications, 1964.) 84s. net.