B. A. D. Stocker

High Infectivity of Salmonella typhimurium newly infected by the colI factor

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.

Mapping of prophage P22 in Salmonella typhimurium.

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.

TRANSFER OF COLE1 AND COLE2 DURING HIGH-FREQUENCY TRANSMISSION OF COLI IN SALMONELLA TYPHIMURIUM.

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.

X-ray and related studies of the flagella of non-motile bacteria

Abstract Flagella specimens from motile and non-motile variants of a single strain of Salmonella typhimurium were examined in the electron microscope and by X-ray diffraction. The results reveal no apparent significant differences between either the form or structure of the two specimens, the same fibrous protein (“flagellin”, a member of the k-m-e-f group) being present in each, and in the same two configurations, α and supercontracted. Parallel biological tests suggested also that the non-motility of the non-motile variant was not due either to lack of coordination between (normal) flagella, or to a generalised body, or cell-wall, defect. It is therefore proposed that the source of the paralysis is located in the link between (apparently) normal body and (apparently) normal flagellum; in the presumed basal granule, in fact.

The occurrence of rare motile bacteria in some non-motile Salmonella strains.

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.

Isolation and Serological Analysis of Mutant Forms of Flagellar Antigen i of Salmonella typhimurium

Summary: Nine spontaneous mutants with altered forms of flagellar antigen i were obtained by picking more rapidly spreading swarms from growth in semi-solid medium containing enough anti-i serum to retard spreading growth. One mutant was in a line of Salmonella typhi given antigen i by transduction, the rest in S. typhimurium strain LT2 adeC-7 proA-46. Two mutants of independent origin were serologically identical and presumably arose by a recurrence of the same mutation. Bacteria expressing the mutant phase-1 antigen were normally motile and the LT2 mutants showed normal phase-variation, to give cultures with an apparently unaltered phase-2 antigen, 1,2,3. Flagellate bacteria with flagella of two of the mutant types, iM6 and iM9, were agglutinated to titres 8--16 by sera from uninoculated rabbits and to titres 50--100 by sera from rabbits immunized with unrelated antigens; suspensions of flagella of these types, but not of others, caused flocculation of indian ink. The residual activity of anti-i (wild type) sera fully absorbed with mutant antigens showed that each mutant antigen had lost some of the serological specificity of the wild-type antigen; the complex pattern of residual activity on mutant and wild-type antigens of anti-i (wild type) sera absorbed with pairs of mutant antigens indicated the existence of at least 13 antigenic factors in the wild-type antigen, and that each ofthe serologically distinct mutant antigens lacked a different combination of these factors. The residual activities on the homologous antigens of antimutant sera fully absorbed with wild-type antigen showed that all the mutant antigens, except perhaps iM6 and iM9, had antigenic specificities absent from the wild-type antigen. Each of the 8 serologically different antigens had a unique new specificity, but antigens iM7, iM10 and iM12 shared some new factors. Attempts to infer the linear order of the presumed sites of amino acid substitution in the polypeptide chain of flagellin from the serological data were unsuccessful; this probably indicates the incorrectness of an assumption involved: namely, that anti-flagellar antibodies have an absolute affinity for, and only for, all of the amino acid side-chains (or all of a reactive subset of them) in a relevant length of polypeptide chain.

Variation in composition of chromosome fragments transduced by phage P22

Abstract 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.