Influence of lipopolysaccharide and protein in the cell envelope on recipient capacity in conjugation of Salmonella typhimurium.

In crosses of Salmonella typhimurium FfinP301 lac+ to F- strains of S. typhimurium in broth, recipient strains which were rough mutants affected in the outer core region of the lipopolysaccharide gave an average of 1.4 Lac+ transconjugants per donor cell and over 50% of the donor and recipient cells in mating aggregates, whereas smooth recipient strains gave 0.08 Lac+ transconjugants and few cells in mating aggregates. Strains with mutations affecting the inner core of the lipopolysaccharide were usually poor recipients. When cells were mated on Millipore membrane filters, both smooth and rough strains gave ca. 1.0 Lac+ transconjugants per donor cell. Plasmids in Inc groups FI, FII, M, J, and I beta gave more transconjugants with rough than smooth strains, but there were no difference in crosses with plasmids in Inc groups T, L, P, N, and W. Strains with mutations in the ompA gene (deficient in Omp Ap = 33K = II* = conjugation protein) yielded only 0.02 Lac+ transconjugants per donor cell and few cells in mating aggregates. There was no indication of a deficiency of Omp Ap in smooth strains compared with rough strains. Reduced fertility of smooth recipients may occur because the O side chains of the lipopolysaccharide shield the recipient and reduce the frequency of stabilization of mating aggregates. However, gradient-of-transmission experiments indicated that once these mating aggregates are stabilized, they are equally stable in both smooth and rough recipients. Fertility was high in crosses of S. typhimurium Flac+ to Escherichia coli K-12 F- (0.75 Lac+ transconjugants per donor cell; over 50% of the cells in mating aggregates). In crosses of E. coli K-12 Flac+ to S. typhimurium smooth F-, ca. 10(-5) Lac+ transconjugants per donor cell were obtained; in crosses to rough recipient strains, fertility was increased 14-fold, and when the recipient was defective in the SA and LT host restriction systems, fertility was increased in additional 100-fold. Thus, both the lipopolysaccharide and the protein in the cell envelope of S. typhimurium were shown to be important in the recipient function in F-mediated conjugation.     

A method for the enumeration of male-specific bacteriophages in sewage

Male-specific bacteriophages adsorb to F-pili and thus can only infect male host strains. A method was developed for the selective enumeration of these phages, based on the observation that in sewage there are few phages capable of infecting F- -salmonellas--usually less than 10 pfu/ml. Using a male Salmonella strain, constructed by the introduction of the plasmid F'42 lac::Tn5 into Salmonella typhimurium phage type 3, plaque counts in secondary effluent were found to be in the range of 60-8200 pfu/ml. Practically all the phages detected had a host range restricted to male Salmonella or Escherichia coli strains, were resistant to chloroform and their infectivity was inhibited by RNase. Electron microscopy of lysates revealed phage particles that were morphologically identical to the male-specific single-strand RNA phages. Similar results were obtained with a strain of Salm. indiana carrying F'42 lac. A derivative of the Salm. typhimurium LT2 strain carrying an F-plasmid (F'42 lac fin P301) derepressed for fertility inhibition by the resident plasmid pSLT was equally sensitive to male-specific phages, but from sewage samples many other phages infecting F- E. coli but not F- Salmonella were isolated using this host strain.     

A method for the enumeration of male-specific bacteriophages in sewage

H avelaar , A.H. & H ogeboom , W.M. 1984. A method for the enumeration of male-specific bacteriophages in sewage. Journal of Applied Bacteriology 56 , 439–447.
Male-specific bacteriophages adsorb to F-pili and thus can only infect male host strains. A method was developed for the selective enumeration of these phages, based on the observation that in sewage there are few phages capable of infecting F^--salmonellas—usually less than 10 pfu/ml. Using a male Salmonella strain, constructed by the introduction of the plasmid F'42 lac::Tn5 into Salmonella typhimu-rium phage type 3, plaque counts in secondary effluent were found to be in the range of 60–8200 pfu/ml. Practically all the phages detected had a host range restricted to male Salmonella or Escherichia coli strains, were resistant to chloroform and their infectivity was inhibited by RNase. Electron microscopy of lysates revealed phage particles that were morphologically identical to the male-specific single-strand RNA phages. Similar results were obtained with a strain of Salm. indiona carrying F'42 lac . A derivative of the Salm. typhimurium LT2 strain carrying an F-plasmid (F'42 lac fin P301) derepressed for fertility inhibition by the resident plasmid pSLT was equally sensitive to male-specific phages, but from sewage samples many other phages infecting F^- E. coli but not F^- Salmonella were isolated using this host strain.     

Electrotransformation in Salmonella typhimurium LT2

Electroporation gives high efficiency of transformation in Salmonella typhimurium LT2, yielding 10(8)-10(9) electrotransformants per microgram of pBR322 DNA. Lipopolysaccharide (LPS) composition has little influence on electrotransformation efficiency by electroporation, unlike Ca2+ shock methods, which give ca. 10(6) transformants/microgram DNA with strains with Rc or Rd2 LPS, 10(4) transformants with most smooth and rough strains, and 10(2) transformants with strains with Re LPS. Thus cell envelope properties are less crucial in electrotransformation than in Ca2+ shock methods. The reciprocal restriction barrier between Escherichia coli K-12 and S. typhimurium LT2 reduces electrotransformation by ca. 100-fold, but host-restriction mutants reduce or eliminate the barrier.     

Isolation of a hybrid F' factor-carrying Escherichia coli lactose region and Salmonella typhimurium histidine region, F42-400 (F' ts114 lac+, his+): its partial characterization and behavior in Salmonella typhimurium.

Episome F' ts114 lac+ (F42-114) was transferred into Salmonella typhimurium carrying an F'his+ (FS400) episome, and fused episome F' ts114 lac+, his+ (F42-400) was obtained. Episome F42-400 could be transferred to S. typhimurium, Escherichia coli and Klebsiella pneumoniae. Identification of the episome was based on: (i) temperature sensitivity of the Lac+ and His+ phenotypes; (ii) the fact that F- segregants, obtained after temperature curing or acridine orange curing, were simultaneously Lac- and His-; and (iii) linkage of lac+ with his+ in episomal transfers to E. coli and S. typhimurium. The frequency of episome transfer was influenced by the genotype of the donor. Plasmid LT2, prevalent in S. typhimurium LT2 strains, was suggested to be responsible for the low fertility of S. typhimurium donors. Episome F42-400 was capable of chromosome mobilization, and the extent of chromosome mobilization was not influenced by the presence or absence of the histidine region on the donor chromosome. Growth in a defined medium with acridine orange was able to cure F42-400. The frequency of curing was increased (the frequency of His+ cells was 0.0001%) if the cells were grown at 40 C in the presence of acridine orange. Selection for temperature-resistant Lac+, His+ derivatives in a strain without histidine deletion yielded Hfr strains. However, similar and stronger selections in strains without the chromosomal histidine region failed to yield Hfr strains. Our inability to obtain Hfr's in strains without the chromosomal histidine region was explained by assuming that the episome F42-400 has lost the F sites involved in integration into the S. typhimurium chromosome.     

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.     

Hemin-Deficient Mutants of Salmonella typhimurium

Nine hemin-deficient mutants of Salmonella typhimurium LT2 were isolated as neomycin-resistant colonies. Five of these mutants could be stimulated by Delta-aminolevulinic acid (Delta-ALA), thus representing hemA mutants. Since S. typhimurium LT2 is not able to incorporate hemin, the identification of the mutants not stimulated by Delta-ALA was made on the basis of the simultaneous loss of catalase activity and cytochromes. The hemA gene was mapped by conjugation in the trp region, probably in the order purB-pyrD-hemA-trp; the episome FT(71)trp does not carry the hemA gene. Transductional intercrosses by phage P22 indicate that hemA 11, 12, 13, and 37 are at very closely linked sites, whereas hemA14 is at a more distant site in the same or an adjacent gene. No joint transduction was detected between hemA and trp or pyrF. The loci affected in the other hemin-deficient mutants were linked in conjugation to the pro(+) marker (frequency of linkage, 88 to 97%), but cotransduction of the two markers could not be obtained. The episome F lac hem purE, which originates from Escherichia coli K-12, could complement these hemin-deficient mutants of S. typhimurium LT2. As a result, the sequence of the markers on the chromosome of S. typhimurium LT2 is probably pro heme purE, analogous to the sequence found in E. coli K-12. Thus, the chromosome of S. typhimurium also possesses two hem regions, with a location similar to that described in E. coli K-12.     

Synthesis of FinP RNA by plasmids F and pSLT is regulated by DNA adenine methylation.

DNA adenine methylase mutants of Salmonella typhimurium contain reduced amounts of FinP, an antisense RNA encoded by the virulence plasmid pSLT. Lowered FinP levels are detected in both Dam- FinO+ and Dam- FinO- backgrounds, suggesting that Dam methylation regulates FinP production rather than FinP half-life. Reduced amounts of F-encoded FinP RNA are likewise found in Dam- mutants of Escherichia coli. A consequence of FinP RNA scarcity in the absence of DNA adenine methylation is that Dam- mutants of both S. typhimurium and E. coli show elevated levels of F plasmid transfer. Inhibition of F fertility by the S. typhimurium virulence plasmid is also impaired in a Dam- background.     

Phage t: a group T plasmid-dependent bacteriophage

Phage t was isolated from sewage from Pretoria. It formed plaques only on Escherichia coli and Salmonella typhimurium strains that carried plasmids belonging to incompatibility group T. Five of six group T plasmids permitted visible lysis of R+ host strains. There was no visible lysis of E. coli J53-2 or S. typhimurium LT2trpA8 carrying the T plasmid Rts1 although the strains supported phage growth as indicated by at least a 10-fold increase in phage titre. The latter strains transferred the plasmid at high frequency to E. coli strain CSH2 and the resulting transconjugants plated the phage. Proteus mirabilis strain PM5006(R402) failed to support phage growth although it transferred the plasmid and concomitant phage sensitivity to E. coli J53-2. The phage was hexagonal in outline, RNA-containing, resistant to chloroform and adsorbed to the shafts of pili determined by T plasmids.     

Molecular analysis of the rfaD gene, for heptose synthesis, and the rfaF gene, for heptose transfer, in lipopolysaccharide synthesis in Salmonella typhimurium.

We report the analysis of three open reading frames of Salmonella typhimurium LT2 which we identified as rfaF, the structural gene for ADP-heptose:LPS heptosyltransferase II; rfaD, the structural gene for ADP-L-glycero-D-manno-heptose-6-epimerase; and part of kbl, the structural gene for 2-amino-3-ketobutyrate CoA ligase. A plasmid carrying rfaF complements an rfaF mutant of S. typhimurium; rfaD and kbl are homologous to and in the same location as the equivalent genes in Escherichia coli K-12. The RfaF (heptosyl transferase II) protein shares regions of amino acid homology with RfaC (heptosyltransferase I), RfaQ (postulated to be heptosyltransferase III), and KdtA (ketodeoxyoctonate transferase), suggesting that these regions function in heptose binding. E. coli contains a block of DNA of about 1,200 bp between kbl and rfaD which is missing from S. typhimurium. This DNA includes yibB, which is an open reading frame of unknown function, and two promoters upstream of rfaD (P3, a heat-shock promoter, and P2). Both S. typhimurium and E. coli rfaD genes share a normal consensus promoter (P1). We postulate that the yibB segment is an insertion into the line leading to E. coli from the common ancestor of the two genera, though it could be a deletion from the line leading to S. typhimurium. The G+C content of the rfaLKZYJI genes of both S. typhimurium LT2 and E. coli K-12 is about 35%, much lower than the average of enteric bacteria; if this low G+C content is due to lateral transfer from a source of low G+C content, it must have occurred prior to evolutionary divergence of the two genera.     

Evaluation of an Escherichia coli host strain for enumeration of F male-specific bacteriophages

A method was developed for the selective enumeration of F male-specific bacteriophages in samples of environmental waters. The host strain for the phages, Escherichia coli HS(pFamp)R, has three antibiotic resistance markers, ampicillin on the Famp plasmid, which codes for pilus production, and streptomycin and nalidixic acid on the chromosome. The strain is resistant to coliphages T2 to T7 and phi X174. More than 95% of the phages from environmental samples which plaqued on the host strain were F male specific. The host bacterium had a higher plaquing efficiency than E. coli K-12 Hfr for F-specific phages in stock suspensions and sewage effluents. The F male-specific phage levels in prechlorinated, secondary-treated sewage effluents generally were about 10(3) to 10(4) PFU/100 ml. The levels in the influents to the sewage treatment plants and in septic tank contents were about 10(5) PFU/100 ml. RNA-containing phages composed about 90% of the total F-specific phage population in sewage effluents.     

Evaluation of an Escherichia coli host strain for enumeration of F male-specific bacteriophages.

A method was developed for the selective enumeration of F male-specific bacteriophages in samples of environmental waters. The host strain for the phages, Escherichia coli HS(pFamp)R, has three antibiotic resistance markers, ampicillin on the Famp plasmid, which codes for pilus production, and streptomycin and nalidixic acid on the chromosome. The strain is resistant to coliphages T2 to T7 and phi X174. More than 95% of the phages from environmental samples which plaqued on the host strain were F male specific. The host bacterium had a higher plaquing efficiency than E. coli K-12 Hfr for F-specific phages in stock suspensions and sewage effluents. The F male-specific phage levels in prechlorinated, secondary-treated sewage effluents generally were about 10(3) to 10(4) PFU/100 ml. The levels in the influents to the sewage treatment plants and in septic tank contents were about 10(5) PFU/100 ml. RNA-containing phages composed about 90% of the total F-specific phage population in sewage effluents.     

Genetic Transfer of Salmonella typhimurium and Escherichia coli Lipopolysaccharide Antigens to Escherichia coli K-12

Escherichia coli K-12 varkappa971 was crossed with a smooth Salmonella typhimurium donor, HfrK6, which transfers early the ilv-linked rfa region determining lipopolysaccharide (LPS) core structure. Two ilv(+) hybrids differing in their response to the LPS-specific phages FO and C21 were then crossed with S. typhimurium HfrK9, which transfers early the rfb gene cluster determining O repeat unit structure. Most recombinants selected for his(+) (near rfb) were agglutinated by Salmonella factor 4 antiserum. Transfer of an F' factor (FS400) carrying the rfb-his region of S. typhimurium to the same two ilv(+) hybrids gave similar results. LPS extracted from two ilv(+),his(+), factor 4-positive hybrids contained abequose, the immunodominant sugar for factor 4 specificity. By contrast, his(+) hybrids obtained from varkappa971 itself by similar HfrK9 and F'FS400 crosses were not agglutinated by factor 4 antiserum, indicating that the parental E. coli varkappa971 does not have the capacity to attach Salmonella O repeat units to its LPS core. It is concluded that the Salmonella rfb genes are expressed only in E. coli varkappa971 hybrids which have also acquired ilv-linked genes (presumably rfa genes affecting core structure or O-translocase ability, or both) from a S. typhimurium donor. When E. coli varkappa971 was crossed with a smooth E. coli donor, Hfr59, of serotype O8, which transfers his early, most his(+) recombinants were agglutinated by E. coli O8 antiserum and lysed by the O8-specific phage, Omega8. This suggests that, although the parental E. coli K-12 strain varkappa971 cannot attach Salmonella-specific repeat units to its LPS core, it does have the capacity to attach E. coli O8-specific repeat units.     

The rfaS gene, which is involved in production of a rough form of lipopolysaccharide core in Escherichia coli K-12, is not present in the rfa cluster of Salmonella typhimurium LT2.

Partial sequencing of the rfa cluster of Salmonella typhimurium LT2 indicated a region of 336 bp between rfaP and rfaB in the site occupied by the rfaS gene in Escherichia coli K-12. This region does not contain a functional rfaS gene, although DNA analysis suggests that the region may have contained an ancestral gene. This conclusion that S. typhimurium LT2 lacks rfaS is supported by its lipopolysaccharide (LPS) gel phenotype, since LT2 does not make the lipooligosaccharide band characteristic of LPS from smooth strains of E. coli K-12.     

Construction and characterization of two lexA mutants of Salmonella typhimurium with different UV sensitivities and UV mutabilities.

Salmonella typhimurium has a SOS regulon which resembles that of Escherichia coli. recA mutants of S. typhimurium have already been isolated, but no mutations in lexA have been described yet. In this work, two different lexA mutants of S. typhimurium LT2 have been constructed on a sulA background to prevent cell death and further characterized. The lexA552 and lexA11 alleles contain an insertion of the kanamycin resistance fragment into the carboxy- and amino-terminal regions of the lexA gene, respectively. SOS induction assays indicated that both lexA mutants exhibited a LexA(Def) phenotype, although SOS genes were apparently more derepressed in the lexA11 mutant than in the lexA552 mutant. Like lexA(Def) of E. coli, both lexA mutations only moderately increased the UV survival of S. typhimurium, and the lexA552 strain was as mutable as the lexA+ strain by UV in the presence of plasmids encoding MucAB or E. coli UmuDC (UmuDCEc). In contrast, a lexA11 strain carrying any of these plasmids was nonmutable by UV. This unexpected behavior was abolished when the lexA11 mutation was complemented in trans by the lexA gene of S. typhimurium. The results of UV mutagenesis correlated well with those of survival to UV irradiation, indicating that MucAB and UmuDCEc proteins participate in the error-prone repair of UV damage in lexA552 but not in lexA11. These intriguing differences between the mutagenic responses of lexA552 and lexA11 mutants to UV irradiation are discussed, taking into account the different degrees to which the SOS response is derepressed in these mutants.     

Silent genes in bacteria: the previously designated 'cryptic'ilvHI locus of 'Salmonella typhimurium LT2' is active in natural isolates

Abstract Gene ilvG in Escherichia coli K-12 and ilvl in ' Salmonella typhimurium LT2' ( S. enterica serotype Typhimurium, strain LT2) are inactive due to frameshift or nonsense mutations, respectively. These inactive genes have been suggested to be part of 'cryptic' genetic systems which are defined as being of long-term regulatory and evolutionary significance. We have shown that the nonsense mutation in ilvI is present only in derivatives of the laboratory strain ' S. typhimurium LT2'. All natural isolates of Salmonella examined have an arginine codon at the corresponding location of their ilvl sequences. Further, two randomly selected natural isolates of serotype Typhimurium are shown to each have an active ALS III isozyme. Our findings strongly suggest that the only Salmonella strains which lack a functional ilvHI locus are LT2 isolates. We suggest that the mutations leading to inactivation of both ilvI in ' S. typhimurium LT2' and ilvG in E. coli K-12 are more likely to have been acquired during laboratory storage and/or cultivation, rather than representing cryptic systems of gene regulation.     

Variant pili produced by mutants of the Flac plasmid

Transfer-proficient Flac mutants with reduced abilities to plate various F-specific phages were isolated, either by selection after mutagenesis, or as revertants of Flac traA mutants. In many of the mutants pilus-related properties were altered, including physical adsorption of R17 phage, the number of pili per cell and the outgrowth/retraction equilibrium. Complementation studies showed that the mutations were in traA, suggesting that specific alterations in the amino-acid sequence of the pilin subunit protein were responsible for the altered pilus properties. Complementation between the Flac traA mutants and the derepressed plasmid R100-1 restored phage sensitivity in some cases, suggesting that the incorporation of both mutant and R100-1 subunits into the pilus structure may result in conformational changes which increase the capacity of the pilus to interact with phages.     

Effect of the R plasmids of Salmonella typhimurium strains of various origins on salmonella virulence

Antibiotic-multiresistant S. typhimurium strains, phagovars 20 and 25, of different origin (isolated in hospital infections transferred through everyday contacts and by alimentary route, as well as from samples taken from open water basins and sewage) have been found to carry both transmissible and nontransmissible R plasmids. The frequency of the transfer of R plasmids to Escherichia coli K-12 C 600 rif varies within 0.6 X 10(-5) and 0.7 X 10(-3) per donor cell. The direct transfer of plasmids to S. typhimurium has been mostly realized only in the presence of the mobilizing plasmid, the transfer frequency being 1.0 X 10(-7) to 2.2 X 10(-4) per donor cell. S. typhimurium transconjugates show decreased virulence in both enteral and intraperitoneal infection of mice.     

Accumulation of 3-(N-morpholino)propanesulfonate by osmotically stressed Escherichia coli K-12.

We found that exogenous morpholinopropanesulfonate (MOPS) is concentrated approximately fivefold in the free volume of the cytoplasm of Escherichia coli K-12 (strain MG1665) when grown at high osmolarity (1.1 OsM) in two different media containing 40 mM MOPS. MOPS was not accumulated by E. coli grown in low-osmolarity MOPS-buffered medium or in 1.1 OsM MOPS-buffered medium containing the osmoprotectant glycine betaine. Salmonella typhimurium LT2 did not accumulate MOPS under any condition examined. We infer that accumulation of MOPS by E. coli K-12 is not due to passive equilibration but rather to transport, possibly involving an as yet uncharacterized porter not present in S. typhimurium. Glutamate and MOPS were the only anionic osmolytes we observed by 13C nuclear magnetic resonance in E. coli K-12 grown in MOPS-buffered medium. The increase in positive charge accompanying the increase in the steady-state amount of K+ in cells shifted from low to high external osmolarity appeared to be compensated for by changes in the amounts of putrescine, glutamate, and MOPS. MOPS is not an osmoprotectant, because its accumulation did not increase cell growth rate.