Gene dosage effects of the structural gene for a lipoprotein of the Escherichia coli outer membrane.

The gene dosage effects of the structural gene (lpp) for the lipoprotein of the Escherichia coli outer membrane were examined. A novel F-prime factor containing the lpp gene was constructed. The amount of the free-form lipoprotein in the merodiploid strain carrying the F-prime factor was found to be about two times as great as that in the corresponding haploid strain. On the other hand, the amount of the bound-form lipoprotein, which is vovalently linked to the peptidoglycan, was not significantly different in the merodiploid strain as compared with the corresponding haploid strain. The present results suggest that the lpp gene is expressed constitutively in contrast to another major protein of the E. coli outer membrane, tolG protein (protein II, D. B. Datta et al., J. Bacteriol. 128:834-841, 1976). The F-prime factor isolated may include a portion of the E. coli chromosome (located between 33 and 36 min on the genetic map) that is not covered by any other F-prime factor.     

pryB mutations as suppressors of arginine auxotrophy in Salmonella typhimurium.

Salmonella typhimurium strains which produce high constitutive levels of aspartate transcarbamoylase due to the pyrH700 mutation were found to grow more slowly in minimal medium than pyrH+ controls. The addition of arginine or citrulline but not ornithine restored normal growth rates. This requirement for arginine was completely suppressed by pyrB mutations and partially suppressed by pyrC and pyrD mutations. No suppression was observed with mutants at the pyrF locus. Introduction of leaky mutation argI2002 resulted in a more extreme arginine requirement and accentuated suppression by pyrB mutations. Suppression by the pyrC and pyrD mutations was reduced as a result of the incorporation of the leaky argI2002 allele. These results indicate that in pyrH700 strains carbamoyl phosphate is preferentially directed toward the formation of intermediates in the pyrimidine biosynthetic pathway. Arginine auxotrophy results from the reduced availability of carbamoyl phosphate for the biosynthesis of arginine. Suppression of this arginine dependence for growth is used as a convenient positive selection technique for pyrB mutations.     

Electron microscope heteroduplex studies of sequence relations among plasmids of Escherichia coli: structure of F100, F152, and F8 and mapping of the Escherichia coli chromosomal region fep-supE-gal-attlambda-uvrB.

The genetic and physical structures of commonly used F-prime factors carrying the galactose region of the Escherichia coli chromosome were analyzed. Deletions in the chromosomal DNA sequences in the F-prime factors were found to be frequent events. A genetic method was developed to reconstruct the original F-prime factors from deletion variants. Heteroduplex analysis of the reconstructed F-prime factors confirmed the derivation of the F-prime factors F100 and F152, from the same Hfr, and finally determined the normal E. coli chromosomal sequence in the region between fep and uvrB, containing about 5 min in genetic units and about 246.5 in kilobase units (kb). This sequence could be connected with the DNA sequences of the lac-purE region, which had been physically determined previously. Together they constituted a total of 528.6 kb. From these combined sequences, the distance from lacPO to galK was calculated to be 412.9 kb, which corresponds to 8.8 min in genetic units.     

Merodiploidy in ESCHERICHIA COLI-SALMONELLA TYPHIMURIUM Crosses: The Role of Unequal Recombination between Ribosomal RNA Genes

Previous workers have shown that intergeneric crosses between Salmonella typhimurium and Escherichia coli produce a high proportion of merodiploid recombinants among the viable progeny. We have examined the unequal cross-over event that was responsible for a number of intergeneric merodiploids. The merodiploids that we studied were all heterozygous for the metB-argH interval and were the products of intergeneric conjugal crosses. We found that when the S. typhimurium donor had its transfer origin closely linked to metB and argH, all recombinants examined were merodiploid, and they generally arose as F-prime factors. Many of these F-prime factors had been created by recombination between flanking rrn genes in the donor. When the S. typhimurium Hfr transfer origin was more distant from the selected markers, quite different results were obtained. Depending on the donor, 19-47% of the recombinants that acquired the donor argH+ or metB+ genes were merodiploid for these loci, but none of the recombinants were F-prime. A majority of the merodiploids had a novel (nonparental) rrn gene, indicating that unequal recombination between nonidentical rrn genes was a prevalent mechanism for establishing the merodiploidy. Both tandem and nontandem duplications were found. Some of the merodiploids duplicated E. coli genes in addition to acquiring S. typhimurium genes. Some merodiploids contained the oriC region from each parent. Of a total of 118 intergeneric merodiploids characterized from all donors, 48 different genotypes were observed, and 38 of the 48 had one or more nonparental rrn operons.     

Expression of a mutation affecting F incompatibility in the integrated but not the autonomous state of F.

Previously we have described a mutant Hfr strain in which incompatibility between the integrated F factor and an autonomous F-prime (F') factor was abolished. The mutation (inc) was located in the integrated F factor. F-prime factors isolated from the mutant Hfr strain have the same incompatibility behavior as those isolated from normal Hfr strains. Reintegration of these F' factors into the chromosome restores the Inc- phenotype characteristic of the mutant Hfr. The inc mutation thus affects incompatibility between integrated F and autonomous F(Fi-Fa incompatibility) but not incompatibility between two autonomous F factors (Fa-Fa incompatibility). The implications of this finding for the mechanism of plasmid incompatibility are discussed.     

Activity of Chi Recombinational Hotspots in SALMONELLA TYPHIMURIUM

Chi sites have previously been shown to stimulate homologous recombination by the Escherichia coli RecBC pathway. To test the activity of Chi in another organism, bacteriophage lambda crosses were carried out in Salmonella typhimurium strains bearing the E. coli lambda receptor protein. Chi is active in these crosses in S. typhimurium, but is less active than in the same crosses carried out in E. coli. The lower Chi activity in S. typhimurium appears to be intrinsic to the S. typhimurium RecBC enzyme, since the Chi activity in E. coli-S. typhimurium hybrids depends on the species of origin of their RecBC enzyme. For these studies we constructed and F' factor and a pBR322-derived plasmid carrying the thyA+ recC+ recB+ argA+ region of the S. typhimurium chromosome.     

Loss of Colicinogeny in Escherichia coli Strains Infected by Certain Resistance Factors

The original observation that in wild-type colicinogenic Escherichia coli strains the introduction of some R factors abolish their colicin production was studied in certain col(+) strains bearing well-defined col factors. Two resistance (R) factors were used and introduced by conjugation in these strains, namely the 222 factor of Watanabe and a Salmonella typhimurium ST factor (coding for resistance to streptomycin and tetracycline only). The introduction of above mentioned R factors abolished the colicin production of col(+) strains most probably by elimination of col factors. All col factors, however, were not equally susceptible to elimination by the R factors tested, since colicin production in ML strains was abolished by infection by the 222 factor but not by the R factor of S. typhimurium ST, which is able to eliminate other col factors.     

Physical map of the Salmonella typhimurium histidine transport operon: correlation with the genetic map.

A detailed restriction map of a 12.4-kilobase EcoRI fragment of Salmonella typhimurium deoxyribonucleic acid (DNA) containing the entire histidine transport operon and the argT gene is presented. Subclones of specific regions of the transport operon of S. typhimurium were constructed in plasmid vectors. An accurate correlation between the restriction map and the location of genetically defined deletions was obtained by hybridizing restriction digests of chromosomal DNA from strains carrying each deletion with cloned transport operon DNA as a probe. These data were used to position the histidine transport genes on the cloned 12.4-kilobase fragment of DNA.     

Phylogeny and molecular identification of vibrios on the basis of multilocus sequence analysis

We analyzed the usefulness of rpoA, recA, and pyrH gene sequences for the identification of vibrios. We sequenced fragments of these loci from a collection of 208 representative strains, including 192 well-documented Vibrionaceae strains and 16 presumptive Vibrio isolates associated with coral bleaching. In order to determine the intraspecies variation among the three loci, we included several representative strains per species. The phylogenetic trees constructed with the different genetic loci were roughly in agreement with former polyphasic taxonomic studies, including the 16S rRNA-based phylogeny of vibrios. The families Vibrionaceae, Photobacteriaceae, Enterovibrionaceae, and Salinivibrionaceae were all differentiated on the basis of each genetic locus. Each species clearly formed separated clusters with at least 98, 94, and 94% rpoA, recA, and pyrH gene sequence similarity, respectively. The genus Vibrio was heterogeneous and polyphyletic, with Vibrio fischeri, V. logei, and V. wodanis grouping closer to the Photobacterium genus. V. halioticoli-, V. harveyi-, V. splendidus-, and V. tubiashii-related species formed groups within the genus Vibrio. Overall, the three genetic loci were more discriminatory among species than were 16S rRNA sequences. In some cases, e.g., within the V. splendidus and V. tubiashii group, rpoA gene sequences were slightly less discriminatory than recA and pyrH sequences. In these cases, the combination of several loci will yield the most robust identification. We can conclude that strains of the same species will have at least 98, 94, and 94% rpoA, recA, and pyrH gene sequence similarity, respectively.     

pyrR identical to pyrH in Salmonella typhimurium: control of expression of the pyr genes.

Mutants of Salmonella typhimurium showing constitutive synthesis of the pyrimidine biosynthetic enzymes coded for by the pyrA-F genes (G. A. O'Donavan and J. C. Gerhart, 1972) have been reinvestigated. The high rate of expression of the pyrB-F genes in these mutants as well as their pyrimidine excretion is shown to be due to mutations in the gene pyrH encoding uridine 5'-monophosphate kinase. Thus, the term pyrR used for these mutants should be replaced by the designation pyrH.     

Evidence of abortive recombination in ruv mutants of Escherichia coli K12

Summary Genetic recombination in Escherichia coli was investigated by measuring the effect of mutations in ruv and rec genes on F-prime transfer and mobilization of nonconjugative plasmids. Mutation of ruv was found to reduce the recovery of F-prime transconjugants in crosses with recB recC sbcA strains by about 30-fold and with recB recC sbcB sbcC strains by more than 300-fold. Conjugative plasmids lacking any significant homology with the chromosome were transferred normally to these ruv mutants. Mobilization of the plasmid cloning vectors pHSG415, pBR322, pACYC184 and pUC18 were reduced by 20- to 100-fold in crosses with ruv rec ⁺ sbc ⁺ strains, depending on the plasmid used. Recombinant plasmids carrying ruv ⁺ were transferred efficiently. With both F-prime transfer and F-prime cointegrate mobilization, the effect of ruv was suppressed by inactivating recA. It is proposed that the failure to recover transconjugants in ruv recA ⁺strains is due to abortive recombination and that the ruv genes define activities which function late in recombination to help convert recombination intermediates into viable products.     

Formation of F''TRP Plasmids in ESCHERICHIA COLI K12

From strains carrying two different F-prime factors, we recovered F' derivatives that acquired the trp chromosomal region. These F'trp plasmids can be isolated at a frequency of 10^-5 to 10^-6. They were characterized genetically by looking at the size of the trp segment they acquired and at the location of that segment in the parental F' plasmid. Results are discussed in relationship to possible transposition mechanisms.     

Infection of Salmonella typhimurium with coliphage Mu d1 (Apr lac): construction of pyr::lac gene fusions.

A procedure was developed for introducing the coliphage Mu d1 (Apr lac) into Salmonella typhimurium in order to construct gene fusions that place the structural genes of the lac operon under the control of the promoter-regulatory region of other genes. To introduce Mu d1 from Escherichia coli K-12 into S. typhimurium, which is normally not a host for Mu, we first constructed an E. coli double lysogen carrying the defective Mu d1 phage and a Mu-P1 hybrid helper phage (MuhP1) that confers the P1 host range. A lysate prepared from this strain was used to infect a P1-sensitive (i.e., galE), restriction-deficient, modification-proficient strain of S. typhimurium, and a double lysogen carrying Mu d1 and MuhP1 was isolated. Induction of the latter strain produced lysates capable of infecting and generating gene fusions in P1-sensitive strains of S. typhimurium. In this paper we describe the construction of pyr::lac fusions by this technique.     

Superinfection with R Factors by Transduction in Escherichia coli and Salmonella typhimurium

Superinfection immunity is found in the conjugal transfer of R factors between two fi(+) R factors and between two fi(-) R factors (fi = fertility inhibition), as we reported previously. In contrast, no reduction in the frequencies of transduction of an fi(+) R factor 222 was caused by the presence of fi(+) R factors in the recipients in transduction systems with phage P1kc in Escherichia coli K-12 and with phage P22 in Salmonella typhimurium LT-2. The absence of superinfection immunity in transduction may be due to the difference in the route of entry of the R factor. The frequencies of transduction of an fi(+) R factor were reduced, although slightly, by the presence of fi(-) R factors in the recipients. This reduction is probably due to host-controlled restriction of the entering fi(+) R factor by the fi(-) R factors in the recipients, since transduction of an fi(+) R factor by the transducing phage propagated on the strain carrying both fi(+) and fi(-) R factors was not reduced by the presence of homologous fi(-) R factors in the recipients. The fi(+) R factor 222, when transduced to the recipient strains carrying other R factors, recombined genetically at high frequencies with these resident R factors, regardless of their fi type.     

Identification of a membrane protein associated with expression of the surface exclusion region of the F transfer operon.

Membrane preparations from radioactively labeled male and female strains of Escherichia coli K-12 were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. An intensely labeled band corresponding to a protein of molecular weight of 24,000 was readily apparent in preparations from Hfr and F-prime strains but not in those from female strains. When preparations from a series of Hfr strains containing transfer operon deletions were examined, presence of the band was found to be associated with retention of the region of the F transfer operon between ilzA and traD. Thus, the band ("protein S") appears to be the product of an F tra operon activity corresponding to traS (the gene for surface or entry exclusion), or an unknown gene in its vicinity. As predicted, protein S was subject to Fin+ control; only a faint band was detectable if the repressed plasmid R100 was also present in the F lac strain. A 24,000-dalton protein was also found in membrane preparations from strains carrying the derepressed plasmids R100-1 and R1-19 but not in those from strains carrying the repressed plasmids R100 or R1. Thus, the appearance of protein S in the membrane may be a general phenomenon resulting from transfer operon expression of F-like plasmids.     

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.     

Genes affecting coliphage BF23 and E colicin sensitivity in Salmonella typhimurium.

Rough strains of Salmonella typhimurium were sensitive to coliphage BF23. Spontaneous mutants resistant to BF23 (bfe) were isolated, and the trait was mapped using phage P1. The bfe gene in S. typhimurium was located between argF (66% co-transducible) and rif (61% co-transducible). The BF23-sensitive S. typhimurium strains were not sensitive to the E colicins. Cells of these rough strains absorbed colicin, as measured by loss of E2 or E3 killing units from colicin solutions and by specific adsorption of 125I-colicin E2 to bfe+ cells. Sensitivity to colicins E1, E2, and E3 was observed in a S. typhimurium strain carrying the F'8 gal+ episome. This episome complemented the tolB mutation of Escherichia coli. We conclude that the bfe+ protein satisfies requirements for adsorption of both phage BF23 and the E colicins. In addition, expression of a gene from E. coli, possibly tolB, is necessary for efficient E colicin killing of S. typhimurium.     

Spontaneous, ultraviolet and ionizing radiation mutagenesis in two auxotrophic strains of Salmonella typhimurium carrying an R plasmid.

Ultraviolet-induced, gamma-induced and spontaneous mutation yields were studied in two different auxotrophic strains of Salmonella typhimurium in the presence and absence of the UV-protecting drug resistance transfer factor R-Utrecht. One strain, carrying the hisC527 (amber) mutation, showed significantly increased spontaneous, UV- and gamma-induced mutability in the presence of the R-Utrecht plasmid. The other strain, carrying the trpD1 mutation (thought to be a missense mutation), also showed significantly increased UV mutability in the presence of the R-Utrecht plasmid. The other strain, carrying the trpD1 mutation (thought to be a missense mutation), also showed significantly increased UV mutability in the presence of the R factor, but appeared to show no significant increase in spontaneous mutability and only a very slight increase in gamma-mutability when carrying the R factor. These results demonstrate that the R-Utrecht plasmid, known to enhance UV-induced mutation yields in S. typhimurium, can also significantly enhance both spontaneous and gamma-induced mutation yields in this species. The latter effects are not so discernible with all markers, however, as shown by the results with strains carrying the trpD1 mutation. Enhancement of spontaneous mutability thus appears to be correlated with enhancement of gamma-mutability rather than UV mutability.     

Two-step cloning and expression in Escherichia coli of the DNA restriction-modification system StyLTI of Salmonella typhimurium.

The StyLTI restriction-modification system is common to most strains of the genus Salmonella, including Salmonella typhimurium. We report here the two-step cloning of the genes controlling the StyLTI system. The StyLTI methylase gene (mod) was cloned first. Then, the companion endonuclease gene (res) was introduced on a compatible vector. A strain of S. typhimurium sensitive to the coliphage lambda was constructed and used to select self-modifying recombinant phages from a Res- Mod+ S. typhimurium genomic library in the lambda EMBL4 cloning vector. The methylase gene of one of these phages was then subcloned in pBR328 and transferred into Escherichia coli. In the second step, the closely linked endonuclease and methylase genes were cloned together on a single DNA fragment inserted in pACYC184 and introduced into the Mod+ E. coli strain obtained in the first step. Attempts to transform Mod- E. coli or S. typhimurium strains with this Res+ Mod+ plasmid were unsuccessful, whereas transformation of Mod+ strains occurred at a normal frequency. This can be understood if the introduction of the StyLTI genes into naive hosts is lethal because of degradation of host DNA by restriction activity; in contrast to most restriction-modification systems, StyLTI could not be transferred into naive hosts without killing them. In addition, it was found that strains containing only the res gene are viable and lack restriction activity in the absence of the companion mod gene. This suggests that expression of the StyLTI endonuclease activity requires at least one polypeptide involved in the methylation activity, as is the case for types I and III restriction-modification systems but not for type II systems.