Genetic and physiological analysis of an envB spherelike mutant of Escherichia coli K-12 and characterization of its transductants.

The envB1 mutation mediating a distorted cell morphology of Escherichia coliK-12 was cotransducible with strA, aroE, aspB, and argG. The mapping data is consistent with a gene location for envB around 62.5 min. In partial diploids envB1 was recessive to its wild-type allele. The original envB mutant contained a second mutation in a locus denoted sloB close to strA. The following gene order is suggested: sloB-strA-aroE-envB-aspB-argG. The sloB1 mutation caused a marked reduction in the growth rate of both envB and envB+ strains. Moreover, this mutation in the presence of envB1 appears to increase the ratio between deoxyribonucleic acid and protein in cells growing in rich medium. The phenotypic properties of envB1, sloB+ and envB+ transductants were characterized. Cells with envB1, sloB+ genotype were hypersensitive to several penicillins including the beta-lactam compound, amidino penicillin. Penicillin hypersensitivity could not be explained by increased outer membrane penetrability. The original envB mutant (envB1,SLOB1), as well as envB1, sloB1 or envB+, SLOB1 transductants were resistant to amidino penicillin. Resistance was explained by the slow growth rate mediated by the sloB1 mutation. The similarity between envB cells and wild-type cells treated with sublethal concentrations of amidino penicillin was emphasized.     

High-level resistance to mecillinam produced by inactivation of soluble lytic transglycosylase in Salmonella enterica serovar Typhimurium

By screening for high-level mecillinam resistant derivatives of a low-level resistant strain (cysB403 galE1922 relA21::Tn10) of Salmonella enterica serovar Typhimurium, a MudJ insertion in the gene for soluble lytic transglycosylase (slt) was isolated. This insertion (slt-1::MudJ) increased the resistance to mecillinam of cysB and cysE strains (MIC: about 20-40 microg mL(-1)) to a strikingly high level (MIC: 160 microg mL(-1)). As in Escherichia coli K-12, the slt mutation slightly increased the sensitivity of the wild type and of several strains that carried mutations that did not increase mecillinam resistance. All the strains acquired a spherical cell shape when treated with mecillinam. The effect of slt-1::MudJ was limited to mecillinam, the response to several other antibiotics remaining unaltered by the insertion. The results presented in this paper demonstrate that soluble lytic transglycosylase performs an important role in the response to mecillinam, which only becomes evident when failure of CysB/CysE function causes medium-level resistance. The results also suggest that soluble lytic transglycosylase interacts with, and is partially inhibited by normal lipopolysaccharide.     

Evidence for a chromosome-borne resistance transposon (Tn916) in Streptococcus faecalis that is capable of "conjugal" transfer in the absence of a conjugative plasmid

Streptococcus faecalis strain DS16 harbors the conjugative hemolysin-bacteriocin plasmid pAD1 (35 megadaltons) and the nonconjugative R-plasmid pAD2 determining resistance to streptomycin, kanamycin, and erythromycin; a tetracycline resistance (Tetr) determinant is located on the chromosome. When strain DS16 was mated (on membrane filters) with the plasmid-free strain JH2-2, Tetr transconjugants could be obtained at a frequency of about 10(-6) per recipient. Analyses of transconjugants showed that some contained the Tetr determinant linked to pAD1. Subsequent studies showed that the Tetr determinant was located on a 10-megaldalton transposon, designated Tn916, which could insert into two hemolysin plasmids: pAM gamma 1 and pOB1. In addition, derivatives of DS16 devoid of pAD1 were capable of transferring Tetr to recipient strains. Transconjugants (plasmid-free) from such matings could subsequently act as donors in the transfer of Tetr. Both transposition and transfer were found to be rec independent.     

Nonadaptive mutations occur on the F' episome during adaptive mutation conditions in Escherichia coli.

One of the most studied examples of adaptive mutation is a strain of Escherichia coli, FC40, that cannot utilize lactose (Lac-) but that readily reverts to lactose utilization (Lac+) when lactose is its sole carbon source. Adaptive reversion to Lac+ occurs at a high rate when the Lac- allele is on an F' episome and conjugal functions are expressed. It was previously shown that nonselected mutations on the chromosome did not appear in the Lac- population while episomal Lac+ mutations accumulated, but it remained possible that nonselected mutations might occur on the episome. To investigate this possibility, a second mutational target was created on the Lac- episome by mutation of a Tn1O element, which encodes tetracycline resistance (Tetr), to tetracycline sensitivity (Tets). Reversion rates to Tetr during normal growth and during lactose selection were measured. The results show that nonselected Tetr mutations do accumulate in Lac- cells when those cells are under selection to become Lac+. Thus, reversion to Lac+ in FC40 does not appear to be adaptive in the narrow sense of the word. In addition, the results suggest that during lactose selection, both Lac+ and Tetr mutations are created or preserved by the same recombination-dependent mechanism.     

Transductional Instability of Tn5-Induced Mutations: Generalized and Specialized Transduction of Tn5 by Bacteriophage P1

Generalized transduction is commonly used to move transposon-induced mutations among bacterial strains by selecting for inheritance of a transposon-encoded resistance determinant. Although complete cotransduction of the resistance determinant and the chromosomal mutation might be expected, it is often found that when Tn5(Kan) insertion mutations are transduced by bacteriophage P1 most of the nonmutant kanamycin-resistant transductants area due to specialized transduction of Tn5. Such P1::Tn5 specialized transducing phage are not found when a mutant Tn5 element lacking a functional transposase is employed.     

Construction of delta aroA his delta pur strains of Salmonella typhi.

Salmonella typhi strains with two deletion mutations, each causing an attenuating auxotrophy, have been constructed from strains Ty2 and CDC 10-80 for possible use as oral-route live vaccines. An aroA(serC)::Tn10 transposon insertion was first transduced from a Salmonella typhimurium donor into each wild-type S. typhi strain. Transductants of the Aro- SerC- phenotype were treated with transducing phage grown on an S. typhimurium strain with an extensive deletion at aroA; selection for SerC+ yielded transductants, some of which were delta aroA. A his mutation was next inserted into a delta aroA strain in each line by two steps of transduction. Two deletions affecting de novo purine biosynthesis were used as second attenuating mutations: delta purHD343, causing a requirement for hypoxanthine (or any other purine) and thiamine, and delta purA155, causing an adenine requirement. The purHD343 deletion was introduced into the delta aroA his derivatives of each strain by cotransduction with purH::Tn10, and the purA155 deletion was introduced into the CDC 10-80 delta aroA his derivative by cotransduction with an adjacent silent Tn10 insertion by selection for tetracycline resistance. Tetracycline-sensitive mutants of each of the three delta aroA his delta pur strains were isolated by selection for resistance to fusaric acid. The tetracycline-sensitive derivative of the CDC 10-80 delta aroA his delta purA155 strain, designated 541Ty, and its Vi-negative mutant, 543Ty, constitute the candidate oral-route live-vaccine strains used in a recent volunteer trail (M. M. Levine, D. Herrington, J. R. Murphy, J. G. Morris, G. Losonsky, B. Tall, A. A. Lindberg, S. Stevenson, S. Baqar, M. F. Edwards, and B. A. D. Stocker, J. Clin. Invest. 79:885-902, 1987). Tetracycline-sensitive mutants of the delta aroA his delta purHD derivative of strains Ty2 and CDC 10-80 may also be appropriate as live vaccines but have not been tested as such.     

Genes aroA and serC of Salmonella typhimurium constitute an operon.

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.     

Two conjugation systems associated with Streptococcus faecalis plasmid pCF10: identification of a conjugative transposon that transfers between S. faecalis and Bacillus subtilis.

The tetracycline resistance plasmid pCF10 (58 kilobases [kb]) of Streptococcus faecalis possesses two separate conjugation systems. A 25-kb region of the plasmid (designated TRA) was shown previously to determine pheromone response and conjugation functions required for transfer of pCF10 between S. faecalis cells (P. J. Christie and G. M. Dunny, Plasmid 15:230-241, 1986). When S. faecalis cells were mixed with Bacillus subtilis in broth, tetracycline resistance was transferred from S. faecalis. The tetracycline-resistant B. subtilis cells contained a 16-kb region of pCF10 (distinct from TRA) that carried the tetracycline resistance determinant (Tetr). This Tetr element was found to transfer between S. faecalis and B. subtilis strains in the absence of plasmids. Genetic and molecular techniques were used to establish locations of the element at several different sites on the B. subtilis chromosome. The Tetr element could be transferred in filter matings from B. subtilis to S. faecalis strains and between recombination-proficient and -deficient S. faecalis strains in the absence of any plasmid DNA. The transfer required direct cell-to-cell contact and was not inhibited by DNase. The Tetr element was shown to transpose from the S. faecalis chromosome to various locations within the hemolysin plasmid pAD1. Together, the data indicate that the Tetr element, termed transposon Tn925, is very similar to the conjugative transposon Tn916 in both structure and function. A derivative of Tn925, containing transposon Tn917 inserted into a site approximately 3 kb from one end, exhibited elevated transfer frequencies and may provide a useful means for delivering Tn917 by conjugation into various gram-positive species.     

Rho and Ribosome Mutation Interaction: Lethality of rho-15 in rpsL or rpsE Strains, and rho-15 Methionine Auxotrophy in rps+ Strains of ESCHERICHIA COLI

The phenotype of Escherichia coli K-12 carrying rho-15 in the genetic background DW319 ilv lacZ::IS1 is described. Seventy-eight percent (70/90) of Ilv+ transductants acquired the following phenotype: temperature-sensitive growth on minimal salts medium, Ts+ growth on complex medium and suppression of the lac polar mutation. At 42 degrees on minimal medium, the rho-15 transductants were cross-fed by a substance diffusing from Rho+ transductants or controls. The requirement for this substance was satisfied by methionine or cystathionine, but not by any other single amino acid or combination of amino acids, by spermidine, or by mono- or divalent cationic salts.--Transduction of rho-15 into four other Ilv- recipients revealed two phenotypic patterns. Recipients with rpsL or rpsE ribosomes yielded rho-15 transductants that were Ts on all media, or Ts on minimal medium whether or not methionine was present. The effect of the ribosome on expression of rho-15 was confirmed by transduction of appropriate rps alleles into DW319, followed by co-transduction of rho-15 with Ilv+. The growth rate of double rho-15 rpsL or rho-15 rpsE strains was severely reduced at 42 degrees in comparison with strains carrying any of these single mutations. Models for rho and ribosome interaction are presented.     

A New Fungal Metabolite and Root Growth-stimulating Activity of Its Methyl Ester

Escherichia coli rodA mutant AOS151 grows as round cells at 30 and 42°C (H. Matsuzawa, K. Hayakawa, T. Sato, and K. Imahori, J. Bacteriol., 115, 436–442 (1973)). The mutant was found to be resistant to mecillinam at both temperatures. lip⁺ transductants were prepared by Pl phage transduction via strain AOS151, the cotransduction frequency of round morphology (Rod^?) at 42°C with the lip gene being about 90%. At 42°C all 54 Rod^? transductants tested were resistant to mecillinam. At 30°C all but two of these Rod^? (at 42°C)-type transductants were rod-shaped, and all were sensitive to mecillinam; the two strains grew as ovoid cells. The original rodA mutant AOS151 probably involves an additional mutation(s), that expresses the round cell shape at lower temperature, whereas the rodA51 mutation alone seems to result in temperature-sensitive expression of round cell morphology and mecillinam resistance. rodA mutant cells cultured at either 30 or 42°C had wild-type penicillin-binding protein 2, judging from penicillin-binding activity, electrophoretic mobility, and thermosensitivity.     

Is the IS1-flanked r-determinant of the R plasmid NR1 a transposon?

The 23 kilobase multiple drug resistance r-determinant (r-det) of the R plasmid NR1 is an IS1-mediated transposon, Tn2671. Drug-resistant Escherichia coli transductants isolated after infection with bacteriophage P1::Tn2671 derivatives carry the intact r-det in their chromosomes. Independently isolated transductants carry the r-det at different locations on the chromosome. From the E. coli chromosome, Tn2671 can transpose to various locations on the phage P7 genome. Throughout these processes, r-det is maintained as a stable unit. Various possible molecular mechanisms, which all might contribute with characteristic frequencies to the transposition of Tn2671, are discussed. The results presented are relevant to the understanding of mechanisms for a wide spreading of drug resistance genes.     

Facilitated transfer of IncP beta R751 derivatives from the chromosome of Bacteroides uniformis to Escherichia coli recipients by a conjugative Bacteroides tetracycline resistance element.

The broad-host-range IncP beta plasmid R751 can mobilize itself from Escherichia coli to Bacteroides spp, but it is not maintained in Bacteroides spp. If R751 carries the Bacteroides transposon Tn4351, it can be integrated into the Bacteroides chromosome. Previously we showed that R751, integrated in the chromosome of Bacteroides uniformis, cannot mobilize itself out of B. uniformis into E. coli or isogenic B. uniformis strains. In this report, we showed that if the Bacteroides conjugative tetracycline resistance element Tcr ERL was coresident with the R751 insertion in B. uniformis, derivatives of R751 were transferred to E. coli, where they were recovered as plasmids. The most common derivatives were R751::Tn4351 and R751::IS4351, but some strains transferred R751 derivatives, containing additional DNA segments ranging in size from 10 to 23 kilobases. These DNA inserts cross-hybridized with chromosomal DNA from B. uniformis which did not carry the Tcr ERL element. Therefore, the inserts appeared to be segments of the wild-type B. uniformis chromosome and were not associated with the Tcr ERL element. The transfer of integrated R751 from B. uniformis was independent of the RecA phenotype of the E. coli recipients and did not appear to be due to transfer of B. uniformis chromosomal DNA, followed by RecA-dependent recombination between homologous IS4351 sequences to form the resultant R751 plasmid derivatives. Consistent with this, no transfer of Tn4351 (associated with the cointegrated R751) from B. uniformis donors to isogenic B. uniformis recipients was detected (< 10(-8)). Our data support the hypothesis that R751 excises from the B. uniformis chromosome by recombination involving flanking Tn4351 or IS4351 sequences and forms nonreplicating circles. The mobilization of these circular forms out of B. uniformis to E.coli is then facilitated by the Tcr ERL element.     

Transformation of a conditionally peptidoglycan-deficient mutant of Staphylococcus aureus with plasmid DNA.

A simple and reliable method for polyethylene glycol-induced plasmid transformation of a temperature-sensitive peptidoglycan-deficient mutant of Staphylococcus aureus is described. The procedure uses strains carrying the tofA372 mutation grown under conditions that yield osmotically fragile cells capable of efficient wall regeneration. The peptidoglycan-deficient cells were transformed with plasmids pE194 and pI258 at frequencies comparable with those obtained with protoplasts prepared with lysostaphin treatment. A readily portable tofA372 mutation was constructed by isolating an insertion of the erythromycin resistance transposon Tn551 adjacent to tofA372. tofA372 was shown by protoplast fusion and transformation analyses to be in the gene order hly-421-omega [Chr::Tn551]1059-tofA372-uraB232-omega [Chr::Tn916]1101-thrB106 on the chromosome of S. aureus NCTC 8325.     

Introduction of the Streptococcus faecalis transposon Tn916 into Bacillus thuringiensis subsp. israelensis

The conjugative Streptococcus faecalis transposon Tn916 was introduced into Bacillus thuringiensis subsp. israelensis by filter matings with S. faecalis. B. thuringiensis transconjugants resistant to tetracycline (Tetr) were detected at a frequency of approximately 7.0 X 10(-7) per recipient cell during filter matings, whereas transfer of Tn916 was not observed in broth matings. The Tetr phenotype in subsp. israelensis was stable in the absence of antibiotic selection. Southern hybridization analysis revealed that Tn916 had inserted into several different sites on the B. thuringiensis subsp. israelensis chromosome but insertion into plasmid DNA was not observed. Movement of Tn916 was demonstrated when Tetr B. thuringiensis transconjugants were mated with isogenic recipients. Southern hybridizations, however, showed that the resulting Tetr isolates contained Tn916 junction fragments that were nearly identical to the donor, suggesting that this movement resulted from transfer of chromosomal DNA from donor to recipient or from a fusion of mating cells, rather than conjugative transposition of the Tn element.     

Genetic recombination of bacterial plasmid DNA: effect of RecF pathway mutations on plasmid recombination in Escherichia coli.

Tn5 insertion mutations in the recN gene, and in what appears to be a new RecF pathway gene designated recO and mapping at approximately 55.4 min on the standard genetic map, were isolated by screening Tn5 insertion mutations that cotransduced with tyrA. The recO1504::Tn5 mutation decreased the frequency of recombination during Hfr-mediated crosses and increased the susceptibility to killing by UV irradiation and mitomycin C when present in a recB recC sbcB background, but only increased the sensitivity to killing by UV irradiation when present in an otherwise Rec+ background. The effects of these and other RecF pathway mutations on plasmid recombination were tested. Mutations in the recJ, recO, and ssb genes, when present in otherwise Rec+ E. coli strains, decreased the frequency of plasmid recombination, whereas the lexA3, recAo281, recN, and ruv mutations had no effect on plasmid recombination. Tn5 insertion mutations in the lexA gene increased the frequency of plasmid recombination. These data indicate that plasmid recombination events in wild-type Escherichia coli strains are catalyzed by a recombination pathway that is related to the RecF recombination pathway and that some component of this pathway besides the recA gene product is regulated by the lexA gene product.     

Transposon Tn10-like tetracycline resistance determinants in Haemophilus parainfluenzae.

Tetracycline resistance (Tcr) determinants from three different strains of Haemophilus parainfluenzae expressed 10-fold higher levels of resistance when mated into Escherichia coli. No plasmid was found in any of the E. coli recipients, even in matings in which a plasmid was identified in the donor Haemophilus sp. The Tcr determinant from Haemophilus sp. caused instability of resident plasmids in the recipient E. coli: all plasmids were lost within 30 generations in antibiotic-free media. However, by serial subculture in antibiotics, stable resident plasmids were obtained which carried the Tcr determinant from Haemophilus sp. and were transferable by conjugation and transformation among E. coli strains. All Haemophilus determinants hybridized with a probe for the Tcr determinant on Tn10, which bears inducible Tcr. However, Haemophilus determinants were constitutively resistant to tetracycline in the Haemophilus donors and in the E. coli recipients. This constitutive expression was recessive to wild-type Tn10 in the same cell, indicating that the constitutive phenotype resulted from the absence of an active repressor. Restrictive enzyme analysis of various E. coli plasmid derivatives bearing a Tcr determinant from Haemophilus sp. demonstrated that the inserted DNA was of similar size (8.95 to 9.35 kilobases), close to that of Tn10. Heteroduplex analysis and DNA:DNA hybridization confirmed that the Tcr determinant from Haemophilus sp. had greater than 90% homology with the Tn10 determinant, including the DNA sequence for the repressor.     

A collection of strains containing genetically linked alternating antibiotic resistance elements for genetic mapping of Escherichia coli.

We present a collection of 182 isogenic strains containing genetically linked antibiotic resistance elements located at approximately 1-min intervals around the Escherichia coli chromosome. At most positions both Tn10 (Tetr) and TN10kan (Kanr) elements are available, so that the collection contains a linked set of alternating antibiotic resistance markers. The map position of each insertion has been aligned to the E. coli genetic map as well as to the Kohara ordered clone bank. These strains are designed to be used in a rapid two-step mapping system in E. coli. In the first step, the mutation is localized to a 5- to 15-min region of the chromosome by Hfr mapping with a set of Hfr strains containing either Tn10 or Tn10kan elements located 20 min from their respective origins of transfer. In the second step, the mutation is localized to a 1-min region by P1 transduction, with a collection of isogenic insertion strains as donors. We discuss the uses of this collection of strains to map and eventually to clone a variety of mutations in E. coli.     

Utilization of a thermosensitive episome bearing transposon TN10 to isolate Hfr donor strains of Erwinia carotovora subsp. chrysanthemi

A thermosensitive episome bearing the transposon Tn10, F(Ts)::Tn10 Lac+, has been successfully transferred from Escherichia coli to several wild strains of the enterobacteria Erwinia carotovora subsp. chrysanthemi, which are pathogenic on Saintpaulia ionantha. In one of these strains, all of the characters controlled by this episome (Lac+, Tetr, Tra+) were expressed, and its replication was stopped at 40 degrees C and above. At 30 degrees C, the episome was easily transferred between strains derived from E. carotovora subsp. chrysanthemi 3937j and to E coli. Hfr donor strains were obtained from a F' strain of 3937j by selecting clones which grew at 40 degrees C on plates containing tetracycline. One of these strains, Hfrq, was examined in more detail: the characters Lac+ and Tetr were stabilized and did not segregate higher than its parental F' strain. The mating was most efficient at 37 degrees C on a membrane. Hfrq transferred its chromosome to recipient strains at high frequency and in a polarized fashion, as evidenced by the gradient of transfer frequencies, the kinetics of marker entry (in interrupted mating experiments), and the analysis of linkage between different markers. The chromosome of Hfrq was most probably transferred in the following sequence: origin...met...xyl...arg...ile...leu...thr...cys...pan...ura...gal...trp...his. ..pur... Moreover, this genetic transfer system proved to be efficient in strain construction.