Cloning and expression of two Streptococcus mutans glucosyltransferases in Escherichia coli K-12

Chromosomal DNA from Streptococcus mutans strain MFe28 (serotype h) was cloned in the bacteriophage vector lambda L47.1. Two classes of recombinants were found which expressed glucosyltransferase activity in phage plaques: (i) gtfS, which expressed a glucosyltransferase synthesizing a water-soluble, dextranase-sensitive glucan, and (ii) gtfI, which expressed a primer-dependent glucosyltransferase synthesizing an insoluble glucan.     

Novel intergenic repeats of Escherichia coli K-12

An online catalog of intergenic DNA repeat sequence elements is added to the EcoGene Escherichia coli K-12 genome sequence annotation and analysis project (bmb.med.miami.edu/EcoGene). A library of noncoding (intergenic) DNA sequences depleted of known intergenic repeat classes was searched for DNA sequence similarities to identify novel DNA repeat sequence classes.     

Expression of Treponema pallidum antigens in Escherichia coli K-12

A colony bank of recombinant plasmids harboring Treponema pallidum DNA inserts has been established in Escherichia coli K-12. By using an in situ immunoassay, we identified four E. coli clones that expressed T. pallidum antigens. Thus, recombinant DNA technology may provide powerful new tools for studying the pathogenesis of T. pallidum infection.     

Mutagenesis and DNA repair for alkylation damages in Escherichia coli K-12.

In this work we report on the isolation of an Escherichia coli K-12 mutation, which confers a high sensitivity to bacteria cells to mutagenesis by simple monofunctional alkylating agents. The mutation emerged spontaneously from a bacterial strain that already proved useful in various mutagenicity studies. By monitoring the influence of such a mutation on the frequency of induced mutation by ethylating (EMS, DES, ENU, ENNG) vs. methylating (MMS, DMS, MNU, MNNG) compounds, and on the in vivo repair capacity for different alkyl-DNA lesions (O6-alkG, N7-alkG, N3-meA), we conclude that the mutation should affect the gene (ogt) that encodes constitutive DNA repair alkyltransferase (ATase). Thus in the presence of ada, differences in mutagenicity were observed only with ethylating agents; the sensitization of cells to both the ethylating and methylating partners requiring, by contrast, the absence of the ada protein. These results support the reported in vitro substrate specificities for both ogt and ada ATases. The parental cells exhibited biphasic dose-response curves in accordance with the idea of low basal level saturation attributed to the uninducible ogt ATase. Deficient bacterial derivatives showed, by contrast, linear mutation induction responses. The in vivo removal of alkylated bases from DNA was measured in bacterial strains deficient in the excision repair pathway (delta uvrB) and unable to induce the adaptive response (ada::Tn10). The very low initial levels for O6-meG and O6-etG (1.1 and 0.2 molecules per cell, respectively) were readily repaired by the parental cells but remained unchanged in the hypermutable derivatives. This result suggests that in the absence of nucleotide excision repair and of the adaptive response, no alternative pathway, other than ogt, is available for the repair of the major mutagenic lesion, O6-alkG, at least during the first 4 hours after alkylation. Comparatively, no differences were found in the capacity to repair the major lethal adduct, N3-meA, in agreement with the fact that no effect on cell survival was detected. In conclusion, we propose that the biological significance of the ogt protein relies mainly on its ability to prevent mutagenesis by low levels of bulkier ethylation products (especially in the absence of uvr excision repair.(ABSTRACT TRUNCATED AT 400 WORDS)     

Competition between congenic Escherichia coli K-12 strains in vivo.

The ability of Escherichia coli to colonize the large bowels of animals is related to many factors inherent to the intestinal environment and the bacterium. The use of germfree mice eliminates the competition between E. coli and the other microflora and allows most E. coli strains to colonize. We found that E. coli K-12 strains differing in chromosomal antibiotic resistance could monoassociate in germfree mice in large numbers. However, when two or more strains were in competition with each other, we detected quantitative differences in the abilities of the strains to colonize. The order of colonizing ability was as follows: nalidixic acid resistance greater than streptomycin resistance greater than rifampin resistance. We also found that a nalidixic acid-resistant strain bearing plasmid pBR322 colonized less efficiently and at lower levels when in competition with the nalidixic acid-resistant strain. Studies of the membrane proteins of the various strains indicated that changes in membrane proteins occurred concomitantly with altered resistance to antimicrobial agents. These results suggest that chromosomally linked alterations in antimicrobial sensitivity may also reflect changes in membrane proteins and a decreased ability to colonize mammalian intestines in otherwise isogenic bacterial strains.     

Mutation frequency decline in MMS-treated Escherichia coli K-12 mutS strains

It has been shown that the decline in mutant frequency (MFD)(argE3_(ochre)Arg+) which occurs in MMS–treated and thentransiently starved AB1157 Escherichia coli K–12 cellsconcerns revertants which arose by supL suppressor formationin a process which is umuDC dependent. Here we have examinedwhether MMS–induced Arg⁺ revertants are susceptible todecline when bacteria are deficient in mismatch repair. We showthat there is an absence of MFD in MMS–treated Ml (mutS)and in EC2416 (mutS umuDC) cells defective in mismatch repairwhich is associated with a change in the spectrum of MMS–inducedmutations formed. In contrast to AB1157, transformation of Ml(mutS) bacteria with plasmids harbouring various combinationsof umuD(D')C genes does not enhance the level of MMS–inducedmutations but may influence the proportion of supL mutations.These supL mutations show MFD. Repair processes under MFD conditionswere confirmed by analysis of plasmid DNA isolated from MMS–treatedbacteria at different stages of their starvation and digestionwith Fpg protein. ¹To whom correspondence should be addressed. Tel: +48 658 4766; Fax: +48 3912 1623; Email: celina{at}ibbrain.ibb.waw.pl     

Immunological analysis of porin polymorphism in Escherichia coli B and K-12

Two sets of monoclonal antibodies (MoF type I and MoF type II) directed against the OmpF protein were used to analyze the immunological reactivity of the major outer membrane porins of E. coli B and K-12. All these antibodies present a specificity to the native OmpF protein. In addition, among the type II antibodies, MoF 18, 19 and 20 could recognize an epitope present on both monomeric and trimeric forms of the porin as demonstrated by immunoblotting analyses. The use of two different screening methods led to the isolation of two different sets of MoF, one specific for a native conformation accessible only on E. coli B strain and the second directed against epitopes present on OmpF of the two strains, B and K-12. These various responses are discussed in relation to the lipopolysaccharide binding to OmpF and with respect to the screening test used.     

Anaerobic growth does not support biofilm formation in Escherichia coli K-12

Association with a surface in a structure known as biofilm is the prevailing microbial lifestyle. Here we show the kinetics of biofilm formation of Escherichia coli W3110 in static cultures growing under aerobic or anaerobic conditions. Aerobically growing cells in LB medium started to produce detectable amounts of biofilm after 4 to 8 h, displaying maximal accumulation of formed biofilm at 24 h, corresponding to the onset of stationary phase. Then an abrupt reduction in the biomass of the biofilm was observed. This decrease was not prevented by external addition of fresh nutrients and coincided with the depletion of oxygen as measured by the enzymatic activity of the AdhE protein. No biofilm formation was detected in cultures grown anaerobically in LB or LB supplemented with nitrate, nitrite, DMSO or fumarate, even after 72 h of incubation, well inside the stationary phase, suggesting that under anaerobic growth conditions E. coli cannot form biofilms.     

Cosmid cloning of Rickettsia prowazekii antigens in Escherichia coli K-12.

Rickettsia prowazekii DNA was partially digested with Sau3A or HindIII, ligated with the cosmid vector pHC79, packaged in vitro, and transduced into Escherichia coli HB101. Cosmid cloning of Sau3A-digested rickettsial DNA yielded 1,288 ampicillin-resistant colonies; 798 cosmid clones resulted with HindIII-digested rickettsial DNA. Chimeric cosmid DNA was extracted from the latter gene bank, digested to completion with HindIII, and compared by agarose gel electrophoresis with a HindIII digest of rickettsial genomic DNA. The two digestion profiles were quite similar in their overall banding patterns, indicating that the clone bank was significantly representative of the rickettsial genome. When both clone banks were screened for expression of rickettsial antigens by enzyme-linked immunosorbent assay with goat anti-R. prowazekii serum, ca. 20% of the clones reacted positively. Two clones were randomly selected for more detailed analysis. Each contained a large chimeric plasmid (40.2 and 38.1 kilobases) which apparently yielded smaller deletion derivatives (13.6 and 12.6 kilobases) when transformed into an E. coli minicell strain. Each recombinant plasmid directed the synthesis of new protein species not observed in control minicells. One of the clones produced a 51,000-dalton protein in minicells, which comigrated with a protein reactive with anti-R. prowazekii serum. This protein was not present in negative controls. When antibodies to this protein were incubated with a Western blot of rickettsial total protein, they bound to a 52,000-dalton polypeptide. Hence, the cloned rickettsial gene product in E. coli corresponds to a protein of similar size in R. prowazekii. This study demonstrates the feasibility of cosmid cloning of rickettsial antigens in E. coli.     

Infection of Escherichia coli K-12 by bacteriophage φX-174

By several different biochemical tests, a previously described isolation procedure was shown to produce host-range mutants of φX-174 capable of infecting some strains of Escherichia coli K-12. One of these mutants (φXK-9) has a wide host range which includes E. coli C, B, and Shigella Y6R. By fragment mapping the site of the host-range mutation was shown to be in gene F.     

Genetic Transfer of Shigella flexneri Antigens to Escherichia coli K-12

The genes controlling synthesis of Shigella flexneri group- and type-specific antigens were transferred to Escherichia coli K-12 recipients by conjugation with an S. flexneri Hfr. After mating E. coli with an Hfr strain of S. flexneri 2a and selecting for his⁺ recombinants, a high proportion of the E. coli hybrids agglutinated in S. flexneri grouping serum. None of these hybrids expressed S. flexneri type-specific antigen II. When an E. coli his⁺ hybrid possessing the S. flexneri group antigen was remated with the same Hfr with selection for pro⁺ hybrids, a high proportion now expressed the type-specific antigen as well as the previously inherited group antigen. If such crosses were performed in reverse order (i.e., pro⁺ followed by his⁺ selection), a different pattern of serological behavior was observed. None of the pro⁺ hybrids showed the type-specific antigen. Subsequent mating for his⁺ resulted in hybrids with both the group- and type-specific antigens. These results show that genes controlling the synthesis of S. flexneri group antigen (linked to the his locus) and type-specific antigen (linked to the pro locus) are widely separated on the chromosome. Expression of the type-specific antigen II depends on the presence of the group antigen.     

Characterization of the Streptococcus mutans plasmid pva318 cloned into Escherichia coli.

We further characterized the cryptic plasmid pVA318 of Streptococcus mutans. It had a contour length of 5.64 +/- 0.26 kilobases and a guanine-plus-cytosine content of 32 to 34 mol %. Upon cloning the pVA318 plasmid into the vector pBR322 in Escherichia coli, we made the following observations. The expression of tetracycline resistance by HindIII-cloned chimeras, where the insert was in the tetracycline resistance promotor, depended on the orientation of the pVA318 insert. Both HindIII-cloned chimeras segregated from polA(Ts) cells at a nonpermissive temperature. Chimeric molecules cloned with PstI initially showed much instability; the reason for this is unknown, although stable variants were obtained. Both HindIII-cloned variants and a PstI-cloned chimera produced a pVA318-specific protein of approximately 20,000 molecular weight in E. coli minicells. The biological function of this protein is not known; it had no bacteriocin activity against S. mutans or group A Streptococcus indicator strains, and it did not appear in the E. coli periplasm. We constructed a map of pVA318 for restriction endonucleases HindIII, HpaI, PstI, and HaeIII. A previously reported BamHI site in pVA318 did not appear in the pVA318 portion of any of our chimeric clones.     

Localization of a streptothricin acetyl transferase in cells of Escherichia coli K-12

Streptothricin acetyl transferase coded for by plasmids pIE636 and pIE637 in Escherichia coli K-12 was found to be located at the inner side of the cytoplasmic membrane.     

Isolation and properties of complement-resistant strains of Escherichia coli K-12.

Several strains that were resistant to the bactericidal action of antibody and complement were isolated from Escherichia coli K-12 W3110/SM by selecting them through the medium containing antiserum and complement. They can be agglutinated by antiserum against the parent strain and showed similar immune adherence reactivity to the parent when sensitized with this antiserum. Few differences were found in the compositions of phospholipids and proteins between both inner and outer membranes of these strains and those of the parent. However, there were fewer short-chain and more long-chain fatty acids in these strains than in the parent. It was also found that unsaturated fatty acide decreased and saturated and cyclopropanoic acids increased in phosphatidylethanolamine and phosphatidylglycerol in both inner and outer membranes of one of these strains when compared with those from the parent. Therefore, the resistance of these strains to the complement-mediated bactericidal action was considered to be due to the rigidity of their membrane structures which might repel the insertion of membrane-attack complement complex C5b-9, although they could fix the earlier complement components up to the step of the formation of C4b,2a,3b complex enzyme.     

Molecular cloning and expression of Treponema pallidum DNA in Escherichia coli K-12.

A gene bank of Treponema pallidum DNA in Escherichia coli K-12 was constructed by cloning SauI-cleaved T. pallidum DNA into the cosmid pHC79. Sixteen of 800 clones investigated produced one or more antigens that reacted with antibodies from syphilitic patients. According to the separation pattern of the antigens produced on sodium dodecyl sulfate-polyacrylamide gels, six different phenotypes were distinguished among these 16 clones. These antigens reacted also with anti-T. pallidum rabbit serum. No antibodies against the cloned antigens were found in normal rabbit serum and in nonsyphilitic human serum. The antigens produced by the E. coli K-12 recombinant DNA clones comigrated in sodium dodecyl sulfate-polyacrylamide gels with antigens extracted from T. pallidum bacteria, suggesting that the treponemal DNA is well expressed in E. coli K-12. Several of the cosmid recombinant plasmids have been subcloned, resulting in smaller T. pallidum recombinant plasmids which are more stably maintained in the cell and produce more treponemal antigen. Monoclonal antibodies were raised against T. pallidum, and one hybridoma produced antibodies that reacted not only with an antigen from T. pallidum but also with the antigen produced by one of the E. coli clones.     

Increased survival in calves of Escherichia coli K-12 carrying an Ent plasmid.

Escherichia coli K-12 strains with and without an Ent plasmid were fed to calves, and the survival of each was monitored by viable bacterial counts of the feces. The E. coli K-12 strain carrying the Ent plasmid survived in the calves at significantly higher levels and for a longer period of time than the E. coli F(-) stain.