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.     

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.     

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.     

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     

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.     

Sensitivity of polA mutants of Escherichia coli K-12 to ozone and radiations

Different polA mutants of Escherichia coli K-12 were exposedto ozone, X-rays and UV radiation, in order to compare the roleof the various enzymatic activities of DNA polymerase I in therepair of damage caused by these agents. As was the case withradiations, the polymerase-deficient mutants were the most sensitiveto ozone, followed by the 5'–3' exonuclease-deficientmutants. The 3'–5' exonuclease activity of pol I appearedto play a minor role in the repair of damage induced by allthese agents.     

A novel role for protein-tyrosine kinase Etk from Escherichia coli K-12 related to polymyxin resistance

The role of protein-tyrosine kinases in bacterial polymyxin resistance was assessed by both genetic and biochemical approaches. Each of the two genes, wzc and etk, encoding protein-tyrosine kinases in Escherichia coli, was knocked out by using the PCR-based method of one-step inactivation of chromosomal genes, and the corresponding mutant strain was assayed in each case for resistance to different concentrations of polymyxin B by measuring the percentage of surviving cells. The resistance of a double knock-out wzc-etk-mutant was also analyzed and complementation experiments were performed by checking the effect of plasmid vectors expressing either Wzc or Etk. Our results concurred in showing that protein-kinase Wzc is not essential for polymyxin resistance, whereas protein-kinase Etk appears to play a key role in such antibiotic resistance. This newly found specific function of Etk reinforces the concept that protein-tyrosine kinases are involved in distinct facets of bacterial physiology.     

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.     

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.     

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.     

Streptococcus mutans Genes That Code for Extracellular Proteins in Escherichia coli K-12

Chromosomal DNA from Streptococcus mutans 6715 (serotype g) was cloned into Escherichia coli K-12 by using the cosmid pJC74 cloning vector and a bacteriophage λ in vitro packaging system. Rabbit antiserum against S. mutans extracellular proteins was used for immunological screening of the clone bank. Twenty-one clones produced weak to strong precipitin bands around the colonies, but only after the λ c1857 prophage was induced by being heated to lyse the E. coli cells. None of the clones expressed enzyme activity for several known S. mutans extracellular enzymes. One of these clones contained a 45-kilobase recombinant plasmid designated pYA721. An 8.5-kilobase fragment of S. mutans DNA from pYA721 was isolated and recloned into the BamHI restriction site of the plasmid vector pACYC184 to construct pYA726. pYA726 contained all, or nearly all, of the gene for a surface protein antigen (the spaA protein) of S. mutans 6715. This was deduced from immunological studies in which extracts of cells harboring pYA726 reacted with antisera against both purified 6715 spaA protein (about 210,000 daltons) and the immunologically similar antigen I/II of serotype c strains of S. mutans. In addition, the S. mutans spaA protein was found to possess at least one antigenic determinant not present on the protein specified by pYA726. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of E. coli clone extracts revealed that pYA726 produced a polypeptide with a molecular mass of about 180,000 daltons which was predominantly found in the periplasmic space of E. coli cells. Antisera to the spaA protein of S. mutans reacted with extracellular protein from representative strains of S. mutans serotypes a, c, d, e, f, and g, but not b.     

Pathogenicity island evaluation in Escherichia coli K1 by crossing with laboratory strain K-12.

In bacterial pathogens, strain-specific chromosomal segments often contain genes encoding strain-specific traits, and because these genes often appear to be dedicated to pathogenic interactions with eucaryotic hosts, the segments containing them may be considered so-called pathogenicity islands (G. Blum, M. Ott, A. Lischewski, A. Ritter, H. Imrich, H. Tschape, and J. Hacker, Infect. Immun. 62:606-614, 1994). We evaluated the contribution to pathogenesis of a recently identified strain-specific chromosomal segment from an Escherichia coli K1 mammalian-newborn sepsis strain: transfer of E. coli K-12 DNA sequences near 64 min, by P1 transduction, into K1 strain RS218 resulted in an RS218-K-12 chimera that (i) contained a shortened NotIotl restriction fragment (relative to wild-type RS218) encompassing the 64-min region; (ii) lacked invasiveness in newborn rats; and (iii) grew in vitro, in both rich and minimal laboratory media, indistinguishably from strain RS218. In addition, genomic DNA from the chimera failed to hybridize with sequences of the K1 capsule genes from strain RS218, suggesting that the chromosomal segment near 64 min which was lost contained these sequences and indeed contained K1-specific virulence genes. Transfer of K-12 sequences resulting in deletion of E. coli pathogen-specific chromosomal segments may afford a general method of detecting genes encoding virulence and/or other distinguishing traits.     

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.     

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)     

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.     

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.