Virulence plasmid-associated sensitivity to rifampicin and novobiocin in Escherichia coli

Introduction of the ColV, I-K94 or ColV-K30 plasmid into Escherichia coli K12 resulted in increased sensitivity to growth inhibition and killing by rifampicin and novobiocin. For increased rifampicin sensitivity, both transfer and colicin components had to be present whereas transfer components plus another unidentified ColV component were needed for novobiocin sensitivity. The Co1 factor Co1B-K98 conferred increased rifampicin sensitivity, an effect not dependent on transfer components, but this plasmid had no effect on the novobiocin sensitivity of E. coli. The effects of ColV, I-K94 on rifampicin sensitivity were fully reversed by magnesium ions but Mg²⁺ had only a slight effect on the novobiocin inhibition of the Co1V, I-K94⁺ strain and the rifampicin inhibition of the Co1B-K98⁺ strain. Prior growth at 25C greatly reduced the effects of Co1V, I-K94 on rifampicin and novobiocin sensitivity but had essentially no effect on rifampicin sensitivity of the Co1B-K98⁺ strain.     

质粒在大肠杆菌对噬菌体抗性中的作用

The introduction of the ColV, I-K94 or R124 plasmid into Escherichia coli K12 resulted in resistance to certain phages. Derivatives of E. coli carrying the plasmid R124 and ColV, I-K94 were resistance to the phages T4, Mel comparing with the plasmid-free parent and the plasmid ColV, I-K94 conferred resistance to the phage Tull*. It suggested that an envelope change caused by the plasmids might be responsible for the resistance because most of the phages fell to absorb to the plasmid-bearing E. coli cells.     

Virulence plasmid-associated adhesion of Escherichia coli and its significance for chlorine resistance

Introduction of the ColV, I-K94 virulence plasmid into strains of Escherichia coli led (for four out of five strains tested) to a marked increase in the ability of organisms to adhere to glass beads. For strain 1829, the plasmid led to increased attachment to other materials including sand, agar, agarose, chitin and cellulose. The increased adhesion to glass beads was due to the presence of the plasmid and not to its introduction into a variant with altered adhesive properties. The plasmid-encoded VmpA protein did not appear to be necessary for the ColV, I-K94-promoted adhesion but adhesion was absolutely dependent on the presence of derepressed levels of transfer components in the ColV+ strains and partially dependent on the presence of colicin components. The extent of the plasmid-promoted adhesion was greatest for organisms grown at 30 degrees, 37 degrees or 42 decrees C and adhesion was almost abolished by growth at 21 degrees or 25 degrees C; this finding is in accord with transfer and colicin components being involved in adhesion. Of several other plasmids tested for their effects on adhesion, those with derepressed transfer properties showed a marked effect as did the RI resistance plasmid. Because of the ease of handling glass bead-attached organisms, such preparations were used as a model for studying the relevance of attachment to the resistance of E. coli to chlorination in the water purification process. Organisms of 1829 ColV, I-K94, attached to glass beads, were more resistant to damage and killing by chlorine than were unattached organisms. Three findings suggest that such chlorine resistance may be significant for survival during water chlorination. Firstly, ColV, I-K94+ bacteria became attached if incubated in sewage effluent with glass beads at 20 degrees C. Secondly, ColV+ organisms already attached to glass beads maintained their attachment during 24 h incubation in effluent at 20 degrees C and thirdly such effluent incubated organisms remained chlorine resistant provided that they retained their attachment.     

Suppression by the ColV,I-K94 plasmid of a growth lesion in ompA mutants of Escherichia coli

Organisms of three independently isolated ompA mutants of Escherichia coli failed to form colonies on glucose minimal agar (glucose MA) at 44 degrees C after growth in glucose minimal salts medium at 37 degrees C, although all three strains formed colonies on nutrient agar at 44 degrees C. Supplementation of the glucose MA with individual amino acids including L-methionine and/or L-cysteine did not allow colony formation at 44 degrees C, although addition of 0.1% Casamino acids was effective; replacement of glucose with other energy sources or ammonium ions with glutamate also did not allow growth at 44 degrees C. The failure to form colonies at 44 degrees C was not due to killing of the organisms, because colonies were formed if plates of the ompA mutant initially incubated at 44 degrees C were shifted to 30 degrees C after 16 h. Introduction of the ColV, I-K94 plasmid into P678-54 ompA, 1131 ompA or an ompC ompA mutant suppressed the 44 degrees C growth lesion, but other plasmids (F lac, R483ColIa, RI, ColB-K98, R124) tested in P678-54 ompA did not. Growth of the ColV, I-K94+ derivative at 44 degrees C was due to a suppressing effect of the plasmid rather than to introduction of the plasmid into a variant with normal or altered OmpA protein. An attempt was made to ascertain which component(s) encoded by ColV, I-K94 was (were) responsible for allowing growth at 44 degrees C. Transfer components appeared unlikely to be involved and plasmids which conferred individual colicins (plus the corresponding immunity component) did not suppress.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sequence rearrangements in the plasmid ColV,I-K94

ColV,I-K94 is a conjugative (F-like) plasmid specifying colicins V and I. Numerous rearrangements were observed in ColV,I-K94 plasmids from three culture collections and in derivatives which specified larger inhibition zones and increased titers of colicin V. Several of the rearrangements in ColV,I-K94 derivatives specifying higher titers of colicin V involved a 1.3 ± 0.08-kb inverted repeat sequence which was separated by a 3.3 ± 0.2-kb sequence. This was rearranged in one derivative, pKH46, such that the 3.3-kb sequence became flanked by a 2.5 ± 0.15-kb inverted repeat which was subsequently altered to form a 2.1 ± 0.1-kb inverted repeat. In two other derivatives, the 3.3-kb sequence was deleted together with all of the flanking inverted repeat (in pKH39-2) or part of each repeat (in pKH39-1). A 0.5 ± 0.1-kb sequence adjacent to one of the 1.3-kb repeats was inverted or substituted in both of these plasmids. A novel 12.9 ± 0.7-kb inverted duplication separated by a 7.3 ± 0.4-kb sequence was present in the ColV,I-K94 derivative, pKH41, and insertion of the γδ element was detected in another derivative, pKH46-1, after this nonconjugative plasmid had been mobilized by the F plasmid. The genes for colicin V and for immunity to colicin V were mapped to a 3.6-kb sequence. The copy numbers of both pKH39-2 and pKH46-1::γδ were greater than those of pKH38 from which they were derived.     

The ColV, I-K94 plasmid and heat sensitivity of Escherichia coli

The ColV, I-K94 plasmid markedly increased the heat sensitivity of Escherichia coli K12 but had no sensitizing effect on a wild E. coli isolate. The plasmid effect on strain K12 appeared to result from ColV-encoded transfer and colicin components possibly due to their effects on membrane properties.     

Effect of ColV plasmids on the hydrophobicity of Escherichia coli

Abstract The hydrophobicity of E. coli strains carrying or lacking the colicin V ( ColV ) plasmids, ColV , I-K94 or ColV -K30 was assayed. ColV ⁺ derivatives of strain 1829, produced by conjugation or transformation, were more hydrophobic than either the original 1829 parental strain or a Col ^- derivative formed by curing 1829 ColV -K30 of its plasmid by an SDS/high temperature growth technique. Transfer of ColV into other E. coli strains also led to increased hydrophobicity. This effect of ColV plasmids was observed for organisms grown at 37°C; ColV ⁺ and ColV^- strains did not differ in hydrophobicity of grown at 21°C. This finding and other studies suggest that sex pili may be involved in the increased hydrophobicity.     

Copper sensitivity in an envelope mutant of Escherichia coli and its suppression by ColV, I-K94

A phage K3-resistant isolate from Escherichia coli P678-54 was devoid of both the OmpA and OmpC proteins but had high levels of the OmpF protein. Associated with these changes, the strain showed increased sensitivity to inhibition by detergents and greatly increased sensitivity to Cu²⁺. Introduction of the ColV, I-K94 plasmid into this mutant produced a derivative with markedly increased resistance to Cu²⁺ ions but unchanged detergent sensitivity. Analysis of membranes showed that the ColV, I-K94⁺ derivative had essentially no OmpF protein in its outer membrane. A ca 36 K outer membrane protein was present which resembled the OmpC protein in size and failure to dissociate in SDS at low temperature. It was distinct from the OmpC protein, however, in failing to allow either tetracycline uptake or the adsorption of T4-type phages. The possible significance of OmpF porin derepression (and its reversal by ColV, I-K94) for enterobacterial survival in aquatic situations is discussed.     

Suppression by the ColV,I-K94 plasmid of inhibitor sensitivities in ompA mutants of Escherichia coli

A series of ompA mutants derived from Escherichia coli K12 strains showed increased sensitivity (compared with the ompA+ parents) to aminoglycoside antibiotics and to other cationic agents including polymyxin B. One tested mutant also showed increased sensitivity to nafcillin and fusidic acid, but not to the hydrophilic ampicillin. All these inhibitor sensitivities in the ompA mutants were suppressed by ColV, I-K94 and by certain other ColV plasmids, but not by any of the other tested large plasmids. Suppression correlated with the production of the VmpA protein, but transfer and colicin components were not needed for suppression. Further comparison of the ompA and vmpA genes and their products was made and it indicated that there is little if any homology between the genes, that the synthesis of their products is regulated by quite different mechanisms, and that regions of these gene products exposed at the cell surface show different susceptibility to protease attack after denaturation.     

finO sequences on conjugally repressed and derepressed F-like plasmids

DNA-DNA hybridization studies have demonstrated the physical relatedness of the fertility inhibition gene, finO, among both FinO+ and FinO- F-like conjugative plasmids, viz. ColV2-K94, R100-1,R1drd19,R1,R6-5, UCR123, R386, p307, R453, R773, and pIP162-1. Furthermore, the data indicate that finO sequences on the FinO- plasmid ColV2-K94 map downstream of the transfer region, within 93.6-95.3 ColV2-K94.     

Decreased DNA damage by acid and increased repair of acid-damaged DNA in acid-habituated Escherichia coli

A study of the conjugal transfer of ColV,I-K94 tn 10 from acid-treated donors suggested that acid-habituated recipients repair acid-damaged plasmid DNA better than those that are not habituated. The presence of an increased repair activity for acid-damaged DNA in habituated cells was confirmed by isolating pBR322 from acid-treated organisms; habituated cells produced more transformants when transformed by it than did non-habituated ones. Additionally, agarose gel electrophoretic studies of pBR322 DNA isolated from acid-damaged cells and tests of its transforming activity both indicated that plasmid DNA in habituated cells is less damaged by extreme acidity than is that in non-habituated organisms.     

Decreased DNA damage by acid and increased repair of acid-damaged DNA in acid-habituated Escherichia coli

A study of the conjugal transfer of ColV,I-K94 tn10 from acid-treated donors suggested that acid-habituated recipients repair acid-damaged plasmid DNA better than those that are not habituated. The presence of an increased repair activity for acid-damaged DNA in habituated cells was confirmed by isolating pBR322 from acid-treated organisms; habituated cells produced more transformants when transformed by it than did non-habituated ones. Additionally, agarose gel electrophoretic studies of pBR322 DNA isolated from acid-damaged cells and tests of its transforming activity both indicated that plasmid DNA in habituated cells is less damaged by extreme acidity than is that in non-habituated organisms.     

Contributions of traT and iss genes to the serum resistance phenotype of plasmid ColV2-K94

Abstract A genetic determinant for serum resistance, designated iss , has been found previously on the colicinogenic plasmid ColV2-K94. In this work we have identified a second serum resistance gene, traT , on ColV2-K94. The serum resistance mediated by derivatives of ColV2-K94 was due to presence of one or both of the iss and traT genes. Plasmid pWS12 (TraT⁺ Iss⁺) contained the kanamycin (Km) resistance transposon Tn 903 inserted near the origin of replication of ColV2-K94, and plasmids pWS15 (TraT⁺), pWS16 (TraT⁺) and pWS18 (TraT⁺ Iss⁺) were deletion derivatives of pWS12 constructed in vitro and in vivo. pWS12 and pWS18 conferred a 20-fold increase in relative resistance to 20% guinea pig serum when introduced into the serum-susceptible, genetically defined recA strain of Escherichia coli K-12, AB2463. Plasmids pWS15 and pWS16, from which iss had been deleted, still conferred 5-fold increases in relative resistance on AB2463. The level of resistance conferred on this strain by the antibiotic resistance plasmid R100–1 (which expresses the traT serum resistance gene) was comparable to that of plasmids pWS15 and pWS16. The 25-kDa traT gene product was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the outer membrane proteins of strain AB2463 carrying ColV2-K94. This protein cross-reacted immunologically with the traT protein expressed by F or R100–1. Our results indicated that both traT and iss are capable of mediating serum resistance in ColV2-K94.     

Second replicon in ColV2-K94 mediates the stable coexistence of two incompatible plasmids.

The colicin-producing plasmid pWS12, a Tn903 derivative of ColV2-K94, was found to be incompatible with the IncFI plasmids KLF1 and R386. It was compatible with the IncFII plasmids R538 and R100. Three overlapping mini-ColV derivatives, pWS15, pWS16 and pWS17, were obtained by restriction digestion of pWS12. Unlike pWS12, pWS16 exhibited incompatibility with both IncFI and IncFII plasmids, whereas the pWS15 and pWS17 plasmids expressed IncFII incompatibility but not the IncFI incompatibility of their parental ColV plasmid. We show that, although pWS12 has an IncFII replicon, Rep1, it does not normally express IncFII incompatibility because a second replicon, Rep2 (homologous to the secondary replicon of F), functions during the stable coexistence of the plasmid with IncFII plasmids. When Rep2 is deleted (as in the mini-ColV plasmids) or made nonfunctional (as in a PolA mutant strain), ColV then behaves as an IncFII plasmid.     

Characterization and sequence analysis of pilin from F-like plasmids.

Conjugative pili are expressed by derepressed plasmids and initiate cell-to-cell contact during bacterial conjugation. They are also the site of attachment for pilus-specific phages (f1, f2, and QB). In this study, the number of pili per cell and their ability to retract in the presence of cyanide was estimated for 13 derepressed plasmids. Selected pilus types were further characterized for reactivity with anti-F and anti-ColB2 pilus antisera as well as two F pilus-specific monoclonal antibodies, one of which is specific for a sequence common to most F-like pilin types (JEL92) and one which is specific for the amino terminus of F pilin (JEL93). The pilin genes from eight of these plasmids were cloned and sequenced, and the results were compared with information on F, ColB2, and pED208 pilin. Six pilus groups were defined: I, was F-like [F, pED202(R386), ColV2-K94, and ColVBtrp]; IIA was ColB2-like in sequence but had a lowered sensitivity to f1 phage due to its decreased ability for pilus retraction [pED236(ColB2) and pED203(ColB4)]; IIB was ColB2-like but retained f1 sensitivity [pED200(R124) and pED207(R538-1)]; III contained R1-19, which had a ColB2-like amino terminus but had an additional lysine residue at its carboxy terminus which may affect its phage sensitivity pattern and its antigenicity; IV was R100-1-like [R100-1 and presumably pED241(R136) and pED204(R6)] which had a unique amino-terminal sequence combined with a carboxy terminus similar to that of F. pED208(Folac) formed group V, which was multipiliated and exhibited poor pilus retraction although it retained full sensitivity to f1 phage. The pED208 pilin gene could not be cloned at this time since it shared no homology with the pilin gene of the F plasmid.     

Characterization of the stability functions and inverted repeat structure X3 of the IncFI plasmid ColV2-K94

A region of the IncFI plasmid ColV2-K94 which showed homology to the sop partitioning genes of F was cloned and characterized in an attempt to study the stability functions of this element. The sop region contained the incD incompatibility determinant common to many IncFI plasmids, but could not confer on ColV2-K94 miniplasmids the same stable inheritance found in the intact ColV2-K94; thus, other functions appear to be required for efficient plasmid maintenance. Adjacent to the area of sop homology was the X3 region, which was found to contain three inverted IS1-like sequences. The X3 region of ColV2-K94 was similar in organization to the aerobactin iron uptake region of ColV3-K30, but ColV2-K94 lacked the ability to synthesize either the aerobactin siderophore or its outer membrane receptor.     

Plasmid Specificity of The Origin of Transfer of Sex Factor F

The ability of F-like plasmids to promote transfer from the F origin of transfer was determined. Chromosome transfer was measured from plasmid derivatives of RecA(-) Hfr deletion strains which had lost all the F transfer genes but which in some cases retained, and in others had also lost, the origin sequence. ColV2 and ColVBtrp could initiate transfer from the F origin, but R100-1, R1-19, and R538-1 drd could not. These results can be correlated with the plasmid specificity of the traI components of the different plasmid transfer systems, supporting the hypothesis that the origin of transfer is the site of action of the traI product. Most F-like plasmids, including R1-19 and R538-1 drd, could transfer ColE1, consistent with previous findings that the (plasmid-specific) traI product is not necessary for ColE1 transfer by Flac; ColE1 transfer may be initiated by a ColE1-or host-determined product. R100-1 and R136fin(-) could not transfer ColE1 efficiently, apparently because of differences residing in their pilus-forming genes.     

Habituation to alkali and increased u.v.-resistance in DNA repair-proficient and -deficient strains of Escherichia coli grown at pH 9.0

Repair-deficient strains of Escherichia coli carrying polAI or recA mutations were more alkali-sensitive than was their repair-proficient parent but, like strain 1829 ColV, I-K94, they showed habituation to alkali (induction of increased resistance) when grown at pH 9.0. Occurrence of such increased alkali resistance in the recA mutant implies that habituation to alkali does not depend on induction of SOS-related repair mechanisms. Organisms of repair-proficient and repair-deficient strains also became more resistant to u.v.-irradiation after growth at pH 9.0; this increased u.v.-resistance also appeared to be RecA-independent.     

Chromosomal genes for ColV plasmid-determined iron(III)-aerobactin transport in Escherichia coli.

Four chromosomal genes, tonA (fhuA), fhuB, tonB, and exbB, were required for the transport of iron(III)-aerobactin specified by the plasmids ColV-K311, ColV-K229, ColV-K328, and ColV-K30. These genes also determine the transport system in Escherichia coli for the iron ionophore ferrichrome. Aerobactin and ferrichrome are both iron ligands of the hydroxamate type, but they are of different structure. The ColV plasmids determine an outer membrane protein that serves as a receptor for cloacin. Cloacin-resistant mutants were devoid of iron(III)-aerobactin transport but were unimpaired in ferrichrome transport. We conclude that for iron(III)-aerobactin transport two outer membrane proteins, the TonA and the cloacin receptor protein, have to interact functionally or structurally or both.     

Nucleotide sequence analysis of the complement resistance gene from plasmid R100

The multiple antibiotic resistance plasmid R100 renders Escherichia coli resistant to the bactericidal action of serum complement. We constructed a plasmid (pOW3) consisting of a 1,900-base-pair-long restriction fragment from R100 joined to a 2,900-base-pair-long fragment of pBR322 carrying ampicillin resistance. E. coli strains carrying pOW3 or R100 were up to 10,000-fold less sensitive to killing by serum complement than were plasmid-free bacteria or bacteria carrying pBR322. Nucleotide sequencing revealed that 875 of the 1,900 bases from R100 correspond exactly to part of the bacterial insertion sequence IS2. The remaining 1,075 bases contained only one sizeable open reading frame; it covered 729 base pairs (243 amino acids) and was preceded by nucleotide sequences characteristic of bacterial promoters and ribosome binding sites. The first 20 amino acids of the predicted protein showed features characteristic of a signal sequence. The remainder of the predicted protein showed an amino acid composition almost identical with that determined for the traT protein from the E. coli F factor. Southern blot analysis showed that the resistance gene from R100 does not hybridize to the serum resistance gene from ColV,I-K94 isolated by Binns et al.; we concluded that these genes are distinct.