Biotic and Abiotic Factors Affecting Plasmid Transfer in Escherichia coli Strains

[This corrects the article on p. 393 in vol. 58.].     

Resistance to Arsenic Compounds Conferred by a Plasmid Transmissible Between Strains of Escherichia coli

A plasmid, R773, which confers resistance to arsenic compounds, is transmissible between strains of Escherichia coli. It is a member of compatibility group F(1).     

Storage of Escherichia coli Strains Containing Plasmid DNA in Liquid Nitrogen

The stability of plasmids and lysogenic bacteriophage in 16 strains of Escherichia coli was studied following storage in liquid nitrogen for periods up to 2 years. A comparison was made with cultures which had undergone lyophilization. Plasmid loss was detected by plating on selective media containing antibiotics to which resistance was conferred by plasmid DNA. Loss of plasmid DNA was not evident following storage in liquid nitrogen or lyophilization.     

Conjugative Plasmid Transfer and Adhesion Dynamics in an Escherichia coli Biofilm

A conjugative plasmid from the catheter-associated urinary tract infection strain Escherichia coli MS2027 was sequenced and annotated. This 42,644-bp plasmid, designated pMAS2027, contains 58 putative genes and is most closely related to plasmids belonging to incompatibility group X (IncX1). Plasmid pMAS2027 encodes two important virulence factors: type 3 fimbriae and a type IV secretion (T4S) system. Type 3 fimbriae, recently found to be functionally expressed in E. coli, played an important role in biofilm formation. Biofilm formation by E. coli MS2027 was specifically due to expression of type 3 fimbriae and not the T4S system. The T4S system, however, accounted for the conjugative ability of pMAS2027 and enabled a non-biofilm-forming strain to grow as part of a mixed biofilm following acquisition of this plasmid. Thus, the importance of conjugation as a mechanism to spread biofilm determinants was demonstrated. Conjugation may represent an important mechanism by which type 3 fimbria genes are transferred among the Enterobacteriaceae that cause device-related infections in nosocomial settings.Bacterial biofilms are complex communities of bacterial cells living in close association with a surface (17). Bacterial cells in these protected environments are often resistant to multiple factors, including antimicrobials, changes in the pH, oxygen radicals, and host immune defenses (19, 38). Biofilm formation is a property of many bacterial species, and a range of molecular mechanisms that facilitate this process have been described (2, 3, 11, 14, 16, 29, 33, 34). Often, the ability to form a biofilm is dependent on the production of adhesins on the bacterial cell surface. In Escherichia coli, biofilm formation is enhanced by the production of certain types of fimbriae (e.g., type 1 fimbriae, type 3 fimbriae, F1C, F9, curli, and conjugative pili) (14, 23, 25, 29, 33, 39, 46), cell surface adhesins (e.g., autotransporter proteins such as antigen 43, AidA, TibA, EhaA, and UpaG) (21, 34, 35, 40, 43), and flagella (22, 45).The close proximity of bacterial cells in biofilms creates an environment conducive for the exchange of genetic material. Indeed, plasmid-mediated conjugation in monospecific and mixed E. coli biofilms has been demonstrated (6, 18, 24, 31). The F plasmid represents the best-characterized conjugative system for biofilm formation by E. coli. The F pilus mediates adhesion to abiotic surfaces and stabilizes the biofilm structure through cell-cell interactions (16, 30). Many other conjugative plasmids also contribute directly to biofilm formation upon derepression of the conjugative function (16).One example of a conjugative system employed by gram-negative Enterobacteriaceae is the type 4 secretion (T4S) system. The T4S system is a multisubunit structure that spans the cell envelope and contains a secretion channel often linked to a pilus or other surface filament or protein (8). The Agrobacterium tumefaciens VirB-VirD4 system is the archetypical T4S system and is encoded by 11 genes in the virB operon and one gene (virD4) in the virD operon (7, 8). Genes with strong homology to genes in the virB operon have also been identified on other conjugative plasmids. For example, the pilX1 to pilX11 genes on the E. coli R6K IncX plasmid and the virB1 to virB11 genes are highly conserved at the nucleotide level (28).We recently described identification and characterization of the mrk genes encoding type 3 fimbriae in a uropathogenic strain of E. coli isolated from a patient with a nosocomial catheter-associated urinary tract infection (CAUTI) (29). The mrk genes were located on a conjugative plasmid (pMAS2027) and were strongly associated with biofilm formation. In this study we determined the entire sequence of plasmid pMAS2027 and revealed the presence of conjugative transfer genes homologous to the pilX1 to pilX11 genes of E. coli R6K (in addition to the mrk genes). We show here that biofilm formation is driven primarily by type 3 fimbriae and that the T4S apparatus is unable to mediate biofilm growth in the absence of the mrk genes. Finally, we demonstrate that conjugative transfer of pMAS2027 within a mixed biofilm confers biofilm formation properties on recipient cells due to acquisition of the type 3 fimbria-encoding mrk genes.     

R6K Plasmid Replication: Influence of Chromosomal Genotype in Minicell-Producing Strains of Escherichia coli K-12

Alkaline sucrose velocity sedimentation and cesium chloride-ethidium bromide equilibrium centrifugation have been used to determine the number of copies per chromosomal equivalent of the relaxedly replicating R6K plasmid (a conjugative plasmid conferring ampicillin and streptomycin resistance) in two minicell-producing strains of Escherichia coli K-12. In one strain, the average number of covalently closed circular R6K molecules per chromosomal equivalent is 13 in log-phase and 35 in stationary-phase cells. In the other strain, there is an average of six covalently closed circular R6K molecules per chromosomal equivalent in both log- and stationary-phase cells. Selection from this strain of spontaneously occurring mutants resistant to high concentrations of ampicillin has been accomplished and such mutants show a two- to threefold increase in the number of R6K copies per chromosomal equivalent. Relative to the parental strain, mutants display the following properties: (i) elevated streptomycin resistance, (ii) a 10-fold increase in R6K conjugal transfer, (iii) a 10-fold increase in the amount of R6K plasmid deoxyribonucleic acid segregated into minicells, and (iv) a two- to threefold increase in R6K-specified beta-lactamase. The mutation(s) responsible for the increase in the number of R6K molecules per chromosomal equivalent is located on the bacterial chromosome. No R6K-linked mutations conferring the above phenotypes have been obtained. The mutations are presumed to be in chromosomal genes which play a role in the regulation of R6K replication in this strain.     

Relation Between Survival and Deoxyribonucleic Acid Replication in Ultraviolet-Irradiated Resistant and Sensitive Strains of Escherichia coli B/r

When arabinose-grown Escherichia coli B/r is ultraviolet (UV) irradiated in the logarithmic phase of growth, the dose inactivation curve for both colony formation and deoxyribonucleic acid (DNA) synthesis (based on the relative rates of synthesis) is exponential in nature. When protein synthesis is inhibited before UV-irradiation, both inactivation curves have a large shoulder. Pre-irradiation inhibition of protein synthesis increases considerably the colony-forming ability of a UV-irradiated Hcr(-) and Rec(-) strain of E. coli B/r. However, with the repair-deficient strains, both the shoulder and slope of the survival curve are affected. We investigated the effect of UV irradiation on DNA synthesis in Hcr(-) bacteria and found that pre-irradiation inhibition of protein synthesis increases UV resistance of DNA replication in this strain also. The results suggest that inhibition of protein synthesis before irradiation increases UV resistance in E. coli B/r by a mechanism which is independent of both the excision and recombination repair systems.     

Selection of dinB Alleles Suppressing Survival Loss upon dinB Overexpression in Escherichia coli

Escherichia coli strains overproducing DinB undergo survival loss; however, the mechanisms regulating this phenotype are poorly understood. Here we report a genetic selection revealing DinB residues essential to effect this loss-of-survival phenotype. The selection uses strains carrying both an antimutator allele of DNA polymerase III (Pol III) α-subunit (dnaE915) and either chromosomal or plasmid-borne dinB alleles. We hypothesized that dnaE915 cells would respond to DinB overproduction differently from dnaE⁺ cells because the dnaE915 allele is known to have an altered genetic interaction with dinB⁺ compared to its interaction with dnaE⁺. Notably, we observe a loss-of-survival phenotype in dnaE915 strains with either a chromosomal catalytically inactive dinB(D103N) allele or a low-copy-number plasmid-borne dinB⁺ upon DNA damage treatment. Furthermore, we find that the loss-of-survival phenotype occurs independently of DNA damage treatment in a dnaE915 strain expressing the catalytically inactive dinB(D103N) allele from a low-copy-number plasmid. The selective pressure imposed resulted in suppressor mutations that eliminated growth defects. The dinB intragenic mutations examined were either base pair substitutions or those that we inferred to be loss of function (i.e., deletions and insertions). Further analyses of selected novel dinB alleles, generated by single-base-pair substitutions in the dnaE915 strain, indicated that these no longer effect loss of survival upon overproduction in dnaE⁺ strains. These mutations are mapped to specific areas of DinB; this permits us to gain insights into the mechanisms underlying the DinB-mediated overproduction loss-of-survival phenotype.     

Gene Transfer in Pasteurella pestis Harboring the F′Cm Plasmid of Escherichia coli

A strain of Pasteurella pestis, harboring the F'Cm plasmid from Escherichia coli, was able to donate its chromosome to auxotrophic recipient strains of P. pestis. The frequency of gene transfer in P. pestis was approximately 10(-6) per donor cell, 100 times less efficient than gene transfer in Pasteurella pseudotuberculosis, but efficient enough to determine entry times for the markers histidine, threonine, and tryptophan and to show linkage to the markers arginine and pigmentation. An attempt to extend the conjugation system to different serotypes of P. pseudotuberculosis and to Yersinia enterocolitica did not succeed.     

Use of Peracetic Acid as a Disinfectant in a Water-Treatment Plant: Effect on the Plasmid Contents of Escherichia coli Strains

Total thermotolerant coliforms (TTC) and Escherichia coli strains were isolated from sewage from a treatment plant before and after peracetic acid (PAA) disinfection. The plasmid profiles of 120 E. coli strains were analyzed. Although PAA disinfection effectively reduced the number of TTC and E. coli strains, the percentage of E. coli strains containing plasmids was not statistically different among water samples. The sizes of the plasmids found ranged from <3 kb to >56 kb, but plasmids of between 3 and 5 kb were encountered most frequently.     

Survival of Escherichia coli in foods

Studies describing the survival of Escherichia coli in foods, more often than not use the O157:H7 serovar as the target organism. Whilst E. coli O157:H7 is undoubtedly the predominant agent of concern for foodborne disease caused by enterohaemorrhagic E. coli (EHEC), a consequence of this concern is the commonly held view that this one serovar is 'atypical' in its response to stress conditions and therefore better able to survive adverse environments. Many of the studies published do not make comparisons with other E. coli (either commensal organisms or other pathogenic types) or other members of the Enterobacteriaceae, that would justify this view. Nevertheless, there has been a great deal of valuable data and information generated describing the fate of E. coli O157:H7 in a range of foods stored under various conditions. In many respects, the results of these studies are not surprising considering the survivability of other closely related pathogens, such as Shigella spp. This ability to survive in foods for long periods of time confirms the need for reliable control measures where contamination is possible or likely, e.g. proper handling and thorough cooking of beefburgers. The factors that may influence survival in different foods are described, with the intention of providing an insight in this area of food safety. Key considerations for carrying out survival studies are identified, with particular reference to methodologies used.     

Chromosome Transfer and Recombinant Formation with Deoxyribonucleic Acid Temperature-Sensitive Strains of Escherichia coli

Haploid recombinant yield is reduced in matings conducted at 42.5 C between deoxyribonucleic acid (DNA) temperature-sensitive [dna⁻(TS)] recipients unable to synthesize DNA at 42.5 C and any of the three major donor types (Hfr, F⁺, F′) of Escherichia coli. No such reduction is observed in matings conducted at 42.5 C when the dna⁻(TS) mutation is in the donor parent. Evidence is presented which indicates that chromosome transfer from donors to recipients unable to replicate DNA at 42.5 C during vegetative growth occurs at normal frequencies when the mating is conducted at 42.5 C. It is concluded that some stage in haploid recombinant formation is adversely affected in dna⁻(TS) recipients mated at the temperature restrictive for DNA synthesis.     

Plasmid genes increase membrane permeability in Escherichia coli

The membrane permeability to o-nitrophenyl beta-D-galactoside is increased in the presence of rifampicin in Escherichia coli cells carrying srnB+ or pnd+ plasmids, but not in the cells carrying srnB- or pnd- mutant plasmids. The same permeability alteration was also observed at 42 degrees C when a rpoC4- mutant strain was used as a host strain in the absence of rifampicin. These results and the blockage of the effects by action of chloramphenicol suggest that the increase of permeability to o-nitrophenyl galactoside was caused by the expression of srnB+ or pnd+ gene, respectively. srnB+ gene expression leads to massive RNA degradation, probably through the activation of the rna+ gene product. In an rna- strain carrying the srnB+ plasmid, the extent of RNA degradation was reduced, whereas the permeability to o-nitrophenyl galactoside was increased to the same level as in the rna+ strain. Also, the increase in permeability to o-nitrophenyl galactoside was observed at 30 degrees C, although high-temperature incubation (42 degrees C) was necessary for the induction of RNA degradation. These results suggest that the alteration in permeability is a more direct effect of the expression of srnB+ or pnd+ gene and that the RNA degradation is a secondary phenomenon caused by the alteration in the membrane.     

Plasmid formation from bacteriophage lambda in Escherichia coli

Summary Among the survivors of Escherichia coli derivatives infected with phage c1 or vir that are unable to establish ordinal lysogeny, clones arise which perpetuate the nondefective phage genome. When the bacteria bears a mutation(s) that makes the cell tolerant to the phage multiplication, such clones appear readily.The bacteria- complex was studied genetically and chemically, and it was concluded that the intact phage genomes, about two to four circular copies per bacterial chromosome, are perpetuated in bacterial cytoplasm as plasmids or in lysogenic state in cytoplasm.Several lines of evidence suggests that the phage genome in the lysogenic state in cytoplasm is under a different regulatory system from that in the normal prophage state on chromosome.     

R-Plasmid Transfer to and from Escherichia coli Strains Isolated from Human Fecal Samples

Strains of Escherichia coli recently isolated from human feces were examined for the frequency with which they accept an R factor (R1) from a derepressed fi⁺ strain of E. coli K-12 and transfer it to fecal and laboratory strains. Colicins produced by some of the isolates rapidly killed the other half of the mating pair; therefore, conjugation was conducted by a membrane filtration procedure whereby this effect was minimized. The majority of fecal E. coli isolates accepted the R factor at lower frequencies than K-12 F⁻, varying from 10⁻² per donor cell to undetectable levels. The frequencies with which certain fecal recipients received the R-plasmid were increased when its R⁺ transconjugant was either cured of the R1-plasmid and remated with the fi⁺ strain or backcrossed into the parental strain. The former suggests the loss of an incompatibility plasmid, and the latter suggests the modification of the R1-plasmid deoxyribonucleic acid (DNA). In general, the fecal R⁺E. coli transconjugants were less effective donors for K-12 F⁻ and heterologous fecal strains than was the fi⁺ K-12 strain, whereas the single strain of Citrobacter freundii examined was generally more competent. Passage of the R1-plasmid to strains of salmonellae reached mating frequencies of 10⁻¹ per donor cell when the recipient was a Salmonella typhi previously cured of its resident R-plasmid. However, two recently isolated strains of Salmonella accepted the R1-plasmid from E. coli K-12 R⁺ or the R⁺E. coli transconjugants at frequencies of 5 × 10⁻⁷ or less.     

Ammonia Inhibition of Plasmid pRmeGR4a Conjugal Transfer between Rhizobium meliloti Strains

We have examined nutritional factors influencing conjugal transfer of the two nonsymbiotic large plasmids, pRmeGR4a and pRmeGR4b, of Rhizobium meliloti GR4. To monitor transfer, each plasmid was tagged with a different antibiotic resistance marker. Transfer of plasmid pRmeGR4b was dependent upon the presence of plasmid pRmeGR4a on the same donor cell. Transconjugants for pRmeGR4b were obtained at frequencies 5-to 10-fold higher than transconjugants carrying both plasmids, indicating that mobilization of pRmeGR4b by pRmeGR4a probably occurred in trans. Conjugal transfer of the tagged plasmids between R. meliloti strains was tested on minimal medium supplemented with single amino acids, nitrate, or ammonium as the single nitrogen source. A higher number of transconjugants was obtained when glutamate was the only nitrogen source, whereas conjugation was virtually undetectable on ammonium. No relationship was found between donor or recipient growth rate and plasmid transfer rate on a given nitrogen source. Furthermore, in media containing both glutamate and ammonium as nitrogen sources, transfer was reduced almost 100-fold compared with that in media containing glutamate alone. Inhibition was readily detected at 2.5 mM or higher concentrations of either ammonium chloride or ammonium sulfate and appeared to be specific for exogenously supplied ammonium. Inhibition of conjugal transfer between R. meliloti strains by ammonium was only observed for rhizobial plasmids, not for a heterologous plasmid such as RP4. Apparently, ammonium did not affect the plasmid-encoded transfer machinery, as it had no influence on rhizobial plasmid transfer from R. meliloti to Agrobacterium tumefaciens. The effect of ammonium seemed to take place on R. meliloti recipient cells, thereby reducing the efficiency of plasmid conjugation, probably by affecting mating pair formation or stabilization.     

Mathematical Model of Plasmid Transfer between Strains of Streptomycetes in Soil Microcosms

A mathematical model was developed and used to simulate the long-term dynamics of growth and plasmid transfer in nutrient-limited soil microcosms of Streptomyces lividans TK24 carrying chromosomal resistance to streptomycin, S. lividans 1326; and S. violaceolatus ISP5438. Donor, recipient, and transconjugant survival was modelled by an extension to the Verhulst logistic equation which takes account of nutrient limitation, and plasmid transfer was modelled by a mass action model. Rate parameters were derived from experimental data on the early stages of the development of sterile systems. The model predicted donor, recipient, and transconjugant populations in 2.4-h (0.1-day) steps and was tested against the long-term behavior of the experimental sterile systems and independent experimental data on nonsterile systems. Bacteria were periodically enumerated onto selective media over a 20-day period. The effects of long-term nutrient-moisture depletion were correctly predicted.     

Influence of the Fertility Episome on the Survival of X-irradiated Escherichia coli

The resistance of Escherichia coli to the lethal effects of X rays was increased by the presence of a fertility episome integrated into the chromosome.