Colicin Resistance Research Articles

Response of Escherichia coli minimal ter operon to UVC and auto-aggregation: pilot study.

AimThe study of minimal ter operon as a determinant of tellurium resistance (TeR) is important for the purpose of confirming the relationship of these genes to the pathogenicity of microorganisms. The ter operon is widespread among bacterial species and pathogens, implicated also in phage inhibition, oxidative stress and colicin resistance. So far, there is no experimental evidence for the role of the Escherichia coli (E. coli) minimal ter operon in ultraviolet C (UVC) resistance, biofilm formation and auto-aggregation. To identify connection with UVC resistance of the minimal ter operon, matched pairs of Ter-positive and -negative E. coli cells were stressed and differences in survival and whole genome sequence analysis were performed. This study was aimed also to identify differences in phenotype of cells induced by environmental stress.MethodsIn the current study, a minimal ter operon(terBCDEΔF) originating from the uropathogenic strain E. coli KL53 was used. Clonogenic assay was the method of choice to determine cell reproductive death after treatment with UVC irradiation at certain time intervals. Bacterial suspensions were irradiated with 254 nm UVC-light (germicidal lamp in biological safety cabinet) in vitro. UVC irradiance output was 2.5 mW/cm2 (calculated at the UVC device aperture) and plate-lamp distance of 60 cm. DNA damage analysis was performed using shotgun sequencing on Illumina MiSeq platform. Biofilm formation was measured by a crystal violet retention assay. Auto-aggregation assay was performed according to the Ghane, Babaeekhou & Ketabi (2020).ResultsA large fraction of Ter-positive E. coli cells survived treatment with 120-s UVC light (300 mJ/cm2) compared to matched Ter-negative cells; ∼5-fold higher resistance of Ter-positive cells to UVC dose (p = 0.0007). Moreover, UVC surviving Ter-positive cells showed smaller mutation rate as Ter-negative cells. The study demonstrated that a 1200-s exposure to UVC (3,000 mJ/cm2) was sufficient for 100% inhibition of growth for all the Ter-positive and -negative E. coli cells. The Ter-positive strain exhibited of 26% higher auto-aggregation activities and was able to inhibit biofilm formation over than Ter- negative strain (**** P < 0.0001).ConclusionOur study shows that Ter-positive cells display lower sensitivity to UVC radiation, corresponding to a presence in minimal ter operon. In addition, our study suggests that also auto-aggregation ability is related to minimal ter operon. The role of the minimal ter operon (terBCDEΔF) in resistance behavior of E. coli under environmental stress is evident.

Activity spectrum of colicins produced by Shigella sonnei and genetic mechanism of colicin resistance in conspecific S. sonnei strains and Escherichia coli.

Colicin-mediated killing is an example of allelopathy, which has been found among several bacteria. Screening of 42 strains of Shigella sonnei isolated from diarrheal patients revealed that 39 (93%) S. sonnei strains were positive for colicin production against Escherichia coli DH5α. In the PCR-based detection of the colicin types, 36 (92.3%) were identified as E3, 2 (5.1%) as E3 and E8, and 1 (2.6%) as E3 and E2. Representative S. sonnei strains producing heterologous colicins exhibited antagonism against diarrheagenic Escherichia coli (DEC) groups. Although it is known that mutation in the colicin receptor renders the host resistant to colicin, there is a dearth of information on the genetic characterization of such mutants. In the fluctuation test, colicin-resistant E. coli mutants were found to occur spontaneously at the rates of 2.51 × 10(-8) and 5.52 × 10(-8) per generation when exposed to colicins E3 and E8 and colicins E3 and E2, respectively. Genotypic characterization of colicin-resistant E. coli (EC(Cr)) and S. sonnei (SS(Cr)) strains displayed mutations in the btuB gene, which encodes the receptor for vitamin B12 uptake. This gene was interrupted by various insertion sequences, such as IS1, IS2, and IS911. Complementation of EC(Cr) and SS(Cr) with plasmid-borne btuB (pbtuB) accomplished restoration of the colicin-susceptible phenotype. The vitamin B12 uptake assay gave an insight into the physiological relevance of the btuB mutation. Our studies provide insights into the latent influence of S. sonnei colicins in governing the existence of some of the shigellae and all of the DEC and the genetic mechanism underlying the emergence of resistance.

Assessment of resistance to colicinogenic Escherichia coli by E. coli O157:H7 strains

To assess a collection of 96 Escherichia coli O157:H7 strains for their resistance potential against a set of colicinogenic E. coli developed as a probiotic for use in cattle. Escherichia coli O157:H7 strains were screened for colicin production, types of colicins produced, presence of colicin resistance and potential for resistance development. Thirteen of 14 previously characterized colicinogenic E. coli strains were able to inhibit 74 serotype O157:H7 strains. Thirteen E. coli O157:H7 strains were found to be colicinogenic and 11 had colicin D genes. PCR products for colicins B, E-type, Ia/Ib and M were also detected. During in vitro experiments, the ability to develop colicin resistance against single-colicin producing E. coli strains was observed, but rarely against multiple-colicinogenic strains. The ability of serotype O157:H7 strains to acquire colicin plasmids or resistance was not observed during a cattle experiment. Escherichia coli O157:H7 has the potential to develop single-colicin resistance, but simultaneous resistance against multiple colicins appears to be unlikely. Colicin D is the predominant colicin produced by colicinogenic E. coli O157:H7 strains. The potential for resistance development against colicin-based strategies for E. coli O157:H7 control may be very limited if more than one colicin type is used.

THE PHENOTYPIC AND FITNESS EFFECTS OF COLICIN RESISTANCE IN ESCHERICHIA COLI K-12.

Previous studies indicate that most natural isolates of Escherichia coli are resistant to most or all colicins (antibiotics produced by E. coli) when assessed in the laboratory. Additionally, resistance to different colicin types appears to arise in a nonindependent manner. One possible mechanism to explain this nonindependence is pleiotropy: Multiple resistances are selected after exposure to a single colicin. This study, which was designed to address the role of pleiotropy in the generation of colicin resistance, revealed that 96% of colicin resistant mutants were resistant to two or more colicins. Mutational class was important because putative translocation mutants (Tol pathway mutants) resisted fewer colicins than putative receptor mutants. To determine whether colicin resistance is costly, the effects of colicin resistance mutations on maximal growth rate in a rich medium were also examined. Relative to the sensitive ancestor, translocation mutations lowered maximal growth rates by 17%, whereas putative receptor mutations did not significantly lower growth rates. Thus, when nutrients are abundant, the most advantageous forms of colicin resistance may not impose a cost. The ecological consequences of pleiotropic colicin resistance could involve population cycling between colicin sensitivity and resistance. Additionally, if the cost of resistance depends on the environment, ecological diversification could result.

The Phenotypic and Fitness Effects of Colicin Resistance in Escherichia coli K-12

The Phenotypic and Fitness Effects of Colicin Resistance in Escherichia coli K-12

HIGH LEVELS OF COLICIN RESISTANCE IN ESCHERICHIA COLI.

Colicins are plasmid-encoded antibiotics that are produced by and kill Escherichia coli and other related species. The frequency of colicinogeny is high, on average 30% of E. coli isolates produce colicins. Initial observations from one collection of 72 strains of E. coli (the ECOR collection) suggest that resistance to colicin killing is also ubiquitous, with over 70% of strains resistant to one or more colicins. To determine whether resistance is a common trait in E. coli, three additional strain collections were surveyed. In each of these collections levels of colicin production were high (from 15 to 50% of the strains produce colicins). Levels of colicin resistance were even higher, with most strains resistant to over 10 colicins. A survey of 137 non-E. coli isolates revealed even higher levels of resistance. We discuss a mechanism (pleiotropy) that could result in the co-occurrence of such high levels of colicin production and colicin resistance.

High Levels of Colicin Resistance in Escherichia coli

High Levels of Colicin Resistance in Escherichia coli

Characterization of a region of the IncHI2 plasmid R478 which protects Escherichia coli from toxic effects specified by components of the tellurite, phage, and colicin resistance cluster.

The IncHI2 plasmid R478 specifies resistance to potassium tellurite (Te(r)), to some bacteriophages (Phi), and to pore-forming colicins (PacB). The genes encoding the three phenotypes are linked, and an 8.4-kb fragment of R478 DNA encoding them cannot be subcloned unless cocloned with a second section of the plasmid. Subclone pKFW4A contains a 5.9-kb BamHI-EcoRI fragment which caused some toxicity when present in Escherichia coli cells. Bacterial cells containing freshly transformed pKFW4A, examined by light microscopy and electron microscopy, had a filamentous morphology consistent with a block in septation. Insertion of transposon Tn1000 into terZ, -A, -B, and -C genes of pKFW4A resulted in the loss of the filamentation phenotype. Deletion of several regions of the clone confirmed that these latter components are involved in the filamentation phenotype. The region specifying protection from toxicity caused by the larger 8.4-kb fragment (encompassing this cluster and the entire 5.9-kb section of pKFW4A) was sequenced and analyzed by T7 polymerase expression and Tn1000 mutagenesis. Three open reading frames, terW, terY, and terX, were identified in a 2.6-kb region. Two polypeptides with approximate molecular masses of 18 and 28 kDa were expressed in CSRDE3 cells and were consistent with TerW (17.1 kDa; 155 amino acids [aa]) and TerY (26.9 kDa; 248 aa), whereas a protein of 213 aa deduced from terX was not observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The terX gene product shows strong identity with the previously identified TerE, TerD, and TerZ polypeptides, and there is a conserved motif of 13 residues, GDN(R/L)TG(E/A)GDGDDE, within this group of polypeptides. Complementation analysis indicated that terW, located approximately 6.0 kb upstream of terZ, brings about protection of cells from toxic effects of components of the Te(r), Phi, and PacB cluster.

Mutation analysis of the receptor for colicins E1-E7. A pilot study.

Thirty eight mutant clones of the colicin indicator strain Escherichia coli K 12 ROW, selected by their insensitivity to any of the colicins E1-E7, were isolated. Comparison of their sensitivity-resistance patterns to colicins E1-E7 enabled us to draw a rough preliminary map of the receptor for E colicins. In this receptor, the highly specific binding site for colicin E1 partially overlaps with the domain shared by all colicins E2 through E7. A specific binding site of this domain appears to be common for colicins E3 and E6; a part of the E3 and E6 binding site is also common for colicins E4 and E5 and a small, least specific, part also for colicins E2 and E7. Using colicin assay experiments, the binding capacity of colicin E receptor mutants could be estimated. A decreased, but not completely lost ability of certain mutants to bind colicins E, correlated to their lowered sensitivity to them, was found. Thus the phenomenon of partial colicin resistance was established, showing that colicin sensitivity--resistance is not a qualitative but a quantitative marker.

Colicin resistance in relation to pathogenicity factors in strains of Escherichia coli isolated from the intestinal tract of piglets

Three hundred and fifteen E. coli strains isolated from the intestine of piglets were examined for K-antigens 88 and 99, enterotoxin production and colicin resistance. Of these strains 308 belonged to one of 3 following different groups: Group 1: 0149, K88, producing heat-labile (LT) and heat-stable (ST) enterotoxins, group 2: 064, K99, producing ST, and group 3: variable O-antigens, no K-antigens or enterotoxin production. Almost 100 % of the E. coli strains were found to be resistant to colicins E1, E3, Ia, H and D+X. Resistance to colicins E2, B+M, V and K+X were found in 91.7 %, 43.8 %, 49.8 % and 62.2 % respectively. E. coli strains in group 1 were always (resistant to colicin E2, while about 87 % of the other strains were resistant to this colicin. E. coli strains in group 2 were more often resistant to colicin B+M, V and K+X (65 %, 94 %, 83 %) than strains in group 1 (37 %, 24 %, 64 %) and strains in group 3 (37 %, 52 %, 46 %). E. coli strains in group 2 showed a high degree of multiresistance, 45.1 % of the strains being resistant to all of the 9 colicins. About 10 % of the other strains were resistant to all of the 9 colicins. E. coli strains harbouring the enteropathogenicity factors K99 antigen and ST production, showed a higher degree of colicin resistance than both the E. coli strains with K88 antigen and ST and LT production, and the E. coli strains lacking enteropathogenicity factors.

Colicin Production in Relation to Pathogenicity Factors in Strains of Escherichia Coli Isolated from the Intestinal Tract of Piglets

Three hundred and fifteen E. coli strains isolated from the intestine of piglets were examined for K-antigens 88 and 99, enterotoxin production and colicin resistance. Of these strains 308 belonged to one of 3 following different groups: Group 1: 0149, K88, producing heat-labile (LT) and heat-stable (ST) enterotoxins, group 2: 064, K99, producing ST, and group 3: variable O-antigens, no K-antigens or enterotoxin production. Almost 100 % of the E. coli strains were found to be resistant to colicins E1, E3, Ia, H and D+X. Resistance to colicins E2, B+M, V and K+X were found in 91.7 %, 43.8 %, 49.8 % and 62.2 % respectively. E. coli strains in group 1 were always (resistant to colicin E2, while about 87 % of the other strains were resistant to this colicin. E. coli strains in group 2 were more often resistant to colicin B+M, V and K+X (65 %, 94 %, 83 %) than strains in group 1 (37 %, 24 %, 64 %) and strains in group 3 (37 %, 52 %, 46 %). E. coli strains in group 2 showed a high degree of multiresistance, 45.1 % of the strains being resistant to all of the 9 colicins. About 10 % of the other strains were resistant to all of the 9 colicins. E. coli strains harbouring the enteropathogenicity factors K99 antigen and ST production, showed a higher degree of colicin resistance than both the E. coli strains with K88 antigen and ST and LT production, and the E. coli strains lacking enteropathogenicity factors.

Escherichia coli K-12 outer membrane protein (OmpA) as a bacteriophage receptor: analysis of mutant genes expressing altered proteins.

The outer membrane protein OmpA of Escherichia coli K-12 serves as a receptor for a number of T-even-like phages. We have isolated a series of ompA mutants which are resistant to such phages but which still produce the OmpA protein. None of the mutants was able to either irreversibly or reversibly bind the phage with which they had been selected. Also, the OmpA protein is required for the action of colicins K and L and for the stabilization of mating aggregates in conjugation. Conjugal proficiency was unaltered in all cases. Various degrees of colicin resistance was found; however, the resistance pattern did not correlate with the phage resistance pattern. DNA sequence analyses revealed that, in the mutants, the 325-residue OmpA protein had suffered the following alterations: Gly-65----Asp, Gly-65----Arg, Glu-68----Gly, Glu-68----Lys (two isolates), Gly-70----Asp (four isolates), Gly-70----Val, Ala-Asp-Thr-Lys-107----Ala-Lys (caused by a 6-base-pair deletion), Val-110----Asp, and Gly-154----Ser. These mutants exhibited a complex pattern of resistance-sensitivity to 14 different OmpA-specific phages, suggesting that they recognize different areas of the protein. In addition to the three clusters of mutational alterations around residues 68, 110, and 154, a site around residue 25 has been predicted to be involved in conjugation and in binding of a phage and a bacteriocin (R. Freudl, and S. T. Cole, Eur. J. Biochem, 134:497-502, 1983; G. Braun and S. T. Cole, Mol. Gen. Genet, in press). These four areas are regularly spaced, being about 40 residues apart from each other. A model is suggested in which the OmpA polypeptide repeatedly traverses the outer membrane in cross-beta structure, exposing the four areas to the outside.

Genetics of colicin E susceptibility inCitrobacter freundii

The insensitivity of Citrobacter freundii to the E colicins is based on tolerance to colicin E1 and resistance to colicins E2 and E3. Spontaneous colicin A resistant mutants of C. freundii also lost their colicin E1 receptor function. Sensitivity to colicin E1 can be induced by F'gal+tol+ plasmids, the tol A+ gene product of which is responsible for this effect. Receptor function for colicins E2 and E3 is induced by the E. coli F'14 bfe+ plasmid, which is also able to enhance notably the receptor capacity for colicin E1. The bfe+ gene product of E. coli, which is responsible for these phenomena, also restores the receptor function for colicin A and E1 in colicin A resistant mutants of C. freundii. All results show that there is a remarkable difference between the E. coli bfe+ gene product and the bfe+ gene product of C. freundii and also between the tol A+ gene products of these strains. The sensitivity to phage BF23 parallels the sensitivity to colicins E2 and E3 and is also induced by the F'14 bfe+ plasmid.

Biosynthesis of the outer membrane receptor for vitamin B12, E colicins, and bacteriophage BF23 by Escherichia coli: kinetics of phenotypic expression after the introduction of bfe+ and bfe alleles.

The bfe locus codes for the cell surface receptor for vitamin B12, the E colicins, and bacteriophage BF23 in the Escherichia coli outer membrane. When the bfe+ allele, which is closely linked to the argH locus, was introduced into an argH bfe recipient by conjugation, arg+ recombinant cells rapidly and simultaneously acquired sensitivity to colicin E3 and phage BF23. In the reciprocal experiment introducing bfe into an argH bfe+ recipient, it was found that colicin E3-resistant, arg+ cells began to appear shortly after the arg+ recombinant population began to divide. This was far earlier than would have been predicted on the basis of 220 receptors per haploid cell. Moreover, there was a lag between the appearance of colicin resistance and the appearance of resistance to killing by phage BF23, and hence a period of time during which some arg+ recombinant cells were sensitive to the phage but resistant to the colicin. Colicin E3 added to cells during this period of time protected against phage killing, indicating that the colicin-resistant cells still had receptors capable of binding colicin on their surface. The modification of the phenotypic expression of colicin and phage resistance by inhibitors of deoxyribonucleic acid, ribonucleic acid, and protein synthesis was also investigated. The results obtained indicate that the receptor protein coded for by the bfe locus can exist on the cell surface in several different functional states.

Characterization of group B colicin-resistant mutants of Escherichia coli K-12: colicin resistance and the role of enterochelin.

Nine classes of group B colicin-resistant mutants were examined to study the role of enterochelin in colicin resistance. Four of the mutants studied (cbt, exbC, exbB, and tonB) hypersecreted enterochelin. Enterochelin hypersecretion was apparently responsible for resistance of the exbC mutant to colicins G and H and for resistance of the exbB mutant to colicins G, H, Ia, Ib, S1, and V. All four mutants scored as colicin B tolerant, even in the absence of enterochelin synthesis. The mutants produced substantially increased amounts of two high-molecular-weight outer membrane polypeptides when grown under limiting iron conditions. The presence of these polypeptides was correlated with increased colicin B-neutralizing activity in the outer membrane preparations.

A function common to iron-enterochelin transport and action of colicins B, I, V in Escherichia coli

the idea that enterochelin is overproduced due to an iron shortage in the cell caused by the lack of a membrane component in the mutants involved in both ironenterochelin uptake and killing of the cells by colicins B, I, V. Here we show that the colicin resistance is indeed independent of the presence of enterochelin and that colicin resistant strains are unable to transport enterochelin. The described function maps outside ton B at about 65 min of the genetic map of E. coli. Tentatively we designate the function and the corresponding gene feu Cferric enterochelin uptake) to distinguish it from the fep function at 13 min which is also involved in transport of ferric enterochelin [ 131.

Genetics of resistance to colicins in Escherichia coli K-12: cross-resistance among colicins of group A.

By using each of the available colicins, we have isolated a large number of colicin-resistant mutants. They included both receptor and tolerant mutants and each was screened for cross-resistance to all other colicins. On the basis of the cross-resistance of these mutants it was possible to place known colicins into two mutually exclusive groups, group A and group B. Mutants selected as resistant to colicins of group A may or may not be cross-resistant to other colicins of group A, BUT Are never resistant to colicins of group B. The reverse also applies. The mutants isolated as resistant to colicins of group A (A, E1, E2, E3, K, L, N, S4, and X) have been divided into 21 phenotypic classes on the basis of their colicin resistance patterns. These include most of the tolerant and receptor mutants previously isolated, some of which were previously shown to also have an increased sensitivity to certain antibiotics and detergents. Type strains from each of the phenotypic classes were therefore tested for sensitivity to a range of antibiotics, detergents, and surfactants that included all those previously used. With these new data, it has been possible to speculate informatively on the mode of action of the different colicins. We have confirmed the position of previously isolated mutations on the Escherichia coli K-12 genetic map, and located approximately the loci conferring colicin resistance in some of the newly isolated mutants.

Novel approaches to the mode of action of colicins.

According to the theory of Fredericq (1949) and Nomura (1964), colicins are attached by specific receptor sites in the cell walls of sensitive bacteria, which mediate their inhibitive effects. During last years, a great variety of experimental data have been accumulated, some of which cannot be easily interpreted in terms of this theory. There exist considerable discrepancies concerning the chemical nature and molecular weight of isolated receptors. The attachment of a colicin onto its receptor need not be irreversible. The inhibition of numerous membrane-associated functions in colicin-tolerant mutants suggests their pleiotropic deletion nature. The difference between colicin resistance and colicin tolerance does not seem to be clear-cut. Cells of stable L-forms of protoplast type, completely devoid of their walls, retain in most cases the same patterns of sensitivity to colicins as rods of the same strains. Experimental changes in the relationship between the cell wall and the cytoplasmic membrane decrease colicin sensitivity of the cells. Colicin E3 has been found to be a specific endoribonuclease, able to cleave a terminal fragment from the 16 S rRNA also in isolated ribosomes in vitro: not only in ribosomes from sensitive bacteria, but also in those from resistant ones and from eukaryotic cells. A destabilization of the DNA helix was induced by colicin E2 in vitro as in vivo. It seems that there exist two distinct types of colicin receptors with different functions: those in the cell wall, and those in the cytoplasmic membrane. Only the contact of colicins with the latter ones is biologically effective and starts both stages of their inhibitive effect: the reversible and the irreversible ones.

Colicin resistance associated with resistance factors in Escherichia coli.

Derivatives ofE. coliK12 which had received a number of different drug resistance factors by conjugation fromSalmonellastrains were found also to have acquired insensitivity to colicins. The insensitivity associated with some R factors was similar in character to immunity conferred by colicinogeny, while that given by others more closely resembled the higher level of resistance due to absence of the colicin receptor. It was, however, not complete. Many of the R factors also restricted the growth of various phages.