Allelopathy of bacteria in a lattice population: Competition between colicin-sensitive and colicin-producing strains

We analyse the population dynamics of two strains of bacteria (Escherichia coli): one strain produces a toxin (called colicin) that increases the mortality of a colicin-sensitive strain in the neighbourhood, but does not harm the colicin-producing strain itself. On the other hand, in the absence of colicin in the environment, the colicin-sensitive strain enjoys a higher population growth rate. The model is closely related to the evolutionary dynamics of social interaction. It has been established previously that a perfectly mixing population shows bistability; that is, whichever strain dominates initially tends to defeat the other. On the other hand, empirical and computer simulation results in lattice structured populations show neither co-existence nor bistability of the two strains. In this paper, we analyse the lattice model based on pair approximation (forming a system of ordinary differential equations of global densities and local densities), and by the direct c omputer simulation of the spatial stochastic model. Both the pair approximation dynamics and the computer simulation show that, for most regions of parameter values, one strain defeats the other, irrespective of the initial abundance. However, the pair approximation analysis also suggests a relatively narrow parameter region of bistability, which should disappear when the model is considered on a lattice of infinitely large size. The biological implications of the results and the relationship of the present model with other models of the evolution of spite or altruistic behaviours are discussed. This suggests that the dynamics reflected by the spatially explicit lattice model may be sufficient, perhaps even necessary, to support the evolution of colicin.     

Translocation of colicin from the receptor to the inner cell membrane: function of the peptidoglycan layer

Sensitivity of spheroplasts (prepared in two ways) of a colicin-sensitive strain, of colicinresistant and of colicin-tolerant mutants and of strains immune to colicins E1 and E2 was estimated and compared. Generally, the removal of the peptidoglycan layer brought about a slight nonspecific support for colicin translocation across the cell wall in sensitive,tolB tolerant and immune bacteria.tolB spheroplasts were colicin E1-sensitive, but E2-insensitive. Spheroplasts were always fragile and lysed spontaneously, especially those produced by lysozyme. Bacteria carryingtolA, tolQ andtolR mutations kept their colicin insensitivity as spheroplasts, just as the resistant ones. Bacteria rendered colicinogenic and hence colicin-immune turned to high colicin sensitivity in spheroplast form. The results indicate a change in plasma membrane associated with the spheroplast formation.     

Derepression of colicin E1 synthesis in the constitutive tif mutant strain (spr tif sfi) and in a tif sfi mutant strain of Escherichia coli K-12.

We show here that expression of the colicin gene of the ColE1 plasmid is greatly derepressed in Escherichia coli K-12 strain DM1187 spr tif sfi, which is a constitutive tif mutant, altered in the lexA gene, and which shows constitutive expression of various pathways of the recA-dependent, lexA-blocked (SOS) repair system. In this strain colicin E1 synthesis is at least 100-fold greater than that observed in uninduced control strains (spr+ tif sfi and spr+ tif+ sfi). This result confirms the regulatory role of the lexA product in colicin E1 synthesis. Colicin yields by the uninduced strain DM1187 are as high as the maximum yields from mitomycin-induced control strains and often are several-fold higher. When the nonconstitutive tif sfi strain GC467 is raised to 43 degrees C to induce the SOS system, a low level of colicin synthesis is observed which is less than one-tenth of the yield obtained by induction with mitomycin C. Addition of adenine at the time of shift-up can increase the colicin yield of tif sfi to about one-third of the yield obtained with mitomycin C. We have also found that colicin overproduction can be detected by altered colony appearance in an overlay assay with colicin-sensitive bacteria. In addition, the lethality of the process of colicin synthesis is observed here without the use of bacteriostatic inducing agents.     

Lipopolysaccharide Defective Mutant of Escherichia coli with Decreased Sensitivity to Colicin E2

Several colicin-sensitivity mutants were isolated from Escherichia coli K-12. The mutants could not form colonies in the presence of colicin E2, but recovered their colony-forming ability on trypsin treatment even after prolonged incubation with the colicin. They showed increased sensitivity to hydrophobic antibiotics and detergents, as well as resistance against P1 and T4 phages, both of which seemed due to structural changes of lipopolysaccharide (LPS). Quantitative analysis by gas-liquid chromatography revealed that the mutant-LPS contained a different stereoisomer of heptose with decreased amounts of neutral sugars (rhamnose, glucose and galactose). LPS extracted from the parental colicin-sensitive strain could neutralize the killing activity of colicin E2 in vitro, but the mutant-LPS could not. The mutant strains retained functional receptor proteins for colicin E2. These observations suggest that LPS plays an important role in the early stage of the interaction of colicin E2 with E. coli cells.     

Isolation and characterization of ColE2 plasmid mutants unable to kill colicin-sensitive cells

Summary After transfer from a mutagenized host, twenty one ColE2 plasmid mutants were isolated after screening 10,000 clones for abnormal colicin production. Analysis by SDS polyacrylamide slab gel electrophoresis of proteins synthesized after mitomycin C-induction of mutant cultures, indicates that all but two of the mutations are in the structural gene for colicin E2. Of these, nine produce fragments of colicin in both whole cells and minicells and some are suppressed by nonsense suppressors.Studies with a nonsense mutant producing only a small colicin E2 fragment (ColE2-421) suggest that colicin E2 is not involved in plasmid DNA replication, in the control of its own synthesis, or required for cell death when cells become committed to colicin production. The two plasmid mutants outside the colicin gene segregate plasmid-free cells at 33°, 37° and 43°. One segregates fairly rapidly (about 4% per generation) though the colicin-producing cells make normal amounts of colicin, whilst the other segregates more slowly and the colicin-producing cells make much reduced amounts of colicin.     

Two new E colicins, E8 and E9, produced by a strain of Escherichia coli

We have isolated a strain of Escherichia coli from chicken caeca which produces two E colicins and colicin M. This strain has seven plasmids, five of which have been transferred to E. coli K12. Two E. coli K12 derivatives which produce the two E colicins separately have been tested against seven standard E colicin producing strains which define seven different immunity groups. Our results indicate that these new E colicins define two further immunity groups, E8 and E9.     

Inhibition of lipopolysaccharide O-antigen synthesis by colicin M

Colicin M inhibits peptidoglycan biosynthesis at the level of the bactoprenyl carrier lipid. Since the synthesis of O-antigen also requires bactoprenyl carrier lipid, the effect of colicin M on O-antigen biosynthesis was studied using a colicin-sensitive strain of Salmonella typhimurium. Determination of O-antigen intermediates by two different methods showed that bactoprenyl-dependent O-antigen biosynthesis was inhibited by colicin M. Synthesis of both O-antigen and peptidoglycan was almost immediately inhibited following colicin addition. This was followed some 20 min later by cell lysis. The only known common step between O-antigen and peptidoglycan synthesis is formation of bactoprenyl phosphate by dephosphorylation of bactoprenyl pyrophosphate. Determination of bactoprenyl phosphates showed an accumulation of bactoprenyl pyrophosphate in colicin-treated cultures. It was concluded that dephosphorylation of the bactoprenyl lipid carrier was inhibited by colicin M, and this in turn prevented both O-antigen and peptidoglycan synthesis.     

In vivo formation of transmissible resistance factor by recombination between nontransmissible resistance factor and Col B factor.

Germ-free swine were artificially contaminated with tetracycline (TC) sensitive strains of Escherichia coli and Klebsiella pneumoniae. One of these strains, E. coli 3306, was infected with a plasmid carrying kanamycin (KM) resistance, i.e., T-kan factor. Another strain, E. coli P-5, carried a conjugally transferable Col B factor. Among the nine strains used, only E. coli P-38 became TC-resistant after TC administration. Three types of TC-resistance E. coli P-38 strains were found; (a) one strain carried nontransferable TC resistance and could not produce colicin, (b) one strain carried TC resistance with a high transmission frequency which could not produce colicin, and (c) one strain carried TC resistance with a low transmission frequency that could produce colicin B. Genetic studies disclosed that the transmissible TC resistance factors, i.e., Rms105 (group b) and Rms104 (group c), were formed by recombination between Col B factor and nontransmissible TC-resistance (tet) determinant which appeared in E. coli P-38 mutants.     

Colicin E2 production and release by Escherichia coli K12 and other Enterobacteriaceae

Previous work has shown that Escherichia coli K12 ColE2+ cells undergo a form of partial lysis and exhibit increases in lysophosphatidylethanolamine (lysoPE) and free fatty acid content due to activation of phospholipase A when induced to produce and release colicin E2. The increase in lysoPE content was assumed to be essential for efficient colicin release. These same characteristics are also presented by some natural ColE2+ isolates, and by other representatives of the Enterobacteriaceae after transformation with derivatives of a ColE2 plasmid. However, Salmonella typhimurium strains carrying ColE2 plasmids released colicin without partial lysis and without increasing their lysoPE content. A previously undetected minor phospholipid, which appeared in these and other strains only when they were induced to produce colicin, may be an important factor in colicin release. In ColE2+ E. coli K12, production of this new lipid was dependent on phospholipase A activation following expression of the ColE2 lysis gene. Some other ColE2+ strains did not respond to induction of colicin production in the same way as ColE2+ E. coli K12. These strains were less sensitive to inducer (mitomycin C) or unable to produce increased amounts of colicin in response to induction, or unable to degrade colicin once it was released. In general, the results suggest that colicin release occurs by the same or similar processes in the various strains tested, and support the continued use of E. coli K12 as the model strain for studying the mechanisms of colicin release.     

Production of colicins V and V2 in vitro and in vivo. Study of their inhibitory action on phagocytosis by peritoneal macrophages]

In recent years, a possible relationship between pathogenicity and colicinogeny in some Escherichia coli colicin V-producing strains had been inferred. In our laboratory, we have elaborated a simple in vitro method for the production of colicin V free of large, non dialyzable macromolecules and presumably of bacterial endotoxin. This allows study of the effects of colicin V in vivo without an undesirable added physiological response of the experimental animal to endotoxin. All the Col V+ strains we have studied displayed a greater ability to survive in the peritoneal cavity of mice than the Col V- strains. Also, we have detected colicin V in peritoneal fluids of agonizing mice injected with Col V+ strains. Phagocytosis by peritoneal macrophages seemed to be inhibited in vitro in the presence of colicin V. Colicin V is not toxic in vivo in low concentration, after intraperitoneal or intravenous injection but it may favor the multiplication and the invasiveness of the strains that produce it.     

Involvement of colicin in the limited protection of the colicin producing cells against bacteriophage

The restriction/modification system is considered to be the most common machinery of microorganisms for protection against bacteriophage infection. However, we found that mitomycin C induced Escherichia coli containing ColE7-K317 can confer limited protection against bacteriophage M13K07 and lambda infection. Our study showed that degree of protection is correlated with the expression level of the ColE7 operon, indicating that colicin E7 alone or the colicin E7-immunity protein complex is directly involved in this protection mechanism. It was also noted that the degree of protection is greater against the single-strand DNA bacteriophage M13K07 than the double-strand bacteriophage(lambda). Coincidently, the K(A) value of ColE7-Im either interacting with single-strand DNA (2.94x10(5)M(-1)) or double-strand DNA (1.75x10(5)M(-1)) reveals that the binding affinity of ColE7-Im with ssDNA is 1.68-fold stronger than that of the protein complex interacting with dsDNA. Interaction between colicin and the DNA may play a central role in this limited protection of the colicin-producing cell against bacteriophages. Based on these observations, we suggest that the colicin exporting pathway may interact to some extent with the bacteriophage infection pathway leading to a limited selective advantage for and limited protection of colicin-producing cells against different bacteriophages.     

Evolution of microparasites in spatially and genetically structured host populations: The example of RHDV infecting rabbits

Several studies have shown that classical results of microparasite evolution could not extend to the case where the host species shows an important spatial structure. Rabbit haemorrhagic disease virus (RHDV), responsible for rabbit haemorrhagic disease (RHD), which recently emerged in rabbits, has strains within a wide range of virulence, thus providing an interesting example of competition between strains infecting a host species with a metapopulation structure. In addition, rabbits may show a genetic diversity regarding RHDV susceptibility. In the present paper we use the example of the rabbit-RHDV interaction to study the competition between strains of a same microparasite in a host population that is both spatially and genetically structured. Using metapopulation models we show that the evolution of the microparasite is guided by a trade-off between its capacity to invade subpopulations potentially infected by other strains and its capacity to persist within the subpopulation. In such a context, host genetic diversity acts by reducing the number of hosts susceptible to each strain, often favouring more persistent—and generally less virulent—strains. We also show that even in a stochastic context where host genes regularly go locally extinct, the microparasite pressure helps maintain the genetic diversity in the long term while reinforcing gene loss risk in the short term. Finally, we study how different demographic and epidemiologic parameters affect the coevolution between the rabbit and RHDV.     

Interaction of Colicins with Bacterial Cells IV. Immunity Breakdown Studied with Colicins Ia and Ib

Colicinogenic cells are immune to the lethal effect of the colicin which they produce. In the presence of very high concentrations of colicin, however, colicinogenic cells are no longer immune to the homologous colicin. This phenomenon, immunity breakdown, was studied with colicins Ia and Ib. The biochemical effects of colicin Ib on Escherichia coli were studied with a standard noncolicinogenic strain. At multiplicities of about 10 or higher, colicin Ib inhibited incorporation of leucine into protein and incorporation of (32)P-inorganic phosphate into deoxyribonucleic acid and ribonucleic acid by more than 95%. Under the same conditions, (32)P incorporation into phospholipid and nucleotide fractions was inhibited only partially (about 80 and 60%, respectively). Inhibition of (32)P incorporation into the terminal phosphorus of adenosine triphosphate was also considerably less than that of macromolecular synthesis (50 to 60%). (32)P incorporation into the nonnucleotide organic phosphate fraction was not inhibited. Respiration was not affected. Colicin Ia showed the same biochemical effects as colicin Ib. A mutant of an Ib-colicinogenic E. coli strain selected for resistance to low concentrations of colicin Ia was shown to be resistant to high concentrations of homologous colicin Ib, whereas the parent Ib-colicinogenic strain is sensitive to high concentrations of colicin Ib. This mutant lost its specific receptors for colicin Ib. Moreover, the biochemical effects of high concentrations of colicin Ib on Ib-colicinogenic cells during immunity breakdown were similar to the effects found in sensitive cells exposed to low concentrations of the same colicin. It is concluded that the killing of colicinogenic cells in the presence of high concentrations of homologous colicin is indeed caused by the homologous colicin molecules.     

Escherichia coli K317, formerly used to define colicin group E2, produces colicin E7, is immune to colicin E2, and carries a bacteriophage-restricting conjugative plasmid.

Escherichia coli strain CL137, a K-12 derivative made E colicinogenic by contact with Fredericq's strain K317, was unaffected by colicin E2-P9, but K-12 carrying ColE2-P9 was sensitive to the E colicin made by strains CL137 and K317. This colicin we named E7-K317 because by the test of colicinogenic immunity it differed from colicins E1-K30, E2-P9, and E3-CA38 and from recently recognized colicins termed E4Horak, E5, and E6. Strain K317 as conjugational donor transmitted E7 colicinogeny; about half the E7-colicinogenic transconjugants were immune to colicin E2-P9. A spontaneous variant of CL137 retained E7 colicinogeny but was sensitive to E2 colicins. We attribute the E2 immunity of strain CL137 and some E7-coliconogeic transconjugants to a "colicin-immunity plasmid," ColE2imm-K317, from strain K317. Tra+ E7-colicinogenic transconjugants restricted phage BF23 in the same way as strains carrying ColIb-P9. We attribute Tra+ and restricting ability to a plasmid, pRES-K317, acquired from strain K317, and related to the ColI plasmids.     

FtsH-dependent processing of RNase colicins D and E3 means that only the cytotoxic domains are imported into the cytoplasm

It has long been suggested that the import of nuclease colicins requires protein processing; however it had never been formally demonstrated. Here we show that two RNase colicins, E3 and D, which appropriate two different translocation machineries to cross the outer membrane (BtuB/Tol and FepA/TonB, respectively), undergo a processing step inside the cell that is essential to their killing action. We have detected the presence of the C-terminal catalytic domains of these colicins in the cytoplasm of target bacteria. The same processed forms were identified in both colicin-sensitive cells and in cells immune to colicin because of the expression of the cognate immunity protein. We demonstrate that the inner membrane protease FtsH is necessary for the processing of colicins D and E3 during their import. We also show that the signal peptidase LepB interacts directly with the central domain of colicin D in vitro and that it is a specific but not a catalytic requirement for in vivo processing of colicin D. The interaction of colicin D with LepB may ensure a stable association with the inner membrane that in turn allows the colicin recognition by FtsH. We have also shown that the outer membrane protease OmpT is responsible for alternative and distinct endoproteolytic cleavages of colicins D and E3 in vitro, presumably reflecting its known role in the bacterial defense against antimicrobial peptides. Even though the OmpT-catalyzed in vitro cleavage also liberates the catalytic domain from colicins D and E3, it is not involved in the processing of nuclease colicins during their import into the cytoplasm.     

A spatial model of the evolution of quorum sensing regulating bacteriocin production

Like any form of cooperative behavior, quorum sensing (QS) inbacteria is potentially vulnerable to cheating, the occurrenceof individuals that contribute less but still profit from thebenefits provided by others. In this paper, we explore the evolutionarystability of QS as a regulatory mechanism of antibiotics productionin a spatially structured population, using cellular automaton(CA) modeling. QSg is supposed to regulate the excretion ofa bacteriocin (anticompetitor toxin) in a population of bacteriapolymorphic for the ability to produce and to be immune to thebacteriocin. Both the social interactions resulting from QSand the competitive interactions resulting from the bacteriocinexcretion are supposed to be only effective at the local scale,that is, restricted to the immediately neighboring cells. Thisimplies a rather diffuse kind of group selection. The CA modelis contrasted to a model assuming no spatial structure but withotherwise identical assumptions. Our analysis predicts thatQS as a regulatory mechanism of bacteriocin excretion is evolutionarilyunstable when the competitive interactions between bacteriocin-producing,resistant, and sensitive strains only involve closely relatedstrains which can share the signaling and responding genes involvedin QS. However, when the competition is between unrelated strainsand the QS alleles can only be carried by the bacteriocin-producingstrains, stable QS may evolve provided its costs are small andthe critical quorum threshold is neither too low nor too high.     

Colicins produced by the Escherichia fergusonii strains closely resemble colicins encoded by Escherichia coli

Plasmid DNA of six Escherichia fergusonii colicinogenic strains (three producers of colicin E1, two of Ib and one of Ia) was isolated and the colicin-encoding regions of the corresponding Col plasmids were sequenced. Two new variants of colicin E1, one of colicin Ib, and one of colicin Ia were identified as well as new variants of the colicin E1 and colicin Ib immunity proteins and the colicin E1 lysis polypeptide. The recombinant Escherichia coli producer harboring pColE1 from E. fergusonii strain EF36 (pColE1-EF36) was found to be only partially immune to E1 colicins produced by two other E. fergusonii strains suggesting that pColE1-EF36 may represent an ancestor ColE1 plasmid.     

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.     

Consequences of host heterogeneity, epitope immunodominance, and immune breadth for strain competition

Consumer-resource dynamics of hosts with their pathogens are modulated by complex interactions between various branches of hosts’ immune systems and the imperfectly perceived pathogen. Multistrain SIR models tend to sweep competitive interaction terms between different pathogen strains into a single parameter representing cross-immunity. After reviewing several hypotheses about the generation of immune responses, we look into the consequences of assuming that hosts with identical immune repertoires respond to new pathogens identically. In particular, we vary the breadth of the typical immune response, or the average number of pathogen epitopes a host perceives, and the probability of perceiving a particular epitope. The latter quantity in our model is equivalent both to the degree of diversity in host responses at the population level and the relative immunodominance of different epitopes. We find that a sharp transition to strain coexistence occurs as host responses become narrow or skewed toward one epitope. Increasing the breadth of the immune response and the immunogenicity of different epitopes typically increases the range of cross-immunity values in which chaotic strain dynamics and competitive exclusion occur. Models attempting to predict the outcomes of strain competition should thus consider the potential diversity and specificity of hosts’ responses to infection.     

Novel colicin Fy of Yersinia frederiksenii inhibits pathogenic Yersinia strains via YiuR-mediated reception, TonB import, and cell membrane pore formation

A novel colicin type, designated colicin Fy, was found to be encoded and produced by the strain Yersinia frederiksenii Y27601. Colicin Fy was active against both pathogenic and nonpathogenic strains of the genus Yersinia. Plasmid YF27601 (5,574 bp) of Y. frederiksenii Y27601 was completely sequenced. The colicin Fy activity gene (cfyA) and the colicin Fy immunity gene (cfyI) were identified. The deduced amino acid sequence of colicin Fy was very similar in its C-terminal pore-forming domain to colicin Ib (69% identity in the last 178 amino acid residues), indicating pore forming as its lethal mode of action. Transposon mutagenesis of the colicin Fy-susceptible strain Yersinia kristensenii Y276 revealed the yiuR gene (ykris001_4440), which encodes the YiuR outer membrane protein with unknown function, as the colicin Fy receptor molecule. Introduction of the yiuR gene into the colicin Fy-resistant strain Y. kristensenii Y104 restored its susceptibility to colicin Fy. In contrast, the colicin Fy-resistant strain Escherichia coli TOP10F' acquired susceptibility to colicin Fy only when both the yiuR and tonB genes from Y. kristensenii Y276 were introduced. Similarities between colicins Fy and Ib, similarities between the Cir and YiuR receptors, and the detected partial cross-immunity of colicin Fy and colicin Ib producers suggest a common evolutionary origin of the colicin Fy-YiuR and colicin Ib-Cir systems.