Cross-resistance between bacteriophages and colicins in Escherichia coli K-12.

Cross-resistance between bacteriophages and colicins was studied using collections of bacteriophage- and colicin-resistant mutants of Escherichia coli K-12. No new examples were found of highly specific one-to-one cross-resistance of the type suggestive of common receptors. However, several groups of mutants showed tolerance to colicins and resistance to bacteriophages. Mutants known to be very defective in lipopolysaccharides composition were found to commonly show tolerance to certain colicins in addition to their bacteriophage resistance. Another group of mutants showed varying patterns of resistance to colicins E2, E3, K, L, A, S4, N, and X and bacteriophages E4, K2, K20, K21, K29, and H+. However, many bacteriophage-resistant mutants were fully colicin sensitive, and most colicin-resistant mutants were fully sensitive to bacteriophages.     

The tol gene products and the import of macronmolecules into Escherichia coli

Genetic studies have identified a number of genes whose products appear to be required for the transport of the group A colicins and the single-stranded DNA of certain filamentous bacteriophages into Escherichia coli. Mutations in these genes allow normal binding of the colicins to their outer-membrane receptors and of the bacteriophage of the tip of specific conjugative pili, but do not allow translocation of the macromolecules to their target. These mutations have been designed 'tolerant' (tol) mutations and the protein products specified by these genes appear to comprise part of a transport system known as the Tol import system. Some of these genes have been isolated, sequenced and their protein products localized to the membranes or periplasm of E. coli. Information is also available regarding the domains of the colicins or phage proteins which interact with the Tol proteins. A preliminary model of the location and possible interactions of the Tol proteins is presented.     

Shielding of Escherichia coli outer membrane proteins as receptors for bacteriophages and colicins by O-antigenic chains of lipopolysaccharide.

The accessibility of several outer membrane proteins for bacteriophages and colicins in isogenic smooth and rough Escherichia coli strains was investigated. The results show that O antigen carrying lipopolysaccharide is able to prevent access of all phages and colicins tested to their outer membrane protein receptors.     

Use of lipophilic cation-permeable mutants for measurement of transmembrane electrical potential in metabolizing cells of Escherichia coli.

Some lipopolysaccharide-defective mutants of Escherichia coli showed, without ethylenediaminetetraacetic acid treatment, a quick and high uptake of lipophilic cations such as triphenylmethylphosphonium and tetraphenylphosphonium. The rate and amount of uptake were comparable to those of an ethylenediaminetetraacetic acid-treated wild type. Transmembrane electrical potential, which was calculated from the distribution of these lipophilic cations between the inside and outside of the mutant cells, was about -150 mV at pH 7.5 and showed a strong dependency on the external pH. One of the E. coli mutants, the acrA mutant, was found to be also permeable to dicyclohexylcarbodiimide, an H+-adenosine triphosphatase inhibitor, and 1-anilino-8-naphthalene sulfonate, a fluorescent dye. The acrA mutant was vigorously motile and highly sensitive to many bacteriophages and colicins. Thus, the acrA mutant is quite useful for the quantitative measurement of transmembrane electrical potential by lipophilic cations in intact and metabolizing cells especially in relation to motility and actions of colicins and bacteriophages.     

Solution structure of the E.coli TolA C-terminal domain reveals conformational changes upon binding to the phage g3p N-terminal domain

The Tol-Pal system of Escherichia coli is a macromolecular complex located in the cell envelope. It is involved in maintaining the integrity of the outer membrane and is required for the uptake of two different types of macromolecules, which are bacteriotoxins (colicins) and DNA of filamentous bacteriophages. The TolA protein plays a central role in these import mechanisms. Its C-terminal domain (TolAIII) is involved in the translocation step via direct interaction with the N-terminal domain of colicins and the N-terminal domain of the phage minor coat gene 3 protein (g3pN1). Extreme behaviours of TolAIII have been previously observed, since the structure of TolAIII either remained unaffected or adopted disordered conformation upon binding to different pore-forming colicins. Here, we have solved the 3D structure of free TolAIII by heteronuclear NMR spectroscopy and compared it to the crystal structure of TolAIII bound to g3pN1 in order to study the effect of g3pN1 on the tertiary structure of TolAIII. Backbone 1H, 15N and 13C resonances of the g3pN1-bound TolAIII were also assigned and used to superimpose the solution structure of free TolAIII on the crystal structure of the g3pN1-TolAIII fusion protein. This allowed us to track conformational changes of TolAIII upon binding. While the global fold of free TolAIII is mainly identical to that of g3pN1-bound TolAIII, shift of secondary structures does occur. Thus, TolAIII, which interacts also in vivo with Pal and TolB, is able to adapt its conformation upon binding to various partners. Possible models for protein binding mechanisms are discussed to explain this so-far unobserved behaviour of TolAIII.     

Cointegrational transduction and mobilization of gentamicin resistance plasmid pWP14a is mediated by IS140

The structures of two R-plasmids pWP14a and pWP12a (Tra-, Ap, Gm; 21 kb) and of several cointegrates they form with bacteriophages P1Cm and P1-15 were analyzed. In each case, replicon fusion was mediated by the element IS140 (about 0.8 kb), one copy of which resides on both plasmids adjacent to the gentamicin resistance determinant (AAC(3)-III). pWP14a cointegrated preferentially into or near the invertible C-loop structure of the P1 genome. Cointegrational mobilization of pWP14a was observed also with several conjugative R-factors. The process of replicon fusion is independent of the host's rec+ functions. Sequences homologous to IS140 are constituents of many R-factors, including RA1, R40a, R124, R144, Rts1, N3, and pJR255. IS140 also shows homology to two other sequences, IS15 delta and Tn2680, but not to other, well studied transposable elements. The ampicillin resistance determinant of pWP14a is within a Tn3-like transposon, Tn3651.     

Import of biopolymers into Escherichia coli: nucleotide sequences of the exbB and exbD genes are homologous to those of the tolQ and tolR genes, respectively.

Escherichia coli with mutations in the exb region are impaired in outer membrane receptor-dependent uptake processes. They are resistant to the antibiotic albomycin and exhibit reduced sensitivity to group B colicins. A 2.2-kilobase-pair DNA fragment of the exb locus was sequenced. It contained two open reading frames, designated exbB and exbD, which encoded polypeptides of 244 and 141 amino acids, respectively. Both proteins were found in the cytoplasmic membrane. They showed strong homologies to the TolQ and TolR proteins, respectively, which are involved in uptake of group A colicins and infection by filamentous bacteriophages. exbB and exbD were required to complement exb mutations. Osmotic shock treatment rendered exb mutants sensitive to colicin M, which was taken as evidence that the ExbB and ExbD proteins are involved in transport processes across the outer membrane. It is concluded that the exb- and tol-dependent systems originate from a common uptake system for biopolymers.     

Allosteric β‐propeller signalling in TolB and its manipulation by translocating colicins

The Tol system is a five‐protein assembly parasitized by colicins and bacteriophages that helps stabilize the Gram‐negative outer membrane (OM). We show that allosteric signalling through the six‐bladed β‐propeller protein TolB is central to Tol function in Escherichia coli and that this is subverted by colicins such as ColE9 to initiate their OM translocation. Protein–protein interactions with the TolB β‐propeller govern two conformational states that are adopted by the distal N‐terminal 12 residues of TolB that bind TolA in the inner membrane. ColE9 promotes disorder of this ‘TolA box’ and recruitment of TolA. In contrast to ColE9, binding of the OM lipoprotein Pal to the same site induces conformational changes that sequester the TolA box to the TolB surface in which it exhibits little or no TolA binding. Our data suggest that Pal is an OFF switch for the Tol assembly, whereas colicins promote an ON state even though mimicking Pal. Comparison of the TolB mechanism to that of vertebrate guanine nucleotide exchange factor RCC1 suggests that allosteric signalling may be more prevalent in β‐propeller proteins than currently realized.     

Spontaneous tandem amplification and deletion of the shiga toxin operon in Shigella dysenteriae 1

Only one species of Shigella, Shigella dysenteriae 1, has been demonstrated to produce Shiga toxin (Stx). Stx is closely related to the toxins produced by Shiga toxin-producing Escherichia coli (STEC). In STEC, these toxins are often encoded on lambdoid bacteriophages and are major virulence factors for these organisms. Although the bacteriophage-encoded stx genes of STEC are highly mobile, the stx genes in S. dysenteriae 1 have been believed to be chromosomally encoded and not transmissible. We have located the toxin genes of S. dysenteriae 1 to a region homologous to minute 30 of the E. coli chromosome, within a 22.4 kbp putative composite transposon bracketed by IS600 insertion sequences. This region is present in all the S. dysenteriae 1 strains examined. Tandem amplification occurs via the flanking insertion sequences, leading to increased toxin production. The global regulatory gene, fnr, is located within the stx region, allowing deletions of the toxin genes to be created by anaerobic growth on chlorate-containing medium. Deletions occur by recombination between the flanking IS600 elements. Lambdoid bacteriophage genes are found both upstream and within the region, and we demonstrate the lysogeny of Shigella species with STEC bacteriophages. These observations suggest that S. dysenteriae 1 originally carried a Stx-encoding lambdoid prophage, which became defective due to loss of bacteriophage sequences after IS element insertions and rearrangements. These insertion sequences have subsequently allowed the amplification and deletion of the stx region.     

Isolation and preliminary characterization of mutants of Escherichia coli K-12 overproducers of 2 exported proteins: beta-lactamase and alkaline phosphatase

Spontaneous mutants of Escherichia coli characterized by the overproduction of two periplasmic proteins, beta-lactamase and alkaline phosphatase were isolated. Such olp (Overproduction of beta-Lactamase and alkaline Phosphatase) mutants were selected for growth in the presence of ampicillin and were identified on the basis of their increased content in alkaline phosphatase activity. Phenotypic analysis of olp mutants (resistance to bacteriophages and colicins) suggest that the organisation of their envelope has been deeply modified. Analysis of their cell envelope protein composition indicated that most mutants have a decreased content of porin proteins OmpF and OmpC. These mutations were mapped near the mtl locus, at minute 81 of the bacterial genetic map.     

Characteristics of Escherichia coli grown in bay water as compared with rich medium

Membrane-filtered bay water can support a certain degree of growth of Escherichia coli organisms isolated from the bay water or from sewage. The effect of the growth medium (bay water versus rich medium) on sensitivities to antimicrobial agents and cell envelope proteins was studied in many of these strains. Bay water-grown cells were less sensitive to bacteriophages and colicins, but were more sensitive to heavy metals and detergents as compared with rich-medium-grown cells. These results indicated that the cell envelope composition of the bay water-grown cells could be modified, resulting in altered susceptibility to various antimicrobial agents. An analysis of cell envelope proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that cells from rich-medium-grown cultures contained two or three major outer membrane proteins, whereas in bay water-grown cells, the OmpF protein was greatly reduced.     

Characteristics of Escherichia coli grown in bay water as compared with rich medium.

Membrane-filtered bay water can support a certain degree of growth of Escherichia coli organisms isolated from the bay water or from sewage. The effect of the growth medium (bay water versus rich medium) on sensitivities to antimicrobial agents and cell envelope proteins was studied in many of these strains. Bay water-grown cells were less sensitive to bacteriophages and colicins, but were more sensitive to heavy metals and detergents as compared with rich-medium-grown cells. These results indicated that the cell envelope composition of the bay water-grown cells could be modified, resulting in altered susceptibility to various antimicrobial agents. An analysis of cell envelope proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that cells from rich-medium-grown cultures contained two or three major outer membrane proteins, whereas in bay water-grown cells, the OmpF protein was greatly reduced.     

Isolation and characterization of extragenic suppressor mutants of the tolA-876 periplasmic-leaky allele in Escherichia coli K-12

tolA mutants of Escherichia coli K-12 release periplasmic proteins into the extracellular medium; they are sensitive to growth inhibitors such as cholic acid and tolerant to group A colicins and filamentous bacteriophage. Suppressor mutants of the tolA-876 allele were isolated by selecting for cholic acid resistant clones that did not release periplasmic ribonuclease I. One class of tolA suppressor strains carried mutations in the staA gene (for suppressor of tolA) located a 41 min. tolA-876 staA strains partially recovered a wild-type phenotype: they exported alkaline phosphatase and beta-lactamase into the periplasm and only released very low amounts of periplasmic proteins; moreover, they were sensitive to E1 and A colicins and more resistant than tolA-876 staA+ strains to various growth inhibitors. Furthermore, tolA-876 staA-2 and tolA+staA-2 mutants were 10- to 2700-times more resistant than staA+ strains to bacteriophages TuIa, TuIb and T4, and TuII whose receptors are major outer membrane proteins OmpF, OmpC and OmpA, respectively. SDS-PAGE analysis suggested that cell envelopes of staA or staA+ strains contained similar amounts of these proteins but characterization of strains carrying ompF (or C or A)-phoA gene fusions showed that mutation stA-2 reduced ompF gene expression by a factor of two. Analysis of double mutants strains carrying mutation staA-2 and a tolA, tolB, excC or excD periplasmic-leaky mutation showed that staA suppression was allele specific which suggested that proteins TolA and StaA might directly interact.     

Serotype-converting bacteriophages and O-antigen modification in Shigella flexneri

O-antigen modification (serotype conversion) in Shigella flexneri, which is an important virulence determinant, is conferred by temperate bacteriophages. Several serotype-converting phages have been isolated and preliminary characterization has identified the genes involved in O-antigen modification, and has also provided insight into the molecular biology of these phages.     

Incidence of lysogenic, colicinogenic and siderophore-producing strains among human non-pathogenicEscherichia coli

The current incidence ofEscherichia coli strains in healthy humans capable of producing the inhibitory exoproducts, such as temperate bacteriophages, corpuscular or HMW (high-molar mass) and proteinaceous or LMW (low-molar mass) colicins and siderophores was determined. Fifty-threeE. coli strains were collected from the colons of 53 healthy human volunteers in Brno (Czechia) and tested for spontaneous and induced production of inhibitory exoproducts in a cross-test against each other. Of the strains tested, 37.7% produced bacteriophages, 41.5% produced from one to several LMW colicins, 11.3% formed HMW colicins and 15.1% (eight strains) produced exocellular siderophores different from enterochelin. Of these, seven strains formed aerobactin and one strain formed an untyped siderophore.E. coli strains differ greatly in the incidence of colicinogeny and lysogeny from its closest systemic relatives in the genusEscherichia and therefore should not be regarded as a model bacterium in this respect. This work was supported by grants from theGrant Agency of the Czech Republic (310/01/0013 and 310/03/1091) and by the institutional support of theMinistry of Education, Youth and Sports of the Czech Republic (MŠM 002 162 2415).     

Production of and sensitivity to colicins among serologically classified strains of Escherichia coli

A significant proportion of 242 serologically classified strains of Escherichia coli of human origin produced colicins (33%) or were inhibited by one or more of six standard colicins (57%). The most common colicins identified were E1, I, and B; colicins B and V had greatest range of activity. Generally, neither the production of, nor sensitivity to, individual colicins was restricted to strains of a single serogroup. The coexistence of strains of one serogroup that were sensitive to the action of a colicin produced by strains of another serogroup was encountered among 2 of 21 fecal specimens containing strains of multiple serogroups. The production of colicins was not a major determinant in the acquistion of, or subsequent changes in, strains of E. coli in the feces of 10 newborn infants.     

AUREMOL-RFAC-3D,combination of R-factors and their use for automated quality assessment of protein solution structures

We present here the computer program AUREMOL-RFAC-3D that is a generalization of the previously published program RFAC for the fully automated estimation of residual indices (R-factors) from 2D NOESY spectra. It is part of the larger AUREMOL software package (www.auremol.de). RFAC-3D calculates R-factors directly from two-dimensional homonuclear NOESY spectra as well as from three-dimensional ¹⁵N or ¹³C edited NOESY-HSQC spectra and thus extends the application range to larger proteins. The fully automated method includes automated peak picking and integration, a Bayesian noise and artifact recognition and the use of the complete relaxation matrix formalism. To enhance the reliability of the calculated R-factors the method is also generalized to calculate combined R-factors from a set of 2D and 3D-spectra. For an optimal combination of the information derived from different sources a plausible formalism had to be derived. In addition, we present a novel direct R-factors based measure that correlates an R-factors as defined in this paper to the root mean square deviation of the actual structure from the optimal structure. The new program has been successfully tested on the histidine containing phosphocarrier protein (HPr) from Staphylococcus carnosus and on the Ras-binding domain (RBD) of the Ral guanine-nucleotide dissociation stimulation factor (RalGDS).     

Diversity and Geographical Distribution of Flavobacterium psychrophilum Isolates and Their Phages: Patterns of Susceptibility to Phage Infection and Phage Host Range

Flavobacterium psychrophilum is an important fish pathogen worldwide that causes cold water disease (CWD) or rainbow trout fry syndrome (RTFS). Phage therapy has been suggested as an alternative method for the control of this pathogen in aquaculture. However, effective use of bacteriophages in disease control requires detailed knowledge about the diversity and dynamics of host susceptibility to phage infection. For this reason, we examined the genetic diversity of 49 F. psychrophilum strains isolated in three different areas (Chile, Denmark, and USA) through direct genome restriction enzyme analysis (DGREA) and their susceptibility to 33 bacteriophages isolated in Chile and Denmark, thus covering large geographical (>12,000 km) and temporal (>60 years) scales of isolation. An additional 40 phage-resistant isolates obtained from culture experiments after exposure to specific phages were examined for changes in phage susceptibility against the 33 phages. The F. psychrophilum and phage populations isolated from Chile and Denmark clustered into geographically distinct groups with respect to DGREA profile and host range, respectively. However, cross infection between Chilean phage isolates and Danish host isolates and vice versa was observed. Development of resistance to certain bacteriophages led to susceptibility to other phages suggesting that “enhanced infection” is potentially an important cost of resistance in F. psychrophilum, possibly contributing to the observed co-existence of phage-sensitive F. psychrophilum strains and lytic phages across local and global scales. Overall, our results showed that despite the identification of local communities of phages and hosts, some key properties determining phage infection patterns seem to be globally distributed.