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.     

Genetics of colicin E susceptibility inCitrobacter freundii

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

A new biofilm-associated colicin with increased efficiency against biofilm bacteria

Formation of bacterial biofilm communities leads to profound physiological modifications and increased physical and metabolic exchanges between bacteria. It was previously shown that bioactive molecules produced within the biofilm environment contribute to bacterial interactions. Here we describe new pore-forming colicin R, specifically produced in biofilms formed by the natural isolate Escherichia coli ROAR029 but that cannot be detected under planktonic culture conditions. We demonstrate that an increased SOS stress response within mature biofilms induces SOS-dependent colicin R expression. We provide evidence that colicin R displays increased activity against E. coli strains that have a reduced lipopolysaccharide length, such as the pathogenic enteroaggregative E. coli LF82 clinical isolate, therefore pointing to lipopolysaccharide size as an important determinant for resistance to colicins. We show that colicin R toxicity toward E. coli LF82 is increased under biofilm conditions compared with planktonic susceptibility and that release of colicin R confers a strong competitive advantage in mixed biofilms by rapidly outcompeting sensitive neighboring bacteria. This work identifies the first biofilm-associated colicin that preferentially targets biofilm bacteria. Furthermore, it indicates that the study of antagonistic molecules produced in biofilm and multispecies contexts could reveal unsuspected, ecologically relevant bacterial interactions influencing population dynamics in natural environments.     

Iron uptake byDesulfovibrio vulgaris outer membrane components in artificial vesicles

Desulfovibrio vulgaris lipopolysaccharide and outer membrane proteins (OMPs) were incorporated into vesicles ofD. vulgaris phospholipid and studied for [⁵⁵Fe]binding activity. Both lipopolysaccharide and an extract of two major OMPs caused large increases in⁵⁵Fe uptake over control (phospholipid only) vesicles. CommercialSalmonella typhimurium lipopolysaccharide gave a similar result, but the effect was inhibited by calcium ions; this was not the case forDesulfovibrio. The lipid A portion ofS. typhimurium lipopolysaccharide had a high iron-binding ability, whereasDesulfovibrio lipid A iron binding was little different from control values;D. vulgaris lipopolysaccharide thus has a specific iron-binding site within its polysaccharide side chain.     

Genetic variation in IncI1-co1Ib plasmids

Nucleotide sequences of portions of three plasmid genes (cib, cir, and abi) present in IncI1-Co1Ib colicin plasmids obtained from strains of Salmonella typhimurium isolated in either 1974 (Barker strains) or between 1935 and 1941 (Murray strains) were examined along with sequences of the chromosomal gene for 6-phosphogluconate dehydrogenase (gnd). Our principal findings were: (1) The plasmid genes were virtually identical to those in IncI1-CoIIb plasmids from E. coli, suggesting that Salmonella and E. coli share overlapping pools of these plasmids. (2) The plasmid genes were much less polymorphic than gnd or any other known chromosomal gene from Salmonella, further suggesting horizontal transfer with rapid transmission and turnover. (3) No characteristic differences were found in either the plasmid genes or the chromosomal gene between the 1974 isolates and the Murray strains, indicating that these plasmids have been stable for at least several decades. (4) There was an excess of amino-acid replacement polymorphisms, relative to synonymous polymorphisms, in the plasmid genes, which is consistent with the hypothesis of diversifying selection among colicin-producing plasmid families. (5) The abi (abortive infection) gene present in each of the plasmids contained two single-nucleotide insertions relative to the published sequence. These result in a putative abi protein of 114 amino acids instead of 89.     

High efficiency transformation of Salmonella typhimurium and Salmonella typhi by electroporation

Summary Salmonella typhimurium and S. typhi were transformd with high efficiency by electroporation. Transformation efficiencies of up to 10¹⁰ transformants per g of pBR322 were obtained. In contrast to chemical transformation methods, neither the smooth lipopolysaccharide of S. typhimurium nor the Vi capsular polysaccharide of S. typhi greatly affected transformation efficiency. The introduction of a galE mutation slightly improved transformation efficiency in S. typhimurium (< tenfold) while the Vi antigen of S. typhi had no detectable effect. The transformation efficiency of S. typhimurium with DNA derived from Escherichia coli was increased greatly by the removal of the hsd restriction system (100-fold). Under these conditions electroporation can be used for the routine and direct transformation of Salmonella strains with partially purified (alkaline lysis) plasmid DNA from E. coli.     

Experimental Salmonellosis Immunizing Effect of Live Vaccine Prepared from Various Mutants of Salmonella Having Different Cell Wall Polysaccharides

Immunizing potencies of vaccines prepared from various strains of Salmonella were graded by comparing the mortality rate of immunized mice after challenge with highly virulent strains of either Salmonella enteritidis or S. typhimurium. The resistance against this challenge infection was shown to be conferred by joint immunization with a specific factor, which was represented by O specific lipopolysaccharide of smooth strains, and cross-protection factor, which was a major potent factor in live vaccine. The distribution of this cross-protection factor in rough mutants of S. typhimurium was found to be limited to strains which possessed a polysaccharide chain longer than that of glucose₁-less mutant. The potency conferring cross-resistance was found to be maintained partly in formalin-killed cells and cell walls of the strains harboring cross-protection factor but not in lipopolysaccharide extracted from such strains.     

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.     

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.     

Excision of a DNA sequence determining kanamycin resistance from a ColE1-Km recombinant plasmid

Summary A 4.8×10⁶ dalton ECoRI-generated fragment of the R-factor R6-5 carrying the gene for kanamycin resistance (Km) was joined in vitro to ECoRI-treated ColE1 plasmid DNA. Transformation ofE. coli with the ColE1-Km recombinant plasmid yielded clones, which were immune to colicin E1, resistant to kanamycin and failed to produce colicin E1. During multiplication of this recombinant plasmid in the presence of chloramphenicol, cells expressed an increased resistance to kanamycin. Transformation studies with the recombinant DNA molecule showed very frequent loss of Km resistance in those cells harbouring a preexisting F'gal plasmid. Since colicin immunity is not affected and the col^- phenotype is still present, one has to test for a remaining DNA sequence further existing in ColE1 DNA by cleaving the plasmid DNA with the ECoRI restriction endonuclease. The full length of ColE1 DNA (6.2 kb) was restored, which confirmed that no deletion of ColE1 DNA sequences had occured. The remaining DNA sequence was identified as a 2.0 or 2.2 kb segment. On the basis of the length of the excised fragment it is proposed that the insertion sequence IS1 and a part of the inverted repeat sequence with coordinates 21.0 to 22.0 of the R6-5 DNA are recognised by a nucleolytic function.     

The properties of UV sensitive mutants of Escherichia coli K12 which are also refractory to colicin E2

Summary Among mutants refractory to colicin E2 at low temperatures but sensitive at high temperatures (designated Ref-II), three strains are described which are also UV sensitive. Although colicin refractivity is temperature dependent UV sensitivity is expressed at all temperatures. Although the UV sensitive lesion appears to be similar in its effects to that in Rec (recombinationless) strains, mutants specifically isolated as Rec strains are in fact more sensitive to E2 than are wild type strains. It is suggested that E2 refractivity and UV sensitivity in the mutants probably reflects the phenotypic expression of distinct although linked genes. It is also suggested that the degradation of DNA stimulated by adsorption of E2 to wild type bacteria may be caused by the same enzyme(s) which causes enhanced breakdown of DNA in some rec ^– mutants after UV irradiation.     

Identification of putative ancestors of the multidrug-resistant <Emphasis Type="Italic">Salmonella enterica</Emphasis> serovar typhimurium DT104 clone harboring the<Emphasis Type="Italic"> Salmonella</Emphasis> genomic island 1

The origin of multidrug-resistant Salmonella enterica serovar typhimurium (S. typhimurium) harboring the Salmonella Genomic Island 1 (SGI1), which was detected for the first time in the mid-1980s is unknown. In this study, we performed microarray genomotyping of four multidrug-resistant SGI1 positive strains and found that unlike the S. typhimurium LT2 strain, the multidrug-resistant strains lacked genes STM0517-0529 allowing the utilization of allantoin as a sole nitrogen source. We extended this observation by PCR screening of additional 120 S. typhimurium field strains and found that this locus was absent in all SGI1 positive and also in 24% of SGI1 negative strains, which were proposed to be the original recipients of SGI1. To prove this hypothesis, we compared the STM0517-0529 negative strains (with or without the SGI1) by PFGE and PCR prophage typing and found that 8 out of 11 of the SGI1 negative strains and 17 out of 22 SGI1 positive strains were of identical PFGE pattern and PCR prophage pattern, while this specific pattern was never observed among STM0517-0529 positive strains. We therefore propose that a lineage of the S. typhimurium DT104 sensitive strain first lost the ability to metabolize allantoin and then acquired SGI1.     

Studies of colicin induction with an imm- col+ mutant of the plasmid colicin E1.

Summary A mutant of a derivative of the colicin E1 plasmid has been isolated that does not confer immunity to colicin E1 on its host (imm^-) although it is still capable of producing colicin (col⁺). Cells carrying the col⁺, imm^- plasmid are capable of forming colonies and grow best in liquid culture in the presence of trypsin. The induction of colicin synthesis by ultraviolet light has been analysed using this mutant plasmid. The results suggest that a) the expression of the col⁺ gene may be delayed for many generations after the inducing stimulus, b) although induced cells are usually killed they can reproduce and c) the capacity to produce colicin can be propagated and segregated into the progeny of an induced cell.     

Growth of coliphage BF23 on rough strains of Salmonella typhimurium: the bfe locus

Summary Coliphage BF23 develops in Salmonella typhimurium rough strains. The phage is neither restricted nor modified by S. typhimurium. The growth patterns of the phage were slightly different in S. typhimurium than in Escherichia coli, although phage propagated on S. typhimurium is identical to the phage propagated in E. coli by several criteria used. Mutants of S. typhimurium resistant to BF23 were isolated and found to map (by P22-and Plmediated transduction) in the same position as bfe mutants of E. coli. The order of genes was: metB-argC-bfe-rif-purD-metA.Phage BF23 does not form plaques on smooth S. typhimurium strains, since the phage fails to adsorb irreversibly to smooth cells. Nevertheless, on solid agar, the phage prevents growth of many (but not all) smooth strains. Moreover, UV-and alkali-inactivated phage BF23, although unable to form plaques on sensitive hosts, retains the ability to prevent growth of the host on solid medium. This ability is sensitive to protease and resistant to DNAse and RNase. Heat treatment of the phage causes rapid loss of the cell-growth-preventing-ability whereas the ability to form plaques is lost much more slowly. These results lead to a proposal that phage BF23 virions carry a colicin-like factor that kills sensitive cells.     

Tumor‐Targeting Salmonella typhimurium A1‐R Promotes Tumoricidal CD8+ T Cell Tumor Infiltration and Arrests Growth and Metastasis in a Syngeneic Pancreatic‐Cancer Orthotopic Mouse Model

The present study determined the effect of the tumor‐targeting strain Salmonella typhimurium A1‐R (S. typhimurium A1‐R) on CD8⁺ tumor‐infiltrating lymphocytes (TILs) in a syngeneic pancreatic‐cancer orthotopic mouse model. The effect of tumor‐targeting S. typhimurium A1‐R on CD8⁺ TILs was determined on the Pan02 murine pancreatic‐adenocarcinoma implanted orthotopically in the pancreatic tail of C57BL/6 immunocompromised mice. Three weeks after orthotopic implantation, mice were randomized as follows G1: untreated control group (n = 8); and G2: S. typhimurium A1‐R‐treatment group (n = 8, 1 × 10⁷ colony forming units [CFU]/body, iv, weekly, 3 weeks). On the 22nd day from initial treatment, all mice were sacrificed and tumors were harvested. The tumor‐volume ratio was defined as ratio of tumor volume on the 22nd day relative to the 1st day. The tumor volume ratio was significantly lower in the S. typhimurium A1‐R‐treated group (G2) (3.0 ± 2.8) than the untreated control (G1) (39.9 ± 30.7, P < 0.01). Hematoxylin and easin (H&E) staining on tumor sections was performed to evaluate tumor destruction which was classified according to the Evans grading system and found to be much greater in the S. typhimurium A1‐R‐treated mice (G2). Six mice in G1 had peritoneal dissemination, whereas no mice showed peritoneal dissemination in G2 (P < 0.01). Immunohistochemical staining with anti‐mouse CD8⁺ antibody was performed in order to detect TILs determined by calculating the average number of CD8⁺ cells in three high power fields (200×) in the treated and untreated tumors. The TIL score was significantly higher in G2 (133.5 ± 32.2) than G1 (45.1 ± 19.4, P < 0.001). The present study demonstrates that S. typhimurium A1‐R promotes CD8⁺ T cell infiltration and inhibition of tumor growth and metastasis. J. Cell. Biochem. 119: 634–639, 2018. © 2017 Wiley Periodicals, Inc.     

Functions related to the receptor protein specified by the tsx gene of Escherichia coli

The receptor protein for the phage T6 and colicin K, coded by the tsx gene, facilitated the diffusion of all nucleosides and deoxynucleosides except cytidine and deoxcytidine through the outer membrane of Escherichia coli K-12 and Escherichia coli B. The tsx protein was coregulated with the nucleoside uptake system. Constitutive cytR and deoR mutants contained higher amounts of this protein than wild type strains. There was a good correlation between the initial rate of nucleoside uptake and the adsorption rate of phage T6. From the observation that nucleosides did not compete with each other in the translocation across the outer membrane and that they did not inhibit T6 adsorption it was concluded that the tsx protein forms a pore to which nucleosides have only little if any binding affinity.A major outer membrane protein specified by the ompA gene influenced the function of the tsx protein. Outer membranes of ompA mutants showed an enhanced binding of colicin K but the strains were colicin K insensitive (tolerant). The T6 phage adsorbed at the same rate and plated with the same efficiency as to ompA ⁺ strains. The uptake rate of thymidine and of adenosine was reduced by 16–33% in ompA mutants.The adsorption rate of phage T6 on mutants with altered lipopolysaccharide was the same or even higher than on wild type strains. However the plating efficiency was reduced ranging from 0–46%. Lipopolysaccharide plays no role in the primary adsorption of phage T6 but it is apparently required in a later step of the infection process.Non Standard Abbreviations LPS lipopolysaccharide - cAMP-CRP complex of cyclic adenosine 3,5-monophosphate (cAMP) and its receptor protein (CRP)     

Conjugation-deficient mutants of Escherichia coli distinguish classes of functions of the outer membrane OmpA protein

Summary Sixty-two E. coli mutants, selected as being deficient as recipients in F factor conjugation, are altered either in the amount or function of the outer membrane OmpA protein or in lipopolysaccharide structure. These two components may function together in conjugation, since the residual conjugation activity of a mutant lacking OmpA protein was unaffected by the additional presence of a lipopolysaccharide defect. Sixty of the strains carried mutations mapping to ompA, and these could be divided into classes depending on the amount of OmpA protein in their membranes. Representatives of these classes of mutant alleles failed to complement in diploids, indicating that they all affect the ompA structural gene and nearby sequences needed for its expression. The properties of these classes distinguish three groups of OmpA protein functions: 1) the structural function in the outer membrane in providing resistance to chelating agents and the hydrophobic antibiotic novobiocin, 2) the receptor functions in phage Tull^* and K3 infection, and 3) the functions of binding cells together during conjugation, facilitating the uptake of receptorbound colicin K or L, and allowing phage Ox2 to infect. Different cellular amounts or sites in OmpA protein are thus required for these three groups of functions.     

Characterization of the cea gene of the ColE7 plasmid.

Summary The complete nucleotide sequence (1731 nucleotides) of the gene encoding colicin E7 (cea) of plasmid CoIE7-K317 was determined. This sequence encoded a deduced polypeptide of 576 amino acids of molecular weight 61349 Da. Comparison of the nucleotide and amino acid sequences ofcea E7 with those of other E-group colicins revealed that colicin E7 was closely related to colicin E2, both in gene sequence and in predicted secondary structure of the deduced protein. Judging from the results of cross-immunity tests, we postulated that CoIE7 is probably a proximate ancestor of Co1E2 and Co1E8. Based on results from colicin production tests on cells harboring a 5 end deleted form of thecea E7 gene, we propose, that a previously unknown, non-inducible promoter may be involved in regulation of the constitutive expression of thecea E7 gene.     

Identification of the sid outer membrane receptor protein in Salmonella typhimurium SL1027.

Summary A protein of molecular weight 78,000 daltons, missing in albomycin and phage ES18 resistant mutants, has been identified in the outer membrane of Salmonella typhimurium SL1027. Mutants with a tonB like resistance and overproduction of outer membrane proteins due to iron shortage were also isolated. The mutation which leads to the protein deficiency maps in the sid gene region, the mutation related to overproduction of proteins maps near trp. Although the S. typhimurium and the E. coli protein mediate translocation of the iron complex ferrichrome and the structurally analogous antibiotic albomycin through the outer membrane no cross-reactivity exists in binding the phages T5, T1 and ES18 or colicin M.