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 0 antigen carrying lipopolysaccharide is able to prevent access of all phages and colicins tested to their outer membrane protein receptors.Outer membrane proteins of Escherichia coli are known to function as receptors for many bacteriophages and colicins (4). This receptor function has generally been demonstrated in rough strains such as E. coli K-12 which lack the 0-antigen part of lipopolysaccharide (LPS). It is conceivable that the long 0-antigenic chains present on most wild-type E. coli strains (5) can sterically hinder access of phages and colicins to their outer membrane protein receptors. This receptors (11). All 12 phages failed to form plaques on the smooth strains U20 and F2, but 10 and 6 phages, respectively, were able to infect derivative strains U20-3 and F2-1 with strongly reduced amounts of 0 antigen (Table 1). Apparently, in strains U20 and F2, the 0-antigenic part of LPS creates a barrier to these outer membrane proteinspecific phages. Resistance of the rough strains to the other phages may be caused by lack of receptors or by digestion of possibility has often been suggested (for example, see references 7, 8, and 12), but never systematically investigated. In the present study, the effect of 0 antigen on the ability of a number of different phages and colicins to reach their outer membrane protein receptor sites has been investigated. Twelve outer membrane protein-specific phages were tested for their ability to plate on four smooth E. coli strains, i.e, strains U20, F2, F3, and F8, and their isogenic 0-antigen-deficient derivatives, strains U20-3, F2-1, F3-2, and F8-5. Strains in the latter group have been isolated as spontaneous mutants resistant to phages which recognize the 0 antigens of strains in the former group as their * Corresponding author. 449the phage DNA by restriction endonucleases. A more complex picture was obtained with strains F3 and F8 (Table 1), which contain less 0 antigen than strains U20 and F2 (Fig. 1). Exclusive plating on the rough strain F3-2 or F8-5 or both was observed only for phages K20 and T6. All other phages that infected the rough strains F3-2 or F8-5 were also capable of plating on the smooth parental strain. Apparently, the amount of 0 antigen produced by strains F3 and F8 is insufficient to create an effective barrier to all of the outer membrane protein-specific phages.We also investigated the ability of eight different colicins to reach their receptor sites on several outer membrane proteins in the presence or absence of 0 antigen (Table 2). In contrast to their rough derivatives, strains U20 and F2 were resistant to all colicins tested. Similarly, five colicins were on May 10, 2018 by guest
To collect information on synthesis and regulation of the peptidoglycan-associated pore-forming outer membrane proteins b and c, mutants resistant to phages Me1 and TuIa were analyzed. Genetic analysis showed three linkage groups, corresponding with the genes tolF (phenotype b-c+), meoA (phenotype b+c-) and ompB (phenotypes b-c-, b-c+, b++c- and b++c+/-). It has recently been described that also a b+c- phenotype can occur in the latter linkage group [Chai, T., Foulds, J., J. Bacteriol. 130, 781-786 (1977)]. Among ompB (b-c+)/meoA (b+c-) double mutants strains were found with the b+c- phenotype, showing that ompB is not the structural gene for protein b. Studies on purified proteins b and c showed profound differences between the two proteins with respect to the electrophoretic mobility of fragments obtained by treatment with cyanogen bromide, trypsin and chymotrypsin. The amino acid in position three of the amino-termini of proteins b and c, isolated from isogenic strains, were identified as isoleucine and valine respectively. Both the genetic and biochemical results are consistent with a model recently published [Ichihara, S., Mizushima, S., J. Biochem. (Japan) 83, 1095-1100 (1978)] which predicts that tolF and meoA are the structural genes for the proteins b and c respectively and that ompB is a regulatory gene whose product regulates the levels of both proteins.
(Rcccived Novcinher 29. 1 9 W M a r c h 5. 1985) --EJB 841257 In order t o study the structure-function relationship o f t h e PhoE protein pore we have isolated five independent. 'I'C'45-resistant, plzoE mutants all of which appeared to produce normal amounts of a n clectrophoretically altered PhoE protein, designated a s PhoE * protein. Nticleotidc sequence analysis of the DNA liagments carrying the mutations showed that the mutations all correspond to a G C to A . 'r transition at the same place within the phoE gene resulting in a deduced change of amino acid residue arginine 158 into histidine. 'This result shows that the arginine 158 residue plays ;in important role in thc interaction of the PhoE protein pore with phage TC45.Moloreover, studies on the channel properties o!' the PhoE * protein showed that the PhoE channel has lost part of its preference for negatively charged solutes. ;IS a result of the arginine to histidine changc. 'l'he results are discussed in terms of the structure-function relationship of PhoE protein ;is well as in terms o f thc topological organization of the protein channel in the outer membrane.
The development and applicability of a dose‐controlled experimental infection with atypical Aeromonas salmonicida in carp is described. The proliferation and clinical manifestations of experimentally induced carp erythrodermatitis mimicked a natural infection. An in‐vivo assay was used to evaluate the lethal properties of cell‐free culture supernatants and a simple serum‐free growth medium was devised for maintaining the virulence of the challenge strain. Depending on the inoculation dose, a sublethal (chronic) to a lethal (acute) infection could be induced, and a dose‐response relation was observed between A. salmonicida inoculum size and carp mortality. The dose‐controlled experimental infection was used as a challenge test for laboratory evaluation of the efficacy of potential vaccine candidates. The vaccine candidates tested, a cell envelope preparation, purified lipopolysaccharide and purified A‐layer (ACE) protein showed no protection or only a feeble one at the best, while formalinized whole cells showed a consistent but only moderate protection. In contrast, when concentrated, detoxified culture supernatant was used, the carp were protected against a subsequent lethal challenge. These observations indicate that immunity against A. salmonicida extracellular products is of prime importance.
Previous studies on the question of whether the PhoE protein pore has a preference for Pi and Pi-containing solutes only or whether it constitutes a general anion-preferring channel, have not given an unequivocal answer either because the presence of the phosphate binding protein was not ascertained or because only arsenate was tested as a non Pi-containing control solute. Permeability properties of PhoE, OmpF and OmpC protein pores for negatively charged solutes were measured in vivo in the presence of phosphate-binding protein. It appeared that the PhoE protein pore is the most efficient channel for the three tested solutes phosphate, succinate and sulphate. Conditions were established to measure the frequency of ethyl methane snlphonate induced mutations as a function of the presence of pore proteins. These results indicate that PhoE protein also forms the most efficient channel for ethyl methane sulphonate. We conclude that the preference of the PhoE protein pore is not restricted to Pi and Pi-containing solutes but also concerns several other negatively charged solutes.
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