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A nonapeptide from IL-1β has been reported to be an immunostimulant and adjuvant. To investigate the possibility of enhancing the immunogenicity of recombinant antigens delivered by live-attenuated Salmonella strains, we inserted an oligonucleotide coding for the nonapeptide from murine IL-1β into the genes of three model proteins: LamB, MalE, and flagellin. The hybrid proteins were expressed and delivered in vivo by Salmonella aroA strains, and serum antibody responses were analyzed. The results showed that the nonapeptide induced an increase in the immune response against Salmonella-delivered flagellin, measured on day 28 post-immunization. However, the adjuvant effect was lost by day 42. In no case was an adjuvant effect detected for Salmonella-delivered LamB or MalE. Thus, by comparing the immune responses raised by purified MalE with and without the peptide, we investigated whether the insertion of the peptide affected the immunogenicity of the protein itself. Also in this case, a modest adjuvant effect was shown only after primary immunization and when very low doses of antigen were used. In conclusion, the immunomodulatory properties of the IL-1β peptide can also be detected when it is delivered in vivo by Salmonella; however, the effect is modest and antigen-dependent. [ABSTRACT FROM AUTHOR]

Logarithmically growing cells of Escherichia coli were fixed with glutaraldehyde and incubated with antimaltose-binding protein F coupled to horseradish peroxidase (molecular weight of the complex 80,000). The position of this complex within the cell envelope was determined by reacting with diaminobenzidine-HO, staining with osmium tetroxide and processing for thin section electron microscopy. The following observations were made: (i) induction of the maltose-binding protein resulted in swelling and staining of the outer membrane; (ii) the swelling and staining was more prominent in short cells, less prominent or absent in long cells; (iii) rare examples exhibited granular staining in the space between the plasma membrane and the peptidoglycan layer. These stainings were observable mainly in pole caps; (iv) a mutant lacking the receptor for phage λ showed altered staining pattern. Treatment of glutaraldehyde-fixed cells with EDTA-lysozyme prevented the specific labelling of the maltose-binding protein. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

ATPase was detected in the membranes of a motile Streptococcus. Maximal enzymic activity was observed at pH 8 and ATP/Mg ratio of 2. Mn and Ca could replace Mg to some extent. Besides ATP, GTP and ITP were substrates. The enzyme was inhibited by N,N′-dicyclohexylcarbodiimide but not by sodium azide, uncouplers or bathophenanthroline. An electrochemical gradient of protons, which was artificially imposed across the membranes of Streptococcus cells by manipulation of either the K diffusion potential or the transmembrane pH gradient, led to ATP synthesis. ATP synthesis was abolished by proton conductors, an inhibitor of the ATPase or an increase in the extracellular K concentration. A comparison between the phosphate potential and the electrochemical proton gradient showed that the data found are in agreement with a stoichiometry of 2 protons translocated per molecule ATP synthesized. [ABSTRACT FROM AUTHOR]

After induction with maltose, 30-40% of the total protein in the osmotic shock fluid consist of maltose-binding protein while the induction ratio (maltose versus glycerol grown cells) for the amount of binding protein synthesized as well as for maltose transport is in the order of 10. Induction of maltose transport does not occur during all times of the cell cycle, but only shortly before cell division. Electronmicroscopic analysis of cells grown logarithmically on glycerol or maltose revealed in the latter the formation of large pole caps. These pole caps arise from an enlargement of the periplasmic space. Small cells contain one pole cap, large cells contain two. Pulse label studies with strain BUG-6, a mutant that is temperature sensitive for cell division reveal the following: Growth at the non-permissive temperature prevents maltose-binding protein synthesis and formation of new transport capacity. After shifting to the permissive temperature the cells regain both functions. Simultaneously, the newly formed cells exhibit pole caps. We conclude that the induction of maltose-binding protein is responsible for the formation of pole caps. In addition, beside the presence of inducer, cell cycle events occuring during division are necessary for the synthesis of maltose-binding protein. [ABSTRACT FROM AUTHOR]

The double-isotopic labelling technique was used to identify comprehensively proteins involved in α-glucan catabolism in Klebsiella pneumoniae NCTC 9633. Cells were grown with either glycerol in the presence of H-leucine or with glycerol plus maltose in the presence of C-leucine. Each labelled culture was then fractionated into the main subcellular components, i.e. the cytoplasm, periplasm, cytoplasmic and outer membrane. Corresponding fractions derived from H-labelled and C-labelled cells were combined, and the proteins were analyzed by polyacrylamide gel electrophoresis under denaturing conditions. Gel slices were then counted for H- and C-radioactivity, a positive deviation from the standard C/H ratio being evidence for the presence of a protein specifically induced by maltose in the culture medium. The protein pattern thus obtained was compared with the properties of proteins comprising a similar pathway for maltodextrin utilization in Escherichia coli K-12. Ample information which has been obtained mainly by genetic analysis is available about maltodextrin-utilizing enzymes in E. coli K-12. [ABSTRACT FROM AUTHOR]

The effects of nigericin, valinomycin and some lipophilic cations on the motile behavior of non-starved and methionine-starved Bacillus subtilis cells were studied. For valinomycin and nigericin a quantitative relationship between the flux in the proton-motive force and the duration of the twiddle response was found. Lipophilic cations bind to the ion gate controlling the twiddle frequency and thereby cause the cells to swim smoothly. To explain the transmission of the chemotactic signal a model is given in which receptors, a hyperpolarizing wave, an ion gate and two methylation sites, viz. methyl-accepting chemotaxis proteins and a further methylation site (MT), play a role. For the transmission of the signal caused by an attractant both the hyperpolarizing wave and an interaction between receptor and methylation site (MT) are needed. The methyl-accepting chemotaxis proteins are involved in the adaptation/deadaptation to altered levels of attractant. Artificial changes in the proton-motive force act directly on the ion gate, which finally controlls the twiddle frequency of the cells. [ABSTRACT FROM AUTHOR]