Fimbriae and their constituent protein (fimbrilin) were purified to homogeneity from the bacterial wash fluid and cell lysate fraction, respectively, of Bacteroides gingivalis 381. Fimbriae, observed by negative staining, were curly, single-stranded filaments with a diameter of ca. 5 nm. The apparent molecular weight of the fimbrilin was 43,000. Fimbriae were resistant to sodium dodecyl sulfate denaturation at 70 degrees C. Heating at 100 degrees C in sodium dodecyl sulfate was needed to completely dissociate them to monomers of fimbrilin. Different sets of antigenic determinants seemed to be exposed on the surfaces of fimbriae and sodium dodecyl sulfate-denatured fimbrilin. Purified fimbriae did not show either hemagglutinating activity or hemagglutination inhibitory activity, although it has been inferred on the basis of circumstantial evidence that fimbriae are correlated to hemagglutinating activity of the organism. Hemagglutinin activity, however, was detected in culture supernatant, and this observation suggests that fimbriae of a different type or a lectin-like protein may be acting as hemagglutinin in B. gingivalis.

Purification and characterization of a novel type of fimbriae from the oral anaerobe Bacteroides gingivalis.

Mutation hisJ5625 has altered the histidine-binding protein J of Salmonella typhimurium such that histidine transport is impaired, even though binding of histidine by the J protein is unimpaired [Kustu, S.G., & Ames, G.F. (1974) J. Biol. Chem. 249, 6976--6983]. We have determined by protein analytical methods that the only effect of this mutation has been the substitution of a cysteine residue for an arginine at a site in the interior of the polypeptide chain. This arginine residue is therefore potentially essential for the transport function of the protein. The mutant protein migrates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis more slowly than the wild type protein, as if its molecular weight were greater by as much as 2000. Since this behavior is apparently due to a single amino acid replacement, a molecular weight difference even between two closely related proteins should not be inferred solely on the basis of sodium dodecyl sulfate gel electrophoresis.

A single amino acid substitution in a histidine-transport protein drastically alters its mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

We determined the DNA sequence of the hag gene of Escherichia coli K-12 and deduced the primary structure of the flagellin consisting of 497 amino acid residues. Comparison of the amino acid sequence with those of other bacterial flagellins revealed a high homology in the NH2- and COOH-terminal regions.

Nucleotide sequence of the hag gene encoding flagellin of Escherichia coli.

Nucleotide Sequence ofthehagGeneEncoding Flagellin of Escherichia coli

Publisher Summary The chapter discusses long established means of identifying Bacillus strains and reviews a newly developed method based on API materials. The chapter presents recent work on instrumented approaches to the identification of the members of this genus. Aerobic endospore-forming rods, which are members of the genus Bacillus , are ubiquitous and because of the resistance of their spores and the ability of the vegetative organisms to degrade many organic materials, the genus is probably the most important group of spoilage and contaminating bacteria affecting the industrial and medical activities of man. The process of identification presupposes the existence of a classification scheme containing taxa with which unknown strains may be identified. Classical methods for the identification of Bacillus are effective if carried out properly, but are slow and the reproducibility of certain tests is not good. Using the API system, greater reproducibility and more rapid identification may be achieved. Instrumented methods hold out the promise of obtaining reliable results even faster, but none is fully developed yet.

12 Identification of Bacillus Species

Purified flagellins derived from 16 strains of Bacillus subtilis were classified into at least five distinct groups on the basis of their reaction with antiflagellar filament antibody and antiflagellin antibody. This classification was in good accord with that derived independently on the basis of amino acid analyses of the flagellins. Flagellar antigenicity appears to provide a useful typological character in classifying B. subtilis strains. Images

J H Shaper

Papers

Classification of Bacillus subtilis flagellins.