Straight mutants of Spirillum volutans can swim

Nonhelical mutant cells of Spirillum volutans ATCC 19554 can swim as fast as the helical cells. Consequently, a helical cell shape is not required for motility of this species, and the function of the polar flagellar fascicles is not merely to cause rotation, and therefore translocation, of the corkscrew-shaped cell.     

The growth of Spirillum volutans ehrenberg in mixed and pure cultures

Summary The growth of Spirillum volutans in mixed culture was studied and liquid media were developed in which it grew rapidly and attained populations of over a million cells per ml. By taking advantage of their rapid and unidirectional motility, spirilla were separated from the mixed population by migration in capillary tubes. Pure cultures were achieved by growing the separated spirilla inside of dialysis sacks in contact with mixed cultures on the outside, and also by growth in an asparagine-mineral salts medium supplemented with an extract of Escherichia coli.Dedicated to Professor E. G. Pringsheim on his 80th birthday.This investigation was supported in part by grant G 9882 from the National Science Foundation.     

Electron microscopic observations of structures associated with the flagella of Spirillum volutans.

Electron microscopy of thin-sectioned Spirillum volutans (ATCC 19554) showed that at the insertion site of the flagellum there was a cylindrical structure with a diameter of ca. 36 nm which extended ca. 19 nm into the cytoplasm. This structure, termed a cytoplasmic flagellar base, enclosed a central rod which was continuous with the hook. There was a continuation of the flagellar base into the peptidoglycan layer, enclosing ringlike structures and the central rod. The flagellar hook and proximal part of the flagellar filament contained a central channel which was large enough to accommodate the flagellin subunit. The flagella of fixed cells may project perpendicularly from the outer membrane in a position corresponding to a trailing, swimming orientation or may bend almost parallel to the membrane in a leading orientation. Maximum bending occurred in the hook region, which may be the structure responsible for executing changes in swimming direction.     

Trailing flagella rotate faster than leading flagella in unipolar cells of Spirillum volutans.

In unipolar cells of Spirillum volutans, the flagellar rotation frequency is halved, approximately, when the flagellar bundle reorientates to rotate about the cell body and reverse the swimming direction. The viscous drag resulting from a concomitant increase in flagellar wave amplitude is probably responsible for the reduced frequency of flagellar rotation.     

Stimulatory effect of dihydroxyphenyl compounds on the aerotolerance of Spirillum volutans and Campylobacter fetus subspecies jejuni.

The aerotolerance of the microaerophilic bacterium Spirillum volutans was greatly stimulated in a defined medium by the presence of dihydroxyphenyl ferric iron-binding compounds such as nor-epinephrine at 10(-5) to 10(-6) M. Dihydroxyphenyl compounds at 2 X 10(-4) M, or iron salts (ferrous or ferric) at high concentration, greatly increased the aerotolerance of a strain of Campylobacter fetus subsp. jejuni when grown on streak plates of Brucella agar. The results suggest that the microaerophilism of these organisms might in part be caused by a failure to synthesize microbial ferric iron-binding compounds at sufficient levels to support aerobic growth.     

Membrane-bound respiratory of Spirillum itersonii.

The membrane-bound respiratory system of the gram-negative bacterium Spirillum itersonii was investigated. It contains cytochromes b (558), c (550), and o (558) and beta-dihydro-nicotinamide adenine dinucleotide (NADH) and succinate oxidase activities under all growth conditions. It is also capable of producing D-lactate and alpha-glycerophosphate dehydrogenases when grown with lactate or glycerol as sole carbon source. Membrane-bound malate dehydrogenase was not detectable under any conditions, although there is high activity of soluble nicotinamide adenine dinucleotide: malate dehydrogenase. When grown with oxygen as the sole terminal electron acceptor, approximately 60% of the total b-type cytochrome is present as cytochrome o, whereas only 40% is present as cytochrome o in cells grown with nitrate in the presence of oxygen. Both NADH and succinate oxidase are inhibited by azide, cyanide, antimycin A, and 2-n-heptyl-4-hydroxyquinoline-N-oxidase at low concentrations. The ability of these inhibitors to completely inhibit oxidase activity at low concentrations and their effects upon the aerobic steady-state reduction levels of b- and c-type cytochromes as well as the aerobic steady-state reduction levels obtained with NADH, succinate, and ascorbate-dichlorophenolindophenol suggest that presence of an unbranched respiratory chain in S. itersonii with the order ubiquinone leads to b leads to c leads to c leads to oxygen.     

Deoxyribonucleic Acid-Mediated Transformation of Spirillum lipoferum

The methodology for deoxyribonucleic acid-mediated transformation of Spirillum lipoferum to resistance to various antimicrobial agents is reported.     

Flagellar protein dynamics in Chlamydomonas

Cilia and flagella appear to be stable, terminal, microtubule-containing organelles, but they also elongate and shorten in response to a variety of signals. To understand mechanisms that regulate flagellar dynamics, Chlamydomonas cells with nongrowing flagella were labeled with (35)S, and flagella and basal body components were examined for labeled polypeptides. Maximal incorporation of label into the flagella occurred within 3 h. Twenty percent of the flagellar polypeptides were exchanged. These included tubulins, dyneins, and 80 other axonemal and membrane plus matrix polypeptides. The most stable flagellar structure is the PF-ribbon, which comprises part of the wall of each doublet microtubule and is composed of tubulin and three other polypeptides. Most (35)S was incorporated into the high molecular weight ribbon polypeptide, rib240, and little, if any, (35)S is incorporated into PF-ribbon-associated tubulin. Both wild-type (9 + 2) and 9 + 0 flagella, which lack central microtubules, exhibited nearly identical exchange patterns, so labeling is not due to turnover of relatively labile central microtubules. To determine if flagellar length is balanced by protein exchange, (35)S incorporation into disassembling flagella was examined, as was exchange in flagella in which microtubule assembly was blocked by colchicine. Incorporation of (35)S-labeled polypeptides was found to occur into flagellar axonemes during wavelength-dependent shortening in pf18 and in fla10 cells induced to shorten flagella by incubation at 33 degrees C. Colchicine blocked tubulin addition but did not affect the exchange of the other exchangeable polypeptides; nor did it induce any change in flagellar length. Basal bodies also incorporated newly synthesized proteins. These data reveal that Chlamydomonas flagella are dynamic structures that incorporate new protein both during steady state and as flagella shorten and that protein exchange does not, alone, explain length regulation.     

Flagellar glycosylation in Clostridium botulinum

Flagellins from Clostridium botulinum were shown to be post-translationally modified with novel glycan moieties by top-down MS analysis of purified flagellin protein from strains of various toxin serotypes. Detailed analyses of flagellin from two strains of C. botulinum demonstrated that the protein is modified by a novel glycan moiety of mass 417 Da in O-linkage. Bioinformatic analysis of available C. botulinum genomes identified a flagellar glycosylation island containing homologs of genes recently identified in Campylobacter coli that have been shown to be responsible for the biosynthesis of legionaminic acid derivatives. Structural characterization of the carbohydrate moiety was completed utilizing both MS and NMR spectroscopy, and it was shown to be a novel legionaminic acid derivative, 7-acetamido-5-(N-methyl-glutam-4-yl)-amino-3,5,7,9-tetradeoxy-D-glycero-alpha-D-galacto-nonulosonic acid, (alphaLeg5GluNMe7Ac). Electron transfer dissociation MS with and without collision-activated dissociation was utilized to map seven sites of O-linked glycosylation, eliminating the need for chemical derivatization of tryptic peptides prior to analysis. Marker ions for novel glycans, as well as a unique C-terminal flagellin peptide marker ion, were identified in a top-down analysis of the intact protein. These ions have the potential for use in for rapid detection and discrimination of C. botulinum cells, indicating botulinum neurotoxin contamination. This is the first report of glycosylation of Gram-positive flagellar proteins by the 'sialic acid-like' nonulosonate sugar, legionaminic acid.     

Cytochrome synthesis and its regulation in Spirillum itersonii

Spirillum itersonii contains b- and c-type cytochromes as well as a carbon monoxide-binding pigment of the cytochrome o type. Synthesis of cytochromes b and c is increased by about two- and fourfold, respectively, when cells are transferred from high to low aeration. The increased concentration of cytochrome is not accompanied by an increase in the respiration rate of the cells. Both cytochrome b and cytochrome c are located in the particulate fraction of cells grown under high or low aeration, and both pigments are fully reducible by succinate. No evidence was found for the accumulation of the protein component of either cytochrome when synthesis of the prosthetic group was limited by iron deficiency, nor did heme or precursors accumulate when protein synthesis was prevented. It was therefore concluded that the formation of the heme prosthetic group is closely integrated with the synthesis of the protein moiety. delta-Aminolevulinate synthase was detected in extracts of the organism. Its activity was correlated with cytochrome synthesis; it was reduced by high aeration and increased under low aeration. The synthase was inhibited by hemin at concentrations of 10 mum or higher. The observations are consistent with a central role for the heme prosthetic group in the regulation of cytochrome synthesis.