- Volume 58, Issue 2, 1969

The base compositions of the deoxyribonucleic acids extracted from 93 cultures of flexibacteria classified in the genera Cytophaga, Flexibacter, Microscilla, Saprospira, Herpetosiphon, Vitreoscilla and Beggiatoa have been determined. The data demonstrate that the guanine plus cytosine contents (GC values) of the representative cultures ranged from 30 to 53 %. A number of the recently proposed nomen species are shown to contain representatives with different GC values.

A computer programme was used to group 85 strains of flexibacteria on the basis of similarity in 84 structural, physiological and biochemical characteristics. The procedure gave 19 groups, the members of which were very similar in their characteristics. The groups could be grouped in seven assemblages whose members showed lesser, but still considerable, similarity. The groups may represent taxonomic entities at the species level; the assemblages, those at the generic level.

About 90 strains of gliding microbes (flexibacteria) have been considered. Data obtained by Lewin & Lounsbery (1969) and Mandel & Lewin (1969) have been used by Fager (1969) in a computer programme designed to indicate affinities and thereby possible relationships. Largely on the basis of Fager’s analysis, a classification of these organisms is here proposed.

Including a few species described elsewhere, 27 species and varieties are distinguished, most of them apparently new. A simplified diagnostic key is presented for their identification. They are assigned to six genera, three (Saprospira, Flexithrix and Herpetosiphon) are recognized primarily on morphological grounds; three others (Cytophaga, Flexibacter and Microscilla), here somewhat re-defined, are based largely on other convenient characteristics.

A method of Adansonian taxonomy was applied to a set of flexuous, gliding bacteria. A clustering of strains which corresponded to Saprospira grandis, Flexibacter flexilis, Saprospira thermalis, Flexibacter giganteus and Flexibacter roseolus was observed from the computed data. High similarity values (> 80 %) were found for strains assigned to the genera Cytophaga, Flexibacter and Microscilla.

The cell wall compositions of 41 strains of oral filamentous bacteria belonging to the genera Fusobacterium, Leptotrichia and Bacterionema were examined by paper chromatography. Leptotrichia walls showed principally alanine, glutamic acid, DAP, glycine and aspartic acid. The genus Bacterionema had the same amino acids but aspartic acid appeared to be a minor component and was not detected in the mucopeptide fraction of the cell walls. The strains of Fusobacterium gave an amino acid pattern characteristic of Gram-negative bacteria; however, lysine was a major component in the mucopeptide. The genus Leptotrichia is thus distinct from the other two genera and should include two species, Leptotrichia buccalis and another organism termed ‘Anaerobic filaments’.

Twenty-two strains of Pseudomonas, representing 15 species, were examined by electron microscopy, utilizing the techniques of negative contrast staining and to a lesser extent shadowing and ultrathin sectioning. Fimbria-like appendages were found on cells of 12 strains, representing 8 species. Polar, fimbria-like filaments were observed on Pseudomonas aeruginosa, P. acidovorans, P. testosteroni, P. maltophilia, P. alcaligenes and P. solanacearum. Peritrichous filaments were found on P. multivorans and P. fragi.

Organisms of two strains of Pseudomonas stizolobii possessed one polar flagellum of unusual thickness. Negative-contrast staining and ultrathin sectioning indicated that the flagella are sheathed and are comparable in structure to the sheathed flagella described in Vibrio and Bdellovibrio. In some instances, flagella displayed sheath and core structure after negative constrast staining. Distal ‘tubules’ and ‘knobs’ apparently consisting entirely of sheath material were also seen. The thickness of the sheath, which in section consisted of an outer dense component and an inner lighter component, was similar to that of the outer double track membrane of the cell wall.

Functional cytochrome(s) and an oxidative phosphorylation system were demonstrated in Streptococcus faecalis grown on media supplemented with haematin. The membrane fraction of cell-free extracts coupled the oxidation of reduced NAD to the formation of ATP from ADP and in organic phosphate. This reaction was sensitive to cyanide, azide and Antimycin a. A b 2 type cytochrome was detected in the membrane fraction with absorption maxima at 412, 425, 529 and 559 nm.

Some interactions of Aspergillus species with Staphylococcus aureus and Pseudomonas aeruginosa, all of which are concerned in otitis externa, are described. The pigments pyocyanine, 1-hydroxyphenazine and the fluorescent green pigments produced by P. aeruginosa inhibited the germination of spores of Aspergillus terreus; the most toxic of these pigments was 1-hydroxyphenazine. P. aeruginosa did not produce fluorescent pigments in Czapek-Dox medium unless the ferrous sulphate was omitted and asparagine added. Following incubation of the Aspergillus species in this medium, however, P. aeruginosa grew and produced fluorescent pigment therein. S. aureus produced antifungal toxic material which was capable of diffusing through cellophan and causing distortion of A. terreus hyphae.

Five fla mutations of Salmonella typhimurium lt2 have been mapped by transduction. They were isolated from five motile (fla +) strains each with a different form of antigen-i, caused by mutation of H1, the structural gene for phase-1 flagellin. H1 was cotransduced (frequency 0·1 to 0·5) with each of the fla mutations. Three-point crosses using a serological selection technique indicate the following order: fla-50—fla-58—fla-55-H1. The position of fla-52 was not discovered. If the fla complementation groups correspond to the fla genes their order is flaB—flaD—flaA—H1.

Of the five H1 mutations four (M5, M20, M11, M12) appear to be very closely linked. The fifth (M6) is outside this cluster. Transductants with functionally and antigenically normal antigen-i were obtained, at a very low frequency, in crosses between i-curly and fla strains: they are attributed to crossing over within H1 between the ‘curly’ mutation and the mutations causing alteration in serological character. Some earlier conclusions (Joys & Stocker, 1963) as to gene order, especially within H1, now seem unjustified.

Analyses, tests with isotopic nitrogen and tests for acetylene and isocyanide reduction, using both continuous and batch cultures, were made with seven strains of putative nitrogen-fixing bacteria and three local isolates. Only two (single strains of Mycobacterium flavum and Pseudomonas azotogensis) fixed nitrogen; the active pseudomonad differed in several respects from the organism originally reported. Other Pseudomonas, Nocardia and Azotomonas species and the three local isolates did not fix; some simulated nitrogen fixation in cultural tests most impressively, but proved simply to be very efficient scavengers of traces of fixed nitrogen.