pu rt m en t of Micro biology , L o i l is ia n ci St N t t. U n i rvr.vity , Bu ton Roil g c., Lo ii isici nu 70803Two strains of Thiothrix niwa were examined to determine their morphological and physiological features. Microculture studies revealed the production of gliding gonidia and possible mechanisms for rosette formation. The strains were able to use only 4 of 41 carbon sources tested, but only if sulfide or thiosulfate was present. Physiologically, Thiothrix may be an obligate mixotroph. The guanineplus-cytosine content of the deoxyribonucleic acid was 52 mol%. Our strains fit the description of T . nivecr, and strain JP2 (= ATCC 31500) was designated as the neotype strain.-~ ~ ~ ~-Thiothrix was first described by Winogradsky in 1888 (23), but little is known about the biology of thi? organism because of a lack of pure cultures. The presence of a sheath on Thiothrix has been verified (1, 3, 9), and the typical rosettes are often seen in sulfide-containing waters. The production of gliding gonidia as reported by Winogradsky (23) has not been adequately verified, and features of the physiology of Thiothri-x have not been examined.The first pure cultures of Thiothrix were obtained recently (9) and were used in this study to (i) verify or refute the properties described by Winogradsky ( 2 3 ) , (ii) determine the physiological features of Thiothrix, and (iii) provide an adequate taxonomic description of the organism. MATERIALS AND METHODSCultures and media. Thi0thri.v nivru strains JP1 and JP2 were isolated from sulfide-containing well water in John Pennycamp State Park in Key Largo, Fla.. by techniques described previously (9). MY (9) and MP (20) media containing either 0.03% sodium sulfide or 0.03% sodium thiosulfate were used routinely as solid media (1.5% agar) or in tubes containing agar butts with liquid overlay5 (biphasic). Incubation was at room temperature (about 22°C) unless stated otherwise.Microculture studies. The life cycle of T. nivecz strain JP2 was examined in microculture. A small amount of melted MP agar was placed into a Whipple counting chamber, inoculated with 1 drop of a 72-h culture, and covered with a cover slip. Rosettes, filaments, and single cells were observed and photographed at intervals for up to 36 h.Morphological features. A Gillet and Sibert phasecontrast microscope fitted with a filar micrometer and a Nikon AFM camera was used to examine and record the morphological features of the mains. Sudan black B was used to detect poly-P-hydroxybutyrate.Growth studies. The ability of each strain to grow at different temperatures was examined by inoculating duplicate tube4 of bipha5ic MY medium and incubating these tubes for up to 2 weeks at the de\iredtemperature. The ability of each strain to grow anaerobically was tested in 125-ml serum bottles containing 20 ml of MY broth supplemented with either sulfide or thiosulfate. The medium was boiled for 5 min, flushed with oxygen-free nitrogen, capped with a butyl rubber stopper, autoclaved, and inoculated with a syringe through the sto...
An accurate most-probable-number enumeration method was developed for counting the number of Beggiatoa trichomes from various freshwater sediments. The medium consisted of extracted hay, diluted soil extract, 0.05% acetate, and 15 to 35 U of catalase per ml. The same enrichment medium, but without the acetate, was the best enrichment medium from which to obtain pure cultures because it supported good growth of the beggiatoas without allowing them to be overgrown by other bacteria. A total of 32 strains of Beggiatoa were isolated from seven different freshwater habitats and partially characterized. The strains were separated into five groups based on several preliminary characteristics. Four of the groups contained cells with trichomes of approximately the same diameter (1.5 to 2.7 ,tm) and may be Beggiatoa leptomitiformis or an unnamed species. The fifth group appeared to be Beggiatoa alba. With the exception of three strains, all of the strains deposited sulfur in the presence of hydrogen sulfide, and all strains grew heterotrophically and deposited poly-fB-hydroxybutyrate and volutin when grown on acetate supplemented with low concentrations of other organic nutrients. Thin sections of sulfur-bearing trichomes indicated that the sulfur granules were external to the cytoplasmic membrane and that they were surrounded by an additional membrane.
The type strains of the three currently recognized species of the genus Microcyclus Brskov 1928 ( M . aquaticus (Drskov, strain ATCC 25396; M . major Gromov, strain BKM 859; and M. f2avus Raj, strain ATCC 232761, together with strains apparently belonging to each species, were examined to determine their taxonomic relationships. There were many differences between the type strains in their morphological and physiological characteristics as well as in the guanine-plus-cytosine (G + C) contents of their deoxyribonucleic acids (DNAs).Therefore, we propose to split the genus Microcyclus into three genera, each containing a single species. M . aquaticus, the type species of Microcyclus, remains as the only species in the genus. It is characterized by the formation of cellular rings which are produced when the ends of the curved cells overlap. It is nonpigmented and is respiratory in its metabolism; the G+C content of its DNA ranges from 66.3 to 68.4 mol%. M . flavus ATCC 22376 and three similar strains were found to possess characters in agreement with those given in the original description of Spirosoma linguale, and they were placed in that species. The name M. flavus Raj 1970 thus becomes a later subjective synonym of Spirosoma linguale (Eisenberg 1891) Migula 1894. This species is characterized by the formation of wavy and coiled filaments, yellow pigmentation on MicrocyclusSpirosoma (MS) agar, and respiratory metabolism. The G+C content of its DNA ranges from 51.0 to 52.9 mol%. Strain DSM 74 is designated as the neotype strain of S . linguale. M . Major BKM 859 and one similar strain were placed in a new genus, Flectobacillus. F . major (Gromov) comb. nov., the type species, is composed of rods which are straight to curved, the degree of curvature varying among individual cells within a culture. The cells may form long, sinuous filaments and rings. Colonies on MS agar are pale pink to rose-colored. Metabolism is respiratory, and the G+C content of its DNA ranges from 39.5 to 40.3 mol%. The type strain ofF. major is strain BKM 859.In the eighth edition of Bergey's Manual (19), bodies of water near Baton Rouge, La., an orgathe genus Microcyclus Brskov 1928 is a heter-nism which resembled M . flavus, another ogenous collection of three species: M . aquati-which resembled M . major, and several strains cus Brskov (the type species), M . flavus Raj, which resembled M . aquaticus. With these adand M. major Gromov. All three species were ditional strains available, a comprehensive described and named on the basis of single study of the taxonomic relationships of these strains, and these strains (see below) are, therefore, according to Rule 18C of the Bacteriological Code (12), type strains by monotypy. The lack of additional strains of these species has been a hindrance in conducting a comprehensive study of this genus. However, Claus (4) determined that two organisms isolated in Europe and designated as Spirosoma sp. were similar to M . flavus. On the basis of the base compositions of the DNAs, Claus et al.
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