Archives of Microbiology (2021) 203:97–105 https://doi.org/10.1007/s00203-020-02006-7 ORIGINAL PAPER The genome sequence of the giant phototrophic gammaproteobacterium Thiospirillum jenense gives insight into its physiological properties and phylogenetic relationships Johannes F. Imhof1 · Terrance E. Meyer2 · John A. Kyndt3 Received: 23 June 2020 / Revised: 21 July 2020 / Accepted: 24 July 2020 / Published online: 5 August 2020 © Springer-Verlag GmbH Germany, part of Springer Nature 2020 Abstract In a conserved culture of the purple sulfur bacterium Thiospirillum jenense DSM216T, cells of this species were easily rec- ognized by cell morphology, large-size spirilla and visible fagellar tuft. The Tsp. jenense genome is 3.22 Mb in size and has a GC content of 48.7 mol%. It was readily identifed as a member of the Chromatiaceae by the complement of proteins in its genome. A whole genome comparison clearly placed Tsp. jenense near Thiorhodovibrio and Rhabdochromatium species and somewhat more distant from Thiohalocapsa and Halochromatium species. This relationship was also found with the sequences of the photosynthetic reaction center protein PufM. The genome sequence supported important properties of this bacterium: the presence of ribulose-bisphosphate carboxylase and enzymes of the Calvin cycle of autotrophic carbon diox- ide fxation but the absence of carboxysomes, an incomplete tricarboxylic acid cycle and the lack of malate dehydrogenase, the presence of a sulfur oxidation pathway including adenylylsulfate reductase (aprAB) but absence of assimilatory sulfate reduction, the presence of hydrogenase (hoxHMFYUFE), nitrogenase and a photosynthetic gene cluster (pufBALMC). The FixNOP type of cytochrome oxidase was notably lacking, which may be the reason that renders the cells highly sensitive to oxygen. Two minor phototrophic contaminants were found using metagenomic binning: one was identifed as a strain of Rhodopseudomonas palustris and the second one has an average nucleotide identity of 82% to the nearest neighbor Rho- doferax antarcticus. It should be considered as a new species of this genus and Rhodoferax jenense is proposed as the name. Keywords Thiospirillum jenense · Thiospirillum genome · Purple sulfur bacteria · Chromatiaceae · New Rhodoferax species Introduction for almost two centuries, especially during the early years of microbiology. The bacterium was found by Ehrenberg It was the immense size of the bacterium, its colored cul- (1838) in calm water of a cove in a small creek near Zie- tures, the content of highly refractive sulfur globules com- genhain (Jena, Germany), where it developed together bined with the active motility and phototactic behavior that with Chromatium okenii. He described this bacterium as made Thiospirillum jenense an interesting study object Ophidomonas jenensis with a cell width of 3.5–4.5 µm and a length of 30–40 up to 100 µm, a giant among bacteria. Cultures of Tsp. jenense are pastel-colored orange-brown Communicated by Erko Stackebrandt. and due to the size, even single cells can be recognized as * Johannes F. Imhof colored in the light microscope. In contrast to many other [email protected] bacteria, the large polar fagellar tuft of Tsp. jenense may be visible in the light microscope (Fig. 1) and already Ehren- 1 GEOMAR Helmholtz Centre for Ocean Research RU Marine Symbioses, Düsternbrooker Weg 20, 24105 Kiel, berg (1838) observed these fagella, for the frst time with a Germany living bacterium. The fagellar tuft pushes the cell forward, 2 Department of Biochemistry, University of Arizona, Tucson, but at reversal of direction it turns down over the cell and AZ, USA then draws it through the water (Buder 1915). It was also 3 College of Science and Technology, Bellevue University, Ehrenberg (1838) who reported on refractive inclusion bod- Bellevue, NE 68005, USA ies that were later recognized to be elemental sulfur globules Vol.:(0123456789)1 3 98 Archives of Microbiology (2021) 203:97–105 and a small bottle was smuggled from the eastern to the western part of Germany across the inner border which was at this time strictly closed. The Thiospirillum thrived for a while in a Winogradsky column set up with sewage sludge and eventually was brought into highly enriched and pure culture (Schlegel and Pfennig 1961). Pfennig discovered that it required the addition of vitamin B 12 and that it also was sensitive to the continuous bright lights in the laboratory and enjoyed a diurnal cycle of 16 h light and 8 h darkness. It would tolerate only 2–3 mM sulfde and had to be fed regularly to achieve dense populations in the cultures. Cultures of Tsp. jenense appear orange in color and the major carotenoids were identifed as lyco- pene and rhodopin (Schmidt 1963; Schmidt et al. 1965). Fig. 1 Brightfeld photomicrograph of Tsp. jenense DSM216 showing Thiospirillum sanguineum on the other hand is a marine intracellular elemental sulfur globules and a polar tuft of fagella. Bar bacterium and is rose-red in color (Paterek and Paynter indicates 10 µm (courtesy of N. Pfennig) 1988), which suggests that the carotenoids may be domi- nated by spirilloxanthin. Pfennig also continued studies on the phototactic behavior (van Niel 1932). The requirement of hydrogen sulfde as well and an impressive movie was produced that is still available as light for the development of Tsp. jenense and other purple online (Pfennig 1963). In this video, the phototactic move- sulfur bacteria was concluded by Buder (1919) from his eco- ment and the reversal of the fagella rotation upon light/dark logical observations on the development of these bacteria. stimuli were demonstrated. Also the collection of cells in a All of the early studies on metabolism, motility and pho- small light frame due to their phototactic response and the totactic responses of Tsp. jenense were made with material accumulation of the cells at light of specifc wavelength, in taken from the natural environment (Winogradsky 1888; particular at the absorption maxima of bacteriochlorophyll Buder 1915, 1919; Schlegel 1956; van Niel 1955). Although a, were observed (Pfennig 1963). However, after this short many attempts had been made in the early years, Tsp. jen- period of active research on this bacterium, for more than ense could not be grown in the laboratory, not even in crude fve decades the problems in cultivation of Tsp. jenense and cultures. It was Buder who was able to keep transient cul- later also the missing availability of cultures have disabled tures of Tsp. jenense according to the Winogradsky method further detailed studies. The original locations, where the (Winogradsky 1888; Buder 1915). The source for many Tsp. jenense was collected from no longer exist and there- years was a pond in Ostrau near Halle, where these bacte- fore, frozen cultures currently represent the only source to ria formed regular mass developments from April to July obtain data on this interesting bacterium. No sequence infor- (Schlegel and Pfennig 1961). This material formed the basis mation of this bacterium, even no 16S rRNA gene sequences, for studies on the photobiology of Tsp. jenense and Chroma- have been available so far. The data presented here for the tium okenii by H.G. Schlegel during his PhD studies in the frst time allow the phylogenetic alignment of Tsp. jenense lab of Johannes Buder (Schlegel 1956). The mechanism of with other purple sulfur bacteria and give insight into its photophobic tactic movements of Tsp. jenense, responsible genomic capability and physiological properties. Despite for the reversal of fagellar rotation upon entering dark zones the lack of pure cultures, bacteria resembling Thiospirillum or dim light, was shown by studies of Buder and Schlegel appear to be more widespread in nature and developments (Buder, 1915, 1919; Schlegel 1956) and demonstrated to be in anaerobic habitats including both marine and freshwater responsible for accumulation of the cells in the light. environments of spiral-shaped bacteria containing diferent Years later Schlegel and Pfennig (1961) in great detail carotenoids have been observed (Paterek and Paynter 1988; described enrichment conditions and culture media for the Briee et al. 2007; Caldwell and Tiedje 1975). This includes a growth of Tsp. jenense. Norbert Pfennig was successful in brackish pond in San Elijo Lagoon near La Jolla (California) the cultivation of Tsp. jenense and other large-cell Chro- were such a development of a pink-colored Thiospirillum matiaceae including Chromatium okenii and Allochroma- was observed and maintained as enrichment for a while in a tium warmingii. As he describes in his memoirs (Pfennig Winogradsky-type column by one of the authors (JFI). The 1993), he used the pond near Ostrau (in particular, well genome sequence of Tsp. jenense should stimulate the search known by H.-G. Schlegel who used material from the same for and the identifcation of other Thiospirillum and related pond during his PhD thesis, and at that time was head bacteria in nature, eventually leading to further isolation and of the Microbiology Institute in Göttingen) as a source characterization of other Thiospirillum species. 1 3 Archives of Microbiology (2021) 203:97–105 99 Materials and methods Metagenomic binning Cultures The sequencing reads were used to perform a metagen- omic binning using the Metagenomic Binning service Cultures of Thiospirillum jenense DSM 216T (Pfennig within PATRIC (Wattam et al. 2017). Paired-end reads 1112, strain Ostrau) were received by JFI from N. Pfen- were used as input and default parameters were used. Sets nig, who at this time (1991) worked at the University of of contig bins were constructed (hits against contigs that Konstanz. Living cultures were maintained for some years have less than fourfold coverage or are less than 400 bp in in the Institute for Microbiology at the University of Bonn length are removed). The contig pool was split into bins and in parallel the cultures were preserved immediately using reference genomes. Quality control of each bin was after transfer to Bonn and kept in liquid nitrogen.