lnternational Journal of Systematic Bacteriology (1 999), 49,479-481 Printed in Great Britain

Phylogenetic position of Chitinophaga pinensis v‘iin the Flexibacter-Bacteroides-Cflophaga phylum

L. 1. Sly,’ M. Taghavi’t and M. Fegan’

Author for correspondence: L. I. Sly. Tel: +61 7 3365 2396. Fax: +61 7 3365 1566. e-mail : [email protected]

Centre for Bacterial Comparison of the 165 rRNA gene sequence determined for Chitinophaga Diversity and pinensis showed that this species is most closely related to Hexibacfer Identification, Department of Microbiology’ and the filiformis in the Flexibacter-BacteroiC~phagaphylum. These two Cooperative Research chitinolytic bacteria, which are characterized by transformation into spherical Centre for Tropical Plant bodies on ageing, belong to a strongly supported lineage that also includes Pathology2, University of Queensland, Brisbane, C’ophaga arvensicola, Flavobacferium ferrugineum and FIexibacfer sancti. The Australia 4072 lineage is distinct from the microcyst-formingspecies Sporoc’ophaga myxococcoides.

Keywords: Chitinophaga pinensis, 16s rRNA, phylogeny, bacterial taxonomy, Flexibacter-Bacteroides-Cytophaga phylum

In 1981, Sangkhobol & Skerman described the species similarities between the morphology of Chitinophaga Chitinophaga pinensis to include five strains of a long, pinensis and Flexibacterfiliformis (Reichenbach, 1992). filamentous, chitinolytic, gliding bacterium that trans- forms on ageing into spherical bodies. The authors Current knowledge of the phylogenetic relationships likened the coccoid bodies to the microcysts of of the flexibacteria derived from 16s rRNA catalogues Sporocytophaga myxococcoides (Leadbetter, 1989). (Paster et al., 1985) shows that Flexibacter filiformis Sangkhobol and Skerman noted that the myxospores (‘Flexibacter elegans’ strain Fx el) and Sporocyt- germinated without any clear evidence of capsule ophaga rnyxococcoides are on phylogenetically distinct shedding as previously described by Leadbetter (1 963) lines within the Flexibac ter- Bacteroides-Cy t ophaga for Sporocytophaga myxococcoides, nor did they ex- phylum. hibit the high refractility of Sporocytophaga myxo- To date, no 16s rRNA sequence has been available for coccoides microcysts under phase microscopy. No Chitinophaga pinensis to determine its phylogenetic comparison with other characteristics of Sporocyto- relationship with the other two species of gliding phaga myxococcoides myxospores such as heat and flexibacteria (Flx.5liformis and Sporocytophaga myxo- desiccation resistance was made. Chitinophaga pinensis coccoides) reported to form spherical bodies (myxo- was differentiated from Sporocytophaga myxo- spores, microcysts or resting stages) on ageing. In this coccoides on the bases of cells more than four times the study we have sequenced the 16s rDNA of the length of Sporocytophaga myxococcoides, an ability to Chitinophaga pinensis type strain ACM 2034T hydrolyse chitin but not cellulose, and a G + C content (= DSM 25MT)and compared this with the available of its genomic DNA of 43-46 mol% compared with sequences of Flexibacter filiformis, Sporocytophaga 36 mol YOfor Sporocytophaga myxococcoides. myxococcoides and other reference sequences in the Skerman (1989) later introduced the term microcyst to Flexibac ter- Bac t ero ides-Cy top haga phylum . describe the spherical coccoid bodies but used this The sequencing of the 16s rDNA of Chitinophaga term interchangeably with the terms myxospores and pinensis and the phylogenetic analysis was carried out resting stage without any clear definitions. Reichen- using methods previously described (Sly et al., 1998). bach (1992) has disputed the formation of microcysts Direct PCR amplification of 16s rDNA was performed by Chitinophaga pinensis and drew attention to the and the PCR products were purified using Promega Magic PCR Prep DNA Purification according to the tPresent address: College of Agriculture, Shiraz University, Shiraz, Iran. manufacturer’s instructions. The PRISM Ready Re- The GenBank accession number for the rDNA sequence of Chitinophaga action DyeDeoxy Terminator Cycle Sequencing Kit pinensis ACM 2034T is AF078775. (Applied Biosystems) was used with the primers 27f,

~ 00867 0 1999 IUMS 479 L. I. Sly, M. Taghavi and M. Fegan

926f, 1114f, 357r, 519r, 907r, 1100r, 1392r and 1525r (Lane, 1991), and 787r and 803f (Stackebrandt & Charfreitag, 1990) to directly sequence the PCR products, following the protocol provided by the

' manufacturer. The reaction mixtures were sequenced 95 Cytophaga hutchinsonii Spo ro cytoph aga myxo co cco ides automatically on an Applied Biosystems 373A DNA - 86 Cyclobacterium marinum Sequencer. 'Microscilla furvescens' Flexibacter flexilis The 16s rRNA sequences were aligned manually using Flexibacter ruber the ae2 editor (Maidak et al., 1997) with representative bacterial 16s rRNA gene sequences from GenBank Runella slithyformis and the rRNA Database Project (Maidak et al., 1997). 79 Flexibacter roseolus Flexibacter lit0 ra lis Positions where length and sequence variations made Spirosoma linguale alignment uncertain were omitted from the analysis. Chitinophaga pinensis Pairwise evolutionary distances were computed using Flexibacter filifo rm is the method of Jukes & Cantor (1969) in the DNADIST Flexibacter sancti PHYLIP - 100 program of the 3.5~software package (Felsen- Cytophaga arvensicola stein, 1993). Phylogenetic dendrograms were con- Flavobacteriurn ferrugineum structed using the neighbour-joining method of Saitou 88 96 Lewinella cohaerens ~ 100 Lewinella nigricans & Nei (1987) in PHYLIP. Bootstrap analyses using Lewinella persica SEQBOOT from PHYLIP 3.5~were performed to de- 99 -99 Saprospira grandis termine the statistical significance of branching - Ha liscomenobacter hydrossis patterns and consensus trees (not shown) were pro- duced using CONSENSE (PHYLIP 3.5~). The dendrogram of relationships (Fig. 1) inferred from a neighbour-joining analysis of the corrected dissimi- larity values over a length of 987 nucleotides (restricted by a short 1263 nucleotide sequence for Cytophaga arvensicola) indicated that Chitinophaga pinensis belongs to a strongly supported lineage in the Flexi- Fig, 1. Phylogenetic neighbour-joining dendrogram showing bacter-Bacteroides-Cytophaga phylum. Members of the relationship of Chitinophaga pinensis with other related the lineage also include Flavobacterium ferrugineum, species in the Flexibacter-Bacteroides-Cytophaga phylum. The Cytophaga arvensicola, Flexibacter sancti, and its sequence of Chlorobium vibrioforme was used as the outgroup. Bootstrap values of 100 analyses are shown at the branch nearest relative, Flexibacter filiforrnis. Nakagawa & points. The scale bar represents 10 nucleotide substitutions per Yamasato (1993) have suggested previously that F. 100 nucleotides of 165 rRNA sequence. The strain numbers and ferruginium, Flexibacter sancti and Cytophaga arvensi- sequence accession numbers (in parentheses) of reference cola may constitute a distinct genus on the bases of sequences used in the analysis are as follows: Bacteroides their phylogentic relationship determined from 16s fragilis (M61006); Chlorobium vibrioforme DSM 260T(M62791); Cyclobacterium rnarinum ATCC 43824 (M62788); Cytophaga rDNA sequence similarities, their strictly respiratory arvensicola IAM 1 2650T (D12657); Cytophaga diffluens ATCC metabolism, MK-7 menaquinone content and a base 23140 (M58765); Cytophaga hutchinsonii ATCC 33406T composition in the range 42.8-48.6 mol %, which is at (M58768); Empedobacter brevis ATCC 14234 (M59052); the high end of the range for the genus Flexibacter Flavobacterium aquatile ATCC 1 1 947T (M62797); Flavobacteriurn ferrugineum ATCC 13524T (M62798); (Reichenbach, 1989). Nakagawa & Yamasato (1996) Flectobacillus major ATCC 29496T (M62787); Flexibacter later showed that Flexibacter filiformis is also a aggre ans ATCC 231 62T (M64628); Flexibacter canadensis ATCC member of this phylogenetic lineage, but on the basis 29591? (M62793); Flexibacter filiformis ATCC 29495T (= Fx el) of bootstrap values showed that the relationship of (M58782); Flexibacter flexilis ATCC 2307gT (M62794); Flexibacter Flavobacterium ferrugineum litoralis ATCC 231 17T (M58784); Flexibacter roseolus ATCC to the other three 23088T (M58787); Flexibacter ruber ATCC 23103T (M58788); members is not strongly supported. The current study Flexibacter sancti ATCC 23092T (M62795) ; Flexithrix dorotheae has added weight to the emerging taxonomic im- ATCC 23 1 63T (AF039296); Haliscomenobacter hydrossis ATCC portance of this deeply branched and well supported 27775T (M58790); Lewinella cohaerens ATCC 23123T lineage which now also includes Chitinophaga pinensis. (AF039262); Lewinella nigricans ATCC 23 1 47T (AF039294); Lewinella persica ATCC 23 1 67T (AF039295) ; 'Microscilla This lineage is well separated from the lineage which aggregans subsp. catalatica' ATCC 231 90 (M58791); 'Microscilla contains Flexibacter jlexilis, the type species of the arenaria' ATCC 231 61 (M60455); Microscilla furvescens', ATCC genus, and supports the transfer of the member species 23129 (M58792); Rhodothermus marinus (X77140); Runella to a new genus. Woese et al. (1990) previously reported slithyformis ATCC 29530T (M62786); Saprospira grandis ATCC Flexibacter 231 lgT (M58795); Spirosoma linguale ATCC 23276 (M62789); the widespread distribution of the species Sporocytophaga myxococcoides ATCC 1001OT (spc.myxoco RDP); in the FBC phylum and showed that Flexibacterflexilis Thermonema lapsum (L11703). and Flexibacter sancti belonged to widely separated lineages. However, there appear to be few phenotypic characteristics which unify the members of the phylo- type. As mentioned earlier, Chitinophaga pinensis and

480 Intern a ti0 na I Jo urnaI of Systematic Bacteriology 49 Chitinophagapinensis

Flexibacter Jiliformis are quite similar in morphology Nakagawa, Y. & Yamasato, K. (1996). Emendation of the genus and share the ability to degrade chitin. Cytophaga Cytophaga and transfer of Cytophaga agarovorans and Cyto- arvensicola and Flexibacter sancti on the other hand phaga salmonicolor to Marinilabilia gen. nov. : phylogenetic cannot degrade chitin, have much shorter cells and do analysis of the Flavobacterium-Cytophagacomplex. Int J Syst Bacteriol46, not form spherical bodies on ageing. In spite of their 599-603. superficial phenotypic similarity, the 95-3% sequence Paster, B. J., Ludwig, W., Weisburg, W. G., Stackebrandt, E., similarity between the 16s rRNA genes of Chitino- Hespell, R. B., Hahn, C. M., Reichenbach, H., Stetter, K. 0. & phuga pinensis Flexibacter Jiliformis Woese, C. R. (1985). A phylogenetic grouping of the bacteroides, and over a length cytophagas, and certain flavobacteria. Syst Appl Microbiol6, of 1445 nucleotides is indicative of their separate 34-42. species status (Stackebrandt & Goebel, 1994). The fact Sporocytophaga myxococcoides Reichenbach, H. (1989). Genus Flexibacter Soriano 1945, 92AL that belongs to a sep- emend. In Bergey’s Manual of Systematic Bacteriology, vol. 3, arate phylogenetic lineage in the Flexibacter- pp. 2061-2071. Edited by J. T. Staley, M. P. Bryant, N. Pfennig Bacteroides-Cytophaga phylum as previously reported & J. G. Holt. Baltimore: Williams & Wilkins. et al., (Paster 1985), indicates that transformation to Reichenbach, H. (1992). The order Cytophagales. In The Pro- spherical bodies is not phylogenetically exclusive and karyotes, 2nd edn, vol. IV, pp. 363 1-3675. Edited by A. Balows, may have been an early evolutionary trait or, more H. G. Triiper, M. Dworkin, W. Harder & K.-H. Schleifer. New likely, that the nature of the spherical body in York: Springer. Chitinophaga pinensis and Flexibacter Jiliformis is Saitou, N. & Nei, M. (1987). The neighbour-joining method: a different to that in Sporocytophaga myxococcoides, as new method for constructing phylogenetic trees. Mol Biol Evol previously suggested by Reichenbach (1992). 4, 406-425. Sangkhobol, V. & Skerman, V. B. D. (1981). Chitinophaga, a new genus of chitinolytic myxobacteria. Int J Syst Bacteriol 31, References 28 5-293. Felsenstein, 1. (1993). PHYLIP (Phylogeny Inference Package) Skerman, V. B. D. (1989). Genus Chitinophaga Sangkhobol and version 3.5~.Seattle : University of Washington. Skerman 1981. In Bergey’s Manual of Systematic Bacteriology, P. Jukes, T. H. & Cantor, C. R. (1969). Evolution of protein vol. 3, pp. 2074-2077. Edited by J. T. Staley, M. Bryant, N. & G. & molecules. In Mammalian Protein Metabolism, pp. 21-1 32. Pfennig J. Holt. Baltimore: Williams Wilkins. Edited by H. N. Munro. New York: Academic Press. Sly, L. I., Taghavi, M. & Fegan, M. (1998). Phylogenetic het- erogeneity within the genus Herpetosiphon : transfer of the Lane, D. J. (1991). 16S/23S rRNA sequencing. In Nucleic Acid marine species Herpetosiphon cohaerens, Herpetosiphon nigri- Techniques in Bacterial Systematics, pp. 115-175. Edited by E. cans, and Herpetosiphon persicus to the genus Lewinella gen. & : Stackebrandt M. Goodfellow. Chichester Wiley. nov. in the Flexibacter-Bacteroides-Cytophaga phylum. Int J Leadbetter, E. R. (1963). Growth and morphogenesis of Sporo- Syst Bacteriol48, 73 1-737. cytophaga. Bacteriol Proc p. 42. Stackebrandt, E. & Charfreitag, 0. (1990). Partial 16s rRNA Leadbetter, E. R. (1989). Genus IV. Sporocytophaga Stanier primary structure of five Actinomyces species : phylogenetic 1940. In Bergey’s Manual of Systematic Bacteriology, vol. 3, pp. implications and development of an Actinomyces israelii- 2061. Edited by J. T. Staley, M. P. Bryant, N. Pfennig & J. G. specific oligonucleotide probe. J Gen Microbioll36, 3743. Holt. Baltimore: Williams & Wilkins. Stackebrandt, E. & Goebel, B. (1994). Taxonomic note: a place Maidak, B. L., Olsen, G. J., Larsen, N., Overbeek, R., McCaughey, for DNA-DNA reassociation and 16s rRNA sequence analysis R. & Woese, C. R. (1997). The RDP (Ribosomal Database in the present species definition in bacteriology. Int J Syst Project). Nucleic Acids Res 25, 109-1 11. Bacteriol44, 846849. Nakagawa, Y. & Yamasato, K. (1993). Phylogenetic diversity of Woese, C. R., Yang, D., Mandelco, L. & Stetter, K. 0. (1990). The the genus Cytophaga revealed by 16s rRNA sequencing and flexibacter-flavobacter connection. Syst Appl Microbiol 13, menaquinone analysis. J Gen Microbioll39, 1155-1 161. 161-165.

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