International Journal of Systematic and Evolutionary Microbiology (2009), 59, 2122–2128 DOI 10.1099/ijs.0.003566-0 Emended descriptions of the genera Myxococcus and Corallococcus, typification of the species Myxococcus stipitatus and Myxococcus macrosporus and a proposal that they be represented by neotype strains. Request for an Opinion Elke Lang and Erko Stackebrandt Correspondence DSMZ – Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. Elke Lang 7B, 38124 Braunschweig Germany [email protected] The genus Corallococcus was separated from the genus Myxococcus mainly on the basis of differences in morphology and consistency of swarms and fruiting bodies of the respective members. Phylogenetic, chemotaxonomic and physiological evidence is presented here that underpins the separate status of these phylogenetically neighbouring genera. Emended descriptions of the two genera are presented. The data also suggest that the species Corallococcus macrosporus belongs to the genus Myxococcus. To the best of our knowledge, the type strains of the species Myxococcus macrosporus and Myxococcus stipitatus are not available from any established culture collection or any other source. A Request for an Opinion is made regarding the proposal that strain Cc m8 (5DSM 14697 5CIP 109128) be formally recognized as the neotype strain for the species Myxococcus macrosporus, replacing the designated type strain Windsor M271T, and that strain Mx s8 (5DSM 14675 5JCM 12634) be formally recognized as the neotype strain for the species Myxococcus stipitatus, replacing the designated type strain Windsor M78T. Introduction The genus Myxococcus was described by Thaxter (1892), Within the family Myxococcaceae, delineation of genera, who first recognized that the myxobacteria, formerly descriptions of species and affiliations of strains to species assigned to the fungi, were in fact bacteria. The species are based primarily on the morphology of vegetative cells, names Myxococcus disciformis, M. flavescens, M. fulvus, M. swarming colonies, fruiting bodies and myxospores. The stipitatus, M. xanthus, M. virescens, M. macrosporus and M. genus Corallococcus was established for strains that build coralloides have since been validly published. M. disciformis fruiting bodies with finger-like projections or of even more was later reclassified as Angiococcus disciformis (Hook et al., bizarre, coralloid shape with cartilaginous consistency. In 1980). In 1981, Reichenbach and Dworkin proposed the contrast, fruiting bodies of Myxococcus species are larger genus name Corallococcus for the former genus and globular, form knobs, either constricted at the base or ‘Chondrococcus’, but the genus name had no standing in on a stalk, and are of a soft consistency (Reichenbach & nomenclature until recently. In his description of the genus Dworkin, 1981). Other properties cited by Reichenbach Corallococcus, Reichenbach (2005c) included the taxa (2005a) as being useful for the differentiation of the two M. coralloides,‘Myxococcus exiguus’ and ‘Chondrococcus genera are that Myxococcus strains are generally sensitive to macrosporus’asCorallococcus coralloides comb. nov., kanamycin, whereas about 60 % of Myxococcus isolates Corallococcus exiguus nom. rev., comb. nov. and grow in the presence of streptomycin, that Corallococcus Corallococcus macrosporus comb. nov., respectively. The strains show opposite reactions towards these antibiotics, genus name was accepted as being validly published, but that Corallococcus strains produce squalene while the species name Corallococcus macrosporus was not Myxococcus strains do not and that Myxococcus strains accepted as being synonymous with Myxococcus macro- are more tolerant of NaCl (up to 2 %). sporus on formal grounds (Reichenbach, 2007; see details below). Profiles of phenotypic characters and fatty acids of individual strains are A small number of taxonomic studies have recently been available with the online version of this paper. published that elucidate the phylogenetic status of the 2122 003566 G 2009 IUMS Printed in Great Britain Request for an Opinion genera Myxococcus and Corallococcus. In a broad 16S rRNA M. stipitatus, for which culturable type strains are not gene sequence-based analysis of almost all myxobacterial available from any established culture collection or any genera described at that time, Spro¨er et al. (1999) found other source. members of the genus Corallococcus (genus name not validly published at the time) branching close but adjacent Differentiation of the genus Corallococcus from to members of the genus Myxococcus. M. macrosporus the genus Myxococcus strains Cc m7 and Cc m8 (5DSM 14697) did not cluster with other members of the genus Corallococcus but within Genomic properties and the composition of whole-cell the radiation of Myxococcus strains. As the 16S rRNA gene fatty acids and physiological properties of several strains of sequence similarities among myxococcal strains were the genus Corallococcus were described previously (Spro¨er higher than 98.2 % and only slightly lower between et al., 1999; Stackebrandt & Pa¨uker, 2005; Stackebrandt myxococci and members of the genus Corallococcus (97– et al., 2007). In this study, these investigations were 98 %), no decision on the taxonomic status of Myxococcus expanded to representatives of different Myxococcus macrosporus was made. species. Published 16S rRNA gene sequences were aligned In a more extensive study on the intrageneric structure of by the ae2 editor (Maidak et al., 1997) and by ARB (Ludwig the genus Corallococcus (Stackebrandt & Pa¨uker, 2005; et al., 2004) and dendrograms were generated using Stackebrandt et al., 2007), 37 strains of Corallococcus, neighbour-joining (Saitou & Nei, 1987) and least-squares Corallococcus macrosporus DSM 14697T and M. xanthus algorithms (De Soete, 1983) as well as maximum- DSM 16526T were analysed with respect to 16S rRNA, gyrB, likelihood analysis (Olsen et al., 1994). Bootstrap analysis rpoB, fusA and lepA gene sequence similarities. In each of was used to evaluate the significance of the tree topology the dendrograms calculated from these genes, Corallococcus statistically by performing 1000 resamplings (Felsenstein, macrosporus DSM 14697T clustered adjacent to M. xanthus 1993). The topologies of all dendrograms thus generated DSM 16526T but significantly outside the cluster defined were very similar (Fig. 1; least-squares analysis) in that by Corallococcus coralloides DSM 2259T and Corallococcus Corallococcus strains and Myxococcus strains formed two exiguus DSM 14696T. This finding was corroborated by well-separated clades supported by bootstrap values of significant differences in fatty acid profiles, Riboprint 99 %. The exception was Corallococcus macrosporus DSM T patterns and metabolic reactions (Stackebrandt et al., 14697 , which clustered within the Myxococcus clade in all 2007). It thus appeared that the morphology-based formal the dendrograms generated, adjacent to the three highly decision to classify M. macrosporus as a member of the related species M. virescens, M. flavescens and M. xanthus. genus Corallococcus is not supported by the chemotaxo- Strains from the DSMZ were used as reference strains for nomic, genetic and metabolic properties analysed. physiological tests and fatty acid analysis (Table 1). The Results of these studies raised afresh the question of cultures were maintained on bakers’ yeast agar (VY/2; DSM whether the separate status of the genera Corallococcus and medium 9). Test media were inoculated with cells 2 Myxococcus is justified and how and where to delineate the suspended in medium MD1 (l 1: 3 g casitone, 0.7 g genus boundaries. The aims of this study were threefold. CaCl2 .2H2O, 2 g MgSO4 .7H2O) after growth on CY agar 21 First, to amend the physiological and chemotaxonomic (l : 3 g casitone, 1 g CaCl2 .2H2O, 1 g yeast extract, 15 g description of Myxococcus species in order to accumulate agar; pH 7.2) for 2 days. Incubations were carried out at evidence as to whether or not the separate status of the two 28 uC. Starch and xylan hydrolysis were tested on overlay genera Myxococcus and Corallococcus is justified, second, to agar. The first layer consisted of water agar (l21:1g support the taxonomic position of Corallococcus macro- CaCl2 .2H2O and 15 g agar agar), the second of starch or sporus as a member of the genus Myxococcus, and third, to xylan agar (l21: 2 g native potato starch or 5 g oat spelt propose neotype strains for the species M. macrosporus and xylan, 0.5 g casitone, 1 g CaCl2 .2H2O, 1 g MgSO4 .7H2O, Fig. 1. Least-squares phylogenetic tree of Myxococcus and Corallococcus strains, based on 16S rRNA gene sequences. Bootstrap values are percentages calculated from 1000 resamplings. Bar, 2 % nucleotide substitutions. http://ijs.sgmjournals.org 2123 E. Lang and E. Stackebrandt Table 1. Myxococcus strains investigated All strains except DSM 16525T, DSM 16526T, DSM 6796, DSM 2260T and DSM 4946T were isolated by H. Reichenbach. The origins and sequence accession numbers of the Corallococcus strains included are listed in Stackebrandt & Pa¨uker (2005). Strain Other strain designation(s) Isolation source Geographical origin Myxococcus fulvus DSM 16525T M17T 5ATCC 25199T Soil Canada DSM 51991 Mx f53 Rabbit faeces Spain, Punta del Hidalgo Myxococcus xanthus DSM 16526T FBT 5ATCC 25232T 5NCIMB 9412T 5NBRC Soil Unknown 13542T 5CCUG 2079T 5CIP 107069T T DSM 6796 FBt/T 5Mx x6 Derivative of strain FB DSM 435 Mx x1 Soil with roots Liberia DSM 52389 Mx x29 Hare faeces