Flavobacteria: recent settlers of the planet Earth Erko Stackebrandt, DSMZ-German Collection of Microorganisms and Cell Cultures GmbH Braunschweig Germany With the introduction of molecular and chemotaxonomic approaches to bacterial systematics, the Cytophaga-Flavobacterium complex, phylum Bacteroidetes, once one oft he dumping grounds in bacteriology, has been dissected and most of its members are today well defined. Only 7 of the 29 species described between 1923 and 1990 are today recognized as authentic Flavobacterium species; the majority of flavobacterial species were described after the advent of 16S rRNA gene sequencing approach (53 in June 2009), 50 of which are still recognized. A full hierarchic system has been provided with the release of Bergey’s Manual of Systematic Bacteriology, 2nd. ed (Ludwig et al., 2008), and the phylogenetic framework of high quality 16S rRNA genes sequences of most type strains are publicly available through the 'living tree' project (Yarza et al., 2008). Together with the availability of “minimal standards (Bernardet et al., 2002) for members of the family Flavobacteriaceae, this information facilitates the description of novel taxa. An increase in cultured diversity can indeed be anticipated, as the past decade already witnessed a dramatic increase in genera of Flavobacteriaceae. While in 1996 the family included 8 genera, the number increased to 61 in 2009. One can assume, that the natural growth requirements of members of this family, like those of other taxa such as Bacillus, Paenibacillus, Pseudomonas or Streptomyces, are well matched by the growth conditions and nutrient supply provided in the laboratory. The 16S rRNA gene sequence tree (Fig. 1) sees the majority aerobic members of the phylum Bacteroidetes cluster together in the family Flavobacteriaceae. The genus Flavobacterium branches next to members of the genus Myroides. Not indicated in this graph is the isolated position of Flavobacterium ceti, a whale associated bacterium, that appears to have a closer relationship to member of Myroides that to other species of Flavobacterium. Members of the genus Flavobacterium have been isolated from a wide range, mainly cold habitats, such as polar freshwater, sea ice and soil, as well as glaciers and freshwater from temperate zones; but also soil and wastewater appear to be a reservoir for these organisms. Warm niches, such as thermophilic springs (F. indicum), are rare. A 2007 survey on partial 16S rRNA gene sequences generated from non-culture studies on natural samples and deposited in public databases resulted in a very similar picture. Most of these sequences originated from glacier, freshwater and Antarctic samples but in addition flavobacterial DNA was also recovered from the Baltic sea, marine sediments and saline lakes and marshes. Apparently the occurrence of flavobacteria is more spread than anticipated from isolation studies, a biased picture of distribution that may be due to emphasis on the ecology of specific sites and biotechnological interests. Fig. 1. 16S rRNA gene sequence tree of genera of Flavobacteriaceae as depicted by the 'living tree' project (Yarza et al., 2008). Though Flavobacterium species are well defined according to the present species definition, the low resolution of most 16S rRNA gene lineages of Flavobacterium species predicts significant changes in phylogenetic placement of most intrageneric clusters once more sequences of type strains will be added to the database. As no publication depicts the position of a new species among the complete dataset of available sequences of type strains the position of individual species may change as it depends on the selection of reference strains. This situation makes it difficult to evaluate intrageneric relationships and even the dendrogram of Flavobacterium in the 'living tree' project is somewhat may not tell the evolutionary correct story, as not only the algorithm used but also the number of aligned sequences in general and those of neighboring genera in particular have an influence on the topology of (any) phylogenetic tree. Nevertheless, in the absence of knowledge about the course of evolution, scientists have to deduce information from the patterns provided by the most reasonable approaches. It appears that Flavobacterium species isolated from cold environments do not have a common origin, though groups of highly related psychrophilic organisms are detected (e.g., the Antarctic F. degerlachii, F. gillisiae, F. frigoris and F. gelidilacus; F. frigidimaris, F. omnivorum and F. fryxellicola; F. frigidimaris and F. micromatii; and F. segetis and F. waeverense) in different regions of the tree. Likewise, neither soil, nor freshwater-born species share a common origin (Fig. 2) Fig. 2 16S rRNA gene sequence tree of type strains of Flavobacterium as depicted by the 'living tree' project (Yarza et al., 2008). Members of Antarctic and glacier origin are in red. The tight phylogenetic relationship among many Flavobacterium species, separated taxonomically mainly on the basis of moderate DNA similarities (as determined by DNA-DNA reassociation) and often superficial differences in phenotypic properties is not unique in bacteriology. Nevertheless, for taxonomists this situation constitutes a dilemma: the ease at which novel flavobacteria can be isolated, together with the current practice in species description will soon lead to a situation where the lack of differentiating properties will prevent the description of novel species despite the presence of significant differences in gene sequences less conserved than 16S rRNA genes. While the genus itself is not a metabolically coherent genus, phylogenetically neighboring species are often also highly related in those characters, used in characterization and differentiation of species. The investigation of BIOLOG GN properties of 47 different type strains (Verbarg, Duckstein, Cousin & Stackebrandt, unpublished) may illustrate this: of the 95 metabolites tested the number of positive reactions per type strain range from 8-11 (F. fryxellicola, F. segetis and F. xinjiangense) to 49-50 (F. gelidilacus and F. hercynium). Note, that even strains from the same environment (Antarctic lakes), e.g., F. fryxellicola and F. gelidilacus, may differ dramatically in their metabolic reactions. Nevertheless, seven of the eight positive reactions in F. fryxellicola are also positive in F. gelidilacus. The 47 type strains share between as little as a single and 46 common positive reactions. Substrates utilized by more than 40 type strains (85%) are dextrin (46 strains), L-glutamic acid (42) and D-glucose (40). Most strains share between 20 and 30 common positive reactions. The differences in metabolic capacities are corroborated by analysis of MALDI-TOF whole cell protein patterns (Ali et al., 2009; Yang & Stackebrandt , unpublished) (Fig. 3). The lack of congruency between phylogenetic distantly related species and MALDI-TOF spectrum analyses has been noted before and is also seen here: while closely related species are also often related by protein spectrum analysis, significant shift are obvious, e.g. the pair F. segetis and F. waeverense, clustering with members of the F. frigidimaris group in 16S rRNA gene sequence analysis (Fig. 2), are well separated members of the F. degerlachii group. While MALDI-TOF is not a phylogenetic method for depicting inter-species relationships it appears a powerful approach to phenotypically characterize intra-species similarities (Cousin et al., 2006) Fig. 3 Distance dendrogram of MALDI-TOF analysis of whole cells of Flavobacterium type strains showing in red and green the position two groups of psychrophilic organisms most of which are also closely related by 16S rRNA gene sequence analysis (note, that not all newly described species are included in Fig. 2). Sphingomonas paucimobilis DSM 30198T and Novosphingobium resinovorum DSM 7478T served as outgroups. The lack of congruency in taxonomic outlines of relatedness and similarities is not new and occurs in basically all species-rich taxa. It will not conclude with the availability of the results of multi-locus sequence analysis of conserved housekeeping genes or even with taxo- genomic analysis of completely sequenced genomes. Genotype and expression of phenotype are not always tightly linked but subject to environmental factors which scientist may not be aware of. The title 'recent settlers' is not used to indicate that the flavobacteria are more recent than other taxa comprising aerobic bacteria. Flavobacteria are just another example of the evolutionary explosion of the phenotype once the oxygen level in the atmosphere raised from 0.2% (the Pasteur point, about 1.2 Gy ago) to 2%, 400 to 500 million years ago. While the 16S rRNA gene is too conserved in function to evolve rapidly, sequences of other genes such as those responsible for metabolic reactions are less constrained. Together with gene shuffling caused by horizontal gene transfer and phage activities the metabolic make-up of a cell changes sufficiently rapidly to distort synchronization between geno- and phenotype (this situation is often reversed in anaerobic bacteria, where the ecological niche put pressure on a conservative phenotype while the underlying gene sequences keep on evolving) The question remains, when did members of Flavobacterium evolve? According to different calibration approaches of 16S rRNA gene sequence divergence with time (Ochman and Wilson, 1987; Stackebrandt 1994), two sequences differing by 1% separated roughly