View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by PubMed Central International Journal of Systematic and Evolutionary Microbiology (2010), 60, 2960–2966 DOI 10.1099/ijs.0.018234-0 Reclassification of the polyphyletic genus Prosthecomicrobium to form two novel genera, Vasilyevaea gen. nov. and Bauldia gen. nov. with four new combinations: Vasilyevaea enhydra comb. nov., Vasilyevaea mishustinii comb. nov., Bauldia consociata comb. nov. and Bauldia litoralis comb. nov. Benjamin Yee,1,2 Gary E. Oertli,1 John A. Fuerst2 and James T. Staley1 Correspondence 1Department of Microbiology, University of Washington, Seattle, WA 98193, USA James T. Staley 2School of Molecular and Microbial Sciences, University of Queensland, Brisbane, Australia [email protected] Species of the genus Prosthecomicrobium are noted for their numerous cellular appendages or prosthecae that extend from the cells. This investigation confirms that the genus is polyphyletic based on an extensive analysis of the 16S rRNA gene sequences of several named species of the genus. The analyses indicate that some Prosthecomicrobium species are more closely related to non-prosthecate genera, including Devosia, Labrenzia, Blastochloris, Methylosinus, Mesorhizobium and Kaistia, than they are to other species of the genus Prosthecomicrobium. For this reason, two of the Prosthecomicrobium clades which are polyphyletic with the type species, Prosthecomicrobium pneumaticum, are renamed as new genera. The currently named species Prosthecomicrobium enhydrum, Prosthecomicrobium mishustinii, Prosthecomicrobium consociatum and Prosthecomicrobium litoralum are reclassified in two new genera, Vasilyevaea gen. nov. and Bauldia gen. nov. with four new combinations: Vasilyevaea enhydra comb. nov. (the type species) and Vasilyevaea mishustinii comb. nov., and Bauldia consociata comb. nov. and Bauldia litoralis comb. nov. (the type species). The type strain of Vasilyevaea enhydra is strain 9bT (5ATCC 23634T 5VKM B-1376T). The type strain of the other species in this genus is Vasilyevaea mishustinii strain 17T (5VKM B-2499T 5CCM 7569T). The type strain of Bauldia litoralis is strain 524-16T (5 NCIB 2233T 5ATCC 35022T). The type strain of the other species in this genus is Bauldia consociata strain 11T (5VKM B-2498T 5CCM 7594T). Bacteria of the genus Prosthecomicrobium, which is a consociatum (Lafitskaya et al., 1976; Vasil’eva et al., 1991). member of the class Alphaproteobacteria, reproduce by The names of the latter two species were recently validly budding and produce several cellular appendages or published (Vasil’eva et al., 2009). prosthecae that extend from each cell in all directions. The primary phenotypic, differentiating features among They were first described from isolates obtained from fresh species of the genus Prosthecomicrobium are morphological, water (Staley, 1968). Subsequently, other species were in particular the number and length of the prosthecae and reported that were isolated from various other habitats whether or not cells are motile. Other differentiating including soil (Vasil’eva et al., 1974), pulp mill aeration features among species of these aerobic bacteria are colony ponds, and brackish and marine water (Stanley et al., 1979; pigmentation, carbon source utilization and presence of Bauld et al., 1983; Schlesner et al., 1989). Species with gas vesicles. validly published names include Prosthecomicrobium pneu- maticum, P. enhydrum (Staley, 1968), P. litoralum (Bauld Phylogenetic analyses of 16S rRNA genes of strains et al., 1983), P. hirschii (Staley, 1984), P. mishustinii and P. representing species of the genus Prosthecomicrobium have revealed the polyphyletic nature of the group, which The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene forms several independent clusters within the class sequences determined in this study are FJ560749, FJ560750 and Alphaproteobacteria (Schlesner et al., 1989; Oertli et al., GQ221761–GQ221768. 2006). Furthermore, chemotaxonomic analysis of fatty 2960 018234 G 2010 IUMS Printed in Great Britain Vasilyevaea and Bauldia, new prosthecate genera acids, in which three identified type species were included, separated from the other named species of the genus also indicated that at least five distinct subgroups exist Prosthecomicrobium by the genera Methylosinus, Blastochloris within the current genus Prosthecomicrobium (Sittig & and Kaistia within the class Alphaproteobacteria. Therefore, Schlesner, 1993). The reason for the polyphyly is not P. pneumaticum is more closely related to the methane- understood; however, two explanations are possible. First, oxidizing bacteria Methylosinus trichosporium and the genes that are responsible for the formation of Methylocystis echinoides (Whittenbury et al., 1970) and the prosthecae may be ancestral to the family that contains phototrophic bacterial genus Blastochloris (Keppen & these bacteria. Then, through evolution, some of the genera Gorlenko, 1975; Hiraishi, 1997) than it is to other species and species lost these genes whereas others retained them. of the genus Prosthecomicrobium. Alternatively, one could postulate that genetic exchange has There is strong bootstrap support in Fig. 1 (.75 % at the occurred in which the genes responsible for prosthecae primary and secondary nodes of the trees) for the formation were transferred from one prosthecate species to clustering of the other species of the genus Pros- another closely related, non-prosthecate species through thecomicrobium with other genera and separate from the horizontal gene transfer. Perhaps the genome sequences of type species P. pneumaticum. Therefore, this is the basis for several representative prosthecate and non-prosthecate the reclassification of several described species of the genus species in this group will aid in resolving this issue. Prosthecomicrobium into new genera. Until now, no attempt has been made to reclassify the Consider the described species P. enhydrum and P. genus Prosthecomicrobium to reflect its polyphyletic nature. mishustinii that form an isolated group with Devosia In this paper, we report the results we have obtained from neptuniae (Fig. 1). The phylogenetic difference between this further comparative phenotypic and phylogenetic analyses. cluster and P. pneumaticum is illustrated by a comparison Based on these findings, we propose the reclassification of of P. pneumaticum and P. enhydrum, which share only several species of the genus Prosthecomicrobium into two 93.3 % 16S rRNA gene sequence similarity (data not new genera. shown). Furthermore, these two species have different 16S rRNA gene reference sequences were selected from phospholipids and fatty acids (Sittig & Schlesner, 1993). representatives of different orders within the class Alpha- For example, the phospholipids of P. pneumaticum include proteobacteria. These were then compared to 16S rRNA large amounts of phosphatidylglycerol, phosphatidyletha- gene sequences of the various species of the genus nolamine and phosphatidyldimethylethanolamine with Prosthecomicrobium including all type strains (Oertli et lower amounts of bisphosphatidylglycerol whereas P. al., 2006) as well as others from the RNA database (Cole et enhydrum contains only phosphatidylglycerol and bispho- al., 2003). The sequence match tool within the RDP-II sphatidylglycerol (Sittig & Schlesner, 1993). Since P. website was used to identify the sequences for alignment enhydrum and P. mishustinii are clustered independently based on best matches. Sequence alignment for phyloge- of P. pneumaticum, we propose a new genus, Vasilyevaea netic tree reconstruction was performed using the NAST gen. nov., with two species, Vasilyevaea enhydra comb. nov. (Nearest Alignment Space Termination; DeSantis et al., and Vasilyevaea mishustinii comb. nov., for this novel 2006) function available at the Greengenes website (http:// cluster. It should be noted that although these two species greengenes.lbl.gov) (DeSantis et al., 2006) and the are quite closely related to one another with 98.9 % 16S alignment was also subjected to Lane masking (Lane, rRNA gene sequence similarity, they are separate species 1991) of ambiguous sites using the column masking tool based upon DNA hybridization analyses (Vasil’eva et al., available at the Greengenes website. 1991). Phylogenetic trees were reconstructed using TreeFinder Similarly, P. litoralum and P. consociatum (with 97.1 % 16S (Jobb et al., 2004) by applying the substitution model of rRNA gene sequence similarity between them) form a clade GTR+I+G, the optimal substitution model selected by that is separate from the type species P. pneumaticum, with Modelgenerator (Keane et al., 2006). A maximum- which they have only 93.7 % and 94.4 % rRNA gene likelihood tree was reconstructed using TreeFinder (Jobb sequence similarity, respectively, as well as from et al., 2004). Distance and maximum-parsimony trees were Vasilyevaea enhydra comb. nov. and other clusters contain- reconstructed using the PHYLIP package (Felsenstein, 2007). ing members of the genus Prosthecomicrobium. A new Bootstrap analysis was performed using 1000 replicates for genus is therefore proposed for this group of prosthecate all trees. Similarity values were calculated using PHYLIP. bacteria, Bauldia gen. nov., with two species, Bauldia litoralis comb. nov. and Bauldia consociata comb. nov. The 16S rRNA gene sequence tree of the genus Prosthecomicrobium and those genera and species to which Prosthecomicrobium hirschii strains