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Microbiol. Cult. Coll. 27(2) :61 ─ 65, 2011 Review Article Phylogenetic studies on the bacterial phylum ‘Verrucomicrobia’ (2011 JSCC Award for Young Scientist) Jaewoo Yoon College of Pharmacy, Keimyung University, 1095 Dalgubeoldaero, Dalseo-Gu, Daegu 704-701, Republic of Korea INTRODUCTION The phylum‘Verrucomicrobia’ (Hedlund et al., 1997) is one of the primary lineages within the domain Bacteria. They represent a distinct lineage within the phylogenetic trees and contain a number of environmental species as well as a small number of cultured species. A number of molecular phylogenetic approaches and culture-independent studies based on 16S rRNA gene sequences (Hugenholtz et al., 1998; O’ Farrell & Janssen, 1999; Rappé & Giovannoni, 2003) revealed that the members of this phylogenetic group have been detected in a very wide range of quite different habitats within the global ecosystem. Especially, recent results of a phylogenetic analysis of soil metagenomic libraries (Liles et al., 2003) indicated that many rRNA genes from the bacterial artificial chromosome (BAC) library had substantially affiliated with the bacterial phylum of ‘Verrucomicrobia’ ; all of the sequences were affiliated with subdivisions that lack cultured representatives. At present, these lineages have been informally classified into five subdivisions numbered 1 to 5 (Hugenholtz et al., 1998) and have also been classified into six monophyletic subdivisions numbered 1 to 6 (Vandekerckhove et al., 2000) of which three are recognized in the second edition of Bergey’ s Manual of Systematic Bacteriology (Garrity & Holt, 2001) as the families Verrucomicrobiaceae (subdivision 1), Opitutaceae (subdivision 4) and‘Xiphinematobacteriaceae’ (subdivision 2). Since the six informal monophyletic subdivisions of this phylogenetic group were first proposed, the names of only a few genera such as Akkermansia, Alterococcus, Opitutus, Prosthecobacter, Rubritalea and Verrucomicrobium (Staley et al., 1976; Schlesner, E-mail: [email protected] 1987; Hedlund et al., 1996, 1997; Shieh & Jean; 1998; Chin et al., 2001; Derrien et al., 2004; Scheuermayer et al., 2006) belonging to subdivisions 1 and 4 have been validly published. Originally, this phylum was known from two genera and five species of prosthecate, aerobic heterotrophs isolated from freshwater environments: Verrucomicrobium spinosum, Prosthecobacter fusiformis, P. debontii, P. vanneervenii and P. dejongeii, although an obligately anaerobic, heterotrophic genus (Opitutus terrae) from rice paddy field soil has recently been isolated and characterized. In addition to the named microorganisms, three taxonomically uncharacterized isolates of “ultramicrobacteria”from rice paddy field soil with 16S rRNA gene sequences closely related to O. terrae have also been described. Alterococcus agarolyticus ADT3T, formerly misclassified as a member of the class Gammaproteobacteria, has been proposed to be included in the family Opitutaceae. The class Opitutae, comprising two orders: Puniceicoccales containing the family Puniceicoccaceae and Opitutales containing the family Opitutaceae, was formally proposed recently for the classification of subdivision 4 (Choo et al., 2007). However, in spite of wide ecological distribution of representatives of the phylum ‘Verrucomicrobia’ in nature, owing to relatively few pure cultivated and characterized species, the classification of this phylum is still ambiguous and informal. For this reason, for formal classification of the phylum‘Verrucomicrobia’ , it is recommended that many verrucomicrobia that thrives in a wide range of terrestrial, aquatic and marine habitats should be isolated and taxonomically investigated. Moreover, the remaining subdivisions must be proposed formally by the rank of class and this phylum should be also formally proposed. ─ 61 ─ Phylogenetic studies on the bacterial phylum‘Verrucomicrobia ’ PHYLOGENETIC STUDIES AND ANALYSIS OF THE CELL WALL COMPONENTS ON THE STRAINS OF THE CLASS OPITUTAE (SUBDIVISION 4) Phylogenetic studies were performed using polyphasic taxonomic approaches on eleven chemoheterotrophic bacteria which were isolated from various marine environments. The phylogenetic tree based on the neighbour-joining (NJ) generated comparisons of the 16S rRNA gene sequences revealed that these isolates were clustered within the family Puniceicoccaceae of the class Opitutae (subdivision 4). Eleven novel strains represented three independent monophyletic lineages that were distinct from species with validly published genera with high bootstrap confidence values (Fig. 1). Furthermore, molecular (DNA-DNA hybridization test and determination of the DNA G+C content), physiological (acid production from carbohydrates, enzyme activities and basic physiological tests) and biochemical and chemotaxonomic analyses (determination of respiratory quinone system and cellular fatty acid content and pigments analysis) were investigated on the novel isolates. In addition, increasing concentrations of the β-lactam antibiotic susceptibility tests and amino acid analysis of the cell wall hydrolysates indicated the absence of muramic acid and mesodiaminopimelic acid and the presence of relatively low molar ratios of alanine and glutamate, which suggested that these strains lack an ordinary Gram negative type of peptidoglycan in the cell wall. Based on the results of phylogenetic analysis and its polyphasic taxonomic evidences, the eleven isolates were considered to represent three novel genera and eight new species belonging to the family Puniceicoccaceae within the class Opitutae (subdivision 4), for which the names Coraliomargarita akajimensis gen. nov., sp. nov. (strain 04OKA010-24T ; Yoon et al., 2007a), Pelagicoccus mobilis gen. nov., sp. nov. (strain 02PA-Ca-133 T ; Yoon et al., 2007b), Pelagicoccus albus sp. nov. (strain YM14-201T; Yoon et al., 2007b), Pelagicoccus litoralis sp. nov. (strains H-MN57T, H-MN48 and MN1-156; Yoon et al., 2007b), Pelagicoccus croceus sp. nov. (strain N5FB36-5T ; Yoon et al., 2007d), Cerasicoccus arenae gen. nov., sp. n o v . ( s t r a i n Y M26-026 T ; Y o o n e t a l . , 2007c ) , Cerasicoccus maritimus sp. nov. (strain YM31-114T; Yoon et al., 2010a) and Cerasicoccus frondicus sp. nov. (strains YM31-066T and YM31-067; Yoon et al., 2010a) are proposed. Jaewoo Yoon PHYLOGENETIC STUDIES ON THE STRAINS OF THE FAMILY VERRUCOMICROBIACEAE (SUBDIVISION 1) AND FORMAL CLASSIFICATION OF THE PHYLUM ‘VERRUCOMICROBIA’ Nineteen chemoheterotrophic bacterial strains were isolated from various marine environments and organisms, and were subjected to phylogenetic investigations using polyphasic taxonomic methods. Phylogenetic analysis based on the neighbour-joining (NJ) generated comparisons of the 16S rRNA gene sequences indicated that these isolates were affiliated to the family Verrucomicrobiaceae (subdivision 1) within the phylum‘Verrucomicrobia’ . Among them, three novel strains had sequences that were related to the genus Rubritalea. Sixteen isolates studied form four different monophyletic clades that were distinct from species with validly published genera with high bootstrap confidence values (Fig. 1). Furthermore, molecular (DNA-DNA hybridization test and determination of the DNA G+C content), physiological (acid production from carbohydrates, enzyme activities and basic physiological tests) and biochemical and chemotaxonomic analyses (determination of respiratory quinone system and cellular fatty acid content and pigments analysis) were investigated on the novel isolates. Some of the novel strains produced carotenoid pigments and squalene. Analysis of the nearly complete 16S rRNA gene sequences from these strains indicated that the Rubritalea strains formed a robust clade having only limited resolution for this very tight group of species. However, the interrelationships deduced from the gyrβ gene-based phylogeny showed better resolution than those based on their 16S rRNA gene sequences for the differentiation of strains at the species level (Yoon et al., 2010b). The genomic DNADNA relatedness values, biochemical and chemotaxonomic data (proportions of cellular fatty acids and menaquinones) also supported the results. On the basis of polyphasic evidences and phylogenetic characteristics, it was concluded that these strains should be classified as four novel genera and fourteen new species of the family Verrucomicrobiaceae within the class Verrucomicrobiae (subdivision 1), for which the names Rubritalea spongiae sp. nov. (strain YM21-132T; Yoon et al., 2007e), Rubritalea tangerina sp. nov. (strain YM27-005 T ; Yoon et al., 2007e), Rubritalea sabuli sp. nov. (strain YM29-052T; Yoon et al., 2008a), Persicirhabdus sediminis gen. nov., sp. ─ 62 ─ Microbiol. Cult. Coll. Dec. 2011 Vol. 27, No. 2 0.02 1118 bp 82 95 95 63 100 87 100 100 T Po1012(DQ302104) Seawater clone 4(DQ302105) T YM27-005(AB297806) T 99 99 YM21-132(AB297805) T HOact23(AB277853) 99 sp. MN1-1006(AB373024) 92 T 100 YM29-052(AB353310) 100 Marine clone SJC3.21(DQ071119) Polychaete clone A 54(AJ441243) 92 T 94 YM20-087(AB331886) YM21-151 (AB331887) 100 T MN1-741(AB331888) T YM27-120(AB331889) 100 T YM26-010(AB331890) 63 YM24-184 (AB331891) 99 99 77 YM20-122(AB331892) 100 Elbe river clone DEV005(AJ401105) Crater lake clone CL500-85(AF316731) 99 T A5J-41-2(AB331893) 100 A5J-40 (AB331894) T 57 A4T-83(AB331895) 99 T YM23-227(AB372852) 98 97 T 93 06SJR1-1(AB372853) 73 T AK18-024(AB372854) T 05IJR53-1(AB372855) T 99 MN1-1037(AB372856) 100 MN1-1047 (AB372857) T ATCC BAA-835(AY271254) 100 Human colonic clone HuCa18(AJ408970) T 99 FC4 ATCC 25309(U60015) T 100 FC1 ATCC 27091(U60012) 100 T FC3i ATCC 700200(U60014) T FC2 ATCC 700199(U60013) 100 T DSM 4136(X90515) 100 ’ Ellin428(AY388649) 100 Bacterium Ellin502 (AY960765) 88 Bacterium Ellin507 (AY960770) Uncultured bacterium clone DA101(Y07576) 92 99 Candidatus (AF217461) Candidatus (AF217460) 100 Candidatus (AF217462) 100 Bacterium Ellin515(AY960778) 100 Bacterium Ellin518(AY960781) Bacterium Ellin5102(AY234519) 92 Freshwateriake clone LD19(AF009974) T PB90-1 DSM 11246(AJ229235) 100 ACB90(AJ229246) 98 46 PB90-3(AJ229239) 84 sp. VeGlc2(X99390) 100 sp. VeCb1 (X99391) 98 Archaeological wood clone pACH90(AY297806) 59 sp. strain VeSm13(X99392) 100 Opitutaceae bacterium TAV1(AY587231) Uncultured bacterium clone BCf3-05(AB062814) 96 82 Opitutaceae bacterium TAV3(AY587233) 82 Opitutaceae bacterium TAV4(AY587234) 100 Opitutaceae bacterium TAV2(AY587232) T BCRC 19135(AF075271) T 91 YM31-114(AB372849) T 100 YM26-026(AB292183) T YM31-066(AB372850) 93 100 YM31-067 (AB372851) 62 T IMCC1545(DQ539046) T 04OKA010-24(AB266750) 37 100 ‘ ’ SI-1234(AB073978) T 54 N5FB36-5(AB297922) T 02PA-Ca-133(AB286015) T YM14-201(AB286016) 100 H-MN48 66 67 MN1-156 100 T H-MN57(AB286017) sp. SA-9(AY695840) 99 Contaminated aquifer clone WCHB1-41(AF050560) Unidentified marine bacterium clone BD2-18(AB015546) Contaminated aquifer clone WCHB1-25 (AF050559) Anoxic marine sediment clone LD1-PA26(AY114317) T ATCC 11775(X80725) 99 The class The order The family Verrucomicrobiaceae (Subdivision 1) The class ‘ (Subdivision 2) ’ Subdivision 3 Subdivision 6 The order The family The class (Subdivision 4) The order The family Subdivision 5 Fig. 1 Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showing the relative positions of the novel strains described in this study among the currently known and cultivated species of the phylum ‘Verrucomicrobia’ . Numbers at the nodes indicate the bootstrap value derived from 1000 replications. Sequences determined in this study are shown in bold. Bar, 2% sequence divergence. ─ 63 ─ Phylogenetic studies on the bacterial phylum‘Verrucomicrobia ’ nov. (strains YM20-087T and YM21-151; Yoon et al., 2008b), Roseibacillus ishigakijimensis gen. nov., sp. n o v . ( s t r a i n M N1-741 T ; Y o o n e t a l . , 2008b ) , Roseibacillus ponti sp. nov. (strain YM27-120T; Yoon et al., 2008b), Roseibacillus persicicus sp. nov. (strains YM26-010T , YM24-184 and YM20-122; Yoon et al., 2008b), Luteolibacter pohnpeiensis gen. nov., sp. nov. (strain A4T-83T ; Yoon et al., 2008b), Luteolibacter algae sp. nov. (strains A5J-41-2T and A5J-40; Yoon et al., 2008b), Haloferula harenae gen. nov., sp. nov. (strain YM23-227T ; Yoon et al., 2008c), Haloferula rosea sp. nov. (strain 06SJR1-1T; Yoon et al., 2008c), Haloferula phyci sp. nov. (strain AK18-024T; Yoon et al., 2008c), Haloferula helveola sp. nov. (strain 05IJR53-1T; Yoon et al., 2008c) and Haloferula sargassicola sp. nov. (strains MN1-1037T and MN1-1047; Yoon et al., 2008c) are proposed. With these isolates, emended descriptions of the class Verrucomicrobiae, the order Verrucomicrobiales and the family Verrucomicrobiaceae are presented. Moreover, to establish the formal classification of the phylum ‘V e r r u c o m i c r o b i a’ , we formally proposed Verrucomicrobia phy. nov., nom. rev. (Yoon et al., 2010a). CONCLUSION We attempted to elucidate the phylogenetic relationships of the thirty novel isolates using polyphasic taxonomic approaches including 16S rRNA and gyrβ gene sequence analysis, together with molecular, physiological, biochemical and chemotaxonomic analyses to characterize the novel strains. Based on these data, it is proposed that these isolates represent seven novel genera and twenty-two new spec i e s w i t h i n t h e p h y l u m‘V e r r u c o m i c r o b i a’ . Furthermore, with these novel strains, the formal classification of the phylum‘Verrucomicrobia’is established. REFERENCES Chin, K.J., Liesack, W. & Janssen, P.H. 2001. 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