Ureaplasma Urease Genes Have Undergone a Unique Evolutionary Process Hiromi Nishida*

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The Open Systems Biology Journal, 2009, 2, 1-7 1 Open Access Ureaplasma Urease Genes have Undergone a Unique Evolutionary Process Hiromi Nishida*

Agricultural Bioinformatics Research Unit, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1- 1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan

Abstract: Ureaplasma, a member of mycoplasmas, has a unique ATP synthesis system, which is coupled to the urea hydrolysis. Urease catalyzes the hydrolysis of urea into carbon dioxide and ammonia. Phylogenetic analyses of the urease genes indicated that Ureaplasma urease genes were not gained by recent horizontal transfer and have a unique evolutionary process. Ureaplasma unique ATP synthesis system leaded to breakdown of the glycolysis pathway. Some glycolytic genes are absent and some glycolytic genes are evolving under relaxed selection in Ureaplasma. Probably glycolytic genes can be used as an indicator of ATP synthesis system. Thus, the organisms that have incomplete glycolysis pathway or glycolytic genes evolving under relaxed selection would have an ATP synthesis system independently of the glycolysis.

Mycoplasmas are widespread in nature as parasites of is coupled to ATP synthesis in Ureaplasma [9]. This unique mammals, reptiles, fishes, arthropods, and plants [1]. During system of Ureaplasma leaded to breakdown of the glycolysis the mycoplasma evolution, gene loss has occurred fre- pathway. For example, Ureaplasma does not have any genes quently, resulting in very small genome size [1-3]. The re- encoding glucose-6-phosphate isomerae [10]. It suggests that ductive evolution of mycoplasmas is still in progress. The the glycolysis system had been important for ATP synthesis genus Ureaplasma is a member of mycoplasmas, which gen- rather than glucose metabolism at least in Ureaplasma (Fig. erates 95% of its ATP using the hydrolysis of urea [4]. 1). Generally the TCA (tricarboxylic acid) cycle is linked to Growth of Ureaplasma is dependent on urea [5]. This unique the glycolysis pathway, which has played an important role ATP synthesis is not found in the other mycoplasmas. In in ATP synthesis of many organisms and conserved in the fact, key enzymes in the glycolytic pathway are absent in course of evolution. On the other hand, the urea hydrolysis is Ureaplasma [6]. In addition, some glycolytic genes of Urea- not linked to the glycolysis pathway. Ureaplasma generates plasma are evolving under relaxed selection [7, 8]. Thus, the ATP through the urea hydrolysis not through the glycolysis. glycolysis pathway is collapsing in Ureaplasma. Probably Ureaplasma glycolysis pathway was not able to be

PAST SYSTEM

Urea hydrolysis Glycolysis ATP synthesis

Urea hydrolysis ATP synthesis Glycolysis ATP synthesis

PRESENT SYSTEM

Urea hydrolysis ATP synthesis Glycolysis

Fig. (1). Model of change of ATP synthesis system in Ureaplasma . In Ureaplasma, the ATP synthesis through the glycolysis had been changed to that through the urea hydrolysis. The glycolysis pathway was broken after the urea hydrolysis was coupled to ATP synthesis.

Urea is hydrolyzed into carbon dioxide and ammonia in broken before the urea hydrolysis was coupled to ATP syn- many organisms. However, it is unique that the urea hydrolysis thesis. Therefore, after the coupling of the urea hydrolysis

and ATP synthesis, dominant ATP synthesis had been

*Address correspondence to this author at the Agricultural Bioinformatics changed from through the glycolysis to through the urea hy- Research Unit, Graduate School of Agricultural and Life Sciences, The drolysis during the Ureaplasma evolution (Fig. 1). University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan; E-mail: [email protected]

1876-3928/09 2009 Bentham Open 2 The Open Systems Biology Journal, 2009, Volume 2 Hiromi Nishida

(A) Rhizobium leguminosarum WSM 2304 88 Rhizobium etli CIAT 652 66 Rhizobium leguminosarum 3841 63 Rhizobium etli CFN 42 Agrobacterium tumefaciens C58 Cereon 22 99 Agrobacterium tumefaciens U.W./Dupont Bradyrhizobium japonicum USDA 110 26 15 Sinorhizobium meliloti 1021 14 99 Sinorhizobium medicae WSM 419 Alphaproteobacteria 10 Mesorhizobium sp. BNC 1 Beijerinckia indica ATCC 9039 54 Jannaschia sp. CCS 1 83 Silicibacter pomeroyi DSS-3 95 Silicibacter sp. TM 1040 26 Dinoroseobacter shibae DFL 12 65 Rhodobacter sphaeroides ATCC 17025 Methylobacterium radiotolerans JCM 2831 Saccharopolyspora erythraea NRRL 2338 Actinobacteria Ureaplasma urealyticum ATCC 33699 42 100 Ureaplasma parvum ATCC 700970 Firmicutes 99 Ureaplasma parvum ATCC 27815 100 Mycobacterium marinum M 85 Mycobacterium ulcerans Agy 99 82 Nocardia farcinica IFM 10152 Actinobacteria 88 Rhodococcus sp. RHA 1 Streptomyces avermitilis MA-4680 37 100 Bordetella bronchiseptica RB 50 Bordetella parapertussis 12822 Betaproteobacteria 88 Shewanella halifaxensis HAW-EB4 Vibrio fischeri MJ 11 95 Gammaproteobacteria 100 Vibrio fischeri ES 114

0.05 (B) Nocardia farcinica IFM 10152 Rhodococcus sp. RHA 1 Streptomyces avermitilis MA-4680 Shewanella halifaxensis HAW-EB4 89 Vibrio fischeri ES 114 72 93 100 Vibrio fischeri MJ 11 Bordetella parapertussis 12822 100 Bordetella bronchiseptica RB 50 Saccharopolyspora erythraea NRRL 2338 47 Ureaplasma urealyticum ATCC 33699 100 100 Ureaplasma parvum ATCC 27815 75 Ureaplasma parvum ATCC 700970

52 Methylobacterium radiotolerans JCM 2831 Beijerinckia indica ATCC 9039 Bradyrhizobium japonicum USDA 110 12 23 Sinorhizobium medicae WSM 419 24 64 27 Sinorhizobium meliloti 1021 24 Mesorhizobium sp. BNC 1 66 Rhizobium etli CFN 42 39 23 Rhizobium leguminosarum WSM 2304 29 67 Rhizobium etli CIAT 652 53 31 Rhizobium leguminosarum 3841 70 Agrobacterium tumefaciens C58 Cereon 100 Agrobacterium tumefaciens U.W./Dupont Jannaschia sp. CCS 1 47 Rhodobacter sphaeroides ATCC 17025 63 Dinoroseobacter shibae DFL 12

83 Silicibacter pomeroyi DSS-3 Silicibacter sp. TM 1040

99 Mycobacterium ulcerans Agy 99 Mycobacterium marinum M Fig. (2). Phylogenetic relationships among Ureaplasma UreA and the homologues. (A) Neighbo r-joining tree. The BLAST program was used to search the GenomeNet website for proteins homologous to Ureaplasma UreA with E-value < 10-23. Ureaplasma UreA and 30 homologous proteins were multiple aligned using the CLUSTAL W program. Phylogenetic tree was reconstructed, based on the multiple-alignment with complete deletion of gap sites using the neighbor-joining method of MEGA software with 1000 bootstrap replicates. (B) Maximum likelihood tree. Phylogenetic tree was reconstructed using the maximum likelihood method of the PHYLIP program with 100 bootstrap replicates. The JTT model was used as the model of amino acid substitution. Number of times to jumble in the PROML program was 2.

Ureaplasma Unique ATP Synthesis System The Open Systems Biology Journal, 2009, Volume 2 3

Staphylococcus aureus MSSA 476 (A) Staphylococcus aureus JH 1 Staphylococcus aureus MRSA 252 Staphylococcus aureus MW 2 Staphylococcus aureus Newman Staphylococcus aureus NCTC 8325 100 Staphylococcus aureus COL Staphylococcus aureus N 315 Firmicutes Staphylococcus aureus JH 9 Staphylococcus aureus Mu 3 97 Staphylococcus aureus Mu 50 Staphylococcus aureus TCH 1516 Staphylococcus aureus RF 122 Staphylococcus aureus USA 300 15 Staphylococcus saprophyticus ATCC 15305 21 Microcystis aeruginosa NIES-843 Synechocystis sp. PCC 6803 100 Anabaena sp. PCC 7120 74 23 Anabaena variabilis ATCC 29413 Nostoc punctiforme PCC 73102 47 Trichodesmium erythraeum IMS 101 Cyanobacteria 5 Synechococcus sp. PCC 7002 10 Prochlorococcus marinus MED 4 90 Prochlorococcus marinus MIT 9215 99 Prochlorococcus marinus MIT 9312 Methylobacillus flagellatus KT Betaproteobacteria 52 Methylobacterium populi BJ 001 Alphaproteobacteria 27 Arthrobacter aurescens TC 1 Corynebacterium urealyticum DSM 7109 Actinobacteria 21 Helicobacter acinonychis Sheeba Epsilomproteobacteria Ureaplasma urealyticum ATCC 33699 21 Ureaplasma parvum ATCC 700970 100 Firmicutes 95 Ureaplasma parvum ATCC 27815

0.05 (B)

Ureaplasma urealyticum ATCC 33699 Ureaplasma parvum ATCC 27815 Ureaplasma parvum ATCC 700970 Staphylococcus saprophyticus ATCC 15305 Staphylococcus aureus Mu 3

2 Staphylococcus aureus JH 9 92 Staphylococcus aureus MW 2 100 Staphylococcus aureus Newman Staphylococcus aureus N 315 100 4 Staphylococcus aureus JH 1

3 Staphylococcus aureus RF 122 Staphylococcus aureus USA 300 Staphylococcus aureus Mu 50 50 2 Staphylococcus aureus MSSA 476 11 3 Staphylococcus aureus COL

36 Staphylococcus aureus MRSA 252 4 Staphylococcus aureus NCTC 8325 Staphylococcus aureus TCH 1516 Synechocystis sp. PCC 6803 Synechococcus sp. PCC 7002 9 38 Prochlorococcus marinus MED 4 98 Prochlorococcus marinus MIT 9312 13 59 Prochlorococcus marinus MIT 9215 26 20 Microcystis aeruginosa NIES-843 100 Trichodesmium erythraeum IMS 101 Nostoc punctiforme PCC 73102 22 52 Anabaena variabilis ATCC 29413 91 Anabaena sp. PCC 7120

51 Methylobacterium populi BJ 001 Methylobacillus flagellatus KT Arthrobacter aurescens TC 1 45 Corynebacterium urealyticum DSM 7109 48 Helicobacter acinonychis Sheeba Fig. (3). Phylogenetic relationships among Ureaplasma UreB and the homologues. (A) Neighbor-joining tree. The BLAST program was used to search the GenomeNet website for proteins homologous to Ureaplasma UreB with E-value < 10-25. Ureaplasma UreA and 32 homologous proteins were multiple aligned using the CLUSTAL W program. Phylogenetic tree was reconstructed, based on the multiple- alignment with complete deletion of gap sites using the neighbor-joining method of MEGA software with 1000 bootstrap replicates. (B) Maximum likelihood tree. Phylogenetic tree was reconstructed using the maximum likelihood method of the PHYLIP program with 100 bootstrap replicates. The JTT model was used as the model of amino acid substitution. Number of times to jumble in the PROML program was 2. 4 The Open Systems Biology Journal, 2009, Volume 2 Hiromi Nishida

(A) Burkholderia mallei SAVP 1 95 Burkholderia mallei ATCC 23344 100 Burkholderia mallei NCTC 10229 Burkholderia mallei NCTC 10247 Betaproteobacteria 100 Burkholderia pseudomallei K 96243 81 Burkholderia multivorans Tohoku 100 Burkholderia multivorans JGI 48 Alcanivorax borkumensis SK 2 Acinetobacter baumannii ATCC 17978 84 Gammaproteobacteria 100 Acinetobacter baumannii AB 0057 75 Acinetobacter baumannii AYE 72 Dinoroseobacter shibae DFL 12 43 100 Mesorhizobium sp. BNC 1 Alphaproteobacteria 38 Herpetosiphon aurantiacus ATCC 23779 Clostridium thermocellum ATCC 27405 Chloroflexi 41 Bacillus halodurans C-125 72 Geobacillus kaustophilus HTA 426 Bacillus amyloliquefaciens FZB 42 Firmicutes 75 100 Bacillus subtilis 168 Clostridium phytofermentans ISDg 41 Arthrobacter sp. FB 24 100 Arthrobacter aurescens TC 1 Actinobacteria Bacillus cereus ATCC 10987 Staphylococcus saprophyticus ATCC 15305 46 100 Staphylococcus epidermidis RP62A Staphylococcus epidermidis ATCC 12228 100 Staphylococcus aureus MRSA 252 65 Staphylococcus aureus JH 1 93 Staphylococcus aureus JH 9 Staphylococcus aureus NCTC 8325 100 Staphylococcus aureus COL Staphylococcus aureus TCH 1516 Staphylococcus aureus Newman Staphylococcus aureus N 315 79 Staphylococcus aureus Mu 50 Firmicutes Staphylococcus aureus USA 300 Staphylococcus aureus MSSA 476 Staphylococcus aureus MW 2 Staphylococcus aureus RF 122 Staphylococcus aureus Mu 3 78 Streptococcus thermophilus LMG 18311 100 Streptococcus thermophilus CNRZ 1066 Streptococcus thermophilus LMD-9 93 Ureaplasma urealyticum ATCC 33699 100 Ureaplasma parvum ATCC 700970 100 Ureaplasma parvum ATCC 27815

0.05 (B)

Staphylococcus aureus TCH 1516 Staphylococcus aureus MW 2 Staphylococcus aureus NCTC 8325 Staphylococcus epidermidis ATCC 12228 100 Staphylococcus epidermidis RP62A Staphylococcus saprophyticus ATCC 15305 Bacillus cereus ATCC 10987 Clostridium phytofermentans ISDg 98 Geobacillus kaustophilus HTA 426 74 Bacillus halodurans C-125 39 Clostridium thermocellum ATCC 27405 6 Herpetosiphon aurantiacus ATCC 23779 Alcanivorax borkumensis SK 2 82 82 67 Burkholderia pseudomallei K 96243 46 100 100 46 Burkholderia mallei NCTC 10247 94 Burkholderia mallei NCTC 10229 76 29 Burkholderia mallei SAVP 1 100 37 18 Burkholderia mallei ATCC 23344 Burkholderia multivorans JGI 63 100 Burkholderia multivorans Tohoku Acinetobacter baumannii ATCC 17978 100 68 56 100 Acinetobacter baumannii AB 0057 32 77 Acinetobacter baumannii AYE Dinoroseobacter shibae DFL 12 100 Mesorhizobium sp. BNC 1 Bacillus amyloliquefaciens FZB 42 100 2 Bacillus subtilis 168 Arthrobacter aurescens TC 1 100 Arthrobacter sp. FB 24 100 Ureaplasma urealyticum ATCC 33699 Ureaplasma parvum ATCC 700970 71 100 Ureaplasma parvum ATCC 27815 Streptococcus thermophilus LMD-9 100 Streptococcus thermophilus LMG 18311 78 Streptococcus thermophilus CNRZ 1066 57 Staphylococcus aureus JH 9 Staphylococcus aureus JH 1 8 Staphylococcus aureus RF 122 Staphylococcus aureus COL 3 Staphylococcus aureus MSSA 476 Staphylococcus aureus Newman 4 Staphylococcus aureus N 315 Staphylococcus aureus MRSA 252 6 Staphylococcus aureus Mu 3 Staphylococcus aureus Mu 50 4 Staphylococcus aureus USA 300 Fig. (4). Phylogenetic relationships among Ureaplasma UreC and the homologues. (A) Neighbor-joining tree. The BLAST program was used to search the GenomeNet website for proteins homologous to Ureaplasma UreC with E-value < 10-170. Ureaplasma UreA and 45 homologous proteins were multiple aligned using the CLUSTAL W program. Phylogenetic tree was reconstructed, based on the multiple- alignment with complete deletion of gap sites using the neighbor-joining method of MEGA software with 1000 bootstrap replicates. (B) Maximum likelihood tree. Phylogenetic tree was reconstructed using the maximum likelihood method of the PHYLIP program with 100 bootstrap replicates. The JTT model was used as the model of amino acid substitution. Number of times to jumble in the PROML program was 2. Ureaplasma Unique ATP Synthesis System The Open Systems Biology Journal, 2009, Volume 2 5

UreD UreE

Staphylococcus aureus N315 Staphylococcus aureus TCH1516 Staphylococcus aureus MW2 Staphylococcus aureus JH9 Staphylococcus aureus JH1 Staphylococcus aureus JH1 Staphylococcus aureus COL 46 Staphylococcus aureus MRSA252 Staphylococcus aureus NCTC8325 Staphylococcus aureus USA300 Staphylococcus aureus Mu50 65 56 Staphylococcus aureus COL Staphylococcus aureus MSSA476 Staphylococcus aureus Newman Staphylococcus aureus Mu3 99 Staphylococcus aureus NCTC8325 Staphylococcus aureus TCH1516 Staphylococcus aureus RF122 99 Staphylococcus aureus RF122 73 Staphylococcus aureus MSSA476 Staphylococcus aureus USA300 Staphylococcus aureus JH9 99 Staphylococcus epidermidis RP62A 63 Staphylococcus aureus Newman 99 Staphylococcus epidermidis ATCC 12228 58 Staphylococcus aureus MRSA252 Staphylococcus saprophyticus 99 Staphylococcus epidermidis RP62A Clostridium phytofermentans 100 Staphylococcus epidermidis ATCC 12228 Corynebacterium urealyticum Staphylococcus saprophyticus Ureaplasma urealyticum ATCC 33699 Geobacillus kaustophilus 99 Ureaplasma parvum ATCC 27815 Ureaplasma urealyticum ATCC 33699 Ureaplasma parvum ATCC 700970 92 100 Ureaplasma parvum ATCC 27815 99 Ureaplasma parvum ATCC 700970

0.1 0.1

UreF UreG

Staphylococcus aureus MSSA476 Staphylococcus aureus Newman Staphylococcus aureus Newman Staphylococcus aureus JH1 Staphylococcus aureus COL Staphylococcus aureus JH9 63 Staphylococcus aureus USA300 Staphylococcus aureus COL Staphylococcus aureus TCH1516 Staphylococcus aureus TCH1516 64 Staphylococcus aureus NCTC8325 Staphylococcus aureus Mu50 100 Staphylococcus aureus MW2 Staphylococcus aureus N315 Staphylococcus aureus JH9 Staphylococcus aureus USA300 100 Staphylococcus aureus N315 Staphylococcus aureus NCTC8325 Staphylococcus aureus Mu3 Staphylococcus aureus MRSA252 47 Staphylococcus aureus JH1 100 Staphylococcus aureus Mu3 61 Staphylococcus aureus MSSA476 Staphylococcus aureus Mu50 33 Staphylococcus aureus MW2 Staphylococcus aureus MRSA252 66 100 Staphylococcus epidermidis ATCC 12228 71 Staphylococcus aureus RF122 33 Geobacillus kaustophilus Staphylococcus saprophyticus 32 Bacillus cereus ATCC 10987 Staphylococcus epidermidis ATCC 12228 Arthrobacter sp. FB24 100 Staphylococcus epidermidis RP62A Arthrobacter aurescens Clostridium phytofermentans 100 Clostridium phytofermentans Ureaplasma urealyticum ATCC 33699 Ureaplasma urealyticum ATCC 33699 Ureaplasma parvum ATCC 27815 100 Ureaplasma parvum ATCC 27815 100 Ureaplasma parvum ATCC 700970 100 68 Ureaplasma parvum ATCC 700970

0.1 0.05 Fig. (5). Phylogenetic relationships among Ureaplasma UreD and the homologues, UreE and the homologues, UreF and the homologues, and UreG and the homologues. The BLAST program was used to search the GenomeNet website for proteins homologous to Ureaplasma UreD, UreE, UreF, and UreG of Ureaplasma parvum ATCC 27815 with E-value < 10-35, 10-25, 10-40, and 10-77, respectively. This search led to identification of 17 UreD homologues, 20 UreE homologues, 20 UreF homologues, and 21 UreG homologues. The 18 UreD, 21 UreE, 21 UreF, and 22 UreG proteins were multiple aligned using the CLUSTAL W program on the GenomeNet website. Phylogenetic tree was reconstructed, based on the multiple-alignment with complete deletion of gap sites using the neighbor-joining method of MEGA software with 1000 bootstrap replicates.

Urease catalyzes the hydrolysis of urea into carbon diox- compared the amino acid sequences of urease genes of ide and ammonia. Urease has three core subunits, UreA, Ureaplasma with the homologous proteins in this study. UreB, and UreC. Generally these three core subunits and the The BLAST program was used to search the GenomeNet related accessory proteins UreD, UreE, UreF, and UreG are website (http://www.genome.jp) for proteins homologous to clustered on the genome. The order of the Ureaplasma (be- UreA, UreB, UreC, UreD, UreE, UreF, and UreG of Urea- longs to the Firmicutes) urease genes in the cluster is identi- -23 -25 -170 plasma parvum ATCC 27815 with E-value < 10 , 10 , 10 , cal to that of the Bacillus sp. TB-90 (belongs to the Fir- -35 -25 -40 -77 10 , 10 , 10 , and 10 , respectively. This search led to micutes) urease genes [11], suggesting that an ancestor of the identification of 30 UreA homologues, 32 UreB homo- Firmicutes had the urease gene cluster. Some species had logues, 45 UreC homologues, 17 UreD homologues, 20 lost some urease genes during the evolution. For example, UreE homologues, 20 UreF homologues, and 21 UreG Bacillus subtilis contains urease structural genes but lacks homologues. The 31 UreA, 33 UreB, 46 UreC, 18 UreD, 21 the accessory genes typically required for the maturation of UreE, 21 UreF, and 22 UreG proteins were multiple aligned urease [12]. On the other hand, the mycoplasmas except for using the CLUSTAL W program on the GenomeNet web- Ureaplasma completely lack urease genes. I have some site. Phylogenetic trees were reconstructed, based on the questions. Was the Ureaplasma urease genes (gene cluster) multiple-alignments with complete deletion of gap sites us- transferred horizontally? Or did the mycoplasmas except for ing the neighbor-joining method of MEGA software [13] Ureaplasma lose urease genes in the course of the myco- with 1000 bootstrap replicates. In addition, phylogenetic plasma evolution? Did the Ureaplasma urease have a unique trees of UreA, UreB, and UreC proteins were reconstructed, evolutionary process? In order to answer these questions, I based on the multiple alignments with complete deletion of 6 The Open Systems Biology Journal, 2009, Volume 2 Hiromi Nishida

Fig. (6). Ureaplasma UreB specific amino acids insertion region. Ureaplasma has a specific 9-amino acids insertion (positions 65-73) in the multiple alignment of 33 UreB proteins.

Fig. (7). Ureaplasma UreC specific amino acids insertion region. Ureaplasma has a specific 25-amino acids insertion (positions 554-578) in the multiple alignment of 33 UreC proteins. Ureaplasma Unique ATP Synthesis System The Open Systems Biology Journal, 2009, Volume 2 7 gap sites using the maximum likelihood method of the REFERENCES PHYLIP program (http://evolution.genetics.washington.edu/ [1] Razin S, Yogev D, Naot Y. Molecular biology and pathogenicity of phylip.html) with 100 bootstrap replicates. The JTT model mycoplasmas. Microbiol Mol Biol Rev 1998; 62: 1094-156. was used as the model of amino acid substitution. Number of [2] Jaffe JD, Stange-Thomann N, Smith C, et al. The complete genome times to jumble in the PROML program was 2. and proteome of Mycoplasma mobile. Genome Res 2004; 14: 1447-61. The phylogenetic analyses showed that the KEGG data- [3] Oshima K, Kakizawa S, Nishigawa H, et al. Reductive evolution base [14] used in this study did not have closely related pro- suggested from the complete genome sequence of a plant- tein to UreA and UreB of Ureaplasma (Figs. 2A, B and 3A, pathogenic phytoplasma. Nat Genet 2004; 36: 27-9. [4] Smith DGE, Russell WC, Ingledew WJ, Thirkell D. Hydrolysis of B). On the other hand, Ureaplasma UreC is closely related to urea by Ureaplasma urealyticum generates a transmembrane poten- Streptococcus UreC with 93% bootstrap support in the tial with resultant ATP synthesis. J Bacteriol 1993; 175: 3253-8. neighbor-joining tree (Fig. 4A) and 71% support in the [5] Kenny GE, Cartwright FD. Effect of urea concentration on growth maximum likelihood tree (Fig. 4B). The diverging points of of Ureaplasma urealyticum (T-strain mycoplasma). J Bacteriol UreA, UreB, and UreC of Ureaplasma are very deep in the 1977; 132: 144-50. [6] Pollack JD. Ureaplasma urealyticum: an opportunity for combina- neighbor-joining trees (Figs. 2A, 3A, 4A), strongly suggest- torial genomics. Trend Microbiol 2001; 9: 169-75. ing that Ureaplasma urease genes (gene cluster) were not [7] Oshima K, Nishida H. Phylogenetic relationships among myco- gained by recent horizontal transfer. It was also supported by plasmas based on the whole genomic information. J Mol Evol the phylogenetic trees of urease accessory proteins (Fig. 5). 2007; 65: 249-58. [8] Oshima K, Nishida H. Detection of the genes evolving under Urea- Interestingly, Ureaplasma UreB and UreC have Urea- plasma-specific selection. J Mol Evol 2008; 66: 529-32. plasma-specific amino acids insertions (Figs. 6, 7). Thus, [9] Romano N, La Licata R, Russo Alesi D. Energy production in Ureaplasma urease gene cluster has a unique evolutionary Ureaplasma urealyticum. Pediatr Infect Dis 1986; 5: S308-12. process and Ureaplasma has a unique urea hydrolysis system [10] Glass JI, Lefkowitz EJ, Glass JS. The complete sequence of the coupling to ATP synthesis. Maybe Ureaplasma ancestor had mucosal pathogen. Ureaplasma urealyticum. Nature 2000; 407: 757-62. used urease in order to live in the urea-rich environment. [11] Neyrolles O, Ferris S, Behbahani N, Montagnier L, Blanchard A. During the evolution, the urea hydrolysis was coupled to Organization of Ureaplasma urealyticum urease gene cluster and ATP synthesis system. After the event, glycolysis pathway expression in a suppressor strain of Escherichia coli. J Bacteriol has not been essential for ATP synthesis in Ureaplasma. 1996; 178: 647-55. [12] Kim JK, Mulrooney SB, Hausinger RP. Biosynthesis of active Some glycolytic genes are absent and some are evolving Bacillus subtilis urease in the absence of known urease accessory under relaxed selection in Ureaplasma. Probaly the estab- proteins. J Bacteriol 2005; 187: 7150-54. lishment of the unique ATP synthesis system triggered those [13] Tamura K, Dudley J, Nei M, Kumar S. MEGA4: Molecular Evolu- changes. If so, glycolytic genes can be used as an indicator tionary Genetics Analysis (MEGA) software version 4.0. Mol Biol of ATP synthesis system. Thus, the organisms that have in- Evol 2007; 24: 1596-99. [14] Kanehisa M, Araki M, Goto S, et al. KEGG for linking genomes to complete glycolysis pathway or glycolytic genes evolving life and the environment. Nucleic Acids Res 2008; 36: D480-D484. under relaxed selection would have a unique ATP synthesis system independently of the glycolysis.

Received: December 1, 2008 Revised: December 16, 2008 Accepted: December 17, 2008

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