MBE Advance Access originally published online on April 4, 2007
Molecular Biology and Evolution 2007 24(7):1439-1442; doi:10.1093/molbev/msm069
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Coexistence of Tubulins and ftsZ in Different Prosthecobacter Species
* Lehrstuhl für Mikrobiologie, Technical University Munich, Freising, Germany
Dipartimento di Biologia, University of Pisa, Pisa, Italy
E-mail: gpetroni{at}biologia.unipi.it.
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Abstract |
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 TOP Abstract Supplementary MaterialAcknowledgementsReferences |
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Prosthecobacter, one of the few cultivable representatives of the bacterial phylum Verrucomicrobia, is of increasing interest to the scientific community due to the presence of tubulin genes in its genome and the apparent absence of the bacterial homologue FtsZ that is normally involved in prokaryotic cell division. These findings suggested the possibility of a vicarious takeover of the FtsZ function through these novel tubulins and opened new scenarios on the possible evolution of bacterial cytoskeleton and cell division. In the present manuscript, we report the characterization of ftsZ and ftsA homologues in different Prosthecobacter species that also possess tubulin genes. Based on these findings, we propose an FtsZ-based cell division mechanism in Verrucomicrobia. The analysis of available genome data of Verrucomicrobia suggests that tubulins are not a feature common to all members of this phylum. Therefore, it can be assumed that Prosthecobacter acquired tubulins through horizontal gene transfer. The functional role of tubulins in Prosthecobacter remains enigmatic.
Key Words: bacterial tubulin (btub) • FtsZ • Prosthecobacter • Verrucomicrobia
The hypothesis that bacteria contain a cytoskeleton that is related to the eukaryote cytoskeleton was first established when the bacterial Z-ring, which plays a key role during bacterial cell division, was visualized using green fluorescent protein–labeled FtsZ. FtsZ is a protein with a secondary structure that mirrors tubulin (Lowe and Amos 1998
; Nogales et al. 1998) and displays in vitro similar dynamic properties (reviewed in Addinall and Holland 2002; Stricker et al. 2002). Although FtsZ is incapable to form microtubule-like structures, the combined structural and functional properties make it unlikely that FtsZ and tubulin proteins evolved twice (Erickson 1998); therefore, eukaryotic tubulin and bacterial FtsZ are considered to be homologous proteins.
Despite microtubule-like structures have been reported several times in bacteria (Bermudes et al. 1994; Petroni et al. 2000), the first molecular indications of the presence of tubulin genes in the bacteria are rather recent. In 2002, during the analysis of the genome sequence (95% completion) of Prosthecobacter dejongeii, Jenkins et al. reported the presence of 2 genes showing a higher similarity to eukaryotic tubulin than to bacterial ftsZ. These genes were referred to as bacterial A tubulin (btubA) and bacterial B tubulin (btubB) because of their apparent similarity to eukaryotic alpha and beta tubulins. However, no FtsZ genes were found in the genome sequence of P. dejongeii (Jenkins et al. 2002). Later biochemical studies showed that BtubA and BtubB are able to associate in vitro into heterodimers that form long filaments. (Schlieper et al. 2005; Sontag et al. 2005).
Prosthecobacter dejongeii is one of the few cultivable representatives of the still poorly investigated bacterial phylum Verrucomicrobia, which is phylogenetically related to Chlamydiae and Planctomycetes (Wagner and Horn 2006). Intriguingly, the latter 2 are the only bacterial phyla that do not possess FtsZ and rely on a yet unknown cell division mechanism (Read et al. 2000; Gloeckner et al. 2003; Horn et al. 2004; Strous et al. 2006).
These findings suggested the possibility of a vicarious takeover of the FtsZ function through the btubs in Verrucomicrobia, thus opening novel scenarios on the evolution of the eukaryotic cell. To evaluate this hypothesis, we accurately screened several Prosthecobacter species and their closest cultivated relative, Verrucomicrobium spinosum, for the presence of ftsZ and tubulin genes.
The complete nucleotide sequence coding for the 2 btub genes, btubA and btubB, of P. dejongeii was already published as well as the partial sequences of these genes in Prosthecobacter vanneervenii and Prosthecobacter debontii (Jenkins et al. 2002). We confirmed the presence of 1 A tubulin and 1 B tubulin gene in P. vanneervenii, and the sequence of both open reading frames together with a connecting spacer was completed. In addition, we could detect and completely sequence 2 further A and B tubulin genes in P. debontii. This finding was also confirmed by Southern blot and hybridization experiments. Prosthecobacter debontii btubA and btubB partial sequences characterized by Jenkins et al. (2002) do not exist as adjacent loci, but each of them is adjacent to the newly identified btub genes. Therefore, we renamed the btub genes in P. debontii. Henceforth, P. debontii btubA (Jenkins et al. 2002) is renamed btubA2 and is followed by the newly characterized btubB2; the newly characterized btubA1 precedes P. debontii btubB (Jenkins et al. 2002) that is renamed btubB1 (table 1). Several combinations of primers were used in polymerase chain reaction (PCR) attempts to detect tubulin genes in V. spinosum but without any success (table 1). This negative result was later confirmed by Blast analysis of V. spinosum genome data (sequence complete and all gaps closed, update 7 May 2005).
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Despite the apparent absence of ftsZ in P. dejongeii (Jenkins et al. 2002
; Staley et al. 2005), we could detect a sequence coding for FtsZ in that organism using consensus degenerate hybrid oligonucleotide primers (Rose et al. 1998) in PCR. Moreover, ftsZ was also identified in P. debontii and P. vanneervenii (accession numbers AJ888907, AJ888908, AM498604). The retrieved sequences were used to detect an open reading frame with protein sequence similarities to FtsZ also in the sequence data of the ongoing V. spinosum DSM 4136 genome project (TIGR_240016, contig 534) (table 1).
Prosthecobacter and Verrucomicrobium FtsZs exhibit most of the typical FtsZ features and some peculiar characteristics. Like typical bacterial FtsZ, they can be divided into the 4 domains (N-terminus, core, spacer, and C-terminus) as defined by Vaughan et al. (2004).
The sequences present the typical features of functional FtsZ. First, 6 out of 6 characteristic motifs of FtsZ were identified by PRINTS fingerprint scan (Attwood et al. 2003) (probability values between 3.4 x 10–49 and 3.9 x 10–44; see table 2 and its extended version in supplementary fig. S1, Supplementary Material online). Second, the tubulin signature motif [S/A/G]GGTG[S/A/T]G (PROSITE motif PS00227) is always present and perfectly conserved (supplementary fig. S1, Supplementary Material online). Third, amino acids which contact guanosine diphosphate (Lowe and Amos 1998; Nogales et al. 1998) are conserved or conservatively exchanged with the exception of position N70H according to Methanocaldococcus jannaschii sequence (supplementary fig. S2, Supplementary Material online). Other nonconservative substitutions in the core domain are 1) position D235G (supplementary fig. S2, Supplementary Material online), a highly conserved position located within the T7-loop which is considered to be important for GTPase activity (Scheffers and Driessen 2001) and FtsZ polymerization (Cordell et al. 2003); and 2) the C-terminal end of the core domain, generally represented by the conserved tripeptide ATG and replaced in Verrucomicrobia by the tripeptide SSL. In all characterized Verrucomicrobia, the substituted amino acids are conserved, thus suggesting that functional constraints are still present at these positions although the substitutions are different from those occurring in other bacteria.
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Residues that have been demonstrated to be involved in protein–protein interaction, for example, with FtsA (Yan et al. 2000
; Haney et al. 2001) are located in the C-terminal domain of FtsZ. These amino acids are arranged in a nonapeptide and are followed by a stretch of variable length, which is rich in basic amino acids (Vaughan et al. 2004). This feature is considered typical of a functionally active FtsZ and is also present in Verrucomicrobia. Moreover, the nonapeptide of the investigated Verrucomicrobia shows a good conservation in comparison to the bacterial consensus sequence (Vaughan et al. 2004) especially in positions which, in Escherichia coli, have been shown to be important for the protein conformation (Mosyak et al. 2000) or are thought to be involved in interactions with FtsA (Haney et al. 2001) (supplementary fig. S3, Supplementary Material online).
Phylogenetic analyses were performed on the core domain protein sequences using the ARB program package (Ludwig et al. 2004). They indicate a steady monophyly of verrucomicrobial FtsZ independently from the applied algorithm. One representative tree is shown in figure 1; the other calculated trees are available in FtsZ_ClustalW ARB database at http://www.arb-home.de. Calculated trees clearly indicate that the phylogenetic information retained by FtsZ is relatively limited and, in most cases, is not sufficient to resolve relationships above the phylum level, as it was also shown in earlier studies (Faguy and Doolittle 1998; Gilson and Beech 2001). Verrucomicrobial FtsZ always cluster together as independent lineage, thus supporting the existence of specific evolutionary constraints for these genes.
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The genomic environment of P. debontii and P. vanneervenii FtsZ was additionally investigated. It shows the presence of an open reading frame similar to ftsA. FtsA is an actin homologue that is also involved in bacterial cell division. Moreover, the V. spinosum genome reveals a cluster of genes involved in cell division, comprising open reading frames with similarities to D-alanine-D-alanine-ligase, ftsQ, ftsA, and ftsZ (fig. 2). This gene order is highly conserved and also found in other distantly related organisms (e.g., E. coli CFT073) (Faguy and Doolittle 1998
).
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The following properties indicate that the identified ftsZ genes are functionally active in Verrucomicrobia: 1) all characteristics typical of functional FtsZ are present; 2) verrucomicrobial FtsZ is evolutionary constrained; and 3) other typical bacterial cell division genes are present in these organisms.
The simultaneous presence of functional FtsZ in Prosthecobacter spp. and Verrucomicrobium together with tubulin genes in the genus Prosthecobacter is a strong indication that FtsZ and not tubulin is the major protein involved in cell division in the Verrucomicrobia.
The comparison of Prosthecobacter tubulins and verrucomicrobial FtsZs shows only a low sequence similarity (see table 2 and its extended version in supplementary fig. S1, Supplementary Material online) and indicates that Prosthecobacter tubulins did not directly derive from Prosthecobacter FtsZ. The apparent absence of tubulin genes in V. spinosum and the great divergence between Prosthecobacter FtsZ and tubulins would favor the hypothesis that tubulin sequences were acquired by Prosthecobacter through horizontal gene transfer as it was already suggested by other authors (Schlieper et al. 2005). In any case, the origin and especially the function of Prosthecobacter tubulins and of those tubulins supposed to be present in other representatives of the phylum, that is, epixenosomes (Rosati et al. 1993; Petroni et al. 2000), remain to be elucidated.
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Supplementary Material |
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Materials and Methods and figures S1–S3 are available at Molecular Biology and Evolution online (http://www.mbe.oxfordjournals.org/). The DNA sequences reported in this work have been deposited in the EMBL nucleotide database (accession numbers AJ888907, AJ888908, AM041148–AM041150, AM498604). ARB database is available at http://www.arb-home.de.
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Acknowledgements |
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TOPAbstractSupplementary MaterialAcknowledgementsReferences |
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This work was supported by a German Research Organization (DFG) grant to K.H.S., W.L., and G.P. Italian Research Ministry (MUR) is acknowledged for additional support to G.P. (PRIN protocol 2006053200_002). The Bayerische Forschungsstiftung is acknowledged for a research and mobility grant to M.P. Christina Eckl is acknowledged for her help with some of the experiments. The authors wish to thank Simone Gabrielli for computer image assistance. Preliminary sequence data were obtained from The Institute for Genomic Research Web site at http://www.tigr.org. Sequencing of V. spinosum DSM4136 was accomplished with support from National Science Foundation.
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Footnotes |
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William Martin, Associate Editor
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