Molecular Evolution of the Actin-Like Mreb Protein Gene Family in Wall
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Biochemical and Biophysical Research Communications 446 (2014) 927–932 Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc Molecular evolution of the actin-like MreB protein gene family in wall-less bacteria ⇑ Chuan Ku a,1, Wen-Sui Lo a,b,c, Chih-Horng Kuo a,b,d, a Institute of Plant and Microbial Biology, Academia Sinica, Taipei 115, Taiwan b Molecular and Biological Agricultural Sciences Program, Taiwan International Graduate Program, National Chung Hsing University and Academia Sinica, Taipei 115, Taiwan c Graduate Institute of Biotechnology, National Chung Hsing University, Taichung 402, Taiwan d Biotechnology Center, National Chung Hsing University, Taichung 402, Taiwan article info abstract Article history: The mreB gene family encodes actin-like proteins that determine cell shape by directing cell wall synthe- Received 26 February 2014 sis and often exists in one to three copies in the genomes of non-spherical bacteria. Intriguingly, while Available online 17 March 2014 most wall-less bacteria do not have this gene, five to seven mreB homologs are found in Spiroplasma and Haloplasma, which are both characterized by cell contractility. To investigate the molecular evolution Keywords: of this gene family in wall-less bacteria, we sampled the available genome sequences from these two gen- Cytoskeleton era and other related lineages for comparative analysis. The gene phylogenies indicated that the mreB Haloplasma homologs in Haloplasma are more closely related to those in Firmicutes, whereas those in Spiroplasma Mollicutes form a separate clade. This finding suggests that the gene family expansions in these two lineages are mreB Spiroplasma the results of independent ancient duplications. Moreover, the Spiroplasma mreB homologs can be clas- Wall-less bacteria sified into five clades, of which the genomic positions are largely conserved. The inference of gene gains and losses suggests that there has been an overall trend to retain only one homolog from each of the five mreB clades in the evolutionary history of Spiroplasma. Ó 2014 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/3.0/). 1. Introduction In Spiroplasma and Haloplasma, two bacterial genera without a cell wall, high copy numbers of mreB homologs have been identi- For a long time, the existence of actin or actin-like proteins in fied. In all the other bacterial taxa characterized to date, at most prokaryotic cells had remained questionable. It was only since three mreB homologs are present [1,9]. However, five to seven the beginning of this century that the presence of diverse bacterial mreB homologs were found in the draft genome sequences of actin homologs and their crucial roles in cell shape determination S. citri [11], S. melliferum [12,13], and H. contractile [14]. In addition have been widely appreciated [1–6]. In both Gram-positive and to being wall-less, these two genera are both characterized by their Gram-negative bacteria such as Bacillus subtilis, Escherichia coli, contractile helical cells [15] or protrusions [16], which are sugges- and Caulobacter crescentus, the mutation or depletion of actin-like tive of the involvement of helical cytoskeletal elements. Based on MreB proteins resulted in disrupted cell shapes [1,7,8]. Experimen- cryo-electron tomography, it has been proposed that MreB possi- tal evidence demonstrated that MreB homologs form helical bly forms a helical ribbon along the length of Spiroplasma cells [17]. filaments beneath the cell envelope that encircle the cell along To investigate the molecular evolution of the mreB gene family its length and determine cell shapes through the control of cell in wall-less bacteria, we took advantage of the recent availability of wall synthesis [1,3,8–10]. This MreB-dependent mechanism of multiple complete genome sequences spanning most of the phylo- cell-shape determination is phylogenetically widespread in genetic diversity within the genus Spiroplasma [18–21] to perform bacteria [1,9]. phylogenomic analysis. These Spiroplasma genomes, together with those of other Mollicutes (Mycoplasma, Mesoplasma, and Achole- plasma), Haloplasma, and two representatives of Firmicutes ⇑ Corresponding author at: Institute of Plant and Microbial Biology, Academia (Table 1), were examined for their gene repertoire of mreB homo- Sinica, 128 Sec. 2, Academia Rd., Nankang, Taipei 115, Taiwan. logs. A species phylogeny based on shared single-copy genes was E-mail address: [email protected] (C.-H. Kuo). reconstructed for Mollicutes, Haloplasma, and Firmicutes, which 1 Present address: Institute of Molecular Evolution, Heinrich Heine University are closely related taxonomic groups based on 16S rRNA [16]. This Düsseldorf, Düsseldorf, Germany. http://dx.doi.org/10.1016/j.bbrc.2014.03.039 0006-291X/Ó 2014 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/3.0/). 928 C. Ku et al. / Biochemical and Biophysical Research Communications 446 (2014) 927–932 Table 1 Genome sequences analyzed in this study. Phylum Class Order Family Taxon Accession numbers Tenericutes Mollicutes Acholeplasmatales Acholeplasmataceae Acholeplasma laidlawii PG-8A NC_010163 Entomoplasmatales Entomoplasmataceae Mesoplasma florum L1T NC_006055 Spiroplasmatacea Spiroplasma apis B31T CP006682 Spiroplasma chrysopicola DF-1T CP005077 Spiroplasma citri GII3-3X AM285301–AM285339 Spiroplasma culicicola AES-1T CP006681 Spiroplasma diminutum CUAS-1T CP005076 Spiroplasma melliferum IPMB4A AMGI01000001–AMGI01000024 Spiroplasma syrphidicola EA-1T CP005078 Spiroplasma taiwanense CT-1T CP005074 Mycoplasmatales Mycoplasmataceae Mycoplasma capricolum ssp. NC_007633 capricolum ATCC 27343T Mycoplasma leachii PG50T NC_014751 Mycoplasma mobile ATCC 43663T NC_006908 Mycoplasma mycoides ssp. NC_005364 mycoides SC PG1T Mycoplasma penetrans HF-2 NC_004432 Mycoplasma putrefaciens KS1T NC_015946 Unclassified Unclassified Haloplasmatales Haloplasmataceae Haloplasma contractile SSD-17BT AFNU01000001–AFNU01000109 Firmicutes Bacilli Bacillales Bacillaceae Bacillus subtilis ssp. subtilis 168 AL009126 Clostridia Clostridiales Clostridiaceae Clostridium botulinum A str. ATCC 3502 AM412317 Proteobacteria Gamma-proteobacteria Enterobacteriales Enterobacteriaceae Escherichia coli str. K-12 substr. MG1655 U00096 species phylogeny provides a framework for our inference of the as the outgroup. The sequences were aligned using MUSCLE v3.8 gains and losses of mreB homologs. [27] with the default settings. A gene tree was inferred using PhyML v3.0 [28] with the WAG + I + G model and six substitution 2. Materials and methods rate categories. Bootstrap supports were estimated from 1000 samples of alignment generated by the SEQBOOT program of PHY- 2.1. Taxon sampling for phylogenetic analyses LIP v3.69 [29]. Another maximum likelihood phylogeny was in- ferred for mreB nucleotide sequences of all eight Spiroplasma Genome sequences of one Haloplasma species (the only species in species and M. florum. The amino acid sequences from these nine this monotypic genus [16]) and eight Spiroplasma species were ob- species were aligned using MUSCLE, converted into nucleotide se- tained from GenBank (Table 1). Because the two S. melliferum strains, quence alignment with PAL2NAL [30], and analyzed using PhyML IPMB4A [13] and KC3 [12], are 99.9% identical at the nucleotide se- with the GTR + I + G model and six substitution rate categories. quence level in single-copy protein-coding genes [13], only the IPM- Bootstrap supports were estimated as described above. B4A genome was used as a representative. Other Mollicutes taxa that cover the diversity of this group were also included: four Myco- 2.4. Species phylogeny plasma species and Mesoplasma florum in the Mycoides-Entomoplas- mataceae clade [22], two Mycoplasma species belonging to two Two species phylogenies were reconstructed, one of which clades outside of the Spiroplasma-Entomoplasmataceae-Mycoides included E. coli K12 (Table 1) as the outgroup for rooting. clade [18], and Acholeplasma laidlawii (representing the Acholeplas- Protein-coding genes were clustered using OrthoMCL as described matales-Anaeroplasma-Phytoplasma clade of Mollicutes). One rep- above. Amino acid sequences of shared single-copy genes were ex- resentative from each of the two major Firmicutes classes was tracted, aligned with MUSCLE, concatenated, and used for PhyML selected: B. subtilis (Bacilli) and Clostridium botulinum (Clostridia). phylogenetic analyses with the WAG + I + G model. Bootstrap sup- ports were estimated from 1000 samples of alignment generated 2.2. Identification of mreB homologs by the SEQBOOT program of PHYLIP. Homologous gene clusters among the genome analyzed were inferred by OrthoMCL [23] with the e-value cutoff set to 3. Results and discussion 1 Â 10À15. Genes that were assigned the KEGG [24,25] Orthology (KO) number K03569 (mreB) and other genes that were clustered 3.1. Numbers of mreB homologs in Spiroplasma and relatives in the same orthologous groups were regarded as mreB homologs (Supplemental Table S1). Multiple homologs of mreB in Spiroplas- A total of 57 mreB homologs in the selected taxa were identified ma were numbered according to their order in the chromosome by a BLAST-based approach (Supplemental Table S1). In Achole- (starting from dnaA). For visualization of the chromosomal plasma and Mycoplasma,nomreB