- Short genome report
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The complete genome sequence and analysis of a plasmid-bearing myxobacterial strain Myxococcus fulvus 124B02 (M 206081)
Standards in Genomic Sciencesvolume 11, Article number: 1 (2016)
Myxobacteria, phylogenetically located in the delta division of the Proteobacteria, are well known for characterized social behaviors and large genomes of more than 9 Mb in size. Myxococcus fulvus is a typical species of the genus Myxococcus in the family Myxococcaceae. M. fulvus 124B02, originally isolated from a soil sample collected in Northeast China, is the one and only presently known myxobacterial strain that harbors an endogenous autonomously replicating plasmid, named pMF1. The endogenous plasmid is of importance for understanding the genome evolution of myxobacteria, as well as for the development of genetic engineering tools in myxobacteria. Here we describe the complete genome sequence of this organism. M. fulvus 124B02 consists of a circular chromosome with a total length of 11,048,835 bp and a circular plasmid of 18,634 bp. Comparative genomic analyses suggest that pMF1 has a longstanding sustention within myxobacteria, and probably contributes to the genome expansion of myxobacteria.
The gliding Gram-negative myxobacteria are characterized by complex social behaviors, e.g. cells moving on solid surfaces in swarms, preying on other microorganisms in a ‘wolf-like’ pattern, and, when nutrients are depleted, developing into myxospores embodied in fruiting bodies [1, 2]. In addition, myxobacteria are able to produce various secondary metabolites and macromolecule degradation enzymes, not only having potential in applications but also probably working as ecological weapons against other living microorganisms [3–5]. Myxobacteria possess large genomes. For instance, the genomes of Myxococcus xanthus DK1622 and the halotolerant M. fulvus HW-1 are 9.14 Mb  and 9.03 Mb  in size, while the genomes of Sorangium cellulosum even reach to 13.03 Mb in strain So ce56  and 14.78 Mb in strain So0157-2 , respectively. The So0157-2 genome is still the largest one reported in prokaryotes.
Extrachromosomal autonomously replicating genetic materials are normally absent from myxobacterial cells. Up to now, pMF1, originally discovered from M. fulvus 124B02 , is still the one and only endogenous plasmid that is able to replicate autonomously in myxobacterial cells. Genome sequencing of M. fulvus 124B02 is thus meaningful for understanding the evolution of myxobacterial genomes, and providing clues for the presence of pMF1 in strain 124B02. Here we report the complete genome sequence and analyses of M. fulvus 124B02.
Classification and features
Strain 124B02 was isolated from a soil sample collected in Northeast China . Vegetative cells of the strain are slender rods with tapering ends, 0.6-0.8 × 4–8 μm. The fruiting bodies are spherical or slightly pear-shaped with a diameter of 50–250 μm and a yellow red color. The strain did not grow expansively, but into membranaceous clumps on CYE solid plates. When grown in liquid CYE medium, the cells grew into spherical clumps. Figure 1 shows morphological characteristics of M. fulvus 124B02. The optimal growth pH for strain 124B02 is in the range of 6.8–7.6, and the optimal growth temperature ranges between 26 °C and 32 °C. The predominant fatty acids of M.fulvus 124B02 cells were determined as iso-C15:0 (33.18 %), C16:1 ω5c (20.19 %), iso-C14:0 3-OH (6.27 %), C16:0 (5.79 %) and C14:0 (5.65 %). 2-hydroxy and 3-hydroxy fatty acids are the major hydroxyl fatty acid components of strain 124B02.
Figure 2 is a phylogenetic tree of the 16S rRNA gene sequences showing the location of M. fulvus 124B02 in the Cystobacterineae suborder of myxobacteria (the GenBank accession number of the 16S rRNA gene sequence of strain 124B02 is EU137665). All three 16S rRNA gene copies in the genome of strain 124B02 are identical, but differ by two nucleotides from the previously published 16S rRNA sequence generated from M.fulvus 124B02 (EU137665). According to the morphological and phylogenetic characteristics, M. fulvus 124B02 was determined as a typical strain of Myxococcus fulvus (Table 1 shows the classification and general features of the strain).
Genome sequencing information
Genome project history
This organism was selected for sequencing because of its evolutionary significance as the only presently known myxobacterial strain bearing an endogenous plasmid. The genome project of M. fulvus 124B02 was deposited in the Genome Online Database and the complete genome sequence of strain 124B02 was deposited in GenBank under the accession number of CP006003. A summary of the project information is shown in Table 2.
Growth conditions and genomic DNA preparation
M. fulvus 124B02 was cultivated in the CTT growth medium containing 1 % casitone, 10 mM Tris–HCl, 1 mM KH2PO4-K2HPO4, 8 mM MgSO4, pH 7.6. The cells were harvested by centrifugation after five days of incubation at 30 °C. DNA was extracted from the cell mass using the methods described previously  with slight modifications. Briefly, approximately 50–100 mg cell pellets were suspended in 500 μl TE buffer, containing 25 mM Tris–HCl (pH 8.0), 25 mM EDTA, and 2 mg/ml lysozyme. The mixture was incubated at 37 °C for 1 h with periodic gentle inversion for cell lysis. Then, 2.5 μl proteinase K was added to a final concentration of 100 μg/ml, and the mixture was incubated at 37 °C for additional 1 h. The total protein was removed with Tris-saturated phenol-chloroform-isoamyl alcohol (25:24:1, pH 8.0). To precipitate DNA, 0.1 volume of 3 M sodium acetate (pH 5.3) and the same volume of isopropyl alcohol were added to the final supernatant. The DNA pellet was washed with 70 % ethanol twice, air-dried, and dissolved in 50 μl ddH2O.
Genome sequencing and assembly
Genome sequencing and assembly were performed in Shanghai Majorbio Bio-Pharm Technology Co., Ltd. The genome was sequenced with a combination of the Roche 454 GS FLX and Illumina GAII sequencing platforms. The 454 pyrosequencing reads, containing 285.8 Mb draft data, were firstly assembled using the Newbler assembler V2.3, producing 51 contigs in 23 scaffolds. This initial assembly was converted into a phrap assembly by making fake reads from the consensus, to collect the read pairs in the 454 paired end library. The clean data from Illumina GAII sequencing were assembled with Velvet assembler and the consensus sequences were shredded into 800-bp overlapped fake reads, which were assembled with the 454 draft data. In total, the combination of the Illumina and 454 sequencing platforms produced 112.5× coverage of the genome. The final assembly contained 738,315 pyro sequences and 12,776,900 Illumina reads. After the shotgun stage, reads were assembled with parallel phrap (High Performance Software, LLC). Then the Phred/Phrap/Consed software package [13–15] was used for quality assessment. Possible misassembles were corrected by sequencing the cloned bridging PCR fragments. We designed primers for the amplification of 76 gap regions to close gaps and to improve the quality of the finished genome. Gaps between contigs were closed by editing in Consed, PCR amplification and 3730 sequencing. The wrong bases were corrected by comparing with Illumina GAII data after the genome cyclization, using BWA (0.7.3a)  and samtools (0.1.19) . The error rate of the completed genome sequence is less than 1 bp in 100,000 bp.
The genome was annotated automatically in GenBank. In addition, we predicted Cluster Regularly Interspaced Short Palindromic Repeats (CRISPRS) with PILER-CR . We analyzed the predicted protein sequences against the National Center for Biotechnology Information (NCBI) non-redundant database, Gene Ontology , KEGG , and COG  databases for functional annotation. The results were summarized with the InterProScan  software. To analyze the COG annotation, hits with an E-value < = 1e-5 were first retained. Then, only the best hit was selected for each protein. Signal peptides and transmembrane helices of all annotated proteins were predicted using SignalP 4.1 Sever  and TMHMM Sever v. 2.0 respectively.
The genome statistics are provided in Table 3, Table 4 and Fig. 3 . M. fulvus 124B02 consists of a circular chromosome with a total length of 11,048,835 bp and a circular plasmid of 18,634 bp. The G+C contents of the chromosome and the plasmid are 69.96 % and 68.7 %, respectively. There were 8,515 predicted coding sequences (CDSs) in the genome, including 9 rRNAs and 80 tRNAs. The protein coding sequences occupied 86.13 % of the whole genome sequence. The majority of the protein-coding genes (5,042, 58.24 % of the total) were assigned putative functions in categories of orthologous group (COG), while the remaining ones were annotated as hypothetical proteins. The distribution of genes in COGs functional categories is presented in Table 5.
Insights from the genome sequence
Until now, 22 myxobacterial genomes have been released in NCBI database. Except for the anaerobic myxobacteria, whose genomes are approximately 5 Mb, all the aerobic myxobacteria have rather large genomes, ranging from 9.03 Mb of M. fulvus HW-1 to 14.78 Mb of S. cellulosum So0157-2. Compared with the other sequenced Myxococcus genomes, i.e. 9.14 Mb of M. xanthus DK1622 , 9.03 Mb of M. fulvus HW-1 , and 10.35 Mb of M. stipitatus DSM 14675 , the genome of M. fulvus 124B02 is rather large. It is known that horizontal gene transfer (HGT) [25, 26] and intra-chromosomal gene duplication (IGD) [27, 28] are two major contributors for the expansion of most prokaryotic genomes. BLASTP searching against the other three sequenced Myxococcus genomes revealed 576 strain-specific duplications in the strain 124B02 genome (the core circle in Fig. 3), accounting for 6.7 % of the total CDSs. The exogenous genetic materials may be introduced into bacterial genomes via plasmids, prophages, virus, integrative conjugative elements, insertion sequence elements or other unclassified elements . Of the total 8,492 CDSs in M. fulvus 124B02 genome, 3926 (46.2 %) were probably derived from plasmids (circles 9 & 10 in Fig. 3), which is similar to that in other myxobacteria [6, 9]. We conducted an all-blast-all analysis using BLASTP program with an E-value cutoff of 1e-5, and the results were transferred into OrthoMCL package to extract the paralogous and orthologous proteins. Interestingly, the phylogenomic analysis indicated that M.fulvus 124B02 is closer to M. stipitatus DSM 14675, rather than M. xanthus DK1622 or M. fulvus HW-1 (Fig. 4a), which was also supported by the genome synteny analysis (Fig. 4b). We found that the major differences between M. fulvus 124B02 and the other three Myxococcus strains were those protein sequences for the metabolism and environment adaption processes [Additional file 1: Table S1, Additional file 2: Table S2, Additional file 3: Table S3] and of those strain-specific genes. For example, according to the COG catalog, the major differences between M. fulvus 124B02 and M. stipitatus DSM 14675 were in the families of lipid transport and metabolism (p-value is 0.0076, Fisher’s exact test, two-tailed test), transcription (p-value is 0.0097, Fisher’s exact test, two-tailed test), secondary metabolites biosynthesis, transport and catabolism (p-value is 0.0015, Fisher’s exact test, two-tailed test) and replication, recombination and repair (p-value is 0.0251, Fisher’s exact test, two-tailed test). M. fulvus 124B02 had approximately 1,230 kb strain-specific fragments, which scattered throughout the whole genome (circles 5 & 6 in Fig. 3). Additional file 4: Table S4 lists the strain specific genes of replication, recombination and repair family, of which the number of M.fulvus 124B02 is less than M.stipitatus DSM 14675.
pMF1 is a low copy number plasmid, containing 23 predicted ORFs . The plasmid has no obvious beneficial genes for persistence in host, such as the genes encoding for antibiotic resistance, virulence, or growth phenotypes. All the predicted genes in pMF1 are of unknown functions, except the replication system (pMF1.13-pMF1.16) and the partitioning system (pMF1.21-pMF1.23), both of which were determined by narrowing-down of sequence fragments [10, 30, 31]. While the pMF1.19 and pMF1.20 genes were on the lagging strand, the others were located on the leading strand (Fig. 5). Interestingly, BLASTP searching against the GenBank database showed that pMF1.19 and pMF1.20 had multiple homologues, mostly in pairs, in different myxobacterial genomes, except that of Anaeromyxobacter . For example, there were at least ten homologues of pMF1.19 and nine of pMF1.20 in M. xanthus DK1622. The predicted protein products of these two genes contained the conserved Pfam09535 and Pfam09533 domains, but lack significant sequence similarities to any known protein families. These function-unknown homologues exist in myxobacteria only. The identities of these homologues ranged from 31 % (WP_011550397, M. xanthus DK1622) to 74 % (MFUL124B02_18095 of M. fulvus 124B02) for pMF1.19 and from 30 % (WP_011550387, M.xanthus DK1622) to 88.0 % (MFUL124B02_18100 of M. fulvus 124B02) for pMF1.20 (Fig. 6a and b are phylogenetic trees of the pMF1.19 and pMF1.20 homologues, respectively). The homologues with highest similarities to pMF1.19 and pMF1.20 are both in M. fulvus 124B02 host genome, which suggested that pMF1 was more closely related with this strain than other myxobacteria. The homologues of pMF1.19 and pMF1.20 in the genome of M. fulvus 124B02 are summarized in an additional file [Additional file 5: Table S5]. In addition to pMF1.19 and pMF1.20, the pMF1.1- pMF1.5 proteins each had a unique homologue in M. stipitatus DSM 14675, from MYSTI_04154 to MYSTI_04158 (YP 007361138.1-YP 007361142.1) (Fig. 6c). It is also noted that, although there is no gene coding for mobility systems , and we have not yet observed conjugative transfer of the plasmid between Myxococcus strains, the pMF1.2 and its homologue MYSTI_04155 had an AAA_10 and TraC-F-type motifs, both were reported to relate to conjugative transfer [33, 34].
M. fulvus 124B02 is a typical strain of Myxococcus fulvus . The complete genome sequence of M. fulvus 124B02 is much larger than the other sequenced genomes of Myxococcus strains. The phylogenomic analysis of total genome sequence indicates that M. fulvus 124B02 is closer to M. stipitatus DSM 14675, rather than M. xanthus DK1622 or M. fulvus HW-1. Multiple copies of the pMF1.19 and pMF1.20 homologues in different myxobacterial strains suggest that myxobacterial genomes are open, not only to being subject to integrate foreign DNA sequences but also to being duplicated by self, in which the pMF1 plasmid played important roles. The bioinformatics analyses, together with the similar G+C contents of pMF1 and myxobacterial genomes, suggested that pMF1 had a longstanding co-adaption with myxobacteria, probably involving in the expansion of myxobacterial genomes.
intra-chromosomal gene duplication
Shimkets LJ. Social and developmental biology of the myxobacteria. Microbiol Rev. 1990;54:473–501.
Reichenbach H. The ecology of the myxobacteria. Environ Microbiol. 1999;1:15–21.
Weissman KJ, Müller R. Myxobacterial secondary metabolites: bioactivities and modes-of-action. Nat Prod Rep. 2010;27:1276–95.
Xiao Y, Wei X, Ebright R, Wall D. Antibiotic production by myxobacteria plays a role in predation. J Bacteriol. 2011;193:4626–33.
Wang Y, Li X, Zhang WY, Zhou XW, Li YZ. The groEL2 gene, but not groEL1, is required for biosynthesis of the secondary metabolite myxovirescin in Myxococcus xanthus DK1622. Microbiology. 2014;160:488–95.
Goldman BS, Nierman WC, Kaiser D, Slater SC, Durkin AS, Eisen JA, et al. Evolution of sensory complexity recorded in a myxobacterial genome. Proc Natl Acad Sci U S A. 2006;103:15200–5.
Li ZF, Li X, Liu H, Liu X, Han K, Wu ZH, et al. Genome sequence of the halotolerant marine bacterium Myxococcus fulvus HW-1. J Bacteriol. 2011;193:5015–6.
Schneiker S, Perlova O, Kaiser O, Gerth K, Alici A, Altmeyer MO, et al. Complete genome sequence of the myxobacterium Sorangium cellulosum. Nat Biotechnol. 2007;25:1281–9.
Han K, Li ZF, Peng R, Zhu LP, Zhou T, Wang LG, et al. Extraordinary expansion of a Sorangium cellulosum genome from an alkaline milieu. Sci Rep. 2013;3:2101.
Zhao JY, Zhong L, Shen MJ, Xia ZJ, Cheng QX, Sun X, et al. Discovery of the autonomously replicating plasmid pMF1 from Myxococcus fulvus and development of a gene cloning system in Myxococcus xanthus. Appl Environ Microbiol. 2008;74:1980–7.
Li YZ, Li J, Zhou L, Zhang Y, Hu W, Chen Q. Isolation and identification of myxobacterial sources in China. Acta Microbiol Sinica. 2000;40:652–6. In Chinese.
Kieser T. Factors affecting the isolation of CCC DNA from Streptomyces lividans and Escherichia coli. Plasmid. 1984;12:19–36.
Ewing B, Hillier LD, Wendl MC, Green P. Base-calling of automated sequencer traces usingPhred. I. Accuracy assessment. Genome Res. 1998;8:175–85.
Ewing B, Green P. Base-calling of automated sequencer traces using phred. II. Error probabilities. Genome Res. 1998;8:186–94.
Gordon D. Viewing and editing assembled sequences using Consed. Curr Protoc Bioinformatics. 2003;12:11–43.
Li H, Durbin R. Fast and accurate short read alignment with Burrows–Wheeler transform. Bioinformatics. 2009;25:1754–60.
Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, et al. The sequence alignment/map format and SAMtools. Bioinformatics. 2009;25:2078–9.
Edgar RC. PILER-CR: fast and accurate identification of CRISPR repeats. BMC Bioinformatics. 2007;8:18.
Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, et al. Gene Ontology: tool for the unification of biology. Nat Genet. 2000;25:25–9.
Ogata H, Goto S, Sato K, Fujibuchi W, Bono H, Kanehisa M. KEGG: Kyoto encyclopedia of genes and genomes. Nucleic Acids Res. 1999;27:29–34.
Tatusov RL, Galperin MY, Natale DA, Koonin EV. The COG database: a tool for genome-scale analysis of protein functions and evolution. Nucleic Acids Res. 2000;28:33–6.
Zdobnov EM, Apweiler R. InterProScan–an integration platform for the signature-recognition methods in InterPro. Bioinformatics. 2001;17:847–8.
Petersen TN, Brunak S, von Heijne G, Nielsen H. SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods. 2011;8:785–6.
Huntley S, Kneip S, Treuner-Lange A, Søgaard-Andersen L. Complete genome sequence of Myxococcus stipitatus strain DSM 14675, a fruiting myxobacterium. Genome Announc. 2013;1:e00100–13.
Gogarten JP, Townsend JP. Horizontal gene transfer, genome innovation and evolution. Nat Rev Microbiol. 2005;3:679–87.
Takeuchi N, Kaneko K, Koonin EV. Horizontal gene transfer can rescue prokaryotes from Muller’s ratchet: benefit of DNA from dead cells and population subdivision. G3 (Bethesda). 2014;4:325–39.
Andersson DI, Hughes D. Gene amplification and adaptive evolution in bacteria. Annu Rev Genet. 2009;43:167–95.
Serres MH, Kerr AR, McCormack TJ, Riley M. Evolution by leaps: gene duplication in bacteria. Biol Direct. 2009;4:46.
Wozniak RA, Waldor MK. Integrative and conjugative elements: mosaic mobile genetic elements enabling dynamic lateral gene flow. Nat Rev Microbiol. 2010;8:552–63.
Sun X, Chen XJ, Feng J, Zhao JY, Li YZ. Characterization of the partitioning system of Myxococcus plasmid pMF1. PLoS One. 2011;6:e28122.
Feng J, Chen XJ, Sun X, Wang N, Li YZ. Characterization of the replication origin of the myxobacterial self-replicative plasmid pMF1. Plasmid. 2012;68:105–12.
Smillie C, Garcillán-Barcia MP, Francia MV, Rocha EP, de la Cruz F. Mobility of plasmids. Microbiol Mol Biol R. 2010;74:434–52.
Marchler Bauer A, Marchler-Bauer A, Derbyshire MK, Gonzales NR, Lu S, Chitsaz F, et al. CDD: NCBI conserved domain database. Nucleic Acids Res. 2015;43(D):222–6.
Christie PJ, Vogel JP. Bacterial type IV secretion: conjugation systems adapted to deliver effector molecules to host cells. Trends Microbiol. 2000;8:354–60.
Lee C, Grasso C, Sharlow MF. Multiple sequence alignment using partial order graphs. Bioinformatics. 2002;18:452–64.
Castresana J. Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Mol Biol Evol. 2000;17:540–52.
Stamatakis A, Hoover P, Rougemont J. A Rapid Bootstrap Algorithm for the RAxML Web Servers. Syst Biol. 2008;57:758–71.
Reddy TBK, Thomas A, Stamatis D, Bertsch J, Isbandi M, Jansson J, et al. The Genomes OnLine Database (GOLD) v.5: a metadata management system based on a four level (meta) genome project classification. Nucleic Acids Res. 2014;43(Database issue):D1099–106.
Field D, Garrity G, Gray T, Morrison N, Selengut J, Sterk P, et al. The minimum information about agenome sequence (MIGS) specification. Nat Biotechnol. 2008;26:541–7.
Woese CR, Kandler O, Wheelis ML. Towards a natural system of organisms: proposal for the domains Archaea, Bacteria, and Eucarya. Proc Natl Acad Sci U S A. 1990;87:4576–9.
Garrity GM, Bell JA, Lilburn T. The revised road map to the manual. In: Bergey’s Manual of Systematic Bacteriology. Springer US, 2005;159–187.
Kuever J, Rainey FA, Widdel F. Class IV. Deltaproteobacteria class nov. In: Garrity GM, Brenner DJ, Krieg NR, Staley JT, editors. Bergey’s Manual of Systematic Bacteriology, vol. 2. 2nd ed. NY: Springer; 2005. p. 922.
Tchan YT, Pochon J, Prévot AR. Etude de systématique bactérienne. VIII. Essai de classification des Cytophaga. Ann Inst Pasteur (Paris). 1948;74:394–400.
Reichenbach H, Garrity GM, Brenner DJ, Krieg NR, Staley JT. Order VIII. Myxococcales. Tchan, Pochon and Prévot 1948, 398AL. Bergey’s manual of systematic bacteriology. Springer, New York, 2005;2(Part C):1059–1072.
Jahn E. Beiträge zur botanischen Protistologie I: Die Polyangiden. Leipzig: Verlag Gebruder Borntraeger; 1924. p. 1–107.
Lang E, Stackebrandt E. Emended descriptions of the genera Myxococcus and Corallococcus, typification of the species Myxococcus stipitatus and Myxococcus macrosporus and a proposal that they be represented by neotype strains. Request for an opinion. Int J Syst Evol Microbiol. 2009;59:2122–8.
Jahn E. Myxobacteriales. Kryptogamenflora der Mark Brandenburg. 1911;5:187–206.
Skerman VBD, McGowan V, Sneath PHA. Approved lists of bacterial names. Int J Syst Bacteriol. 1980;30:225–30.
Agents B. Technical rules for biological agents. www.baua.de TRBA 466.
Yi H, Jin L. Co-phylog: an assembly-free phylogenomic approach for closely related organisms. Nucleic Acids Res. 2013;41(7):e75.
Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol. 2011;28:2731–9.
This work was financially supported by grants from the National Natural Science Foundation of China (grants 31471183 and 31130004).
The authors declare that they have no competing interests.
XJC, KH, LZ and YJL participated in genome sequencing analysis, bioinformatics analysis, drafted the original manuscript, and participated in the revision process. JF and XJC extracted the total genome for sequencing. XJC and YZL wrote the paper. YZL conceived the study, provided funding for the project. All the authors read and approved the final manuscript.
Comparison of COG assignments of the genes between M. fulvus 124B02 and M. stipitatus DSM 14675. (XLSX 11 kb)
Comparison of COG assignments of the genes between M. fulvus 124B02 and M. fulvus HW-1. (XLSX 11 kb)
Comparison of COG assignments of the genes between M. fulvus 124B02 and M. xanthus DK1622. (XLSX 11 kb)
Strain-specific genes in the family of DNA replication, recombination and repair in M. fulvus 124B02, compared with DSM 14675. (XLSX 10 kb)
Homologues of pMF1.19 and pMF1.20 in M. fulvus 124B02. (XLSX 9 kb)