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Complete genome sequence of Microbulbifer sp. CCB-MM1, a halophile isolated from Matang Mangrove Forest, Malaysia

Standards in Genomic Sciences volume 12, Article number: 36 (2017) Cite this article

Abstract

Microbulbifer sp. CCB-MM1 is a halophile isolated from estuarine sediment of Matang Mangrove Forest, Malaysia. Based on 16S rRNA gene sequence analysis, strain CCB-MM1 is a potentially new species of genus Microbulbifer. Here we describe its features and present its complete genome sequence with annotation. The genome sequence is 3.86 Mb in size with GC content of 58.85%, harbouring 3313 protein coding genes and 92 RNA genes. A total of 71 genes associated with carbohydrate active enzymes were found using dbCAN. Ectoine biosynthetic genes, ectABC operon and ask_ect were detected using antiSMASH 3.0. Cell shape determination genes, mreBCD operon, rodA and rodZ were annotated, congruent with the rod-coccus cell cycle of the strain CCB-MM1. In addition, putative mreBCD operon regulatory gene, bolA was detected, which might be associated with the regulation of rod-coccus cell cycle observed from the strain.

Introduction

Microbulbifer sp. CCB-MM1 is a halophile isolated from an estuarine sediment sample taken from Matang Mangrove Forest, Malaysia. The genus Microbulbifer was proposed by González [1] with the description of Microbulbifer hydrolyticus which was isolated from marine pulp mill effluent. Microbulbifer are typically found in high-salinity environments including marine sediment [2], salt marsh [3], costal soil [4] as well as mangrove soil [5]. They were known for their capability to degrade a great variety of polysaccharides including cellulose [1, 5], xylan [1, 5, 6], chitin [1, 5, 6], agar [3, 6] and alginate [7]. Microbulbifer strains are potential sources of carbohydrate active enzymes with biotechnological interest. One of the species, Microbulbifer mangrovi had been reported with the ability to degrade more than 10 different polysaccharides [7].

Polysaccharides have a broad range of industrial applications. The most common storage polysaccharide, starch, can be used as food additives [8], excipients [9] and substrates in fermentation process to produce bioethanol [10]. Structural polysaccharides such as cellulose, chitosan and chitin, on the other hand, can be used to develop high-performance materials due to their renewability, biodegradability, biological inertness and low cost [11,12,13]. However, polysaccharides from natural sources are often not suitable for direct application. Chemical modifications involving the reactive groups (carboxyl, hydroxyl, amido, and acetamido groups) on the backbone of polysaccharide are required to alter their chemical and physical properties to suit the application purposes [14]. In the past years, explorations and researches are in favor of enzymatic method using carbohydrate active enzymes [15]. This alternative method offers the advantages of substrate specificity, stereospecificity, and environment friendly [16]. Hence, the discovery of novel carbohydrate active enzymes has great biotechnological interest and Microbulbifer strains are potential sources of these enzymes.

Therefore, we sequenced the genome of Microbulbifer sp. CCB-MM1 with primary objective to identify potential carbohydrate active enzyme coding genes. The genome insights will serve as baseline for downstream analyses including enzyme activity assays and functional elucidation of these genes. To date, there are seven genomes of Microbulbifer publicly available from GenBank, namely Microbulbifer agarilyticus S89 (NZ_AFPJ00000000.1) [17], Microbulbifer variabilis ATCC 700307T (NZ_AQYJ00000000.1), Microbulbifer elongatus HZ11 (NZ_JELR00000000.1) [18], Microbulbifer sp. ZGT114 (LQBR00000000.1), Microbulbifer thermotolerans DAU221 (CP014864.1) [19], Microbulbifer sp. Q7 (LROY00000000.1) and Microbulbifer sp. WRN-8 (LRFG00000000.1). All of the Microbulbifer genomes are assembled to draft assembly only except the Microbulbifer thermotolerans DAU221 genome. Here we present the complete genome of Microbulbifer sp. CCB-MM1 and some insights from comparative analysis with seven other Microbulbifer genomes.

Organism information

Classification and features

Microbulbifer sp. strain CCB-MM1 was isolated from mangrove sediment obtained from Matang Mangrove Forest. The isolation was done using the method previously described [20] with the use of H-ASWM (2.4% artificial sea water, 0.5% tryptone, 10 mM HEPES, pH 7.6) [21]. CCB-MM1 is a Gram-negative, aerobic, non-spore-forming and halophilic bacterium (Table 1). Its shape appears to be associated with its growth phases where it is rod-shaped at exponential phase (Fig. 1a) and cocci-shaped at stationary phase (Fig. 1b). The rod-shaped cell size ranges from approximately 1.3 to 2.5 μm in length and 0.3 μm in width while the diameter of coccus cells is approximately 0.6 μm. The colonies observed on agar plate are white in colour, circular, and raised with entire edge.

Table 1 Classification and general features of Microbulbifer sp. CCB-MM1 [69]
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Fig. 1
figure 1

Scanning electron micrograph of Microbulbifer sp. CCB-MM1 at (a) exponential and (b) stationary phase

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The 16S rRNA gene sequence of CCB-MM1 was amplified and sequenced using the universal primer pair 27F and 1492R [22]. The 16S rRNA gene sequence analysis was performed by using BLASTN [23] against NCBI 16S ribosomal RNA (Bacteria and Archaea) database. BLAST report revealed that the closely related strains include Microbulbifer rhizosphaerae Cs16bT (98.1%), Microbulbifer taiwanensis CC-LN1-12T (97.3%), Microbulbifer maritimus TF-17T (97.4%), Microbulbifer pacificus SPO729T (97.3%), and Microbulbifer gwangyangensis GY2T (97.3%). Based on the threshold of Proteobacteria -specific 16S rRNA gene sequence similarity at 98.7% [24], the analysis suggests that CCB-MM1 is a new species belonging to the genus Microbulbifer . To reconstruct a phylogenetic tree of Microbulbifer , the 16S rRNA sequences of other Microbubifer type strains were downloaded from GenBank. Then, these sequences were aligned using MUSCLE [25, 26] and MEGA6 [27] was used to reconstruct a neighbour-joining tree [28] with 1000 replications of bootstrap method test [29]. As shown in Fig. 2, CCB-MM1 formed a cluster with M. rhizosphaerae Cs16bT in the phylogenetic tree.

Fig. 2
figure 2

Neighbor-joining phylogenetic tree highlighting the position of Microbulbifer sp. CCB-MM1 relative to other type strains within the genus Microbulbifer, built using MEGA6 based on 16S rRNA sequences with their GenBank accession numbers indicated in parentheses

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Genome sequencing information

Genome project history

Genome of CCB-MM1 was sequenced in October 2015. The whole genome sequencing and annotation were done by Centre for Chemical Biology (Universiti Sains Malaysia). The complete genome sequence is available in GenBank under the accession number CP014143. The project information is summarized in Table 2.

Table 2 Project information
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Growth conditions and genomic DNA preparation

CCB-MM1 was cultured aerobically in 100 mL of H-ASWM for overnight (16 h) at 30 °C with shaking. The genomic DNA was extracted using modified phenol-chloroform method [30]. The integrity of extracted genomic DNA was assessed by gel electrophoresis using 0.7% agarose gel and the quantification was done using NanoDrop 2000 Spectrophotometer (Thermo Scientific, USA).

Genome sequencing and assembly

The whole genome of CCB-MM1 was sequenced using PacBio RS II platform with P6-C4 chemistry (Pacific Biosciences, USA). Two SMRT Cells were used and 2,674,097,380 pre-filter polymerase read bases were obtained, which was approximately 692X coverage of the genome. The reads were assembled using HGAP3 protocol [31] on SMRT Portal v2.3.0 with reads more than 25,000 bp in length being used as seed bases. The assembly result was a circular chromosome with the size of 3,864,326 bp, average base coverage of 431X and 100% base calling. The assembled sequence was polished twice using the resequencing protocol until the consensus concordance reached 100%.

Genome annotation

The genome was annotated using Prokka 1.11 pipeline [32]. The pipeline uses Prodigal [33], RNAmmer [34], Aragorn [35], SignalP [36] and Infernal [37] to predict the coding sequences (CDS), ribosomal RNA genes, transfer RNA genes, signal leader peptides and non-coding RNAs, respectively. In addition, the translated CDS output by Prokka were used to BLAST against protein databases including non-redundant protein database (nr) from GenBank, Swiss-Prot and TrEMBL from UniProt [38], and KEGG database [39]. COG functional categories assignment was done using RPS-BLAST [40] search against the COG database [41]. In addition, antiSMASH 3.0 [42] was used to identify biosynthetic gene clusters and dbCAN [43] was used to identify carbohydrate active enzymes.

Genome properties

CCB-MM1 only contains one circular chromosome and no plasmid. The size of the chromosome is 3,864,326 bp with an overall of 58.85% G + C content (Table 3). The complete genome consists of 3313 ORFs, 79 tRNA, 12 rRNA and 1 tmRNA genes. Of all the 3313 predicted ORFs, 2030 of them can be assigned with functional prediction and 2563 of them can be assigned to COG functional categories (Table 4). The circular map of the genome generated using CGView Comparison Tool [44] is depicted in Fig. 3.

Table 3 Genome statistics
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Table 4 Number of genes associated with general COG functional categories
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Fig. 3
figure 3

Circular map of the genome of Microbulbifer sp. CCB-MM1 generated using CGView Comparison Tool [44]. Circles (from outside) representing the following: 1. COG functional categories for forward coding sequence; 2. Forward sequence features; 3. Reverse sequence features; 4. COG functional categories for reverse coding sequence; 5. GC content; 6. GC skew

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Insights from the genome sequence

Comparative genomics

There are seven genomes of Microbulbifer strains publicly available in GenBank to date. To assess the relatedness between CCB-MM1 and publicly available Microbulbifer genomes, ANI values between the genomes were calculated using method based on MUMmer alignment [45]. Based on the results (Table 5), the ANI values ranged from 85.58% ( Microbulbifer sp. ZGT114 and Microbulbifer sp. WRN-8) to 83.45% (Microbublfer thermotolerans DAU221). These ANI values fall below 95% [46], suggesting that CCB-MM1 represents a different species from the other seven sequenced species. Interestingly, the ANI value between genomes of Microbulbifer sp. ZGT114 and Microbulbifer sp. WRN-8 is 99.99%, which suggests that these two strains belong to the same species. The circular map comparing CCB-MM1 genome and seven other Microbulbifer genomes is shown in Fig. 4.

Table 5 ANI value(%) between Microbulbifer sp. CCB-MM1 genome and seven other Microbulbifer genomes calculated using ANIm [45]
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Fig. 4
figure 4

Circular map comparing strain CCB-MM1 genome and seven other Microbulbifer genomes generated using CGView Comparison Tool [44]. The two outermost rings represent forward and reverse sequence features respectively. The remaining seven rings show the regions of sequence similarity detected by BLAST comparisons conducted between nucleotide sequences from the CCB-MM1 genome and seven other Microbulbifer genomes with the order (from outside) as follow: Microbulbifer elongatus HZ11, Microbulbifer sp. Q7, Microbulbifer sp. WRN-8, Microbulbifer sp. ZGT114, Microbulbifer agarilyticus S89, Microbulbifer thermotolerans DAU221 and Microbulbifer variabilis ATCC 700307T

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Carbohydrate active enzymes

dbCAN [43] was used to predict carbohydrate-active enzyme coding genes present in CCB-MM1 genome, particularly genes belonging to glycoside hydrolase and polysaccharide lyase families that could provide us the insights on carbohydrate degrading capability of CCB-MM1. The analysis was done by running HMMER3 [47] scan using HMMs profile downloaded from dbCAN (version: dbCAN-fam-HMMs.txt.v4) with an e-value cut off of 1e-18 and coverage cut off of 0.35. A total of 71 carbohydrate-active genes were detected and further analysis of these genes using SignalP predicted that 25 of them contain signal peptides. As shown in Table 6, we had found 29 genes associated with GH families including GH3, GH5, GH13, GH16, GH20, GH23, GH31, GH38, GH103 and GH130, however, we found no genes associated with PL families in the genome. Annotation of the GH genes revealed that CCB-MM1 genome possesses genes encoding cellulase (GH5), alpha-amylase, pullulanase (GH13) and beta-glucanase (GH16) with potential interest for biotechnological applications. While gene coding for beta-hexosaminidase, one of the chitinolytic enzymes [48], is present in the genome of CCB-MM1, gene that codes for chitinase was not detected. This suggests that CCB-MM1 lacks the ability to degrade chitin, although further assays are required to confirm the phenotype.

Table 6 GH enzyme coding genes found in CCB-MM1 genome
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Rod-coccus cell cycle

Microbulbifer were found to demonstrate rod-coccus cell cycle, in association with different growth phases [49]. This cell cycle was also observed in CCB-MM1. In CCB-MM1 genome, we found genes which are known to be involved in determining and maintaining the rod shape of bacteria, including mreBCD [50] (AUP74_00016, AUP74_00017 and AUP74_00018), rodA [51] (AUP74_01706) and rodZ [52] (AUP74_01850). BLAST analysis showed that these genes are present in all other Microbulbifer genomes. In addition, we detected the presence of general stress response gene, bolA, in all Microbulbifer genomes. It has been demonstrated that the overexpression of bolA in E.coli inhibited cell elongation and reduced the transcription of mreBCD operon [53]. The gene, mreB, and its product, actin homolog have been studied for their functions in several species of bacteria. This protein lies beneath the cell surface, forming actin-like cables which function as guidance for the synthesis of longitudinal cell wall [54]. While MreB is not essential in E. coli [55], it is found to be essential for Streptomyces coelicolor [56], Rhodobacter sphaeroides [57] and Bacillus subtilis [58]. In E. coli, depletion of MreB caused cells to change from rod-like to spherical shape but these cells were able to survive [59]. In contrast, the spherical-shaped B. subtilis cells eventually lyse. For CCB-MM1, the spherical-shaped cells do not lyse but grow into rod-shaped again after being transferred into fresh medium. We infer that mreB gene may have important functions in determining Microbulbifer cell shape and the rod-coccus cycle of Microbulbifer is likely regulated by BolA through inhibition of mreB transcription when triggered by stress.

Secondary metabolites, ectoine

Ectoine and hydroxyectoine are compatible solutes found primarily in halophilic bacteria. When triggered by osmotic stress, bacteria produce and accumulate them intracellularly to balance the osmotic pressure [60]. Apart from osmotic stress, they were also protectants against temperature stress [61]. A cluster of genes responsible for the biosynthesis of ectoine [62] has been identified in CCB-MM1 genome using antiSMASH 3.0 [42]. These genes encode for aspartate kinase (Ask_Ect) (AUP74_00280), L-ectoine synthase (EctC) (AUP74_00281), diaminobutyrate-2-oxoglutarate transaminase (EctB) (AUP74_00282), L-2,4-diaminobutyric acid acetyltransferase (EctA) (AUP74_00283) and HTH transcriptional regulator (AUP74_00284). The lack of the gene ectD, ectoine hydroxylase, in CCB-MM1 genome suggests that it only has the ability to synthesize ectoine but not hydroxyectoine. By using BLASTP, we searched and found similar gene cluster in other Microbulbifer genomes except Microbulbifer variabilis ATCC 700307 T. While the reason for the absence of these genes in Microbulbifer variabilis ATCC 700307 T is unknown, our findings suggest that Microbulbifer utilized only ectoine instead of ectoine/hydroxyectoine mixture. The transcriptional regulator of ectoine operon, EctR, found in Methylophaga thalassica belongs to MarR family [63]. HTH transcriptional regulator (AUP74_00284) in CCB-MM1 also contains the conserved domain of MarR family. This implies that the HTH transcriptional regulator is likely the putative transcriptional regulator of ectoine operon in Microbulbifer . Ectoine has attracted considerable biotechnological interest due to its stabilizing effects that extend from proteins [64], nucleic acids [65] to whole cells [66]. Such properties allow it to be used in skin care product as cell protectants [66], protein stabilizers [67] and medical application as cryoprotectants in cryopreservation of human cells [68].

Conclusion

In this study we presented the complete genome sequence of Microbulbifer sp. CCB-MM1 with genome size of 3.86 Mb and G + C content of 58.85%. We discussed some insights on its phenotypic characteristics from the genomic perspective, covering carbohydrate active enzymes, rod-coccus cell cycle and secondary metabolite, ectoine. The genome sequence provides valuable information for functional elucidations of novel enzymes for both biotechnological application and fundamental research purposes.

Abbreviations

ANI:

Average nucleotide identity

antiSMASH:

Antibiotics & Secondary Metabolite Analysis Shell

CCB:

Centre for Chemical Biology

dbCAN:

Database for automated carbohydrate-active enzyme annotation

GH:

Glycoside hydrolase

H-ASWM:

High nutrient artificial seawater media

MM:

Matang Mangrove

PL:

Polysaccharide lyase

References

  1. Gonzalez JM, Mayer F, Moran MA, Hodson RE, Whitman WB. Microbulbifer hydrolyticus gen. nov., sp. nov., and Marinobacterium georgiense gen. nov., sp. nov., two marine bacteria from a lignin-rich pulp mill waste enrichment community. Int J Syst Bacteriol. 1997;47:369–76.

    Article  CAS  PubMed  Google Scholar 

  2. Zhang DS, Huo YY, Xu XW, Wu YH, Wang CS, Xu XF, et al. Microbulbifer marinus sp. nov. and Microbulbifer yueqingensis sp. nov., isolated from marine sediment. Int J Syst Evol Microbiol. 2012;62:505–10.

    Article  CAS  PubMed  Google Scholar 

  3. Yoon J-H, Kim I-G, Shin D-Y, Kang KH, Park Y-H. Microbulbifer salipaludis sp. nov., a moderate halophile isolated from a Korean salt marsh. Int J Syst Evol Microbiol. 2003;53:53–7.

    Article  CAS  PubMed  Google Scholar 

  4. Kampfer P, Arun AB, Young CC, Rekha PD, Martin K, Busse HJ, et al. Microbulbifer taiwanensis sp. nov., isolated from coastal soil. Int J Syst Evol Microbiol. 2012;62:2485–9.

    Article  CAS  PubMed  Google Scholar 

  5. Baba A, Miyazaki M, Nagahama T, Nogi Y. Microbulbifer chitinilyticus sp. nov. and Microbulbifer okinawensis sp. nov., chitin-degrading bacteria isolated from mangrove forests. Int J Syst Evol Microbiol. 2011;61:2215–20.

    Article  CAS  PubMed  Google Scholar 

  6. Miyazaki M, Nogi Y, Ohta Y, Hatada Y, Fujiwara Y, Ito S, et al. Microbulbifer agarilyticus sp. nov. and Microbulbifer thermotolerans sp. nov., agar-degrading bacteria isolated from deep-sea sediment. Int J Syst Evol Microbiol. 2008;58:1128–33.

    Article  CAS  PubMed  Google Scholar 

  7. Vashist P, Nogi Y, Ghadi SC, Verma P, Shouche YS. Microbulbifer mangrovi sp. nov., a polysaccharide-degrading bacterium isolated from an Indian mangrove. Int J Syst Evol Microbiol. 2013;63:2532–7.

    Article  CAS  PubMed  Google Scholar 

  8. Jobling S. Improving starch for food and industrial applications. Curr Opin Plant Biol. 2004;7:210–8.

    Article  CAS  PubMed  Google Scholar 

  9. Avérous L, Halley PJ. Starch polymers: from the field to industrial products. In: Avérous L, Halley PJ, editors. Starch polymers. Amsterdam: Elsevier; 2014. p. 3–10.

    Chapter  Google Scholar 

  10. Bai FW, Anderson WA, Moo-Young M. Ethanol fermentation technologies from sugar and starch feedstocks. Biotechnol Adv. 2008;26:89–105.

    Article  CAS  PubMed  Google Scholar 

  11. Sen G, Sharon A, Pal S. Grafted polysaccharides: smart materials of the future, their synthesis and applications. In: Kalia S, Avérous L, editors. Biopolymers: biomedical and environmental applications. Hoboken: Wiley; 2011. p. 99–127.

    Chapter  Google Scholar 

  12. Reddy K, Mohan GK, Satla S, Gaikwad S. Natural polysaccharides: versatile excipients for controlled drug delivery systems. Asian J Pharm Sci. 2011;6:275–86.

    Google Scholar 

  13. Tizzotti M, Charlot A, Fleury E, Stenzel M, Bernard J. Modification of polysaccharides through controlled/living radical polymerization grafting—Towards the generation of high performance hybrids. Macromol Rapid Commun. 2010;31:1751–72.

    Article  CAS  PubMed  Google Scholar 

  14. D’Ayala G, Malinconico M, Laurienzo P. Marine derived polysaccharides for biomedical applications: chemical modification approaches. Molecules. 2008;13:2069.

    Article  PubMed  Google Scholar 

  15. Karaki N, Aljawish A, Humeau C, Muniglia L, Jasniewski J. Enzymatic modification of polysaccharides: mechanisms, properties, and potential applications: a review. Enzym Microb Technol. 2016;90:1–18.

    Article  CAS  Google Scholar 

  16. Gübitz GM, Paulo AC. New substrates for reliable enzymes: enzymatic modification of polymers. Curr Opin Biotechnol. 2003;14:577–82.

    Article  PubMed  Google Scholar 

  17. Oh C, De Zoysa M, Kwon YK, Heo SJ, Affan A, Jung WK, et al. Complete genome sequence of the agarase-producing marine bacterium strain s89, representing a novel species of the genus Alteromonas. J Bacteriol. 2011;193:5538.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Sun C, Chen YJ, Zhang XQ, Pan J, Cheng H, Wu M. Draft genome sequence of Microbulbifer elongatus strain HZ11, a brown seaweed-degrading bacterium with potential ability to produce bioethanol from alginate. Mar Genomics. 2014;18:83–5.

    Article  PubMed  Google Scholar 

  19. Lee YS, Choi YL. Complete genome sequence of cold-adapted enzyme producing Microbulbifer thermotolerans DAU221. J Biotechnol. 2016;229:31–2.

    Article  CAS  PubMed  Google Scholar 

  20. Dinesh B, Lau N-S, Furusawa G, Kim S-W, Taylor TD, Foong SY, et al. Comparative genome analyses of novel Mangrovimonas-like strains isolated from estuarine mangrove sediments reveal xylan and arabinan utilization genes. Mar Genomics. 2016;25:115–21.

    Article  PubMed  Google Scholar 

  21. Furusawa G, Lau NS, Shu-Chien AC, Jaya-Ram A, Amirul AAA. Identification of polyunsaturated fatty acid and diterpenoid biosynthesis pathways from draft genome of Aureispira sp. CCB-QB1. Mar Genomics. 2015;19:39–44.

    Article  PubMed  Google Scholar 

  22. Weisburg WG, Barns SM, Pelletier DA, Lane DJ. 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol. 1991;173:697–703.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol. 1990;215:403–10.

    Article  CAS  PubMed  Google Scholar 

  24. Meier-Kolthoff JP, Göker M, Spröer C, Klenk H-P. When should a DDH experiment be mandatory in microbial taxonomy? Arch Microbiol. 2013;195:413–8.

    Article  CAS  PubMed  Google Scholar 

  25. Edgar RC. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 2004;32:1792–7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Edgar RC. MUSCLE: a multiple sequence alignment method with reduced time and space complexity. BMC Bioinform. 2004;5:113.

    Article  Google Scholar 

  27. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol. 2013;30:2725–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Saitou N, Nei M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol. 1987;4:406–25.

    CAS  PubMed  Google Scholar 

  29. Felsenstein J. Confidence limits on phylogenies: an approach using the bootstrap. Evolution. 1985;39:783–91.

    Article  PubMed  Google Scholar 

  30. Fulton J, Douglas T, Young AM. Isolation of viruses from high temperature environments. Methods Mol Biol. 2009;501:43–54.

    Article  CAS  PubMed  Google Scholar 

  31. Chin C-S, Alexander DH, Marks P, Klammer AA, Drake J, Heiner C, et al. Nonhybrid, finished microbial genome assemblies from long-read SMRT sequencing data. Nat Meth. 2013;10:563–9.

    Article  CAS  Google Scholar 

  32. Seemann T. Prokka: rapid prokaryotic genome annotation. Bioinformatics. 2014;30:2068–9.

    Article  CAS  PubMed  Google Scholar 

  33. Hyatt D, Chen G-L, LoCascio PF, Land ML, Larimer FW, Hauser LJ. Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC Bioinform. 2010;11:1–11.

    Article  Google Scholar 

  34. Lagesen K, Hallin P, Rodland EA, Staerfeldt HH, Rognes T, Ussery DW. RNAmmer: consistent and rapid annotation of ribosomal RNA genes. Nucleic Acids Res. 2007;35:3100–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Laslett D, Canback B. ARAGORN, a program to detect tRNA genes and tmRNA genes in nucleotide sequences. Nucleic Acids Res. 2004;32:11–6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Petersen TN, Brunak S, von Heijne G, Nielsen H. SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Meth. 2011;8:785–6.

    Article  CAS  Google Scholar 

  37. Kolbe DL, Eddy SR. Fast filtering for RNA homology search. Bioinformatics. 2011;27:3102–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Bairoch A, Apweiler R, Wu CH, Barker WC, Boeckmann B, Ferro S, et al. The universal protein resource (UniProt). Nucleic Acids Res. 2005;33:154–9.

    Article  Google Scholar 

  39. Kanehisa M, Goto S. KEGG: kyoto encyclopedia of genes and genomes. Nucleic Acids Res. 2000;28:27–30.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Marchler-Bauer A, Zheng C, Chitsaz F, Derbyshire MK, Geer LY, Geer RC, et al. CDD: conserved domains and protein three-dimensional structure. Nucleic Acids Res. 2013;41:348–52.

    Article  Google Scholar 

  41. Galperin MY, Makarova KS, Wolf YI, Koonin EV. Expanded microbial genome coverage and improved protein family annotation in the COG database. Nucleic Acids Res. 2014;43:D261–9.

  42. Weber T, Blin K, Duddela S, Krug D, Kim HU, Bruccoleri R, et al. antiSMASH 3.0—a comprehensive resource for the genome mining of biosynthetic gene clusters. Nucleic Acids Res. 2015;43:W237–43.

  43. Yin Y, Mao X, Yang J, Chen X, Mao F, Xu Y. dbCAN: a web resource for automated carbohydrate-active enzyme annotation. Nucleic Acids Res. 2012;40:445–51.

    Article  Google Scholar 

  44. Grant JR, Arantes AS, Stothard P. Comparing thousands of circular genomes using the CGView Comparison tool. BMC Genomics. 2012;13:1–8.

    Article  Google Scholar 

  45. Richter M, Rosselló-Móra R. Shifting the genomic gold standard for the prokaryotic species definition. Proc Natl Acad Sci U S A. 2009;106:19126–31.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Kim M, Oh H-S, Park S-C, Chun J. Towards a taxonomic coherence between average nucleotide identity and 16S rRNA gene sequence similarity for species demarcation of prokaryotes. Int J Syst Evol Microbiol. 2014;64:346–51.

    Article  CAS  PubMed  Google Scholar 

  47. Eddy SR. Accelerated profile HMM searches. PLoS Comput Biol. 2011;7:e1002195.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Patil RS, Ghormade V, Deshpande MV. Chitinolytic enzymes: an exploration. Enzym Microb Technol. 2000;26:473–83.

    Article  CAS  Google Scholar 

  49. Nishijima M, Takadera T, Imamura N, Kasai H, An K-D, Adachi K, et al. Microbulbifer variabilis sp. nov. and Microbulbifer epialgicus sp. nov., isolated from Pacific marine algae, possess a rod–coccus cell cycle in association with the growth phase. Int J Syst Evol Microbiol. 2009;59:1696–707.

    Article  CAS  PubMed  Google Scholar 

  50. Levin PA, Margolis PS, Setlow P, Losick R, Sun D. Identification of Bacillus subtilis genes for septum placement and shape determination. J Bacteriol. 1992;174:6717–28.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Henriques AO, Glaser P, Piggot PJ, Moran CP Jr. Control of cell shape and elongation by the rodA gene in Bacillus subtilis. Mol Microbiol. 1998;28:235–47.

    Article  CAS  PubMed  Google Scholar 

  52. Alyahya SA, Alexander R, Costa T, Henriques AO, Emonet T, Jacobs-Wagner C. RodZ, a component of the bacterial core morphogenic apparatus. Proc Natl Acad Sci U S A. 2009;106:1239–44.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Freire P, Neves Moreira R, Arraiano CM. BolA inhibits cell elongation and regulates MreB expression levels. J Mol Biol. 2009;385:1345–51.

    Article  CAS  PubMed  Google Scholar 

  54. Soufo HJ, Graumann PL. Actin-like proteins MreB and Mbl from Bacillus subtilis are required for bipolar positioning of replication origins. Curr Biol. 2003;13:1916–20.

    Article  PubMed  Google Scholar 

  55. Kruse T, Bork-Jensen J, Gerdes K. The morphogenetic MreBCD proteins of Escherichia coli form an essential membrane-bound complex. Mol Microbiol. 2005;55:78–89.

    Article  CAS  PubMed  Google Scholar 

  56. Burger A, Sichler K, Kelemen G, Buttner M, Wohlleben W. Identification and characterization of the mre gene region of Streptomyces coelicolor A3(2). Mol Gen Genet. 2000;263:1053–60.

    Article  CAS  PubMed  Google Scholar 

  57. Slovak PM, Wadhams GH, Armitage JP. Localization of MreB in Rhodobacter sphaeroides under conditions causing changes in cell shape and membrane structure. J Bacteriol. 2005;187:54–64.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Jones LJ, Carballido-Lopez R, Errington J. Control of cell shape in bacteria: helical, actin-like filaments in Bacillus subtilis. Cell. 2001;104:913–22.

    Article  CAS  PubMed  Google Scholar 

  59. Kruse T, Moller-Jensen J, Lobner-Olesen A, Gerdes K. Dysfunctional MreB inhibits chromosome segregation in Escherichia coli. EMBO J. 2003;22:5283–92.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Louis P, Galinski EA. Characterization of genes for the biosynthesis of the compatible solute ectoine from Marinococcus halophilus and osmoregulated expression in Escherichia coli. Microbiology. 1997;143:1141–9.

    Article  CAS  PubMed  Google Scholar 

  61. Ofer N, Wishkautzan M, Meijler M, Wang Y, Speer A, Niederweis M, et al. Ectoine biosynthesis in Mycobacterium smegmatis. Appl Environ Microbiol. 2012;78:7483–6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Peters P, Galinski EA, Trüper HG. The biosynthesis of ectoine. FEMS Microbiol Lett. 1990;71:157–62.

    Article  CAS  Google Scholar 

  63. Mustakhimov II, Reshetnikov AS, Fedorov DN, Khmelenina VN, Trotsenko YA. Role of EctR as transcriptional regulator of ectoine biosynthesis genes in Methylophaga thalassica. Biochem Mosc. 2012;77:857–63.

    Article  CAS  Google Scholar 

  64. Lippert K, Galinski EA. Enzyme stabilization be ectoine-type compatible solutes: protection against heating, freezing and drying. Appl Microbiol Biotechnol. 1992;37:61–5.

    Article  CAS  Google Scholar 

  65. Kurz M. Compatible solute influence on nucleic acids: many questions but few answers. Saline Syst. 2008;4:6.

    Article  PubMed  PubMed Central  Google Scholar 

  66. Graf R, Anzali S, Buenger J, Pfluecker F, Driller H. The multifunctional role of ectoine as a natural cell protectant. Clin Dermatol. 2008;26:326–33.

    Article  PubMed  Google Scholar 

  67. Lentzen G, Schwarz T. Extremolytes: natural compounds from extremophiles for versatile applications. Appl Microbiol Biotechnol. 2006;72:623–34.

    Article  CAS  PubMed  Google Scholar 

  68. Grein TA, Freimark D, Weber C, Hudel K, Wallrapp C, Czermak P. Alternatives to dimethylsulfoxide for serum-free cryopreservation of human mesenchymal stem cells. Int J Artif Organs. 2010;33:370–80.

    CAS  PubMed  Google Scholar 

  69. Field D, Garrity G, Gray T, Morrison N, Selengut J, Sterk P, et al. The minimum information about a genome sequence (MIGS) specification. Nature Biotechnol. 2008;26:541–7.

    Article  CAS  Google Scholar 

  70. 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.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  71. Garrity GM, Bell JA, Lilburn T. Phylum XIV. Proteobacteria phyl. Nov. In: Garrity GM, Brenner DJ, Krieg NR, Staley JT, editors. Bergey’s manual of systematic bacteriology. 2nd Ed. Volume 2, part B. New York: Springer; 2005. p. 1.

    Google Scholar 

  72. Garrity GM, Bell JA, Lilburn T. Class III. Gammaproteobacteria class. nov. In: Garrity GM, Brenner DJ, Krieg NR, Staley JT, editors. Bergey’s manual of systematic bacteriology. 2nd Ed. Volume 2, part B. New York: Springer; 2005. p. 1.

    Google Scholar 

  73. Spring S, Scheuner C, Göker M, Klenk H-P. A taxonomic framework for emerging groups of ecologically important marine gammaproteobacteria based on the reconstruction of evolutionary relationships using genome-scale data. Front Microbiol. 2015;6:281.

    Article  PubMed  PubMed Central  Google Scholar 

  74. Oren A, Garrity GM. Validation list no. 164. List of new names and new combinations previously effectively, but not validly, published. Int J Syst Evol Microbiol. 2015;65:2017–25.

    Article  Google Scholar 

  75. Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, et al. Gene ontology: tool for the unification of biology. The Gene ontology consortium. Nat Genet. 2000;25:25–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We would like to thank Balachandra Dinesh for isolating Microbulbifer sp. CCB-MM1 and Ka Kei Sam for extracting the genomic DNA. N.-S. Lau and G. Furusawa gratefully acknowledge the post-doctoral fellowships granted by Universiti Sains Malaysia. T.H. Moh also acknowledges the financial support provided by Ministry of Higher Education Malaysia (MOHE) through MyBrain15 MyMaster scholarship.

Funding

This work was conducted as part of the mangrove project supported by Research University (RU) mangrove project grant (1001/PCCB/870009) to Centre for Chemical Biology, Universiti Sains Malaysia.

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Authors and Affiliations

  1. Centre for Chemical Biology, Universiti Sains Malaysia, 11900, Penang, Malaysia

    Tsu Horng Moh, Nyok-Sean Lau, Go Furusawa & Al-Ashraf Abdullah Amirul

  2. School of Biological Sciences, Universiti Sains Malaysia, 11800, Penang, Malaysia

    Al-Ashraf Abdullah Amirul

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  1. Tsu Horng Moh
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  2. Nyok-Sean Lau
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  3. Go Furusawa
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  4. Al-Ashraf Abdullah Amirul
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Contributions

TH performed the genome assembly, annotation, bioinformatics analyses and wrote the manuscript. NS and GF designed the experiments and revised the manuscript. AAA coordinated the project and determined the project direction. All authors read and approved the final manuscript.

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Correspondence to Al-Ashraf Abdullah Amirul.

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Moh, T.H., Lau, NS., Furusawa, G. et al. Complete genome sequence of Microbulbifer sp. CCB-MM1, a halophile isolated from Matang Mangrove Forest, Malaysia. Stand in Genomic Sci 12, 36 (2017). https://doi.org/10.1186/s40793-017-0248-0

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Keywords

Environmental Microbiome

ISSN: 2524-6372