Comparative genomics study of polyhydroxyalkanoates (PHA) and ectoine relevant genes from Halomonas sp. TD01 revealed extensive horizontal gene transfer events and co-evolutionary relationships
- Lei Cai†1,
- Dan Tan†1,
- Gulsimay Aibaidula2,
- Xin-Ran Dong3,
- Jin-Chun Chen1,
- Wei-Dong Tian3Email author and
- Guo-Qiang Chen1Email author
© Cai et al; licensee BioMed Central Ltd. 2011
Received: 8 August 2011
Accepted: 1 November 2011
Published: 1 November 2011
Halophilic bacteria have shown their significance in industrial production of polyhydroxyalkanoates (PHA) and are gaining more attention for genetic engineering modification. Yet, little information on the genomics and PHA related genes from halophilic bacteria have been disclosed so far.
The draft genome of moderately halophilic bacterium, Halomonas sp. TD01, a strain of great potential for industrial production of short-chain-length polyhydroxyalkanoates (PHA), was analyzed through computational methods to reveal the osmoregulation mechanism and the evolutionary relationship of the enzymes relevant to PHA and ectoine syntheses. Genes involved in the metabolism of PHA and osmolytes were annotated and studied in silico. Although PHA synthase, depolymerase, regulator/repressor and phasin were all involved in PHA metabolic pathways, they demonstrated different horizontal gene transfer (HGT) events between the genomes of different strains. In contrast, co-occurrence of ectoine genes in the same genome was more frequently observed, and ectoine genes were more likely under coincidental horizontal gene transfer than PHA related genes. In addition, the adjacent organization of the homologues of PHA synthase phaC1 and PHA granule binding protein phaP was conserved in the strain TD01, which was also observed in some halophiles and non-halophiles exclusively from γ-proteobacteria. In contrast to haloarchaea, the proteome of Halomonas sp. TD01 did not show obvious inclination towards acidity relative to non-halophilic Escherichia coli MG1655, which signified that Halomonas sp. TD01 preferred the accumulation of organic osmolytes to ions in order to balance the intracellular osmotic pressure with the environment.
The accessibility of genome information would facilitate research on the genetic engineering of halophilic bacteria including Halomonas sp. TD01.
Polyhydroxyalkanoates (PHA) were firstly discovered in prokaryotes as carbon and energy storage materials . They are one of the most promising members of biodegradable polymers, which are considered as environmentally friendly substitutes of petrochemical-derived plastics . Poly-3-hydroxybutyrate (PHB) was the earliest commercially available product. However, poly(3-hydroxybutyrate-co-hydroxyvalerate) (PHBV) possessed more favourable thermomechanical properties for wider applications as medical materials (sutures and bone-nails/pins), films products (mulch films, shopping bags and compost bags), disposable items (pens and tableware) and packaging materials (especially for food packaging) than PHB . As the market for green plastics has been growing rapidly, demand for a more productive and low cost PHA production process is evident . Although some microorganisms, such as Ralstonia eutropha and genetically engineered Escherichia coli, were thoroughly investigated as PHA industrial producers with high productivity, the research and development of strains and methods with further reduced cost were still necessary for PHA commercialization [3–6].
Halophiles are referred to some organisms, which are able to grow optimally in 5% (w/v) and survive in no less than 10% salt medium [7, 8]. Halophiles have shown advantages for lowering fermentation costs for PHB and PHBV production, thus gaining attention from many researchers. As early as 1972, PHA granules were detected in a haloarchaea Haloarcula marismortui, and then numerous halophiles were identified as producers to accumulate PHB or PHBV . Halophiles were widely spread in the three domains of life: archaea, bacteria, and eukarya . According to the optimal growth concentration of salt tolerated by cells, they could be roughly divided into two groups, moderate and extreme halophiles . Recently, the application of some halophiles for PHA production and copolymer characterization were evaluated [4, 7, 10, 11]. The application of halophiles as PHA producers significantly reduced the costs of fermentation and recovery processes: high salt concentrations were able to minimize the possibility of contamination by non-halophilic microorganisms, and thus the cost and energy consumption for sterilization can be decreased; haloarchaeal cells were able to be lyzed conveniently through osmotic shock treatment with salt-deficient water, and thus the cost for polymers recovery also can be decreased . Moreover, the residual salt in the broth post-fermentation, could be concentrated and recycled, which circumvents environmental problems from the exhaust of waste products and further lowered the production cost . When hydrolyzed whey was used as carbon source, the cost for the production of P(3HB-co3HV) by Haloferax mediterranei was about 30% lower than that for the production of PHA by recombinant E. coli. Our recent study showed a new isolated halophilic strain termed Halomonas sp. TD01, has great potential for PHA production, accumulating over 80 wt% PHB under a continuous fermentation process without sterilization .
PHA synthases, the crucial enzymes for PHA biosynthesis, have received more attention for elucidating their characteristics in these microorganisms. PHA synthases were classified into four groups according to their substrate specificities and subunits organization . Although class I and II PHA synthases comprise enzymes consisting exclusively of one PhaC subunit, they prefer to synthesize short chain length (3-5 carbon atoms) and medium chain length (6-14 carbon atoms) PHA, respectively. Class III and IV PHA synthases comprise enzymes consisting of two different types of subunits, PhaC and PhaE (III) or PhaR (IV, such as that in Bacillus megaterium) subunits; both of them prefer to utilize CoA thioesters to produce short chain length PHA . The study on PHA synthases isolated from PHA accumulating halophiles began with the characterization of PhaC from Halopiger aswanensis, which showed some interesting properties including high thermostability, narrow substrate specificity and tolerance against high salt concentration . A novel subclass of PhaCs was proposed through systematic and detailed studies on the PHA synthases from haloarchaea, which were in high similarity with the class III enzymes from bacteria . However, the H. mediterranei PhaC subunit was larger, and the PhaE subunit was smaller than its bacterial counterparts .
Osmoregulatory mechanisms of halophiles against the high salt concentration condition were typically divided into two strategies: extreme halophiles, i.e. halophilic archaea, were able to absorb KCl to balance the osmotic pressure discrepancy across the cytoplasmic membrane; most moderate halophiles were able to accumulate osmolytes (compatible solutes), which mainly consisted of organic compounds with low molecular weight, including amino acids, amino acid derivatives, sugars, and other polyols . Organic compounds did not notably disturb the essential metabolism for cell survival, and protected proteins by mitigating detrimental effects of freezing, drying and high temperatures . Ectoine is one of the most widespread osmolytes, which are also well known as commercial protectants for enzymes, DNA and whole cells .
In this study, the draft genome of the moderately halophilic bacterium Halomonas sp. TD01 was obtained and analyzed. Several genes relevant to PHA and osmolytes biosynthesis were elucidated and phylogenetically analyzed. Moreover, the predicted proteome was analyzed and compared with that of other species. The results provided invaluable clues, not only to the understanding of the evolution and genes transfer, but also to the strategic guidance of the genetic engineering of halophilic Halomonas sp. TD01 for co-production of PHA and ectoine.
Strain and genome DNA preparation
Halomonas sp. TD01 was isolated from a salt lake in Xinjiang, China, and grown optimally in glucose mineral medium under 5% (w/v) NaCl and pH 9.0 . And it was deposited in China General Microbiological Culture Collection Center (CGMCC No.4353). To construct the insert libraries for sequencing, high-quality total cellular DNA was prepared with the help of the E.Z.N.A. bacterial DNA kit (Omega Bio-Tek Inc. USA).
Two random genomic DNA libraries with insert sizes of 500 and 2,530 base pairs (bp) were constructed, respectively. The sequencing of these libraries was carried out following the Solexa sequencing protocols (Illumina, Inc. USA) in the Beijing Genomics Institute (BGI, Beijing, China). Eliminating the low-quality results and adapter contamination, raw data was assembled into contigs and scaffolds with SOAPdenovo software (v1.04, BGI, Beijing, China).
Glimmer 3.0 software  was adopted to predict genes de novo. For annotation, the alignments of predicted proteins against databases, including KEGG , COG , Swiss-Prot , TrEMBL  and nr (at NCBI, National Center for Biotechnology Information), were carried out with the program blastall (version 2.2.21) . Genes encoding transfer RNA (tRNA) and their secondary structure were predicted using tRNAscan . Genes encoding ribosomal RNA (rRNA) were predicted through RNAmmer  or homologous comparison. Genes encoding other RNA, including microRNA (miRNA), small RNA (sRNA) and small nuclear RNA (snRNA) were predicted by Rfam . Transposons were identified through RepeatMasker and RepeatProteinMasker .
After the assembly, global guanine-cytosine composition of the whole non-redundant sequence was calculated with the in-house program (BGI, Beijing, China). The predicted open reading frames (ORFs) inferred by Glimmer were also counted for GC content and length distribution.
Promoter and signal peptide prediction
The promoter, including -35, -10 and transcription start site (TSS) regions, were predicted through Neural Network Promoter Prediction  and Sequence Alignment Kernel  methods. The signal peptide for secreted proteins was predicted by SignalP .
Multiple sequences alignments and identification of protein motifs
Multiple alignments of predicted proteins with conserved sequences were performed with Constraint-based Multiple Alignment Tool (COBALT)  at NCBI. Conserved motifs were identified by searching sequences against the COG database.
Evolutionary and phylogenetic analysis
The distances between conserved sequences were calculated from the multiple alignments with ClustalW . Neighbor-joining tree phylograms were constructed, bootstrapped (500 replications), and drawn in MEGA (version 5.03) .
With the evolutionary distances, the evolutionary pressures imposed on keeping any two genes in the genome were inspected by computing a Pearson correlation coefficient for any two genes. The missing genes (designated as "ND") were useful in inspecting the co-evolutionary relationships, and usually defined as zero. However, here, the smaller the evolutionary distance, the closer a homologue is to the gene in TD01. To make use of the missing genes, we transformed the evolutionary distances into similarity scores with e(-1*distance) and defined the missing genes (ND) as zero, i.e. a shorter distance relates to an increased similarity score. Then a Pearson correlation coefficient was computed for any two genes using the converted similarity scores.
Calculation of isoelectric point values
FASTA format protein sequences, either predicted from Halomonas sp. TD01 or retrieved from NCBI were submitted to ExPASy Proteomics server (http://web.expasy.org/compute_pi/). The isoelectric point (pI) distribution of each strain was counted with an interval of 0.2 differences from 3 to 13.
Putative PHA and ectoine relevant genes in the genome of Halomonas sp. TD01
Calculated Molecular mass (kDa)b
Polyhydroxyalkanoate synthesis repressor
L-2,4-diaminobutyric acid acetyltransferase
L-2,4-diaminobutyric acid transaminase
Overview of sequencing and gene prediction
Sequencing was conducted with Solexa technology. Using SOAPdenovo software, the clean raw data was assembled into 26 scaffolds varying from 511 to 918,836 bp with a total length of 4,092,837 bp. The estimated percentages of genome coverage were 102.64% and 99.56% based on k-mer analysis and reads comparison, respectively. 3,894 Open Reading Frames (ORF) predicted by Glimmer had occupied 89.18% of the whole assembled sequences. The GC content within coding sequences was 53.23%, which was a little higher than that of the whole sequences (52.57%). Most putative proteins were distributed within the length ranges between 200 and 300 amino acids (aa) (Additional file 2, Figure S1). In addition, the putative proteins were classified according to the COG function (Additional file 3, Figure S2A) and KEGG pathway (Additional file 3, Figure S2B). The comparison of the predicted protein sequence set of Halomonas sp. TD01 with the nr database (NCBI) revealed its close relationship with Halomonas elongata DSM 2581, Chromohalobacter salexigens DSM 3043 and Aromatoleum aromaticum EbN1. Although the sequence of the scaffolds of Halomonas sp. TD01 have not been confirmed, the alignment between the chromosome of C. salexigens and the scaffolds of Halomonas sp. TD01 showed distinguishable co-linearity (Additional file 4, Figure S3).
Identification and evolutionary analysis of PHA relevant genes from Halomonas sp. TD01
The genes relevant to PHA were identified through homologous alignments against the public annotation databases, including KEGG, COG, Swiss-Prot, TrEMBL and nr, using the BLAST program. As inferred from the similarity results, there might be two putative genes encoding PHA synthases (PhaC), three encoding PHA depolymerases (PhaZ), one encoding phasin (PhaP) and one encoding PHA synthesis repressor/regulator (PhaR, which showed 45% identities with YP_725943 of Ralstonia eutropha H16) in the genome of halophilic PHBV producing strain Halomonas sp. TD01 (Table 1). In addition, although the putative PhaC1 shared only 19% identity with PhaC2, both of them hit COG (clusters of orthologous groups) 3243 in the conserved domain database (CDD) at NCBI, implying that they belonged to PHA synthases . Similarly, all the three PhaZs deduced from the genome of Halomonas sp. TD01 shared remarkably the same hits on COG3509 and pfam10503 in CDD, which strongly proposed their functions as PHB depolymerases. Phylogenetic trees clearly illustrated that the putative PhaZ, PhaP and PhaR from Halomonas sp. TD01 had close relationships with the corresponding, well-characterized enzymes from non-halophiles, which presented clear, in silico evidence for their function in PHA degradation and regulation (not shown).
The detailed analysis through multiple sequences alignments of putative PhaC1 and PhaC2 with their homologues was performed. The most similar homologues of both PhaC1 and PhaC2 came from H. elongata DSM 2581 (78%, 73% identities, respectively), C. salexigens DSM 3043 (67%, 67% identities, respectively) and A. aromaticum EbN1 (48%, 69% identities, respectively) (Additional file 5, Table S2). The multiple sequences alignments showed strong conservation between these protein sequences and the well-characterized PHA synthases (Figure 2). The conserved lipase-like box residues were recognized in all of these proteins (Figure 2). In addition, three residues (cysteine, aspartic acid and histidine), composing the conserved catalytic triad of PHA synthases, were also identified in these proteins (Figure 2). However, it is interesting to find that, instead of the traditional lipase-box pattern "GxCxG", all PhaC1 homologues (listed in Figure 2, except for YP_159076 from non-halophilic A. aromaticum EbN1), have "SxCxG", and all the PhaC2 homologues (listed in Figure 2) have "GxCxA". These interesting patterns may imply the existence of a new class of PHA polymerases among the halophilic bacteria. Yet, more experimental data are needed to elucidate whether these patterns are related to the hypersaline environments.
With the progress on the research of PhaCs from halophiles, our vision on PHA synthesis in these strains was amazingly broadened . In contrast, few efforts had been devoted to the studies on other enzymes involved in PHA metabolism excluding PhaC. The putative PhaZ3 was supposed to be a secreted protein through the prediction of signal peptides (Additional file 6, Figure S4), whereas the other two PHA depolymerases (PhaZ1 and PhaZ2) lack signal peptides and may be responsible for the intracellular PHA degradation.
Osmoregulatory mechanisms of Halomonas sp. TD01 inferred from the genome information
The pI values provide simple indication of protein acidity. To compare the differences of protein acidity between halophiles and non-halophiles, the pI distribution of representative proteome from halophilic bacterium Halomonas sp. TD01 and Halomonas elongata DSM 2581, halophilic arhaeon Haloarcula marismortui ATCC 43049 and non-halophilic E. coli MG1655, was calculated with the interval of 0.2 pI from 3 to 13. Bimodal distribution of pI appears clearly on the plot for each strain (Additional file 7, Figure S5). Both acidic peaks of Halomonas sp. TD01 and H. elongata DSM 2581 were at 5.1, which were a little lower than the non-halophilic E. coli MG1655 (at 5.5), but much higher than the extremely halophilic archaeon H. marismortui (at 4.3). While H. marismortui only showed a small peak representing basic proteins, the other three strains shared the distinct basic peaks at around pI 9.5. The median pI values of Halomonas sp. TD01, H. elongata, H. marismortui and E. coli were 5.67, 5.51, 4.46 and 6.17, respectively, which displayed a similar pattern with the pI distribution.
The evolutionary analysis of PHA and ectoine relevant proteins from Halomonas sp. TD01
As revealed through the analysis of the osmoregulatory mechanism, Halomonas sp. TD01 was possibly able to synthesize ectoines with commercial interest as protectants against proteolysis . The existence of PHA and ectoine synthesis genes qualifies Halomonas sp. TD01 as a candidate for the combined production of PHA and osmolytes . Moreover, it was interesting to observe that many species other than halophiles possessed both homologues of putative PHA and ectoine relevant enzymes from Halomonas sp. TD01 (Additional file 1, Table S1). Based on the evolutionary distances of 16S rDNA (Figure 4), PHA and ectoine relevant protein sequences, Halomonas sp. TD01 shared closest relationship with Chromohalobacter salexigens DSM 3043 and Halomonas elongata DSM 2581, which were also known as halophilic bacteria and belonged to the same family Halomonadaceae as the strain TD01 (Additional file 5, Table S2). It was interesting to find that, with regard to PHA relevant enzymes, similarities (the average evolutionary distance of PhaCs: 0.666) between TD01 and rhizobia strains, which fix nitrogen and are symbiotic with plant roots, were significant (Additional file 5, Table S2). The homologues of ectoine synthesis and PHA degradation (PhaZs) enzymes from Halomonas sp. TD01 were present in some species belonging to the class Actinobacteria, are a group of Gram-positive bacteria with high G+C content (Additional file 5, Table S2). In addition, it was noticed that only proteins from some γ-proteobacteria species exhibited significant similarity with the PhaP from strain TD01 (Additional file 5, Table S2), which implies this PhaP possibly possesses a relative independent evolution pathway in the γ-proteobacteria. However, it seemed that the other homologues of strain TD01 spread in α-, β-, γ-proteobacteria (Additional file 5, Table S2), suggesting that PHA and ectoine related enzymes from the halophilic strain TD01 shared common ancestors with the non-halophiles.
So far, there have been relatively few studies on the PHA synthases from halophilic microorganisms . Recently, the detailed studies on the PHA synthases from the halophilic archaea have extended our insights on their classification, providing solid evidences to support the existence of a novel subclass (IIIA) synthases with distinguished features from the subclass (IIIB) isolated from bacteria . The widespread novel PHA synthases in halophilic archaea consist of two subunits, PhaC and PhaE, both of which are required for their activities . However, these novel PHA enzymes are clustered separately from the ones isolated from the halophilic bacteria, including Halomonas sp. TD01, H. elongata, C. salexigens, and Halorhodospira halophila, etc (Figure 1). As illustrated by the phylogenetic analysis, PHA synthases (especially the putative PhaC1) from halophilic bacteria, shared the significant similarities with class I enzymes, which were different from the well-studied enzymes from halophilic archaea (Figure 1) . PhaCs in Halomonas sp.TD01 were able to synthesize PHB with a molecular weight of 600 kDa in fed-batch fermentation, which was in accordance with the characteristics of class I PHA synthases [1, 13]. The conserved catalytic residues, firstly discovered in the PHA synthases of Chromatium vinosum and R. eutropha, were also recognized in PhaC1 and PhaC2 from Halomonas sp. TD01 (Figure 2), indicating that they share similar catalytic mechanisms and substrate specificities [7, 45]. In addition, PHBV was detected when glucose and propionic acid (or valeric acid) were supplemented as carbon sources in the medium, which also agreed with the features of class I synthases . Under the catalysis of putative PhaC1 and/or PhaC2, PHBV accumulation was able to reach 80 wt% of cell dry weight consisting of 30% 3-hydroxyvalerate 3HV fraction when supplemented with valeric acid as additional carbon source, which qualified Halomonas sp. TD01 as a strain of industrial interest for PHBV production . Nevertheless, physiological and enzymatic characterization of the PHA synthases from halophilic bacteria are still required to elucidate whether the putative PhaC2 from Halomonas sp. TD01 and its homologues form their own class of PHA synthases, as implied by the phylogenetic studies and multiple sequences alignment of putative PhaC2 (Figure 2).
While PHA synthases from halophiles have received attentions, the studies on PHA depolymerase, regulator/repressor and phasin from halophiles, which also play crucial roles in the PHA cycle, remained unclear . Evolutionary analysis demonstrated that the PhaC, PhaZ, PhaP, PhaR and ectoine relevant enzymes from halophilic Halomonas sp. TD01 share close relationships (the average evolutionary distance: 0.945) with those from non-halophilic strains (Additional file 5, Table S2). It is very clear that further systematic and detailed studies on these genes from halophiles would improve our understanding on their functions and evolution, potentially leading to enhancing PHA production at reduced costs. Moreover, genome-scale metabolic analysis would also advance the progress of PHA production by halophiles .
The intervals lengths between the co-linear phaP and phaC1 genes from Halomonas sp. TD01 and their homologues in other microorganisms
Halomonas elongata DSM 2581
Chromohalobacter salexigens DSM 3043
Marinomonas sp. MED121
Hahella chejuensis KCTC 2396
Vibrio alginolyticus 12G01
Vibrio brasiliensis LMG 20546
Vibrio caribbenthicus ATCC BAA-2122
Vibrio cholerae 1587
Vibrio coralliilyticus ATCC BAA-450
Vibrio furnissii CIP 102972
Vibrio harveyi 1DA3
Vibrio metschnikovii CIP 69.14
Vibrio mimicus VM573
Vibrio orientalis CIP 102891
Vibrio parahaemolyticus RIMD 2210633
Vibrio shilonii AK1
Vibrio sinaloensis DSM 21326
Vibrio sp. Ex25
Vibrio sp. RC586
Vibrio splendidus LGP32
Vibrio vulnificus CMCP6
Vibrionales bacterium SWAT-3
Grimontia hollisae CIP 101886
Photobacterium angustum S14
Photobacterium damselae subsp. damselae CIP 102761
Photobacterium leiognathi subsp. mandapamensis svers.1.1.
Photobacterium profundum 3TCK
Photobacterium profundum SS9
Photobacterium sp. SKA34
Enhydrobacter aerosaccus SK60
Shewanella halifaxensis HAW-EB4
Shewanella pealeana ATCC 700345
Marinobacter aquaeolei VT8
Marinobacter sp. ELB17
Aeromonas hydrophila subsp. hydrophila ATCC 7966
Aeromonas salmonicid a subsp. salmonicida A449
Wild-type cells require PHA synthesis and degradation cycle to maintain carbon supply balance under changing environments . However, for industrial production of PHA, degradation is not favored, because it leads to the waste of carbon sources and consequently reduces PHA accumulation [47, 48]. For example, it has been reported that the knockout of phaZ in P. putida KT2442 inhibited the decrease of PHA content during batch fermentation processes . For Halomonas sp. TD01, the presence of three putative PHA depolymerases likely decreased PHA accumulation in batch fermentation processes (unpublished observations). Thus, identification of these phaZ genes could help the intentional knockout of PHA degradation pathways in Halomonas sp TD01, in the hope of promoting more PHA accumulation. In addition, distinguishing the intracellular (PhaZ1 and PhaZ2) and extracellular (PhaZ3) depolymerases allow the possibility to identify targets of most importance, reducing the time and labor required for gene knockout.
To maintain the balance of osmotic pressure across the cell membrane, high concentrations of ions or organic osmolytes are accumulated in the cells . Neutral proteins would become denatured in hypersaline condition, while acidic ones, with more negatively charged amino acid residues on the surfaces, were able to stay functional through the binding of hydrated salt ions in the cytoplasm . It was reported that the proteomes of extremely halophilic archaea, such as H. marismortui, showed a notable general tendency towards acidity, which was illustrated by the pI distribution (Additional file 7, Figure S5) . Contrary to this, less acidic proteomes from halophilic bacteria, including Halomonas sp. TD01 and H. elongata, were observed and implied their decreased dependence on the absorption of salt to maintain ionic balance in hypersaline environments . To survive in hypersaline conditions, it was believed that halophilic Halomonas sp. TD01 adopted another universal strategy, accumulating organic compatible solutes instead of inorganic ions to balance the osmotic pressure with a surprisingly broad salt concentration range . This was illustrated by the widespread of biosynthesis and transporter genes for multiple organic osmolytes over the genome. Even though halophilic microorganisms seemed to optimize their metabolism to minimize the energetic cost for osmotic adaptation, the de novo biosynthesis of osmolytes was less favorable with respect to either energy consumption or flexibility than their absorption from environment . In addition, there are many transporters for betaine, which possibly suggested that betaine was also one of the compatible solutes utilized by Halomonas sp. TD01 . However, a complete comprehension of the occurrence and distribution of organic osmoregulatory solutes in Halomonas sp. TD01 remain to be determined. Moreover, as the environmental factors, especially the nutritional ones, are able to strongly influence the production of PHA, the effects of both environmental factors and genetic background of Halomonas sp. TD01 on the production of PHA should be further studied.
In conclusion, the disclosure of the genome sequences of Halomonas sp. TD01 improves our understanding on the metabolism and evolutionary relationship of PHA and osmoregulatory solutes from halophilic bacteria. Detailed and systematic in silico analysis on the PHA and osmolytes relevant genes provides abundant insights on their classifications, functions and phylogeny. Osmoregulatory mechanisms were also discussed through the comparison of pI distribution. The availability of genomic information would inevitably pave new ways for the application of numerous post-genomic technologies and accelerate our work in the genetic optimization of Halomonas sp. TD01 for the industrial production of PHA, and possibly accompanied with the co-production of compatible solute ectoine.
We are grateful to Ian Wimpenny from Keele University, for his help with the improvement of writing. This research was financially supported by 973 Basic Research Fund (Grant No. 2011CBA00807) and Natural Science Foundation of China (Grant No 31170099) as well as by National High Tech 863 Grant (Project No. 2010AA101607),
- Rehm BHA: Polyester synthases: natural catalysts for plastics. Biochem J. 2003, 376: 15-33. 10.1042/BJ20031254View ArticleGoogle Scholar
- Potter M, Steinbüchel A: Poly(3-hydroxybutyrate) granule-associated proteins: Impacts on poly(3-hydroxybutyrate) synthesis and degradation. Biomacromolecules. 2005, 6: 552-560. 10.1021/bm049401nView ArticleGoogle Scholar
- Chen GQ: A microbial polyhydroxyalkanoates (PHA) based bio- and materials industry. Chem Soc Rev. 2009, 38: 2434-2446. 10.1039/b812677cView ArticleGoogle Scholar
- Joshi AA, Kanekar PP, Kelkar AS, Sarnaik SS, Shouche Y, Wani A: Moderately halophilic, alkalitolerant Halomonas campisalis MCM B-365 from Lonar Lake, India. J Basic Microbiol. 2007, 47: 213-221. 10.1002/jobm.200610223View ArticleGoogle Scholar
- Budde CF, Riedel SL, Willis LB, Rha C, Sinskey AJ: Production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from plant oil by engineered Ralstonia eutropha strains. Appl Environ Microbiol. 2011, 77: 2847-2854. 10.1128/AEM.02429-10View ArticleGoogle Scholar
- Park SJ, Choi JI, Lee SY: Short-chain-length polyhydroxyalkanoates: Synthesis in metabolically engineered Escherichia coli and medical applications. J Microbiol Biotechnol. 2005, 15: 206-215.Google Scholar
- Quillaguamán JH, Guzmán H, Van-Thuoc D, Hatti-Kaul R: Synthesis and production of polyhydroxyalkanoates by halophiles: current potential and future prospects. Appl Microbiol Biotechnol. 2010, 85: 1687-1696. 10.1007/s00253-009-2397-6View ArticleGoogle Scholar
- Oren A: Microbial life at high salt concentrations: phylogenetic and metabolic diversity. Saline Systems. 2008, 4: 2-View ArticleGoogle Scholar
- DasSarma P, DasSarma S: On the origin of prokaryotic "species": the taxonomy of halophilic Archaea. Saline Systems. 2008, 4: 5- 10.1186/1746-1448-4-5View ArticleGoogle Scholar
- Kulkarni SO, Kanekar PP, Nilegaonkar SS, Sarnaik SS, Jog JP: Production and characterization of a biodegradable poly (hydroxybutyrate-co-hydroxyvalerate) (PHB-co-PHV) copolymer by moderately haloalkalitolerant Halomonas campisalis MCM B-1027 isolated from Lonar Lake, India. Bioresour Technol. 2010, 101: 9765-9771. 10.1016/j.biortech.2010.07.089View ArticleGoogle Scholar
- Kulkarni SO, Kanekar PP, Jog JP, Patil PA, Nilegaonkar SS, Sarnaik SS, Kshirsagar PR: Characterisation of copolymer, poly (hydroxybutyrate-co-hydroxyvalerate) (PHB-co-PHV) produced by Halomonas campisalis (MCM B-1027), its biodegradability and potential application. Bioresour Technol. 2011, 102: 6625-6628. 10.1016/j.biortech.2011.03.054View ArticleGoogle Scholar
- Choi J, Lee SY: Factors affecting the economics of polyhydroxyalkanoate production by bacterial fermentation. Appl Microbiol Biotechnol. 1999, 51: 13-21. 10.1007/s002530051357. 10.1007/s002530051357View ArticleGoogle Scholar
- Tan D, Xue YS, Aibaidula G, Chen GQ: Unsterile and continuous production of polyhydroxybutyrate by Halomonas TD01. Biorescour Technol. 2011, 102: 8130-8136. 10.1016/j.biortech.2011.05.068. 10.1016/j.biortech.2011.05.068View ArticleGoogle Scholar
- Hezayen FF, Steinbüchel A, Rehm BHA: Biochemical and enzymological properties of the polyhydroxybutyrate synthase from the extremely halophilic archaeon strain 56. Arch Biochem Biophys. 2002, 403: 284-291. 10.1016/S0003-9861(02)00234-5View ArticleGoogle Scholar
- Han J, Hou J, Liu HL, Cai SF, Feng B, Zhou JA, Xiang H: Wide distribution among halophilic archaea of a novel polyhydroxyalkanoate synthase subtype with homology to bacterial type III synthases. Appl Environ Microbiol. 2010, 76: 7811-7819. 10.1128/AEM.01117-10View ArticleGoogle Scholar
- Lu QH, Han J, Zhou LG, Zhou J, Xiang H: Genetic and biochemical characterization of the poly(3-hydroxybutyrate-co-3-hydroxyvalerate) synthase in Haloferax mediterranei. J Bacteriol. 2008, 190: 4173-4180. 10.1128/JB.00134-08View ArticleGoogle Scholar
- Roberts MF: Organic compatible solutes of halotolerant and halophilic microorganisms. Saline Systems. 2005, 1: 5- 10.1186/1746-1448-1-5View ArticleGoogle Scholar
- Kolp S, Pietsch M, Galinski EA, Gütschow M: Compatible solutes as protectants for zymogens against proteolysis. Biochim Biophys Acta. 2006, 1764: 1234-1242.View ArticleGoogle Scholar
- Delcher AL, Bratke KA, Powers EC, Salzberg SL: Identifying bacterial genes and endosymbiont DNA with Glimmer. Bioinformatics. 2007, 23: 673-679. 10.1093/bioinformatics/btm009View ArticleGoogle Scholar
- 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. 10.1093/nar/27.1.29View ArticleGoogle Scholar
- Tatusov RL, Fedorova ND, Jackson JD, Jacobs AR, Kiryutin B, Koonin EV, Krylov DM, Mazumder R, Mekhedov SL, Nikolskaya AN, Rao BS, Smirnov S, Sverdlov AV, Vasudevan S, Wolf YI, Yin JJ, Natale DA: The COG database: an updated version includes eukaryotes. BMC Bioinformatics. 2003, 4: 41- 10.1186/1471-2105-4-41View ArticleGoogle Scholar
- Schneider M, Lane L, Boutet E, Lieberherr D, Tognolli M, Bougueleret L, Bairoch A: The UniProtKB/Swiss-Prot knowledgebase and its Plant Proteome Annotation Program. J Proteomics. 2009, 72: 567-573. 10.1016/j.jprot.2008.11.010View ArticleGoogle Scholar
- Boeckmann B, Bairoch A, Apweiler R, Blatter MC, Estreicher A, Gasteiger E, Martin MJ, Michoud K, O'Donovan C, Phan I, Pilbout S, Schneider M: The SWISS-PROT protein knowledgebase and its supplement TrEMBL in 2003. Nucleic Acids Res. 2003, 31: 365-370. 10.1093/nar/gkg095View ArticleGoogle Scholar
- Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ: Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997, 25: 3389-3402. 10.1093/nar/25.17.3389View ArticleGoogle Scholar
- Schattner P, Brooks AN, Lowe TM: The tRNAscan-SE, snoscan and snoGPS web servers for the detection of tRNAs and snoRNAs. Nucleic Acids Res. 2005, 33: W686-W689. 10.1093/nar/gki366View ArticleGoogle Scholar
- Lagesen K, Hallin P, Rødland EA, Stærfeldt HH, Rognes T, Ussery DW: RNAmmer: consistent and rapid annotation of ribosomal RNA genes. Nucleic Acids Res. 2007, 35: 3100-3108. 10.1093/nar/gkm160View ArticleGoogle Scholar
- Gardner PP, Daub J, Tate J, Moore BL, Osuch IH, Griffiths-Jones S, Finn RD, Nawrocki EP, Kolbe DL, Eddy SR, Bateman A: Rfam: Wikipedia, clans and the "decimal" release. Nucleic Acids Res. 2011, 39: D141-D145. 10.1093/nar/gkq1129View ArticleGoogle Scholar
- Tarailo-Graovac M, Chen N: Using RepeatMasker to identify repetitive elements in genomic sequences. Curr Protoc Bioinformatics. 2009, 4: 10-Google Scholar
- Reese MG: Application of a time-delay neural network to promoter annotation in the Drosophila melanogaster genome. Comput Chem. 2001, 26: 51-56. 10.1016/S0097-8485(01)00099-7View ArticleGoogle Scholar
- Gordon JJ, Towsey MW, Hogan JM, Mathews SA, Timms P: Improved prediction of bacterial transcription start sites. Bioinformatics. 2006, 22: 142-148. 10.1093/bioinformatics/bti771View ArticleGoogle Scholar
- Bendtsen JD, Nielsen H, von Heijne G, Brunak S: Improved prediction of signal peptides: SignalP 3.0. J Mol Biol. 2004, 340: 783-795. 10.1016/j.jmb.2004.05.028View ArticleGoogle Scholar
- Papadopoulos JS, Agarwala R: COBALT: constraint-based alignment tool for multiple protein sequences. Bioinformatics. 2007, 23: 1073-1079. 10.1093/bioinformatics/btm076View ArticleGoogle Scholar
- Chenna R, Sugawara H, Koike T, Lopez R, Gibson TJ, Higgins DG, Thompson JD: Multiple sequence alignment with the Clustal series of programs. Nucleic Acids Res. 2003, 31: 3497-3500. 10.1093/nar/gkg500View ArticleGoogle Scholar
- 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: 2731-2739. 10.1093/molbev/msr121View ArticleGoogle Scholar
- Sayers EW, Barrett T, Benson DA, Bolton E, Bryant SH, Canese K, Chetvernin V, Church DM, DiCuccio M, Federhen S, Feolo M, Fingerman IM, Geer LY, Helmberg W, Kapustin Y, Landsman D, Lipman DJ, Lu Z, Madden TL, Madej T, Maglott DR, Marchler-Bauer A, Miller V, Mizrachi I, Ostell J, Panchenko A, Phan L, Pruitt KD, Schuler GD, Sequeira E, Sherry ST, Shumway M, Sirotkin K, Slotta D, Souvorov A, Starchenko G, Tatusova TA, Wagner L, Wang Y, Wilbur WJ, Yaschenko E, Ye J: Database resources of the National Center for Biotechnology Information. Nucleic Acids Res. 2010, 38: D5-D16. 10.1093/nar/gkp967View ArticleGoogle Scholar
- Rehm BHA, Antonio RV, Spiekermann P, Amara AA, Steinbüchel A: Molecular characterization of the poly(3-hydroxybutyrate) (PHB) synthase from Ralstonia eutropha: in vitro evolution, site-specific mutagenesis and development of a PHB synthase protein model. Biochim Biophys Acta. 2002, 1594: 178-190. 10.1016/S0167-4838(01)00299-0View ArticleGoogle Scholar
- Schubert P, Krüger N, Steinbüchel A: Molecular analysis of the Alcaligenes Eutrophus poly(3-Hydroxybutyrate) biosynthetic operon: Identification of the N-terminus of poly(3-Hydroxybutyrate) synthase and identification of the promoter. J Bacteriol. 1991, 173: 168-175.Google Scholar
- Han J, Lu Q, Zhou L, Zhou J, Xiang H: Molecular characterization of the phaECHm genes, required for biosynthesis of poly(3-hydroxybutyrate) in the extremely halophilic archaeon Haloarcula marismortui. Appl Environ Microbiol. 2007, 73: 6058-6065. 10.1128/AEM.00953-07View ArticleGoogle Scholar
- Hanley SZ, Pappin DJC, Rahman D, White AJ, Elborough KM, Slabas AR: Re-evaluation of the primary structure of Ralstonia eutropha phasin and implications for polyhydroxyalkanoic acid granule binding. FEBS Lett. 1999, 447: 99-105. 10.1016/S0014-5793(99)00235-5View ArticleGoogle Scholar
- Wieczorek R, Pries A, Steinbüchel A, Mayer F: Analysis of a 24-kilodalton protein associated with the polyhydroxyalkanoic acid granules in Alcaligenes Eutrophus. J Bacteriol. 1995, 177: 2425-2435.Google Scholar
- Schwibbert K, Marin-Sanguino A, Bagyan I, Heidrich G, Lentzen G, Seitz H, Rampp M, Schuster SC, Klenk HP, Pfeiffer F, Oesterhelt D, Kunte HJ: A blueprint of ectoine metabolism from the genome of the industrial producer Halomonas elongata DSM 2581T. Environ Microbiol.Google Scholar
- Peter H, Weil B, Burkovski A, Krämer R, Morbach S: Corynebacterium glutamicum is equipped with four secondary carriers for compatible solutes: Identification, sequencing, and characterization of the proline/ectoine uptake system, ProP, and the ectoine/proline/glycine betaine carrier, EctP. J Bacteriol. 1998, 180: 6005-6012.Google Scholar
- Chen CL, Beattie GA: Characterization of the osmoprotectant transporter OpuC from Pseudomonas syringae and demonstration that cystathionine-β-synthase domains are required for its osmoregulatory function. J Bacteriol. 2007, 189: 6901-6912. 10.1128/JB.00763-07View ArticleGoogle Scholar
- Rabus R, Jack DL, Kelly DJ, Saier MH: TRAP transporters: an ancient family of extracytoplasmic solute-receptor-dependent secondary active transporters. Microbiology. 1999, 145: 3431-3445.View ArticleGoogle Scholar
- Jia Y, Yuan W, Wodzinska J, Park C, Sinskey AJ, Stubbe J: Mechanistic studies on class I polyhydroxybutyrate (PHB) synthase from Ralstonia eutropha: Class I and III synthases share a similar catalytic mechanism. Biochemistry. 2001, 40: 1011-1019. 10.1021/bi002219wView ArticleGoogle Scholar
- Sohn SB, Kim TY, Park JM, Lee SY: In silico genome-scale metabolic analysis of Pseudomonas putida KT2440 for polyhydroxyalkanoate synthesis, degradation of aromatics and anaerobic survival. Biotechnol J. 2010, 5: 739-50. 10.1002/biot.201000124View ArticleGoogle Scholar
- Cai L, Yuan MQ, Liu F, Jian J, Chen GQ: Enhanced production of medium-chain-length polyhydroxyalkanoates (PHA) by PHA depolymerase knockout mutant of Pseudomonas putida KT2442. Bioresour Technol. 2009, 100: 2265-2270. 10.1016/j.biortech.2008.11.020View ArticleGoogle Scholar
- York GM, Lupberger J, Tian J, Lawrence AG, Stubbe J, Sinskey AJ: Ralstonia eutropha H16 encodes two and possibly three intracellular poly[D-(-)-3-hydroxybutyrate] depolymerase genes. J Bacteriol. 2003, 185: 3788-3794. 10.1128/JB.185.13.3788-3794.2003View ArticleGoogle Scholar
- Goo YA, Roach J, Glusman G, Baliga NS, Deutsch K, Pan M, Kennedy S, DasSarma S, Ng WV, Hood L: Low-pass sequencing for microbial comparative genomics. BMC Genomics. 2004, 5: 3- 10.1186/1471-2164-5-3View ArticleGoogle Scholar
- Bonneté F, Madern D, Zaccai G: Stability against denaturation mechanisms in halophilic malate-dehydrogenase "adapt" to solvent conditions. J Mol Biol. 1994, 244: 436-447. 10.1006/jmbi.1994.1741View ArticleGoogle Scholar
- Kennedy SP, Ng WV, Salzberg SL, Hood L, DasSarma S: Understanding the adaptation of Halobacterium species NRC-1 to its extreme environment through computational analysis of its genome sequence. Genome Res. 2001, 11: 1641-1650. 10.1101/gr.190201View ArticleGoogle Scholar
- Maskow T, Babel W: Calorimetrically obtained information about the efficiency of ectoine synthesis from glucose in Halomonas elongata. Biochim Biophys Acta. 2001, 1527: 4-10. 10.1016/S0304-4165(01)00115-5View ArticleGoogle Scholar
- Naughton LM, Blumerman SL, Carlberg M, Boyd EF: Osmoadaptation among Vibrio species and unique genomic features and physiological responses of Vibrio parahaemolyticus. Appl Environ Microbiol. 2009, 75: 2802-2810. 10.1128/AEM.01698-08View ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.