Evolutionary patterns of carbohydrate transport and metabolism in Halomonas boliviensis as derived from its genome sequence: influences on polyester production
© Guzmán et al; licensee BioMed Central Ltd. 2012
Received: 2 January 2012
Accepted: 17 April 2012
Published: 17 April 2012
Halomonas boliviensis is a halophilic bacterium that is included in the γ-Proteobacteria sub-group, and is able to assimilate different types of carbohydrates. H. boliviensis is also able to produce poly(3-hydroxybutyrate) (PHB) in high yields using glucose as the carbon precursor. Accumulation of PHB by microorganisms is induced by excess of intracellular NADH.
The genome sequences and organization in microorganisms should be the result of evolution and adaptation influenced by mutation, gene duplication, horizontal gen transfer (HGT) and recombination. Furthermore, the nearly neutral theory of evolution sustains that genetic modification of DNA could be neutral or selected, albeit most mutations should be at the border between neutrality and selection, i.e. slightly deleterious base substitutions in DNA are followed by a slightly advantageous substitutions.
This article reports the genome sequence of H. boliviensis. The chromosome size of H. boliviensis was 4 119 979 bp, and contained 3 863 genes. A total of 160 genes of H. boliviensis were related to carbohydrate transport and metabolism, and were organized as: 70 genes for metabolism of carbohydrates; 47 genes for ABC transport systems and 43 genes for TRAP-type C4-dicarboxylate transport systems. Protein sequences of H. boliviensis related to carbohydrate transport and metabolism were selected from clusters of orthologous proteins (COGs). Similar proteins derived from the genome sequences of other 41 archaea and 59 bacteria were used as reference. We found that most of the 160 genes in H. boliviensis, c.a. 44%, were obtained from other bacteria by horizontal gene transfer, while 13% of the genes were acquired from haloarchaea and thermophilic archaea, only 34% of the genes evolved among Proteobacteria and the remaining genes encoded proteins that did not cluster with any of the proteins obtained from the reference strains. Furthermore, the diversity of the enzymes derived from these genes led to polymorphism in glycolysis and gluconeogenesis. We found further that an optimum ratio of glucose and sucrose in the culture medium of H. boliviensis favored cell growth and PHB production.
Results obtained in this article depict that most genetic modifications and enzyme polymorphism in the genome of H. boliviensis were mainly influenced by HGT rather than nearly neutral mutations. Molecular adaptation and evolution experienced by H. boliviensis were also a response to environmental conditions such as the type and amount of carbohydrates in its ecological niche. Consequently, the genome evolution of H. boliviensis showed to be strongly influenced by the type of microorganisms, genetic interaction among microbial species and its environment. Such trend should also be experienced by other prokaryotes. A system for PHB production by H. boliviensis that takes into account the evolutionary adaptation of this bacterium to the assimilation of combinations of carbohydrates suggests the feasibility of a bioprocess economically viable and environmentally friendly.
Cellular evolution and adaptation have imprinted patterns in microbial genomes through mutation, gene duplication, horizontal gen transfer (HGT) and recombination [1, 2]. The genomes of microorganisms of the three domains of life have experienced such genetic modifications to succeed on their permanence in a particular habitat, where environmental conditions and the size of the microbial populations might influence the organization and number of genes in a particular species throughout the time [1, 3]. Furthermore, the nearly neutral theory of evolution points out that genetic modification of DNA could be neutral or selected, albeit most mutations should be at the border between neutrality and selection, i.e. slightly deleterious base substitutions in DNA are followed by a slightly advantageous substitutions .
The increasing number of genome sequences of different organisms is helping to discern how microbial species diverged. Recent reports on the evolutionary traits followed by different bacteria and archaea have demonstrated that the transfer of genes among these organisms, also referred as horizontal gene transfer, has led to net-like relationships among their genomes [2, 4, 5]. Nevertheless, the phylogenetic association among prokaryotes derived from the sequences of proteins encoded by 102 different genes was consistent to the taxonomic differentiation observed when 16 rRNA sequences of microorganisms are analyzed . The 102 proteins were mainly related to translation and transcription, although proteins involved in the transport and metabolism of amino acids, metal ions and carbohydrates revealed such taxonomic information as well .
The aforementioned studies included the genome sequences of extremely halophilic archaea such as Haloarcula marismortui, Haloquadratum walsbyi and a Halobacterium sp. . These studies on the genome sequences did not include halophilic bacteria. However, a report on the genes of poly(3-hydroxybutyrate) (PHB) polymerases, PHB depolymerases and ectoine synthesis by Halomonas sp. TD01, a halophilic bacterium, suggested that HGT has a role to play on the genome organization of the microorganism . Halophilic microorganisms require salt (NaCl) to grow; a halophile should grow optimally at NaCl concentrations of 5% (w/v) or higher, and tolerate at least 10% (w/v) salt . There are five genome sequences of halophilic bacteria available in public data bases. The sequences of Chromohalobacter salexigens and Halorhodospira halophila were first published followed by the sequence of Halomonas elongata [8, 9], Halomonas sp. TD01  and Halomonas sp. HAL1 . Chromohalobacter and Halomonas species are included in the family Halomonadaceae within the γ-Proteobacteria subgroup. The family Halomonadaceae contains only halophilic and halotolerant aerobic heterotrophs; some of them are able to grow in media with up to 30% (w/v) NaCl . Halophilic bacteria maintain low concentrations of salt intracellularly by accumulating organic compounds of low molecular weight, also known as osmolytes or "compatible solutes" such as ectoine .
Understanding the evolution and levels of polymorphism among genes is attracting much attention in evolutionary biology and biotechnology. Evolution of energy-producing pathways, particularly glycolysis and gluconeogenesis, posses relevance since they determine the type of carbon sources that a species is able to assimilate, and link to metabolic routs that may generate compounds of biotechnological interest . Theories on the evolution of the metabolisms of organisms consider that enzyme polymorphism--alleles for the different enzymes or allozymes--in metabolic pathways was related to genetic mutations [12–14]. A proposal states that the fitness of the pathways associated with an increasing flux is influenced by selected mutations of genes that enhance enzyme activities, albeit enzyme improvements do not continue indefinitely [12, 14]. Mutations will reach a point at which the incremental gains of fitness for a new mutation will be equaled by the noise caused by the random genetic variation [12, 14]. At this stage, the genes or enzymes might evolve under a nearly neutral trend [12, 14]. Moreover, metabolic control in the organisms is also to regulate molecular evolution as well [12, 14]. The proposal assumes no contextual changes such as a change in the functional conditions of an enzyme originated by either epistasis or the environment; or a change in the effective population size of the species .
Halomonas boliviensis is a halophilic bacterium that can develop under a wide range of NaCl concentrations (i.e. 0-25% (w/v)), pH (5-11) and temperatures (0-45°C) . It can also assimilate several carbohydrates as carbon source for growth . Bioprocesses have been designed to attain high productivities of a polyester and osmolytes by H. boliviensis using glucose as the carbon precursor [16, 17]. The polyester accumulated by the bacterium is poly(3-hydroxybutyrate) (PHB), which is used as carbon and energy reservoir . PHB is synthesized by several bacteria from acetyl-CoA when an excess of NADH is present in the bacterial cytoplasm . Such excess can be generated when a high concentration of a carbon source is added to a culture medium and cell growth is limited by the depletion of an essential nutrient, e.g. nitrogen, oxygen, trace elements among others . PHB is attracting much attention in biotechnology because it is a biodegradable plastic-like material, and possesses potential in biomedical applications such as tissue engineering, organ transplants and drug delivery systems . Moreover, the efficiency and economics of the manufacturing process of PHB are determined by the carbon source, fermentation process, and downstream processing of the polymer. The development of cultivation conditions for microorganisms that allow high PHB content and productivity from cheap and renewable carbon sources is therefore important [21, 22].
The present research work reports the genome sequence of H. boliviensis. It also depicts the evolutionary trends that proteins of H. boliviensis have experienced to allow the transport of carbohydrates and their assimilation to achieve acetyl-CoA. The conclusions drawn from these studies were used to create an alternative production system of PHB by H. boliviensis using a combination of carbohydrates. This system should lead to a more economically and environmentally beneficial bioprocess.
The fine high coverage genome sequence, gene prediction, repetitive sequence, COGs and KEGG annotation of Halomonas boliviensis LC1T (= DSM 15516T) were obtained at BGI-Hongkong Co., Hong Kong. For this, Illumina HiSeq 2000 technology was used to conduct paired-end sequencing for DNA samples, and constructed a 1,000 bp library with extended data of 500 Mb. Genome coverage based on k-mer was 95.4%, and genome coverage based on reads mapping was 99.9%. Glimmer 3.0 software package was used to conduct de novo gene prediction . The functional annotation was accomplished by analysis of protein sequences. Genes of H. boliviensis were aligned to others in databases to attain its corresponding functional annotation. To ensure the biological meaning, only one high-quality information as annotation to the genes from many results was chosen. BLAST was used to accomplish functional annotation combined with different databases. BLAST version: blastall 2.2.21 software (provided by the National Center for Biotechnology Information, NCBI) was used for these studies. Alignment results were obtained using the following databases: KEGG, COG, SwissProt, TrEMBL, NR. This whole genome shotgun project was deposited at DDBJ, EMBL and GenBank under the accession number AGQZ00000000. The version described in this paper is the first version, AGQZ01000000.
A total of 6,901 alignments of clusters of orthologous proteins (COGs) of 59 bacteria and 41 archaea, as classified in COGs  and EggNOG  data bases, were gently provided by Puigbò, Wolf and Koonin (2009). The protein sequences of these 100 microorganisms were used as reference for the evolutionary analysis. Protein sequences of H. boliviensis related to carbohydrate transport and metabolism were selected and aligned along with the references for each corresponding COG (Additional file 1: Table S1, supplementary data) using the Muscle program  included in the MEGA 5 software package  with default parameters. Unrooted maximum likelihood phylogenetic trees were constructed using MEGA 5 under a WAG with frequencies (+F) model, with uniform mutation rates among amino acid sites and complete deletion of gaps and missing data.
Analysis and assembly of supernetworks
Supernetworks were constructed by combining the phylogenetic trees of proteins of the glycolysis and gluconeogenesis metabolisms in H. boliviensis and reference strains using the SplitsTree4 program [28, 29] with default parameters. Three analyses were performed for these studies: 1) A supernetwork obtained from three COGs (0126, 0149 and 0837). Both COG0126 and COG0149 are considered among the 102 genes that contain taxonomic information that discriminate well bacteria and archaea in already known families and genera ; 2) A supernetwork obtained after combining six COGs (0126, 0149, 0837, 0469, 0696 and 837); and 3) A supernetwork obtained after combining twenty two COGs (0057, 0126, 0148, 0149, 0166, 0191, 0205, 0235, 0365, 0469, 0508, 0696, 0837, 1012, 1063, 1109, 1249, 1454, 1866, 2017, 2609 and 4993). Supernetworks were analyzed according to method described by Huson et al. in 2006 .
Culture media composition
Seed culture and PHB production media were formulated as described previously . Seed culture contained% (w/v): NaCl, 2.5; MgSO4•7H2O, 0.25; K2HPO4, 0.05; NH4Cl, 0.23; FeSO4•7H2O, 0.005; sucrose 1; monosodium glutamate (MSG), 0.3 and TRIS, 1.5. The PHB production medium included % (w/v): NaCl, 2.5; MgSO4•7H2O, 0.5; K2HPO4, 0.22; NH4Cl, 0.4; FeSO4•7H2O, 0.005; MSG, 0.2; and the following concentration of carbohydrates % (w/v): 1) 2.5 sucrose, 2) 2.0 sucrose and 0.5 glucose, 3) 1.5 sucrose and 1 glucose, 4) 1.0 sucrose and 1.5 glucose, 5) 0.3 sucrose, 0.7 glucose and 1.5 dried molasses and 6) 2.5 dried molasses for 6 different assays, respectively. The composition of the molasses used was 78.1% sucrose, 15.3% glucose and 6.6% of other uncharacterized solids. A low amount of MSG is added to the production medium to induce its depletion by H. boliviensis during the cultivation.
H. boliviensisgrowth and PHB production in flasks
H. boliviensis was grown in 100 ml of seed culture medium in 1,000-ml flasks with rotary shaking at 220 rpm, 30°C for 13 h. The pH of the medium was adjusted to 7.5 using concentrated HCl. Subsequently, 5 ml of the seed culture were inoculated in 1,000-ml Erlenmeyer flasks containing 95 ml of PHB production medium. The pH of the PHB production medium was initially adjusted to 7.5 using 5 M NaOH. The cultures were incubated at 35°C with shaking at 220 rpm, and samples were withdrawn at different time intervals during the cultivation.
Cell dry weight (CDW) and PHB content in H. boliviensis were determined as reported previously . Residual cell mass (RCM) concentration was calculated as the difference between the CDW and PHB concentration, while PHB content (wt%) was obtained as the percentage of the ratio of PHB concentration to the CDW as defined by Lee et al. in 2000 . All analyses were performed in triplicate.
Glutamate concentration was determined by high performance liquid chromatography (HPLC) analysis, as described previously , using a Perkin-Elmer HPLC system with an Aminex HPX-87 C column (Biorad) and a UV detector at 65°C. Calcium chloride solution (5 mM) was used as mobile phase at a flow rate of 0.5 ml/min. Glutamate was monitored at 210 nm. Glucose and sucrose were determined using the same HPLC system with a Polypore CA column (Perkin-Elmer), a RI detector at 80°C and water as mobile phase at a flow rate of 0.3 ml/min.
Results and discussion
Genome of H. boliviensis
Genome of H.boliviensis
DNA, total number of bases
% G+C content
4 119 979
Number of genes
Length occupied by genes (bp)
3 673 824
% G+C content in the gene region
Length occupied by the intergenic region (bp)
% G+C content in the intergenic region
% Intergenic length/Chromosome
Inferring the evolution of proteins involved in the uptake and metabolism of carbohydrates
Protein sequences of H. boliviensis related to carbohydrate transport and metabolism were obtained from clusters of orthologous proteins (COGs), as classified in COGs and EggNOG data bases [24, 25]. A total of 160 genes of H. boliviensis encoded proteins for these clusters: 70 genes were related to the metabolism of carbohydrates; 47 genes were related to ABC transport systems and encoded 14 permease proteins, 23 ATPase proteins and 10 periplasmic proteins; and 43 genes were related to TRAP-type C4-dicarboxylate transport systems and encoded 15 large permease proteins, 11 small permease proteins and 17 periplasmic proteins (Additional file 1: Table S1, supplementary data). Similar proteins were selected from COGs derived from the genome sequences of other 41 archaea and 59 bacteria. To perform evolutionary analyses, unrooted phylogenetic trees were constructed based on a maximum likelihood approach using the sequences of the proteins of H. boliviensis and proteins of other 100 microorganisms for each corresponding COG.
Under the neutral mutation-random drift theory, it is assumed that a certain fraction of new mutation are free of constraint or are selectively neutral, while the rest have deleterious effects and are selectively eliminated . Nevertheless, Figures 1 and 4 imply that most mutations found in proteins related to carbohydrate transport and metabolism were a result of HGT, which agree on some criteria that point out that genetic drift is not sufficient for claiming neutrality , and on a resent observation that estimated that about 60% of the genome evolution of prokaryotes is dominated by HGT . Furthermore, HGT can be related to adaptation of H. boliviensis to its environment (Figures 1, 2) and might, therefore, be selected to attain an optimum physiological response of the species to its habitat . Yet, nearly neutral mutations could be inferred from Figure 3 and Additional file 1: Table S1, suggesting a continuous evolution of the proteins .
Metabolic assimilation of carbohydrates by H. boliviensis
Relationship of the enzymes involved in glycolysis and gluconeogenesis among Prokaryotes
Use of combination of carbohydrates for the production of PHB by H. boliviensis
Three alleles of the PHB synthases were found in the genome of H. boliviensis (Additional file 5: Figure S1). The three alleles are closely related to PHB synthases of Proteobacteria. Moreover, H. boliviensis A2 was clustered with two alleles of PHB polymerases of Halomonas sp. TD01 (Figure S1); one of these alleles (phaC1) was previously reported . However, a third allele of Halomonas sp. TD01 (named phaC2) showed a distant phylogenetic relationship to the PHB synthases of Proteobacteria (Figure S1); phaC2 might have been acquired by HGT . Research on the PHB polymerization and depolymerization pathways in H. boliviensis is in progress. PHB production by H. boliviensis was performed in shake flask experiments under nitrogen limitation conditions (i.e. a low concentration sodium glutamate was added to the culture medium to limit the cell growth). When sucrose was used as the sole carbon source, the accumulation of PHB in H. boliviensis (7.2 wt%) and cell growth (2.7 g/L) were low compared to those obtained with combinations of sucrose and glucose (Figures 11,12). Cell growth increased as the amount of glucose was higher in the medium to reach 8.6 g/L (Figure 11), while the maximum PHB content in H. boliviensis was 52.7 wt% when 1.5% (w/v) sucrose and 1.0% (w/v) glucose were included in the medium composition (Figure 12). The use of molasses enhanced to some extent the cell growth, c.a. 9.4 g/L, but the PHB accumulated in the cells was lower, 43.9 wt% (Figures 11, 12). Both the cell density and the maximum PHB yield attained by H. boliviensis are higher to those reported using glucose as carbon source, i.e. 5.3 g/L and 45 wt% respectively, under similar culture conditions .
The maximum PHB concentration and volumetric productivity reached by H. boliviensis were 4.3 g/L and 0.13 g/L/h, respectively; they are comparable to those reached by Cupriavidus necator, i.e. 5.1 g/L and 0.11 g/L/h , and to those reported for a recombinant E. coli strain, c.a. 7.2 g/L and 0.15 g/L/h . The medium for C. necator and E. coli contained glucose as the carbon source for experiments performed in shake flasks. Under similar culture conditions, Azotobacter vinelandii led to a PHB concentration of 7.5 g/L and a productivity of 0.30 g/L/h . These bacteria attained among the highest productions of PHB, and are recognized for their potential utilization at industrial scales [19, 21].
The viability of the commercialization of PHB is dependent upon the reduction of the total production costs . The price of the carbon source supplied in the culture medium may account up to 40% of the total production costs . Sucrose is at least two times cheaper than glucose while molasses are cheaper than sucrose. The results obtained for the production of PHB by H. boliviensis (Figures 11, 13) suggest that an agricultural surplus such as molasses could be used during the bioprocess scale up to stimulate the cell growth; furthermore an optimum ratio of sucrose and glucose should be added in the culture medium of the largest bioreactor used in a process to induce a high polymer production. Replacing partially glucose by sucrose and molasses should surly reduce the production costs of the polymer and lead also to an environmentally friendly bioprocess. Nevertheless, fed-batch cultivations systems are yet to be performed with H. boliviensis using combinations of carbohydrates to reveal their potential in a large scale process.
The genome size and number of genes found in H. boliviensis were similar to those determined for other halophilic bacteria of the family Halomonadaceae. The ability of H. boliviensis to grow on different carbon sources is explained by the high number of genes related to the carbohydrate uptake and metabolism. Interestingly, most of these genes were obtained from other bacteria by HGT, only 34% of the genes evolved as proteins belonging to Proteobacteria, while 13% of the genes were transferred from haloarchaea and thermophilic archaea. Furthermore, the diversity of enzymes that have the same physiological function led to polymorphism in the metabolic routs. Results obtained in this article depict that most genetic modifications and enzyme polymorphism in the genome of H. boliviensis were mainly influenced by HGT rather than nearly neutral mutations. Molecular adaptation and evolution experienced by H. boliviensis were also a response to environmental conditions such as the type and amount of carbohydrates in its ecological niche. Consequently, the genome evolution of H. boliviensis showed to be strongly influenced by the type of microorganisms, genetic interaction among microbial species and its environment. Such trend should also be experienced by other prokaryotes. A system for PHB production by H. boliviensis that takes into account the evolutionary adaptation of this bacterium to the assimilation of combinations of carbohydrates suggests the feasibility of a bioprocess economically viable and environmentally friendly.
Halomonas boliviensisgenome sequence
This whole genome shotgun project was deposited at DDBJ, EMBL and GenBank under the accession number AGQZ00000000. The version described in this paper is the first version, AGQZ01000000.
The authors would like to thank the Swedish International Development Cooperation Agency (Sida) for supporting our research work.
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