ABSTRACT
Brown algae are one of the largest groups of oceanic primary producers for CO2 removal and carbon sinks for coastal regions. However, the mechanism for brown alga assimilation remains largely unknown in thermophilic microorganisms. In this work, a thermophilic alginolytic community was enriched from coastal sediment, from which an obligate anaerobic and thermophilic bacterial strain, designated Alg1, was isolated. Alg1 shared a 16S rRNA gene identity of 94.6% with Defluviitalea saccharophila LIND6LT2T. Phenotypic, chemotaxonomic, and phylogenetic studies suggested strain Alg1 represented a novel species of the genus Defluviitalea, for which the name Defluviitalea phaphyphila sp. nov. is proposed. Alg1 exhibited an intriguing ability to convert carbohydrates of brown algae, including alginate, laminarin, and mannitol, to ethanol and acetic acid. Three gene clusters participating in this process were predicted to be in the genome, and candidate enzymes were successfully expressed, purified, and characterized. Six alginate lyases were demonstrated to synergistically deconstruct alginate into unsaturated monosaccharide, followed by one uronic acid reductase and two 2-keto-3-deoxy-d-gluconate (KDG) kinases to produce pyruvate. A nonclassical mannitol 1-phosphate dehydrogenase, catalyzing d-mannitol 1-phosphate to fructose 1-phosphate in the presence of NAD+, and one laminarase also were disclosed. This work revealed that a thermophilic brown alga-decomposing system containing numerous novel thermophilic alginate lyases and a unique mannitol 1-phosphate dehydrogenase was adopted by the natural ethanologenic strain Alg1 during the process of evolution in hostile habitats.
INTRODUCTION
Brown algae (Phaeophyceae) are a large group of marine vegetation that have abundant amounts of the yellow-brown pigment fucoxanthin (1). Their huge biomass makes them one of the largest oceanic primary producers for CO2 removal and carbon storage for coastal regions (2). Moreover, as an emerging feedstock for liquid biofuel production, recently the bioconversion of brown algae has been reported frequently (1, 3–6). They have a complex sugar composition, mainly including alginate, mannitol, and laminarin (6). Alginate is a unique structural polysaccharide in brown algae, and it is abundant in the cell wall for mechanical protection (1). It is a linear polysaccharide consisting of two uronic acids, α-l-guluronate (G) and β-d-mannuronate (M) (7). The content of alginate varies by species from 20% to 40% dry cell weight (8, 9). Mannitol is a sugar alcohol form of mannose. Laminarin, a storage polysaccharide in many brown algae, is a linear polysaccharide of β-1,3-linked glucose with small amounts of β-1,6-linkages (10). Mannitol and laminarin are mostly accumulated in the summer, and their content could reach as high as 25% and 30% in the species Laminaria hyperborean (11).
The microbial degradation of brown algae involves the decomposition of the structural polysaccharides (mainly alginate and laminarin) and then the catabolism of the resulting monosaccharides (glucose and uronic acid) and mannitol. Glucose was produced by the hydrolysis of glucan and could be assimilated easily through glycolysis. Mannitol needs additional steps for catabolism before entering glycolysis. In bacteria, the known pathway for mannitol assimilation includes two key enzymes (mannitol degradation I; MetaCyc Pathway Database [http://metacyc.org/]) (12). One is d-mannitol phosphotransferase (PTS) permease, which transports d-mannitol into cells with the formation of d-mannitol 1-phosphate, and the other one is mannitol 1-phosphate dehydrogenase (MPDH), which oxidizes mannitol 1-phosphate to fructose 6-phosphate in a reversible reaction. Fructose 6-phosphate then was assimilated through glycolysis. Alginate degradation has been characterized in several bacteria, and the mechanism varies. For example, Sphingomonas sp. strain A1 can directly transport alginate into its cytoplasm through a superchannel, while most other bacteria, for example, Zobellia galactanivorans, first secrete extracellular alginate lyases to degrade alginate (13, 14). Although strategies differ among bacteria, alginate lyases involved in the degradation process shared similar catalytic functions. According to the classic pathways, alginate first was degraded to oligoalginate by alginate lyase, and oligosaccharides were further exolytically cleaved into unsaturated monosaccharide (spontaneously rearranged into 4-deoxy-l-erythro-5-hexoseulose uronic acid, or DEH) by oligoalginate lyases (15, 16). Subsequently, DEH was converted into 2-keto-3-deoxy-d-gluconate (KDG) by DEH reductase. A kinase then catalyzed KDG to 2-keto-3-deoxy-6-phosphogluconate (KDPG), which was directly assimilated through the Entner-Doudoroff (ED) pathway. For brown alga degradation, a microorganism should not only possess a whole set of enzymes, particularly some polysaccharide-degrading enzymes for depolymerization, but also have a well-evolved redox system to balance the reducing equivalents produced from metabolism processes, especially under anaerobic fermentation conditions (5, 17, 18). As far as we know, an integrated system of brown alga degradation has never been reported in single natural strains.
Alginate lyases and oligoalginate lyases catalyze the depolymerization of alginate into oligomers and monomers through β-elimination reactions (19). Their crucial roles in alginate degradation, as well as their biological applications, make them be widely screened and studied. Alginate lyases have been isolated from various sources, such as marine algae, marine mollusks, fungi, bacteria, and viruses. Most of the characterized alginate lyases were from bacteria, including Zobellia, Agrobacterium, Alteromonas, Azotobacter, Bacillus, Enterobacter, Flavobacterium, Klebsiella, Pseudoalteromonas, Pseudomonas, Sphingomonas, and Vibrio (14, 19–22). To our knowledge, all of these species are mesophilic bacteria, and the optimum temperatures of most of the alginate lyases are below 50°C, with the exception of A1-II (70°C) from Sphingomonas sp. strain A1 (23) (http://www.brenda-enzymes.org/index.php).
In previous work, we demonstrated that the coastal marine environment harbored a diversity of thermophilic cellulolytic bacteria (24). These bacteria showed extremely low 16S rRNA gene identities to their closest relatives, indicating an untapped thermophilic microbial resource. In this work, by using kelp powder as a carbon resource, we enriched the same sediment sample collected from Qingdao coast, and a novel bacterial strain, Alg1, was isolated. Community survey and strain characterization were conducted, and an integrated brown alga-degrading system in Alg1 was revealed based on genome analysis and key enzyme verification.
MATERIALS AND METHODS
Enrichment protocol and survey of enriched community.Samples were collected from marine sediment of a coastal region of the Yellow Sea (36°5′N, 120°32′E), China, in May 2013. One gram of sediment was used as the inoculum in 100 ml of basal medium (BM) at an initial pH of 7.4 and containing 1 g of kelp (Saccharina japonica) powder as the carbon source at 60°C under anaerobic conditions. BM consisted of 0.1 g/liter of KH2PO4, 0.1 g/liter of K2HPO4, 1 g/liter of NaHCO3, 2 g/liter of NH4Cl, 30 g/liter sea salt, 0.5 g/liter of l-cysteine, 1 g/liter yeast extract, and 0.0001 (wt/vol) resazurin. Vitamins were added at the following concentrations (in milligrams per liter): pyridoxamine dihydrochloride, 1; p-aminobenzoic acid (PABA), 0.5; d-biotin, 0.2; vitamin B12, 0.1; thiamine-HCl-2H2O, 0.1; folic acid, 0.2; pantothenic acid calcium salt, 0.5; nicotinic acid, 0.5; pyridoxine-HCl, 0.1; thioctic acid, 0.5; riboflavin, 0.1.
Cultures growing in the presence of kelp were transferred 10 times to get a relatively stable community. The community was surveyed by constructing a 16S rRNA gene clone library as described previously (24). PCR amplifications targeting the 16S rRNA gene used the universal oligonucleotide primers 27F and 1492R (see Table S1 in the supplemental material), and the PCR amplicons were cloned into a pMD18T vector. Two vector-specific primers (M13-47 and RV-M) (see Table S1) were used for the amplification and verification of the DNA inserts.
Strain isolation and characterization.Strain isolation from the community was carried out by plating the serially diluted consortium culture on an anaerobic agar plate containing 0.5% alginate in BM (ABM) with 1.5% agar in an anaerobic chamber. The temperature, salinity, and pH ranges for cell growth were determined in ABM by following the methods of Wang et al. (25). The optical density at 600 nm (OD600) was used to test cell growth for determining the optimal temperature, salinity, and pH for strain Alg1. The utilization of the following substrates as carbon and energy sources was tested in BM with a concentration of 0.5% (wt/vol): acetate, starch, pyruvate, ribose, fructose, lactate, glucose, maltose, xylose, peptone, lactose, galactose, mannose, raffinose, sucrose, arabinose, cellobiose, glycerol, mannitol, rhamnose, peptone, Casamino Acids, laminarin, and alginate.
The cell shape of Alg1 was observed using a scanning electron microscope (S-4800; Hitachi, Tokyo, Japan) (25). Chemotaxonomic characteristics were determined from cells grown at 60°C for 2 days in BM containing 0.5% mannitol. Fatty acids were extracted and analyzed according to the standard protocol of the MIDI (Microbial Identification) system. The G+C content of the DNA was determined by using high-performance liquid chromatography (HPLC) (Waters, Milford, MA) (26). The concentrations of fermentation products were analyzed by HPLC using an Aminex HPX-87H column (Bio-Rad, Hercules, CA).
Phylogenetic analyses.For the 16S rRNA gene clone library, DNAStar Lasergene software was used for manual editing of the amplified 16S rRNA gene sequences. The definition of operational taxonomic units (OTU) at 97% sequence identity was determined using the DOTUR software package (27). The identification of phylogenetic neighbors and the calculation of pairwise 16S rRNA gene sequence identities were achieved by a BLAST search in the EzTaxon-e database and nucleotide databases of the National Center for Biotechnology Information (NCBI) (28). Phylogenetic analysis was performed by the software package MEGA, version 5.0, after multiple alignment of data by CLUSTALX (29). The phylogenetic tree was constructed using neighbor-joining (NJ) methods.
Genome sequencing and sequence analysis.Genomic DNA of strain Alg1 was prepared by following a procedure described previously (30). The genome was sequenced using the Illumina HiSeq 2000 system after constructing the Illumina paired-end DNA library with 170- and 500-bp inserts. The numerous reads were assembled into hundreds of contigs by Velvet (V1.2.03), which were resorted to predict gene functions consequently using Glimmer, GeneMark, and Zcurve. The genes were annotated through NCBI, KEGG, and SEED databases, classified through the CDD database, and constructed into metabolic pathways through the KEGG database. Signal peptides were predicted with SignalP 4.1 (31). Conserved domains within a coding nucleotide sequence were analyzed using CD-search (32).
DNA sequence analysis of the three gene clusters involved in brown alga degradation was performed to identify patterns that mediate transcription, i.e., promoters and Rho-independent terminators, by using the BPROM online program and ARNold online program (33–35).
Enzyme expression, purification, and identification.For the purification of the enzyme encoded by dp0100, strain Alg1 was grown to stationary phase in BM supplemented with 1% alginate. Cells and residual alginate were removed by centrifugation for 20 min at 10,000 × g and 4°C. The supernatant was passed through a 0.22-μm filter, brought to the same volume of cold (−20°C) acetone, and centrifuged for 20 min at 12,000 × g. The precipitate was resuspended with 7 ml 50 mM Tris-HCl (pH 7.3) (buffer A) and was dialyzed using a dialysis bag (Solarbio, Beijing, China) with a permeability molecular weight range of 8,000 to 14,000 in buffer A. The dialyzed protein was injected into an anion exchange column (HiPrep 16/10 Q FF; 20 ml; GE Healthcare, Little Chalfont, United Kingdom) equilibrated with the same buffer. Proteins were eluted at 5 ml min−1 with a 400-ml linear gradient of 0 to 1 M NaCl in buffer A. Each fraction (15 ml) was assessed for alginate lyase activity. An active fraction was obtained at around 300 mM NaCl and then was loaded onto a gel filtration column (HiPrep 16/60 and Sephacryl S-200 HR; GE Healthcare) and eluted with buffer A. The eluted fractions were analyzed by SDS-PAGE. All chromatography procedures were performed on an ÄKTA purifier system (GE Healthcare). The purified proteins were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis (36). All putative brown alga degradation-related genes except dp0100 were selected for heterogeneous expression by following the method of Zhang et al. (36).
Enzyme activity assays.Alginate lyase activity was assayed by measuring the increase in absorbance at 235 nm (A235) of the reaction products (unsaturated uronates) for 3 min at 65°C in a quartz cuvette containing 2 ml of 0.2% alginate in 100 mM acetate-sodium acetate buffer (pH 5.8) and 10 μl purified enzymes. One unit of activity was defined as an increase of 0.1 in the A235 per minute. Laminarinase activity was assayed by incubating laminarin solution (1%, wt/vol, in buffer A, pH 7.3) with purified enzyme at 65°C for 3 min. The release of reducing sugars was measured by the dinitrosalicylic colorimetric (DNS) method (37). One unit of laminarinase activity was defined as the amount of enzyme that released 1 μmol of reducing sugar per min. Protein concentrations were determined by using the Bradford assay kit (Biomed, Beijing, China) with bovine serum albumin as the standard (38).
The measurement of 2-keto-3-deoxy-d-gluconate 6-dehydrogenase (KdgD) activity followed the method previously described (14). The substrate DEH was produced by the exotype alginate lyase Dp1761E. Two hundreds microliters of purified Dp1761E (367 μg ml−1) was added to an 800-μl reaction mixture containing 0.5% (wt/vol) alginate sodium salt and 100 mM acetate-sodium acetate buffer (pH 5.8). After incubation at 60°C for 24 h, the mixture was centrifuged at 12,000 × g for 30 min and the supernatant was conserved as the DEH solution. The reaction mixture for the KdgD assay consisted of 50 μl of purified Dp1759E (28 μg ml−1), 200 μl of DEH solution, 100 μl of NADH (1 mM), and 400 μl potassium phosphate buffer (50 mM, pH 6.0). The enzyme kinetic was determined at 60°C, and activity was monitored by measuring the decrease of the absorbance at 340 nm (A340). KDPG aldolase (KdpgA) activity was determined by using an enzyme-coupled spectrophotometric assay (14). KDG was produced from DEH in the same reaction system as that mentioned above. After 30 min at 60°C, the reaction was stopped by heating the mixture at 100°C for 5 min. After cooling to 60°C, 50 μl of Dp1703E (176 μg ml−1) or Dp1704E (189 μg ml−1) and 20 μl ATP (100 mM) were added to the reaction for another 30 min to allow the formation of ADP. The amount of ADP in the solution was measured by a coupling enzyme assay at 35°C containing 50 mM potassium phosphate buffer (pH 7.4), 5 mM MgCl2, 1 mM phosphoenol pyruvate, 0.3 mM NADH, and 1.5 U lactate dehydrogenase–1.0 U pyruvate kinase (both from rabbit muscle). The reaction was initiated by adding 200 μl ADP-containing reaction mixture. The reduction of fructose 1-phosphate (F1P; >98% purity; Carbosynth Ltd., Berkshire, United Kingdom) and fructose 6-phosphate (F6P; ≥98% purity; Sigma-Aldrich, St. Louis, MO) by MPDH was assayed in 800 μl sodium phosphate buffer (50 mM, pH 6) with 0.2 mM NADH and 50 μl of Dp0124E (149 μg ml−1). F1P or F6P (1 mM) was used to initiate the reaction. MPDH activities were determined from the rate of NADH oxidation at 60°C by measuring the absorbance at 340 nm. The reversible reaction would indicate the oxidation product from mannitol 1-phosphate. For Dp1703E, Dp1704E, Dp1759E, and Dp0124E, 1 U of activity was defined as 1 μmol NADH oxidized or produced per min.
Accession numbers.The nucleotide sequences of 16S rRNA genes from the clone libraries have been deposited in the GenBank database under accession numbers KF775581 to KF775589 and KJ411294 to KJ411335. The accession number of the 16S rRNA gene sequence of Alg1 is KJ411293. The whole-genome shotgun project of Alg1 has been deposited at DDBJ/EMBL/GenBank under the accession number JWID00000000. The version described in this paper is version JWID01000000.
RESULTS
Survey and isolation of the community.From the generated clone library of the enriched kelp-degrading community, a total of 52 clones from the 80 positive transformants were verified as 16S rRNA genes and were used for OTU analysis. Eleven OTUs were found to be represented in the clone library under the definition of 97% sequence identity (Fig. 1). OTU1 to OTU8 represented 94% (49/52) of the sequenced clones and were classified in one large branch with one uncultured species from Arctic sediment (GenBank accession number FN396782), which together formed a parallel branch with Defluviitalea saccharophila LIND6LT2T and some uncultured species from various thermophilic sources (39, 40). OTU9 and OTU10 were closely related to an extremely alkaliphilic bacterium, Alkaliphilus transvaalensis, isolated from a deep South African gold mine (41). OTU11 was found to be close to an uncultured bacterium of clone 27 (GenBank accession number JQ741987) from our former research of a thermophilic cellulolytic community, with 16S rRNA gene identities of 95% (24). All of the clones from the brown alga-degrading community belonged to the order Clostridiales.
Phylogenetic analysis of the clones from the constructed 16S rRNA gene library and their relatives from the GenBank database after multiple alignments of data. The numbers in brackets represent the number of clones in the OTU.
Dozens of colonies with uniform size were formed on the agar plate in the process of strain isolation. Individual colonies were picked up for examination, and all of them shared very similar 16S rRNA gene sequences with the clones in OTU1 (data not shown). One isolate, designated strain Alg1, was used for further study.
Strain characterization.Strain Alg1 was an obligate anaerobic and Gram-negative bacterium with a fermentative metabolism, forming a translucent colony on plates. Alg1 can utilize the main components of brown algae, including alginate, mannitol, and laminarin, as sole carbon sources with main products of ethanol and acetic acid (Table 1) and can utilize fructose and ribose but not sucrose and xylose (Table 2). The growth temperature was from 45°C to 65°C, with the optimum around 55 to 60°C. Alg1 did not show any growth with NaCl instead of sea salt. The tolerance of salinity was from 1 to 5%, with the optimal salinity at 3%. The pH tolerance was from pH 6 to 9, and the optimum pH was between 7 and 8. The cell morphology of Alg1 was rod-shaped, with differences in alginate medium (long rod) and in mannitol medium (short rod) (Fig. 2). The cellular fatty acid composition of the strain Alg1 was analyzed. The major fatty acids were C16:0 (63%), C17:1 iso w5c (24%), and C18:0 (5.2%). The DNA G+C content of strain Alg1 was 28 mol%.
Ethanol and acetic acid production of Alg1 from alginate, mannitol, and glucose after 120 h of fermentation
Physiological, biochemical, and chemotaxonomic characteristics of Alg1
Cell morphologies of Alg1 by scanning electron microscopy (SEM), utilizing alginate (A) and mannitol (B) as carbon sources.
The 16S rRNA gene identification demonstrated that the closest neighbors of strain Alg1 with validly published names were from the order Clostridiales in the phylum Firmicutes, with 94.6% and 89.3% sequence identities to D. saccharophila LIND6LT2T and Vallitalea guaymasensis Ra1766G1(T), respectively (42). An NJ phylogenetic tree based on the 16S rRNA gene revealed that strain Alg1 clustered with D. saccharophila but with a clear phylogenetic distinction, indicating that Alg1 is a new member of the genus Defluviitalea (Fig. 3). Based on the phenotypic, chemotaxonomic, and phylogenetic distinctiveness, strain Alg1 is considered to represent a novel species of the genus Defluviitalea, named Defluviitalea phaphyphila. The type strain is Alg1T (the same as CGMCC 1.5199T and JCM 30481T).
Phylogenetic tree of the 16S rRNA gene of Alg1 and its closest related species from GenBank. Bootstrap values were calculated based on 1,000 replicates.
Genome analysis of genes responsible for brown alga degradation.After assembly and mapping of the sequenced data from the constructed clone libraries of the genomic DNA of strain Alg1, a total of 76 scaffolds containing 129 contigs with a length of 2.54 Mb were obtained. The G+C content of the genome is 28%, which was consistent with the results of strain characterization.
Through genome analysis, three gene clusters and numerous key genes involved in alginate, laminarin, and mannitol degradation were found (Fig. 4). One of the most important phenotypic properties of strain Alg1 is the ability to depolymerize alginate under thermophilic conditions. Genome analysis revealed six genes encoding alginate lyases (Table 3). Dp0084 was one of the predicted alginate lyases with an N-terminal secretion signal, which contained a polysaccharide lyase (PL) 6 family domain (cd14251) conserved in both alginate lyase (EC 4.2.2.3) and chondroitinase B (EC 4.2.2.19). Dp0100, Dp1059, Dp1761, and Dp1770 were predicted to contain a heparinase II/III-like protein domain (pfam07940) (Table 3), which was found in both heparinase II and alginate lyase (43, 44). Dp2072 was predicted to contain an alginate_lyase 2 domain (pfam08787) and shared the highest amino acid identity (52%) with the alginate lyase from Corynebacterium species (45). Dp0100 and Dp2072 both carry an N-terminal secretion signal, as predicted by SignalP 4.1, and are believed to be extracellular alginate lyases. Dp0100 is a multidomain protein, and its catalytic domain (heparinase II/III-like protein domain) shared 18% identity with exotype alginate lyase Atu3025 from Agrobacterium tumefaciens (22). Other domains all were predicted as the substrate binding domains. Dp1761 shared 21% identity with an exo-oligoalginate lyase of PL 17 from Saccharophagus degradans 2-40 (46), and Dp1770 was closely related to an alginate lyase of PL 15 from Agrobacterium tumefaciens C58 (22). Both Dp1761 and Dp1770 contained no signal sequences; therefore, they were hypothesized to be intracellular oligoalginate lyases. In addition to alginate lyases, Dp1703, Dp1704, Dp1705, and Dp1759 also were believed to play important roles in alginate utilization according to genome analysis. Dp1759 was predicted to be a DEH reductase and shared 38% amino acid identity with an NADH-dependent reductase of Sphingomonas sp. strain A1 (47). A small gene cluster including dp1703-dp1705 was involved in the conversion of KDG to pyruvate (Fig. 4B). Dp1703 and Dp1704 both were predicted to have a conserved domain of 2-keto-3-deoxyglucononate kinase (KdgK), while Dp1705 contained a conserved domain of KdpgA (Table 3).
Putative pathways and gene clusters involved in brown alga degradation deduced from the genome of Alg1. (A) Predicted pathways participating in brown alga degradation. (B) Three gene clusters involved in deconstruction of brown algae in the genome of Alg1. The functions of the proteins are color-coded: green, alginate degradation-related genes; yellow, mannitol and fructose metabolism-related genes; black, putative regulation factor; blue, unknown function. PI1, PII1, PII2, PII3, PIII1, PIII2, PIII3, and PIII4 are predicted promoters, and TI, TII, and TIII are predicted Rho-independent terminators. SDR, short-chain dehydrogenase/reductase.
Key genes of the proposed pathways for alginate, laminarin, and mannitol degradation and enzyme activities
Genes for mannitol and laminarin metabolism also were analyzed. The predicted genes involved in mannitol catabolism were found to be located in gene cluster I, which included dp0124-dp0126 (Fig. 4B). Dp0126 was annotated as a mannitol-specific transporter and shared 64% identity with the PTS system mannitol-specific transporter subunit IICBA from Alkaliphilus metalliredigens. Dp0124 contained a medium-chain reductase/dehydrogenase (MDR) domain and was hypothesized to be MPDH. Dp0125 contained a FruK_PfkB_like domain (cd01164) and shared a high identity of 53% with a 1-phosphofructokinase (PFK1) from Alkaliphilus metalliredigens; thus, it was considered PFK1. Laminarin can be hydrolyzed readily by glucosidases. Two glucosidase-encoding genes were annotated through genome analysis, dp0614 and dp1433. Dp0614 displays an N-terminal secretion signal and contains a GH16_laminarinase_like domain, which shares 47% amino acid sequence identity with the glucan endo-1,3-beta-d-glucosidase (EC 3.2.1.39). Dp1433 shared 52% amino acid sequence identity with β-glucosidase (EC 3.2.1.21) and had no secreting signal.
Biochemical characterization of the key enzymes involved in brown alga degradation.Genome analysis revealed at least 15 genes involved in brown alga degradation, which included 10 alginate degradation-related genes, 3 mannitol utilization-related genes, and 2 laminarin degradation-related genes (Table 3). dp0100 has a nucleotide sequence of 5,478 bp and is difficult to express heterogenously. Thus, direct protein purification from the fermentation supernatant of Alg1 was conducted. Alginate lyases in the culture supernatant were purified with an anion exchange column followed by a filtration column, and two absorption peaks were detected for alginate lyase activity. Two peak fractions (A and B) were digested with trypsin and were identified by mass spectrometry. These peptides perfectly matched Dp2072 and Dp0100, with estimated molecular masses of 37 kDa and 205 kDa and covering 69% and 68%, respectively, of their whole sequences (see Fig. S2 in the supplemental material). Heterogenous expression was applied for all other genes except dp0100. Ten recombinant enzymes were successfully expressed in a soluble form, whereas Dp1433 and Dp1705 failed to form soluble proteins in Escherichia coli BL21(DE3) (see Fig. S1). Dp0084, Dp0100, Dp1059, Dp1761, Dp1770, and Dp2072 were found to be able to degrade alginate, with specific activities of 3,592, 2,850, 9, 58, 5, and 210 U mg−1 (Table 3), respectively. Moreover, Dp1761 was confirmed to act in exo mode on alginate, and DEH released by Dp1761 was further used as the substrate to evaluate the activity of the putative DEH reductase Dp1759E. After the addition of purified Dp1759 to the reaction mixture, an immediate decrease of absorbance at 340 nm was observed, indicating the oxidation of NADH (see Fig. S3). Therefore, Dp1759 was confirmed as a KdgD member catalyzing DEH to KDG. The kinase activities of Dp1703 and Dp1704 were detected using KDG produced by Dp1759 as the substrate. After the enzyme-coupled assay system was initiated, significant enzyme reactions were observed as decreases in the A340 for both Dp1703 and Dp1704 (see Fig. S3). Consequently, the conversion of KDG to KDPG was phosphorylated by Dp1703 and Dp1704. Dp0614 showed high hydrolyase activity (95 U mg−1) against laminarin and was confirmed as an active laminarinase.
For the measurement of the dehydrogenase activity of Dp0124, no reaction was observed in the presence of F6P. In contrast, after the addition of F1P, the absorbance at 340 nm observed was significantly decreased with the oxidation of NADH and the formation of NAD+ (see Fig. S3 in the supplemental material). This result indicated that F1P is the oxidation product from mannitol 1-phosphate.
DISCUSSION
The alginolytic community was enriched from a coastal sediment sample at low temperature, and it showed a relatively homogeneous bacterial structure compared to the structure of a cellulolytic community we previously identified (24). We found the members of the alginolytic community all were from the order Clostridiales, and a high proportion of the clones (94%; 49/52) was phylogenetically related to D. saccharophila LIND6LT2T. In contrast, only 76% of the clones of the cellulolytic community were from Clostridiales, and 62% of the clones were phylogenetically related to the predominant species (24), suggesting the deconstruction of lignocellulose is a more complex process than that of brown algae. To our surprise, all of the species from both communities turned out to be novel species with low 16S rRNA gene sequence (<95%) identities to their closest relatives. The thermophilic properties of these species raised the interesting topic of the distribution of marine thermophiles, which normally were found to be widely distributed in high-temperature habitats, including deep-sea hydrothermal vents, subsurface petroleum reservoirs, and hot springs. Increasing amounts of experimental evidence have indicated that the distribution of thermophiles is not limited to such geothermal areas. For example, Hubert et al. demonstrated the diversity of dormant thermophilic bacterial spores that become active at much higher temperatures than they do in situ in Arctic marine sediments (40, 48). The thermophilic alginolytic and cellulolytic communities, which were enriched from coastal low-temperature environments, have provided exciting evidence for the wide distribution of thermophiles (24), suggesting untapped novel microbial resources exist in coastal environments.
Hubert et al. explained the origins of the thermophiles in Arctic sediment with a theory that thermophiles from warm subsurface petroleum reservoirs and ocean crust ecosystems distribute into the cold ocean through seabed fluid flow (40). From the phylogenetic analysis of the alginolytic community, some clones showed high 16S rRNA gene sequence identities of 95% to 99% to the clones from Arctic sediment, suggesting these clones shared similar origins from marine thermal ecosystems. Moreover, the coastal thermophilic species also showed close proximity to species from some terrigenous environments, including hot springs, deep mines, and oil fields, which suggested the involvement of other origins (24). Until now, few thermophilic bacterial pure cultures have been isolated from low-temperature marine environments. Alg1, as one of the few representatives, may give us some direct information on this mysterious population.
Alg1 is a member of spore-forming species from the phylum Firmicutes. The vital requirement of sea salt and the versatile ability in degrading brown algae observed in Alg1 indicate that it is a real marine bacterium that has adapted to the nutritional conditions of the marine environment. Taken together, these results conveyed an inspiring message that novel marine thermophiles with functional and metabolic diversities could be acquired from the coastal environment. The genome analysis and biochemical characterization of key enzymes involved in the deconstruction of brown algae demonstrated that Alg1 contains a full set of genes participating in the catabolism of brown algae (Fig. 4). Moreover, the main products from brown alga catabolism were ethanol and acetic acid. To our knowledge, Alg1 is the first reported fermentative natural bacterium capable of degrading brown algae.
As an anaerobic and thermophilic bacterial strain, Alg1 was determined to have the ability to utilize alginate, a characteristic mostly found in mesophilic bacteria. Two gene clusters and numerous other genes were found to be directly involved in the metabolism of alginate (Fig. 4). dp1759, dp1761, and dp1770 were located in gene cluster III, which was predicted to play important roles in the intracellular metabolism of alginate (from oligoalginate to KDG). Three carbohydrate transporter-encoding genes (dp1764, dp1768, and dp1769) were assumed to transport oligoalginate to the cytoplasm, while their functions need further experimental verification. Furthermore, cluster III was predicted to be a transcriptional unit containing four promoters upstream and one terminator downstream (see Table S2 in the supplemental material). A complex operon like this could ensure the genes located in the cluster closely cooperate for oligoalginate assimilation. Cluster II also was predicted as an operon with multiple promoters. Dp1703 and Dp1704, two KdgK members, showed only 29% identity to each other, indicating different origins. A similar situation was found in the marine alginolytic bacterium Z. galactanivorans, and two KdgK genes (zg4703 and zg2614) were characterized from its genome (14). Dp1705 was predicted to encode KdpgA, which catalyzes KDPG to pyruvate and glyceraldehyde 3-phosphate (G3P) in the ED pathway. The enzymes in cluster II play crucial roles in the complete degradation of alginate into pyruvate, and the predicted multiple promoters could regulate the expression level of individual genes more flexibly to meet the necessities of the cells. The enzymatic degradation of laminarin was common in quite a few of microorganisms, including bacteria, actinomycetes, and fungi (10), and its degradation pathway is much simpler than that of alginate (Fig. 4A). Dp0614 can endolytically cleave laminarin into oligolaminarin, which was further degraded to glucose by a predicted β-glucosidase (Dp1433).
After the cell structure of brown algae was disrupted by the secreted alginate lyases and laminarinase, mannitol was released into the medium. Although mannitol could be utilized by a number of microorganisms, it cannot be fermented under strictly anaerobic conditions by some ethanol-producing microorganisms, such as Zymobacter palmae and Saccharomyces cerevisiae (49). This is attributed mainly to the excess electrons (NADH) produced during mannitol metabolism. Under anaerobic conditions, these excess reducing equivalents could not be neutralized by oxygen, especially when the microorganisms lack transhydrogenase (an enzyme converting the catabolic reducing equivalent NADH to the anabolic reducing equivalent NADPH) (17, 49). The vigorous growth of Alg1 using mannitol as the carbon substrate under obligate anaerobic conditions indicated that Alg1 has a well-balanced system to process the excess reducing equivalents. However, the precise regulation for reducing equivalents still needs further investigation. A mannitol-specific PTS system encoded by dp0126 is responsible for the transport of mannitol into cytosol, and mannitol is converted to mannitol 1-phosphate during this process (Fig. 4A). MPDH encoded by dp0124 then converts mannitol 1-phosphate to fructose 1-phosphate with the production of one NADH molecule. Fructose 1-phosphate was phosphorylated by PFK1 (Dp0125) to produce fructose 1,6-bisphosphate, which was further assimilated through glycolysis (Fig. 4A). In contrast to the classical mannitol degradation pathway found in bacteria in which mannitol 1-phosphate was converted into fructose 6-phosphate, the mannitol degradation pathway possessed by Alg1 showed distinct features in converting mannitol 1-phosphate to fructose 1-phosphate. Actually, Dp0124 first was annotated as an l-sorbose 1-phosphate reductase; however, no activity was detected in the presence of d-sorbitol 6-phosphate and NAD(P)+ (data not shown). After the formation of pyruvate from the glycolysis and ED pathways in metabolizing alginate, laminarin, and mannitol, ethanol synthesis was initiated.
Taxonomy.Defluviitalea phaphyphila (pha.phy. phi'la. Gr. n. phaiós-ophyta, brown algae; N.L. adj. philus -a -um [from Gr. adj. philos -ê -on], friend, loving; N.L. fem. adj., phaphyphila, brown algae-loving).
Cells are Gram-negative, rod-shaped, 0.4 to 0.5 μm by 1 to 5 μm. Forming half-transparent colony on ABM agar plate. Obligately anaerobic with fermentative metabolism. Utilize alginate, mannitol, laminarin, fructose, glucose, mannose, cellobiose, and ribose but not sucrose, xylose, acetate, starch, pyruvate, ribose, fructose, lactate, maltose, xylose, peptone, lactose, galactose, raffinose, arabinose, glycerol, rhamnose, peptone, and Casamino Acids. Growth temperature covers from 45 to 65°C, with an optimum at 55 to 60°C. Alg1 did not show any growth with NaCl instead of sea salt. The tolerance of salinity was from 1 to 5%, with an optimal salinity of 3%. The pH tolerance was from pH 6 to 9, and the optimum pH was between pH 7 and 8. The major fatty acids were C16:0 (63%), C17:1 iso w5c (24%), and C18:0 (5.2%). The G+C content of the genomic DNA in strain Alg1 was 28 mol%. The type strain is Alg1 (the same as CGMCC 1.5199T and JCM 30481T), which was isolated from coastal sediment of an amphioxus breeding zone in Qingdao, China (36°5′ N, 120°32′ E).
ACKNOWLEDGMENTS
We thank Chen Li from the Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, for her help with determination of the cellular fatty acids.
FOOTNOTES
- Received 8 October 2015.
- Accepted 15 November 2015.
- Accepted manuscript posted online 20 November 2015.
Supplemental material for this article may be found at http://dx.doi.org/10.1128/AEM.03297-15.
- Copyright © 2016, American Society for Microbiology. All Rights Reserved.
