Phasin Proteins Activate Aeromonas caviae Polyhydroxyalkanoate (PHA) Synthase but Not Ralstonia eutropha PHA Synthase

In this study, we performed in vitro and in vivo activity assays of polyhydroxyalkanoate (PHA) synthases (PhaCs) in the presence of phasin proteins (PhaPs), which revealed that PhaPs are activators of PhaC derived from Aeromonas caviae (PhaC_Ac). In in vitro assays, among the three PhaCs tested, PhaC_Ac was significantly activated when PhaPs were added at the beginning of polymerization (prepolymerization PhaC_Ac), whereas the prepolymerization PhaC_Re (derived from Ralstonia eutropha) and PhaC_Da (Delftia acidovorans) showed reduced activity with PhaPs. The PhaP-activated PhaC_Ac showed a slight shift of substrate preference toward 3-hydroxyhexanoyl-CoA (C₆). PhaP_Ac also activated PhaC_Ac when it was added during polymerization (polymer-elongating PhaC_Ac), while this effect was not observed for PhaC_Re. In an in vivo assay using Escherichia coli TOP10 as the host strain, the effect of PhaP_Ac expression on PHA synthesis by PhaC_Ac or PhaC_Re was examined. As PhaP_Ac expression increased, PHA production was increased by up to 2.3-fold in the PhaC_Ac-expressing strain, whereas it was slightly increased in the PhaC_Re-expressing strain. Taken together, this study provides evidence that PhaPs function as activators for PhaC_Ac both in vitro and in vivo but do not activate PhaC_Re. This activating effect may be attributed to the new role of PhaPs in the polymerization reaction by PhaC_Ac.     

Enhanced Accumulation and Changed Monomer Composition in Polyhydroxyalkanoate (PHA) Copolyester by In Vitro Evolution of Aeromonas caviae PHA Synthase

By in vitro evolution experiment, we have first succeeded in acquiring higher active mutants of a synthase that is a key enzyme essential for bacterial synthesis of biodegradable polyester, polyhydroxyalkanoate (PHA). Aeromonas caviae FA440 synthase, termed PhaC_Ac, was chosen as a good target for evolution, since it can synthesize a PHA random copolyester of 3-hydroxybutyrate and 3-hydroxyhexanoate [P(3HB-co-3HHx)] that is a tough and flexible material compared to polyhydroxybutyrate (PHB) homopolyester. The in vitro enzyme evolution system consists of PCR-mediated random mutagenesis targeted to a limited region of the phaC_Ac gene and screening mutant enzymes with higher activities based on two types of polyester accumulation system by using Escherichia coli for the synthesis of PHB (by JM109 strain) (S. Taguchi, A. Maehara, K. Takase, M. Nakahara, H. Nakamura, and Y. Doi, FEMS Microbiol. Lett. 198:65-71, 2001) and of P(3HB-co-3HHx) {by LS5218 [fadR601 atoC(Con)] strain}. The expression vector for the phaC_Ac gene, together with monomer-supplying enzyme genes, was designed to synthesize PHB homopolyester from glucose and P(3HB-co-3HHx) copolyester from dodecanoate. Two evolved mutant enzymes, termed E2-50 and T3-11, screened through the evolution system exhibited 56 and 21% increases in activity toward 3HB-coenzyme A, respectively, and consequently led to enhanced accumulation (up to 6.5-fold content) of P(3HB-co-3HHx) in the recombinant LS5218 strains. Two single mutations in the mutants, N149S for E2-50 and D171G for T3-11, occurred at positions that are not highly conserved among the PHA synthase family. It should be noted that increases in the 3HHx fraction (up to 16 to 18 mol%) were observed for both mutants compared to the wild type (10 mol%).     

Alteration of Chain Length Substrate Specificity of Aeromonas caviae R-Enantiomer-Specific Enoyl-Coenzyme A Hydratase through Site-Directed Mutagenesis

Aeromonas caviae R-specific enoyl-coenzyme A (enoyl-CoA) hydratase (PhaJ_Ac) is capable of providing (R)-3-hydroxyacyl-CoA with a chain length of four to six carbon atoms from the fatty acid β-oxidation pathway for polyhydroxyalkanoate (PHA) synthesis. In this study, amino acid substitutions were introduced into PhaJ_Ac by site-directed mutagenesis to investigate the feasibility of altering the specificity for the acyl chain length of the substrate. A crystallographic structure analysis of PhaJ_Ac revealed that Ser-62, Leu-65, and Val-130 define the width and depth of the acyl-chain-binding pocket. Accordingly, we targeted these three residues for amino acid substitution. Nine single-mutation enzymes and two double-mutation enzymes were generated, and their hydratase activities were assayed in vitro by using trans-2-octenoyl-CoA (C₈) as a substrate. Three of these mutant enzymes, L65A, L65G, and V130G, exhibited significantly high activities toward octenoyl-CoA than the wild-type enzyme exhibited. PHA formation from dodecanoate (C₁₂) was examined by using the mutated PhaJ_Ac as a monomer supplier in recombinant Escherichia coli LS5218 harboring a PHA synthase gene from Pseudomonas sp. strain 61-3 (phaC1_Ps). When L65A, L65G, or V130G was used individually, increased molar fractions of 3-hydroxyoctanoate (C₈) and 3-hydroxydecanoate (C₁₀) units were incorporated into PHA. These results revealed that Leu-65 and Val-130 affect the acyl chain length substrate specificity. Furthermore, comparative kinetic analyses of the wild-type enzyme and the L65A and V130G mutants were performed, and the mechanisms underlying changes in substrate specificity are discussed.     

Structure Reveals Regulatory Mechanisms of a MaoC-Like Hydratase from Phytophthora capsici Involved in Biosynthesis of Polyhydroxyalkanoates (PHAs)

Background

Polyhydroxyalkanoates (PHAs) have attracted increasing attention as “green plastic” due to their biodegradable, biocompatible, thermoplastic, and mechanical properties, and considerable research has been undertaken to develop low cost/high efficiency processes for the production of PHAs. MaoC-like hydratase (MaoC), which belongs to (R)-hydratase involved in linking the β-oxidation and the PHA biosynthetic pathways, has been identified recently. Understanding the regulatory mechanisms of (R)-hydratase catalysis is critical for efficient production of PHAs that promise synthesis an environment-friendly plastic.

Methodology/Principal Findings

We have determined the crystal structure of a new MaoC recognized from Phytophthora capsici. The crystal structure of the enzyme was solved at 2.00 Å resolution. The structure shows that MaoC has a canonical (R)-hydratase fold with an N-domain and a C-domain. Supporting its dimerization observed in structure, MaoC forms a stable homodimer in solution. Mutations that disrupt the dimeric MaoC result in a complete loss of activity toward crotonyl-CoA, indicating that dimerization is required for the enzymatic activity of MaoC. Importantly, structure comparison reveals that a loop unique to MaoC interacts with an α-helix that harbors the catalytic residues of MaoC. Deletion of the loop enhances the enzymatic activity of MaoC, suggesting its inhibitory role in regulating the activity of MaoC.

Conclusions/Significance

The data in our study reveal the regulatory mechanism of an (R)-hydratase, providing information on enzyme engineering to produce low cost PHAs.     

Crystal structure of the (R)-specific enoyl-CoA hydratase from Aeromonas caviae involved in polyhydroxyalkanoate biosynthesis

The (R)-specific enoyl coenzyme A hydratase ((R)-hydratase) from Aeromonas caviae catalyzes the addition of a water molecule to trans-2-enoyl coenzyme A (CoA), with a chain-length of 4-6 carbons, to produce the corresponding (R)-3-hydroxyacyl-CoA. It forms a dimer of identical subunits with a molecular weight of about 14,000 and is involved in polyhydroxyalkanoate (PHA) biosynthesis. The crystal structure of the enzyme has been determined at 1.5-A resolution. The structure of the monomer consists of a five-stranded antiparallel beta-sheet and a central alpha-helix, folded into a so-called "hot dog" fold, with an overhanging segment. This overhang contains the conserved residues including the hydratase 2 motif residues. In dimeric form, two beta-sheets are associated to form an extended 10-stranded beta-sheet, and the overhangs obscure the putative active sites at the subunit interface. The active site is located deep within the substrate-binding tunnel, where Asp(31) and His(36) form a catalytic dyad. These residues are catalytically important as confirmed by site-directed mutagenesis and are possibly responsible for the activation of a water molecule and the protonation of a substrate molecule, respectively. Residues such as Leu(65) and Val(130) are situated at the bottom of the substrate-binding tunnel, defining the preference of the enzyme for the chain length of the substrate. These results provide target residues for protein engineering, which will enhance the significance of this enzyme in the production of novel PHA polymers. In addition, this study provides the first structural information of the (R)-hydratase family and may facilitate further functional studies for members of the family.     

Identification of the Polyhydroxyalkanoate (PHA)-Specific Acetoacetyl Coenzyme A Reductase among Multiple FabG Paralogs in Haloarcula hispanica and Reconstruction of the PHA Biosynthetic Pathway in Haloferax volcanii

Genome-wide analysis has revealed abundant FabG (β-ketoacyl-ACP reductase) paralogs, with uncharacterized biological functions, in several halophilic archaea. In this study, we identified for the first time that the fabG1 gene, but not the other five fabG paralogs, encodes the polyhydroxyalkanoate (PHA)-specific acetoacetyl coenzyme A (acetoacetyl-CoA) reductase in Haloarcula hispanica. Although all of the paralogous fabG genes were actively transcribed, only disruption or knockout of fabG1 abolished PHA synthesis, and complementation of the ΔfabG1 mutant with the fabG1 gene restored both PHA synthesis capability and the NADPH-dependent acetoacetyl-CoA reductase activity. In addition, heterologous coexpression of the PHA synthase genes (phaEC) together with fabG1, but not its five paralogs, reconstructed the PHA biosynthetic pathway in Haloferax volcanii, a PHA-defective haloarchaeon. Taken together, our results indicate that FabG1 in H. hispanica, and possibly its counterpart in Haloarcula marismortui, has evolved the distinct function of supplying precursors for PHA biosynthesis, like PhaB in bacteria. Hence, we suggest the renaming of FabG1 in both genomes as PhaB, the PHA-specific acetoacetyl-CoA reductase of halophilic archaea.Several haloarchaeal species belonging to the genera Haloferax, Haloarcula, Natrialba, and Haloquadratum are capable of synthesizing short-chain-length polyhydroxyalkanoates (SCL-PHAs) (6, 8, 14, 16), a large family of biopolymers with desirable biodegradability, biocompatibility, and thermoplastic features (31). Although the metabolic pathways of PHAs in bacteria have been characterized in detail (10, 15, 20, 25, 26, 37), the genes involved in PHA biosynthesis in haloarchaea were not recognized until recently, when the PHA synthase genes were identified and characterized for Haloarcula marismortui, Haloarcula hispanica, and Haloferax mediterranei (6, 19). These archaeal PHA synthases are all composed of two subunits, PhaE and PhaC. They are homologous to the class III PHA synthases from bacteria but have a longer C-terminal extension in the PhaC subunit. Nevertheless, the pathway of supplying the PHA precursors has not yet been clarified for any haloarchaeal strain.Both H. mediterranei and H. hispanica are able to synthesize poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) from unrelated carbon sources, despite the content of the (R)-3-hydroxyvalerate (3-HV) monomer of PHBV in H. mediterranei (10 to 13 mol%) (4, 19) being much higher than that in H. hispanica (∼3 mol%) (19). Conversely, the bacteria Ralstonia eutropha and Synechocystis sp. strain PCC6803, which possess class I and III PHA synthases, respectively, accumulate just poly(3-hydroxybutyrate) (PHB) when the 3-HV-related carbon sources (i.e., propionate and valerate) are not supplied (30). In these two bacteria, the biosynthesis of the (R)-3-hydroxybutyrate coenzyme A [(R)-3-HB-CoA] precursor is conducted by two steps. First, two acetyl-CoA molecules are condensed into one acetoacetyl-CoA molecule by the enzyme β-ketothiolase (PhaA). The acetoacetyl-CoA is then reduced to (R)-3-HB-CoA by a PHA-specific acetoacetyl-CoA reductase (PhaB). The resulting (R)-3-HB-CoA is subsequently incorporated into PHB, catalyzed by PHA synthases (26, 36).Both PhaB and FabG belong to the short-chain dehydrogenase/reductase (SDR) superfamily, whose members are homologous in sequence and have several conserved motifs (27, 29). Interestingly, although FabGs naturally reduce 3-ketoacyl-ACP to form (R)-3-hydroxyacyl-ACP in fatty acid biosynthesis, a few FabGs also recognize 3-ketoacyl-CoA and hence function in PHA biosynthesis. For example, the FabG proteins of Escherichia coli and Pseudomonas aeruginosa have been demonstrated to supply precursors for PHA biosynthesis in recombinant E. coli cells (21, 22, 32, 35). In addition, several FabG paralogs may have evolved a distinct function, to be responsible only for PHA accumulation. This situation was observed in Synechocystis sp. strain PCC6803, where the originally annotated FabG (12) was renamed PhaB after an understanding of its function in PHA biosynthesis (36).Genome-wide analysis of H. marismortui ATCC 43049 (1) revealed eight FabG paralogs in this haloarchaeon. Similarly, multiple fabG paralog genes (fabG1 to fabG6) were also observed in the newly sequenced genome of H. hispanica (our unpublished data). In this study, we demonstrate that fabG1, but not the other five fabG paralogs, encodes the PHA-specific acetoacetyl-CoA reductase in H. hispanica. It is responsible for providing (R)-3-HB-CoA for PHA biosynthesis in Haloarcula species, and interestingly, this enzyme also functions well in Haloferax volcanii, endowing this PHA-defective strain with the ability to accumulate PHA when cotransformed with PHA synthase genes.     

Some Aspects of the Enzyme Activities Involved in Coenzyme A Biosynthesis in Various Microorganisms

The distribution of the enzyme activities relating to CoA biosynthesis from pantothenic acid in various microorganisms and the effect of CoA on these activities are described.

High activities of partial reactions involved in CoA biosynthesis were surveyed in various type culture strains involving bacteria, actinomycetes, lactic acid bacteria, molds, and yeasts. Generally, higher activities were found in bacteria. CoA inhibited the phosphorylation of pantothenic acid, and resulted in a decrease of CoA production in all the CoA producing strains, while only a little inhibition by CoA was observed in the other reactions, and CoA production from pantothenic acid 4′-phosphate by Brevibacterium ammoniagenes IFO 12071 was not repressed even in the presence of 4mm of CoA. Extracellular excretion of the enzymes of CoA biosynthesis was observed when cells were in contact with sodium lauryl sulfate. Degrading activity against CoA and that against AMP were relatively lower in CoA producing strains when compared with those in other strains. It was confirmed that Brown’s route of CoA biosynthesis operates in Brevibacterium ammoniagenes IFO 12071.     

Novel Rhamnosyltransferase Involved in Biosynthesis of Serovar 4-Specific Glycopeptidolipid from Mycobacterium avium Complex

Glycopeptidolipids (GPLs) are one of the major glycolipid components present on the surface of Mycobacterium avium complex (MAC) that belong to opportunistic pathogens distributed in the natural environment. The serovars of MAC, up to around 30 types, are defined by the variable oligosaccharide portions of the GPLs. Epidemiological studies show that serovar 4 is the most prevalent type, and the prognosis of pulmonary disease caused by serovar 4 is significantly worse than that caused by other serovars. However, little is known about the biosynthesis of serovar 4-specific GPL, particularly the formation of the oligosaccharide portion that determines the properties of serovar 4. To investigate the biosynthesis of serovar 4-specific GPL, we focused on one segment that included functionally unknown genes in the GPL biosynthetic gene cluster of a serovar 4 strain. In this segment, a putative hemolytic protein gene, hlpA, and its downstream gene were found to be responsible for the formation of the 4-O-methyl-rhamnose residue, which is unique to serovar 4-specific GPL. Moreover, functional characterization of the hlpA gene revealed that it encodes a rhamnosyltransferase that transfers a rhamnose residue via 1→4 linkage to a fucose residue of serovar 2-specific GPL, which is a key pathway leading to the synthesis of oligosaccharide of serovar 4-specific GPL. These findings may provide clues to understanding the biological role of serovar 4-specific GPL in MAC pathogenicity and may also provide new insights into glycosyltransferase, which generates structural and functional diversity of GPLs.The genus Mycobacterium has a unique feature in the cell envelope that contains a multilayered structure consisting of peptidoglycan, mycolyl-arabinogalactan complex, and surface glycolipids (8, 12). It is known that these components play a role in protection from environmental stresses, such as antimicrobial agents and host immune responses (8, 12). Some of them are recognized as pathogenic factors related to mycobacterial diseases, such as tuberculosis and leprosy (8, 12). In case of nontuberculous mycobacteria that are widely distributed in the natural environment as opportunistic pathogens, glycopeptidolipids (GPLs) are abundantly present on the cell envelope as surface glycolipids (34). GPLs have a core structure in which a fatty acyl-tetrapeptide is glycosylated with 6-deoxy-talose (6-d-Tal) and O-methyl-rhamnose (O-Me-Rha) (2, 5, 13). This structure is common to all types of GPLs, and GPLs with this structure that have not undergone further glycosylation are termed non-serovar-specific GPLs (nsGPLs) (2, 5, 13). Structural diversity generated by further glycosylations, such as rhamnosylation, fucosylation, and glucosylation, is observed for the oligosaccharide portion linked to the 6-d-Tal residue of nsGPLs from Mycobacterium avium complex (MAC), a member of the nontuberculous mycobacteria consisting of two species, M. avium and M. intracellulare (2, 5, 34). Consequently, these nsGPLs with varied oligosaccharides lead to the formation of the serovar-specific GPLs (ssGPLs) that define around 30 types of MAC serovars (10).The properties of MAC serovars are known to be notably different from each other and also to be closely associated with the pathogenicity of MAC (3, 6, 18, 30, 31, 32). Various epidemiological studies indicate that serovar 4 is the most prevalent type and is also one of the serovars frequently isolated from AIDS patients (1, 20, 33, 36). Additionally, pulmonary MAC disease caused by serovar 4 is shown to exhibit a poorer prognosis than that caused by other serovars (23). With respect to host immune responses to MAC infection, serovar 4-specific GPL is reported to have characteristic features that are in contrast to those of other ssGPLs (21, 30). Structurally, serovar 4-specific GPL contains a unique oligosaccharide in which the oligosaccharide of serovar 2-specific GPL is further glycosylated with 4-O-methyl-rhamnose (4-O-Me-Rha) residue through a 1→4 linkage (Table ​(Table1)1) (24). Therefore, it is thought that the presence of 4-O-Me-Rha and its linkage position are important in exhibiting the specificity of biological activities. The biosynthesis of the oligosaccharide portion in several ssGPLs is currently being clarified (15, 16, 17, 25, 26), while that of serovar 4-specific GPL is still unresolved. In this study, we have focused on the genomic region predicted to be associated with GPL biosynthesis in the serovar 4 strain and explored the key genes responsible for the formation of 4-O-Me-Rha that might determine the specific properties of MAC serovar 4.

TABLE 1.

Oligosaccharide structures of serovar 2- and 4-specific GPLs

辅酶Q10的生物合成及其高表达研究进展

辅酶Q10(CoQ10)不仅是呼吸链上的电子传递体,同时也具有抗氧化功能。目前全球市场上的CoQ10正处于一种供不应求的状态。我们简要论述了CoQ10的结构、性质、功能及其生物合成过程,同时概括总结了现阶段为提高CoQ10产量而采用的新型技术手段。     

Characterization of cytotoxin-producing Aeromonas caviae (strain HT10) isolated from a sulfur spring in Orissa, India

During a study of microbial diversity, a bacterial strain designated HT10, was isolated from sediment collected from an unexplored sulfur spring at Athamallik, Orissa, India. Various biochemical tests and 16S rDNA sequence analysis revealed that strain HT10 is Aeromonas caviae. The growth temperature of this strain ranged from 12 to 43 degrees C and the optimum temperature was 30 degrees C. The strain HT10 showed cytotoxic and alpha-hemolytic activity. This is the first report on the isolation of Aeromonas caviae from sulfur spring.     

Dynamics of Aeromonas hydrophila, Aeromonas sobria, and Aeromonas caviae in a sewage treatment pond

The spatiotemporal dynamics of Aeromonas spp. and fecal coliforms in the sewage treatment ponds of an urban wastewater center were studied after 20 months of sampling from five stations in these ponds. Isolation and identification of 247 Aeromonas strains were undertaken over four seasons at the inflow and outflow of this pond system. The hemolytic activity of these strains was determined. The Aeromonas spp. and the fecal coliform distributions showed seasonal cycles, the amplitude of which increased at distances further from the wastewater source, so that in the last pond there was an inversion of the Aeromonas spp. cycle in comparison with that of fecal coliforms. The main patterns in these cycles occurred simultaneously at all stations, indicating control of these bacterial populations by seasonal factors (temperature, solar radiation, phytoplankton), the effects of which were different on each bacterial group. The analysis of the Aeromonas spp. population structure showed that, regardless of the season, Aeromonas caviae was the dominant species at the pond system inflow. However at the outflow the Aeromonas spp. population was dominated by A. caviae in winter, whereas Aeromonas sobria was the dominant species in the treated effluent from spring to fall. Among the Aeromonas hydrophila and A. sobria strains, 100% produced hemolysin; whereas among the A. caviae strains, 96% were nonhemolytic.     

Identification and Recombinant Expression of a Mycobacterium avium Rhamnosyltransferase Gene (rtfA) Involved in Glycopeptidolipid Biosynthesis

The Mycobacterium avium complex is a source of disseminated infections in patients with advanced AIDS. This group of mycobacteria is distinguished by the presence of highly antigenic, surface-exposed glycopeptidolipids, and these glycolipids possess variant oligosaccharide structures that are the chemical basis of the 28 distinct serovars of the M. avium complex. We previously described the ser2 gene cluster, encoding the synthesis of the haptenic oligosaccharide (2,3-dimethylfucose-rhamnose-6-deoxytalose-) of the serovar 2-specific glycopeptidolipid, and revealed a locus (ser2A) encoding a putative rhamnosyltransferase. Sequencing of the ser2A locus demonstrated the presence of three open reading frames, two of which yielded significant homology to several glycosyltransferases, and the deduced amino acid sequences of these two putative glycosyltransferases had 63% identity. These two genes were expressed in Mycobacterium smegmatis, and the resulting recombinant glycopeptidolipids were characterized by thin-layer chromatography and gas chromatography-mass spectrometry. These analyses demonstrated that only one of these genes, termed rtfA, encoded the rhamnosyltransferase responsible for the transfer of rhamnose to 6-deoxytalose. The identification of rtfA will permit further evaluation of glycopeptidolipid biosynthesis and the construction of isogenic mutants of multiple M. avium complex serovars. Moreover, such mutants will help define the role of glycopeptidolipids in the intracellular survival of these bacteria.     

Dynamics of Aeromonas hydrophila, Aeromonas sobria, and Aeromonas caviae in a sewage treatment pond.

The spatiotemporal dynamics of Aeromonas spp. and fecal coliforms in the sewage treatment ponds of an urban wastewater center were studied after 20 months of sampling from five stations in these ponds. Isolation and identification of 247 Aeromonas strains were undertaken over four seasons at the inflow and outflow of this pond system. The hemolytic activity of these strains was determined. The Aeromonas spp. and the fecal coliform distributions showed seasonal cycles, the amplitude of which increased at distances further from the wastewater source, so that in the last pond there was an inversion of the Aeromonas spp. cycle in comparison with that of fecal coliforms. The main patterns in these cycles occurred simultaneously at all stations, indicating control of these bacterial populations by seasonal factors (temperature, solar radiation, phytoplankton), the effects of which were different on each bacterial group. The analysis of the Aeromonas spp. population structure showed that, regardless of the season, Aeromonas caviae was the dominant species at the pond system inflow. However at the outflow the Aeromonas spp. population was dominated by A. caviae in winter, whereas Aeromonas sobria was the dominant species in the treated effluent from spring to fall. Among the Aeromonas hydrophila and A. sobria strains, 100% produced hemolysin; whereas among the A. caviae strains, 96% were nonhemolytic.     

Site-Directed Mutagenesis of <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis> Enoyl Coenzyme A Hydratase Enhancing 3-Hydroxyhexanoate Fractions of Poly(3-hydroxybutyrate-<Emphasis Type="Italic">co</Emphasis>-3-hydroxyhexanoate)

The aim of this study is to enhance 3-hydroxyhexanoate (3HHx) fractions of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate), abbreviated as PHBHHx, through site-directed mutagenesis of Aeromonas hydrophila enoyl Coenzyme A hydratase (PhaJ_Ah). Two amino acids (Leu-65 and Val-130) were selected as a substitutional site based on the structural information of PhaJ_Ah. The purified proteins from the wild-type enzyme and mutants were used to determine hydratase activities. Hydratase activities of four single-mutation enzymes were similar to those of the wild type PhaJ_Ah, while hydratase activities of two double-mutation enzymes were much lower. In addition, the mutated phaJ _Ah was individually co-transformed into E. coli BL21 (DE3) together with pFH21, which carried the PHA synthase (PhaC_Ah) gene from A. hydrophila. The recombinant E. coli harboring plasmid pETJ1 (L65A), pETJ2 (L65V) or plasmid pETJ3 (V130A) synthesized the enhanced 3HHx fractions of PHBHHx from dodecanoate, indicating that Leu-65 and Val-130 of PhaJ_Ah play an important role in determining the acyl chain length substrate specificity. The mutated PhaJ_Ah (L65A, L65V, or V130A) provided higher 3HHx precursors for PHA synthase, resulting in the enhanced 3HHx fractions of PHBHHx. It is possible to change the acyl chain length substrate specificity of PhaJ through site-directed mutagenesis and produce PHBHHx with a wider range of alterable monomer composition.     

Rapid and sensitive method for the detection of Aeromonas caviae and Aeromonas trota by polymerase chain reaction

A 16S rDNA-based polymerase chain reaction (PCR) method was developed for the detection of Aeromonas caviae and Aeromonas trota . These two species were identified from other Aeromonas spp. and closely related species by primers set (AER1 and AER2). The amplified product was 316 bp. The identity of the amplified product was confirmed by DNA–DNA hybridization. Two sets of primers (AER8 and AER9) were used for specific identification of Aer. caviae . Amplifying the 260 bp fragment of 16S rRNA gene region and digesting it with Alu I restriction enzyme, yielded 180- and 80-bp fragments. For PCR assay, template DNA was released by mixing equal volumes of homogenized seeded crab meat with Aer. caviae and Chelex 100 (6%) incubated for 10 min at 56°C followed by addition of an equal volume of 0·1% Triton-X-100 and boiled for 10 min. The detection limit was between 50 and 100 cells g⁻¹ of crab meat. This method is very rapid and obviates the need for DNA isolation from complex food matrices and is specific for detecting two Aeromonas species.     

Role of Genetic Redundancy in Polyhydroxyalkanoate (PHA) Polymerases in PHA Biosynthesis in Rhodospirillum rubrum

This study investigated the apparent genetic redundancy in the biosynthesis of polyhydroxyalkanoates (PHAs) in the Rhodospirillum rubrum genome revealed by the occurrence of three homologous PHA polymerase genes (phaC1, phaC2, and phaC3). In vitro biochemical assays established that each gene product encodes PHA polymerase. A series of single, double, and triple phaC deletion mutants were characterized with respect to PHA production and growth capabilities on acetate or hexanoate as the sole carbon source. These analyses establish that phaC2 contributes the major capacity to produce PHA, even though the PhaC2 protein is not the most efficient PHA polymerase biocatalyst. In contrast, phaC3 is an insignificant contributor to PHA productivity, and phaC1, the PHA polymerase situated in the PHA biosynthetic operon, plays a minor role in this capability, even though both of these genes encode PHA polymerases that are more efficient enzymes. These observations are consistent with the finding that PhaC1 and PhaC3 occur at undetectable levels, at least 10-fold lower than that of PhaC2. The monomers in the PHA polymer produced by these strains establish that PhaC2 is responsible for the incorporation of the C₅ and C₆ monomers. The in vitro characterizations indicate that heteromeric PHA polymerases composed of mixtures of different PhaC paralogs are more efficient catalysts, suggesting that these proteins form complexes. Finally, the physiological role of PHA accumulation in enhancing the fitness of R. rubrum was indicated by the relationship between PHA content and growth capabilities of the genetically manipulated strains that express different levels of the PHA polymer.