Algoriphagus algorifonticola sp. nov., a marine bacterium isolated from cold spring area of South China Sea.

A Gram-stain-negative, aerobic, non-motile, short-rod-shaped bacterium, designated strain hg1T, was isolated from marine sediment within the cold spring area of South China Sea and subjected to a polyphasic taxonomic investigation. Colonies were circular and 1.0-2.0 mm in diameter, coral in colour, convex and smooth after growth on marine agar at 28 °C for 3 days. Strain hg1T was found to grow at 4-40 °C (optimum, 35-37 °C), at pH 6.5-9.0 (optimum, pH 7.5) and with 0-8 % (w/v) NaCl (optimum, 1.5-2 %). Chemotaxonomic analysis showed the sole respiratory quinone was MK-7, and the principal fatty acids are iso-C15 : 0, summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c), and iso-C16 : 0. The major polar lipids are phosphatidylethanolamine, an unidentified phospholipid and five unidentified glycolipids. The DNA G+C content of strain hg1T was 39.6 mol% based on the genome sequence. The comparison of 16S rRNA gene sequence similarities showed that hg1T was closely related to Algoriphagus ornithinivorans DSM 15282T (98.6 % sequence similarity), Algoriphagus zhangzhouensis MCCC 1F01099T (97.9 %) and Algoriphagus vanfongensis DSM 17529T (97.2 %); it exhibited 97.0 % or less sequence similarity to the type strains of other species of the genus Algoriphagus with validly published names. Phylogenetic trees reconstructed with the neighbour-joining, maximum-parsimony and maximum-likelihood methods based on 16S rRNA gene sequences showed that strain hg1T constituted a separate branch with A. ornithinivorans, A. zhangzhouensis, A. vanfongensis in a clade of the genus Algoriphagus. OrthoANI values between strain hg1T and A. ornithinivorans, A. zhangzhouensis and A. vanfongensis were 94.3, 74.1, 73.2 %, respectively, and in silico DNA-DNA hybridization values were 56.2, 18.5 and 18.3 %, respectively. Differential phenotypic properties, together with phylogenetic distinctiveness, demonstrated that strain hg1T is clearly distinct from recognized species of genus Algoriphagus. On the basis of these features, we propose that strain hg1T (=MCCC 1K03570T=KCTC 72111T) represents a novel species of the genus Algoriphagus with the name Algoriphagus algorifonticola sp. nov.

Overproduction of gentamicin B in industrial strain Micromonospora echinospora CCTCC M 2018898 by cloning of the missing genes genR and genS.

In pharmaceutical industry, isepamicin is mainly manufactured from gentamicin B, which is produced by Micromonospora echinospora as a minor component of the gentamicin complex. Improvement of gentamicin B production through metabolic engineering is therefore important to satisfy the increasing demand for isepamicin. We hypothesized that gentamicin B was generated from gentamicin JI-20A via deamination of the C2' amino group. Using kanJ and kanK as the gene probes, we identified the putative deamination-related genes, genR and genS, through genome mining of the gentamicin B producing strain M. echinospora CCTCC M 2018898. Interestingly, genR and genS constitute a gene cassette located approximately 28.7 kb away from the gentamicin gene cluster. Gene knockout of genR and genS almost abolished the production of gentamicin B in the mutant strain, suggesting that these two genes, which are responsible for the last steps in gentamicin B biosynthesis, constitute the missing part of the known gentamicin biosynthetic pathway. Based on these finding, we successfully constructed a gentamicin B high-yielding strain (798 mg/L), in which an overexpression cassette of genR and genS was introduced. Our work fills the missing piece to solve the puzzle of gentamicin B biosynthesis and may inspire future metabolic engineering efforts to generate gentamycin B high-yielding strains that could eventually satisfy the need for industrial manufacturing of isepamicin.

4-Hydroxyphenylpyruvate Dioxygenase Thermolability Is Responsible for Temperature-Dependent Melanogenesis in Aeromonas salmonicida subsp. salmonicida.

Aeromonas salmonicida subsp. salmonicida is a major pathogen affecting fisheries worldwide and is a well-known pigmented member of the Aeromonas genus. This subspecies produces melanin at ≤22°C. However, melanogenesis decreases as the culture temperature increases and is completely suppressed at 30°C to 35°C, while bacterial growth is unaffected. The mechanism and biological significance of this temperature-dependent melanogenesis remain unclear. Heterologous expression of an A. salmonicida subsp. salmonicida 4-hydroxyphenylpyruvate dioxygenase (HppD), the most critical enzyme in the homogentisic acid (HGA)-melanin synthesis pathway, results in thermosensitive pigmentation in Escherichia coli, suggesting that HppD plays a key role in this process. In this study, we demonstrated that the thermolability of HppD is responsible for the temperature-dependent melanization of A. salmonicida subsp. salmonicida Substitutions of three residues, S18T, P103Q, and L119P, in A. salmonicida subsp. salmonicida HppD increased the thermostability of this enzyme and resulted in temperature-independent melanogenesis. Moreover, the replacement of the corresponding residues in HppD from Aeromonas media strain WS, which forms pigment independent of temperature, with those of A. salmonicida subsp. salmonicida HppD resulted in thermosensitive melanogenesis. A structural analysis suggested that mutations at these sites, especially at position P103, strengthen the secondary structure of HppD and greatly improve its thermal stability. Additionally, we found that the HppD sequences of all A. salmonicida subsp. salmonicida isolates were identical and that two of the three residues were clearly distinct from those of other Aeromonas strains.IMPORTANCEAeromonas salmonicida subsp. salmonicida is the causative agent of furunculosis, a bacterial septicemia of cold-water fish of the Salmonidae family. Although other Aeromonas species can produce melanin, A. salmonicida subsp. salmonicida is the only member of this genus that has been reported to exhibit temperature-dependent melanization. Here, we demonstrated that thermosensitive melanogenesis in A. salmonicida subsp. salmonicida strains is due to the thermolability of 4-hydroxyphenylpyruvate dioxygenase (HppD). Additionally, we confirmed that this thermolabile HppD exhibited higher activity at low temperatures than its mesophilic homologues, suggesting this as an adaptive strategy of this enzyme to the psychrophilic lifestyle of A. salmonicida subsp. salmonicida The strictly conserved hppD sequences among A. salmonicida subsp. salmonicida isolates and the specific possession of P103 and L119 residues could be used as a reference for the identification of A. salmonicida subsp. salmonicida isolates.

Identification of YfiH and the Catalase CatA As Polyphenol Oxidases of Aeromonas media and CatA as a Regulator of Pigmentation by Its Peroxyl Radical Scavenging Capacity.

Pyomelanin is the major constituent of pigment in melanogenic Aeromonas strains of bacteria. However, eumelanin, synthesized from tyrosine via L-DOPA and polyphenol oxidases (PPOs), may also be present in this genus since L-DOPA is frequently detected in culture fluids of several species. To address this question, we used a deletion mutant of Aeromonas media strain WS, in which pyomelanin synthesis is completely blocked under normal culture conditions. When tyrosine was supplied to the medium, we observed residual melanin accumulation, which we interpret as evidence for existence of the DOPA-melanin pathway. We traced enzymatic activity in this bacterium using native-polyacrylamide gel electrophoresis. Two PPOs: YfiH, a laccase-like protein, and CatA, a catalase, were identified. However, neither protein was critical for the residual pigmentation in pyomelanin-deficient mutant. We speculate that eumelanin synthesis may require other unknown enzymes. Deletion of yfiH did not affect pigmentation in A. media strain WS, while deletion of the CatA-encoding gene katE resulted in a reduction of melanin accumulation, but it started 9 h earlier than in the wild-type. Since catalases regulate reactive oxygen species levels during melanogenesis, we speculated that CatA affects pigmentation through its peroxyl radical scavenging capacity. Consistent with this, expression of the catalases Hpi or Hpii from Escherichia coli in the katE deletion strain of A. media strain WS restored pigmentation to the wild-type level. Hpi and Hpii also exhibited PPO activity, suggesting that catalase may represent a new class of PPOs.

Identification and molecular characterization of the homogentisate pathway responsible for pyomelanin production, the major melanin constituents in Aeromonas media WS.

The pigmentation of many Aeromonas species has been thought to be due to the production of a L-DOPA (L-3,4-dihydroxyphenylalanine) based melanin. However, in this study we found that although L-DOPA synthesis occurs in the high-melanin-yielding Aeromonas media strain WS, it plays a minor, if any, role in pigmentation. Instead, the pigmentation of A. media strain WS is due to the production of pyomelanin through HGA (homogentisate). Gene products of phhA (encodes phenylalanine hydroxylase), tyrB and aspC (both encode aromatic amino acid aminotransferase), and hppD (encodes 4-hydroxyphenylpyruvate dioxygenase) constitute a linear pathway of converting phenylalanine to HGA and disruption of any one of these genes impairs or blocks pigmentation of A. media strain WS. This HGA biosynthesis pathway is widely distributed in Aeromonas, but HGA is only detectable in the cultures of pigmented Aeromonas species. Heterologous expression of HppD from both pigmented and non-pigmented Aeromonas species in E. coli leads to the production of pyomelanin and thus pigmentation, suggesting that most Aeromonas species have the critical enzymes to produce pyomelanin through HGA. Taken together, we have identified a widely conserved biosynthesis pathway of HGA based pyomelanin in Aeromonas that may be responsible for pigmentation of many Aeromonas species.

Draft genome sequence of high-melanin-yielding Aeromonas media strain WS.

We sequenced the genome of the high-melanin-yielding Aeromonas media strain WS and then analyzed genes potentially involved in melanin formation. The 4.2-Mb draft genome carries multiple genes responsible for pyomelanin synthesis and other candidate genes identified in our separate study, which have no homolog in other strains of Aeromonas species.

Molecular and biochemical characterization of a distinct tyrosinase involved in melanin production from Aeromonas media.

A new tyrosinase was isolated from Aeromonas media strain WS and purified to homogeneity. The purified tyrosinase, termed TyrA, had a molecular mass of 58 kDa and an isoelectric point of 4.90. It exhibited optimal monophenol and diphenol oxidase activities under basic conditions (pH>8.0). TyrA had a relatively higher affinity to diphenol substrate L-dihydroxyphenylalanine (L-dopa) than many other tyrosinases. EDTA or glutathione notably inhibited the enzymatic activities of TyrA, whereas Triton X-100 and SDS activated them. The full-length TyrA gene was cloned, and it encodes a 518 amino acid protein with little similarities to other reported tyrosinases. However, the purified recombinant TyrA expressed in Escherichia coli demonstrated tyrosinase activity. These results suggest that TyrA is the first reported distinct tyrosinase involved in melanin production in the genus Aeromonas.