A novel extracellular alkaline phosphatase/phosphodiesterase from the structural protein family PhoD that encoded by the genome sequence of the marine bacterium Cobetia amphilecti KMM 296 (CamPhoD) has been expressed in Escherichia coli cells. The calculated molecular weight, the number of amino acids, and the isoelectric point (pI) of the mature protein’s subunit are equal to 54832.98 Da, 492, and 5.08, respectively. The salt-tolerant, bimetal-dependent enzyme CamPhoD has a molecular weight of approximately 110 kDa in its native state. CamPhoD is activated by Co²⁺, Mg²⁺, Ca²⁺, or Fe³⁺ at a concentration of 2 mM and exhibits maximum activity in the presence of both Co²⁺ and Fe³⁺ ions in the incubation medium at pH 9.2. The exogenous ions, such as Zn²⁺, Cu²⁺, and Mn²⁺, as well as chelating agents EDTA and EGTA, do not have an appreciable effect on the CamPhoD activity. The temperature optimum for the CamPhoD activity is 45 °C. The enzyme catalyzes the cleavage of phosphate mono- and diester bonds in nucleotides, releasing inorganic phosphorus from p-nitrophenyl phosphate (pNPP) and guanosine 5′-triphosphate (GTP), as determined by the Chen method, with rate approximately 150- and 250-fold higher than those of bis-pNPP and 5′-pNP-TMP, respectively. The Michaelis–Menten constant (K_m), V_max, and efficiency (k_cat/K_m) of CamPhoD were 4.2 mM, 0.203 mM/min, and 7988.6 S⁻¹/mM; and 6.71 mM, 0.023 mM/min, and 1133.0 S⁻¹/mM for pNPP and bis-pNPP as the chromogenic substrates, respectively. Among the 3D structures currently available, in this study we found only the low identical structure of the Bacillus subtilis enzyme as a homologous template for modeling CamPhoD, with a new architecture of the phosphatase active site containing Fe³⁺ and two Ca²⁺ ions. It is evident that the marine bacterial phosphatase/phosphidiesterase CamPhoD is a new structural member of the PhoD family.

A highly active alkaline phosphatase (ALP) of the protein structural family PhoA, from a mussel gut-associated strain of the marine bacterium Cobetia amphilecti KMM 296 (CmAP), was found to effectively dephosphorylate lipopolysaccharides (LPS). Therefore, the aim of this work was to perform a comprehensive bioinformatics analysis of the structure, and to suggest the physiological role of this enzyme in marine bacteria of the genus Cobetia. A scrutiny of the CmAP-like sequences in 36 available Cobetia genomes revealed nine homologues intrinsic to the subspecies C. amphilecti, whereas PhoA of a distant relative Cobetia crustatorum JO1^T carried an inactive mutation. However, phylogenetic analysis of all available Cobetia ALP sequences showed that each strain of the genus Cobetia possesses several ALP variants, mostly the genes encoding for PhoD and PhoX families. The C. amphilecti strains have a complete set of four ALP families’ genes, namely: PhoA, PafA, PhoX, and two PhoD structures. The Cobetia marina species is distinguished by the presence of only three PhoX and PhoD genes. The Cobetia PhoA proteins are clustered together with the human and squid LPS-detoxifying enzymes. In addition, the predicted PhoA biosynthesis gene cluster suggests its involvement in the control of cellular redox balance, homeostasis, and cell cycle. Apparently, the variety of ALPs in Cobetia spp. indicates significant adaptability to phosphorus-replete and depleted environments and a notable organophosphate destructor in eco-niches from which they once emerged, including Zostera spp. The ALP clusterization and degree of similarity of the genus-specific biosynthetic genes encoding for ectoine and polyketide cluster T1PKS, responsible for sulfated extracellular polysaccharide synthesis, coincide with a new whole genome-based taxonomic classification of the genus Cobetia. The Cobetia strains and their ALPs are suggested to be adaptable for use in agriculture, biotechnology and biomedicine.

A strictly aerobic, Gram-stain-negative, rod-shaped, and motile bacterium, designated strain KMM 296, isolated from the coelomic fluid of the mussel Crenomytilus grayanus, was investigated in detail due to its ability to produce a highly active alkaline phosphatase CmAP of the structural family PhoA. A previous taxonomic study allocated the strain to the species Cobetia marina, a member of the family Halomonadaceae of the class Gammaproteobacteria. However, 16S rRNA gene sequencing showed KMM 296’s relatedness to Cobetia amphilecti NRIC 0815^T. The isolate grew with 0.5–19% NaCl at 4–42 °C and hydrolyzed Tweens 20 and 40 and L-tyrosine. The DNA G+C content was 62.5 mol%. The prevalent fatty acids were C_18:1 ω7c, C_12:0 3-OH, C_18:1 ω7c, C_12:0, and C_17:0 cyclo. The polar lipid profile was characterized by the presence of phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, and also an unidentified aminolipid, phospholipid, and a few unidentified lipids. The major respiratory quinone was Q-8. According to phylogenomic and chemotaxonomic evidence, and the nearest neighbors, the strain KMM 296 represents a member of the species C. amphilecti. The genome-based analysis of C. amphilecti NRIC 0815^T and C. litoralis NRIC 0814^T showed their belonging to a single species. In addition, the high similarity between the C. pacifica NRIC 0813^T and C. marina LMG 2217^T genomes suggests their affiliation to one species. Based on the rules of priority, C. litoralis should be reclassified as a later heterotypic synonym of C. amphilecti, and C. pacifica is a later heterotypic synonym of C. marina. The emended descriptions of the species C. amphilecti and C. marina are also proposed.

Marine bacteria of the genus Cobetia, which are promising sources of unique enzymes and secondary metabolites, were found to be complicatedly identified both by phenotypic indicators due to their ecophysiology diversity and 16S rRNA sequences because of their high homology. Therefore, searching for the additional methods for the species identification of Cobetia isolates is significant. The species-specific coding sequences for the enzymes of each functional category and different structural families were applied as additional molecular markers. The 13 closely related Cobetia isolates, collected in the Pacific Ocean from various habitats, were differentiated by the species-specific PCR patterns. An alkaline phosphatase PhoA seems to be a highly specific marker for C. amphilecti. However, the issue of C. amphilecti and C. litoralis, as well as C. marina and C. pacifica, belonging to the same or different species remains open.

A new member of the DegP-type periplasmic serine endoproteases of the S1C family from the marine bacterium Cobetia amphilecti KMM 296 (CamSP) was expressed in Escherichia coli cells. The calculated molecular weight, number of amino acids, and isoelectric point (pI) of the mature protein CamSP are 69.957 kDa, 666, and 4.84, respectively. The proteolytic activity of the purified recombinant protease CamSP was 2369.4 and 1550.9 U/mg with the use of 1% bovine serum albumin (BSA) and casein as the substrates, respectively. The enzyme CamSP exhibited maximum activity at pH 6.0–6.2, while it was stable over a wide pH range from 5.8 to 8.5. The optimal temperature for the CamSP protease activity was 50 °C. The enzyme required NaCl or KCl at concentrations of 0.3 and 0.5 M, respectively, for its maximum activity. The Michaelis constant (K_m) and V_max for BSA were determined to be 41.7 µg/mL and 0.036 µg/mL min⁻¹, respectively. The metal ions Zn²⁺, Cu²⁺, Mn²⁺, Li²⁺, Mg²⁺, and Ca²⁺ slightly activated CamSP, while the addition of CoCl₂ to the incubation mixture resulted in a twofold increase in its protease activity. Ethanol, isopropanol, glycerol, and Triton-X-100 increased the activity of CamSP from two- to four-times. The protease CamSP effectively degraded the wheat flour proteins but had no proteolytic activity towards soybean, corn, and the synthetic substrates, α-benzoyl-Arg-p-nitroanilide (BAPNA) and N-Succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenylalanine 4-nitroanilide (SAPNA).

Advances in the computational annotation of genomes and the predictive potential of current metabolic models, based on more than thousands of experimental phenotypes, allow them to be applied to identify the diversity of metabolic pathways at the level of ecophysiology differentiation within taxa and to predict phenotypes, secondary metabolites, host-associated interactions, survivability, and biochemical productivity under proposed environmental conditions. The significantly distinctive phenotypes of members of the marine bacterial species Pseudoalteromonas distincta and an inability to use common molecular markers make their identification within the genus Pseudoalteromonas and prediction of their biotechnology potential impossible without genome-scale analysis and metabolic reconstruction. A new strain, KMM 6257, of a carotenoid-like phenotype, isolated from a deep-habituating starfish, emended the description of P. distincta, particularly in the temperature growth range from 4 to 37 °C. The taxonomic status of all available closely related species was elucidated by phylogenomics. P. distincta possesses putative methylerythritol phosphate pathway II and 4,4′-diapolycopenedioate biosynthesis, related to C30 carotenoids, and their functional analogues, aryl polyene biosynthetic gene clusters (BGC). However, the yellow-orange pigmentation phenotypes in some strains coincide with the presence of a hybrid BGC encoding for aryl polyene esterified with resorcinol. The alginate degradation and glycosylated immunosuppressant production, similar to brasilicardin, streptorubin, and nucleocidines, are the common predicted features. Starch, agar, carrageenan, xylose, lignin-derived compound degradation, polysaccharide, folate, and cobalamin biosynthesis are all strain-specific.