In-Depth Genome Characterization and Pan-Genome Analysis of Strain KMM 296, a Producer of Highly Active Alkaline Phosphatase; Proposal for the Reclassification of Cobetia litoralis and Cobetia pacifica as the Later Heterotypic Synonyms of Cobetia amphilecti and Cobetia marina, and Emended Description of the Species Cobetia amphilecti and Cobetia marina.

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 0815T. 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 C18:1 ω7c, C12:0 3-OH, C18:1 ω7c, C12:0, and C17:0 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 0815T and C. litoralis NRIC 0814T showed their belonging to a single species. In addition, the high similarity between the C. pacifica NRIC 0813T and C. marina LMG 2217T 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.

Rhodoalgimonas zhirmunskyi gen. nov., sp. nov., a Marine Alphaproteobacterium Isolated from the Pacific Red Alga Ahnfeltia tobuchiensis: Phenotypic Characterization and Pan-Genome Analysis.

A novel Gram-staining negative, strictly aerobic, rod-shaped, and non-motile bacterium, designated strain 10Alg 79T, was isolated from the red alga Ahnfeltia tobuchiensis. A phylogenetic analysis based on 16S rRNA gene sequences placed the novel strain within the family Roseobacteraceae, class Alphaproteobacteria, phylum Pseudomonadota, where the nearest neighbor was Shimia sediminis ZQ172T (97.33% of identity). However, a phylogenomic study clearly showed that strain 10Alg 79T forms a distinct evolutionary lineage at the genus level within the family Roseobacteraceae combining with strains Aquicoccus porphyridii L1 8-17T, Marimonas arenosa KCTC 52189T, and Lentibacter algarum DSM 24677T. The ANI, AAI, and dDDH values between them were 75.63-78.15%, 67.41-73.08%, and 18.8-19.8%, respectively. The genome comprises 3,754,741 bp with a DNA GC content of 62.1 mol%. The prevalent fatty acids of strain 10Alg 79T were C18:1 ω7c and C16:0. The polar lipid profile consisted of phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, an unidentified aminolipid, an unidentified phospholipid and an unidentified lipid. A pan-genome analysis showed that the unique part of the 10Alg 79T genome consists of 13 genus-specific clusters and 413 singletons. The annotated singletons were more often related to transport protein systems, transcriptional regulators, and enzymes. A functional annotation of the draft genome sequence revealed that this bacterium could be a source of a new phosphorylase, which may be used for phosphoglycoside synthesis. A combination of the genotypic and phenotypic data showed that the bacterial isolate represents a novel species and a novel genus, for which the name Rhodoalgimonas zhirmunskyi gen. nov., sp. nov. is proposed. The type strain is 10Alg 79T (=KCTC 72611T = KMM 6723T).

Expression of membrane beta-barrel protein in E. coli at low temperatures: Structure of Yersinia pseudotuberculosis OmpF porin inclusion bodies.

The recombinant OmpF porin of Yersinia pseudotuberculosis as a model of transmembrane protein of the ß-barrel structural family was used to study low growth temperature effect on the structure of the produced inclusion bodies (IBs). This porin showed a very low expression level in E. coli at a growth temperature below optimal 37 °C. The introduction of a N-terminal hexahistidine tag into the mature porin molecule significantly increased the biosynthesis of the protein at low cultivation temperatures. The recombinant His-tagged porin (rOmpF-His) was expressed in E. coli at 30 and 18 °C as inclusion bodies (IB-30 and IB-18). The properties and structural organization of IBs, as well as the structure of rOmpF-His solubilized from the IBs with urea and SDS, were studied using turbidimetry, electron microscopy, dynamic light scattering, optical spectroscopy, and amyloid-specific dyes. IB-18, in comparison with IB-30, has a higher solubility in denaturants, suggesting a difference between IBs in the conformation of the associated polypeptide chains. The spectroscopic analysis revealed that rOmpF-His IBs have a high content of secondary structure with a tertiary-structure elements, including a native-like conformation, the proportion of which in IB-18 is higher than in IB-30. Solubilization of the porin from IBs is accompanied by a modification of its secondary structure. The studied IBs also contain amyloid-like structures. The results obtained in this study expand our knowledge of the structural organization of IBs formed by proteins of different structural classes and also have a contribution into the new approaches development of producing functionally active recombinant membrane proteins.

Studies on the Structure and Properties of Membrane Phospholipase A1 Inclusion Bodies Formed at Low Growth Temperatures Using GFP Fusion Strategy.

The effect of cultivation temperatures (37, 26, and 18 °C) on the conformational quality of Yersinia pseudotuberculosis phospholipase A1 (PldA) in inclusion bodies (IBs) was studied using green fluorescent protein (GFP) as a folding reporter. GFP was fused to the C-terminus of PldA to form the PldA-GFP chimeric protein. It was found that the maximum level of fluorescence and expression of the chimeric protein is observed in cells grown at 18 °C, while at 37 °C no formation of fluorescently active forms of PldA-GFP occurs. The size, stability in denaturant solutions, and enzymatic and biological activity of PldA-GFP IBs expressed at 18 °C, as well as the secondary structure and arrangement of protein molecules inside the IBs, were studied. Solubilization of the chimeric protein from IBs in urea and SDS is accompanied by its denaturation. The obtained data show the structural heterogeneity of PldA-GFP IBs. It can be assumed that compactly packed, properly folded, proteolytic resistant, and structurally less organized, susceptible to proteolysis polypeptides can coexist in PldA-GFP IBs. The use of GFP as a fusion partner improves the conformational quality of PldA, but negatively affects its enzymatic activity. The PldA-GFP IBs are not toxic to eukaryotic cells and have the property to penetrate neuroblastoma cells. Data presented in the work show that the GFP-marker can be useful not only as target protein folding indicator, but also as a tool for studying the molecular organization of IBs, their morphology, and localization in E. coli, as well as for visualization of IBs interactions with eukaryotic cells.