Jeotgalibacillus aurantiacus sp. nov., a novel orange-pigmented species with a carotenoid biosynthetic gene cluster, isolated from wetland soil.

A Gram-stain-positive, orange-pigmented, rod-shaped and flagellated bacterial strain T12T was isolated from wetland soil in Kunyu Mountain Wetland in Yantai, China. The strain was able to grow at 15-40 °C (optimum 37 °C), at 0.0-9.0% NaCl (optimum 2%, w/v) and at pH 5.5-9.0 (optimum 8.5). A phylogenetic analysis based on the 16S rRNA gene sequence indicated that strain T12T is a member of the family Planococcaceae, sharing 97.6% and 97.1% sequence similarity with the type strains of Jeotgalibacillus salarius and Jeotgalibacillus marinus, respectively. Genome-based analyses revealed a genome size of 3,506,682 bp and a DNA G + C content of 43.7%. Besides, the genome sequence led to 55.0-74.6% average amino acid identity values and 67.8-74.7% average nucleotide identity values between strain T12T and the current closest relatives. Digital DNA-DNA hybridization of strain T12T with the type strains of Jeotgalibacillus proteolyticus and J. marinus demonstrated 19.0% and 20.3% relatedness, respectively. The chemotaxonomic analysis showed that the sole quinone was MK-7. The predominant cellular fatty acids were iso-C15:0, anteiso-C15:0, C16:1ω7c alcohol and iso-C14:0. The polar lipids consisted of an unidentified aminolipid, phosphatidylglycerol, diphosphatidylglycerol and two unidentified phospholipids. Based on the polyphasic characterization, strain T12T is considered to represent a novel species, for which the name Jeotgalibacillus aurantiacus sp. nov. is proposed. The type strain is T12T (= KCTC 43296 T = MCCC 1K07171T).

Insights into the Influence of Signal Peptide on the Enzymatic Properties of Alginate Lyase AlyI1 with Removal Effect on Pseudomonas aeruginosa Biofilm.

Most reports on signal peptides focus on their ability to affect the normal folding of proteins, thereby affecting their secreted expression, while few studies on its effects on enzymatic properties were published. Therefore, biochemical characterization and comparison of alginate lyase rALYI1/rALYI1-1 (rALYI1: without signal peptides; rALYI1-1:with signal peptides) were conducted in our study, and the results showed that the signal peptide affected the biochemical properties, especially in temperature and pH. rALYI1 (32.15 kDa) belonging to polysaccharide lyase family 7 was cloned from sea-cucumber-gut bacterium Tamlana sp. I1. The optimum temperature of both rALYI1 and rALYI1-1 was 40 °C, but the former had a wider optimum temperature range and better thermal stability. The optimum pH of rALYI1 and rALYI1-1 were 7.6 and 8.6, respectively. The former was more stable and acid resistant. Noticeably, rALYI1 was a salt-activated enzyme and displayed remarkable salt tolerance. Alginate, an essential polysaccharide in algae and Pseudomonas aeruginosa biofilms, is composed of α-L-guluronate and ß-D-mannuronate. It is also found in our study that rALYI1 is also effective in removing mature biofilms compared with controls. In conclusion, the signal peptide affects several biochemical properties of the enzyme, and alginate lyase rALYI1 may be an effective method for inhibiting biofilm formation of Pseudomonas aeruginosa.

Oceaniglobus trochenteri sp. nov., isolated from the gut microflora of top shell (Trochus maculatus Linnaeus).

A Gram-stain negative, non-flagellated, beige-pigmented, circular, catalase-positive, oxidase-positive bacterium, designated G4T, was isolated from gut microflora of top shell (Trochus maculatus Linnaeus) collected from Diwanggong market, Weihai, People's Republic of China. The novel isolate was able to grow at 4-42 °C (optimum 25-33 °C), pH 7.0-9.0 (optimum 6.5-7.0) and with 0.0-11.0% NaCl (optimum 2.0-3.0%, w/v). Analysis of 16S rRNA gene sequence revealed that strain G4T shared the highest 16S rRNA gene sequence similarities with Oceaniglobus ichthyenteri YLY08T (96.6%), followed by Oceaniglobus indicus 1-19bT (95.3%). The genome of strain G4T, with 32 assembled contigs, was 4.5 Mb long with a G+C content of 65.3 mol%. DNA-DNA hybridization values of the isolate against the closely related type strains were far below the 70% limit for species delineation. The average amino acid identity, average nucleotide identity and digital DNA-DNA genome hybridization relatedness between strain G4T and the closely related members of the genus Oceaniglobus, Oceaniglobus indicus1-19bT and Oceaniglobus ichthyenteri YLY08T were 71.3, 76.4 and 20.0%, and 75.0, 76.3 and 19.4%. The major cellular fatty acid was summed feature 8 (C18:1ω7c and/or C18:1ω6c). The sole respiratory quinone was Q-10. The polar lipid profile consisted of diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol and phosphatidyldimethylethanolamine. The results of phenotypical, phylogenetic and biochemical analyses indicated that strain G4T represents a novel species in genus Oceaniglobus within the family Rhodobacteraceae, for which the name Oceaniglobus trochenteri sp. nov. is proposed. The type strain is G4T (= MCCC 1K04356T = KCTC 82506T).

Pseudaestuariivita rosea sp. nov., isolated from Acmaea sp., a marine mollusk.

A Gram-stain-negative, pink-pigmented, aerobic, non-motile and rod-shaped bacterium, designated as strain H15T, was isolated from Acmaea sp., collected from Weihai, Shandong Province, China. The novel isolate was able to grow at 4-37 °C (optimum 33 °C), pH 5.5-9.0 (optimum 7.0) and with 0.0-7.0% NaCl (optimum 4%, w/v). Phylogenetic analysis based on 16S rRNA gene sequence comparisons revealed that the strain belonged to the family Rhodobacteraceae and was associated to the type strain of Pseudaestuariivita atlantica (96.7%). Genome analysis showed that the genome size was 3,893,398 bp and the DNA G + C content obtained from the draft genome sequence was 56.7%. The secondary metabolites predicated that the strain H15T contained one cluster of lasso peptide, one cluster of bacteriocin, two clusters of terpene production, two clusters of homoserine lactone and one cluster of beta lactone. The average amino acid identity, average nucleotide identity and digital DNA-DNA hybridization values between genome sequences of strain H15T and all the related strains compared were lower than 63.1, 72.0 and 19.7%, respectively. Based on the analysis of chemical components, the predominant cellular fatty acids were summed featured 8 (C18:1ω7c/ω6c, 46.1%), C20:1 ω7c (17.1%), the major polar lipids contained phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine and an unidentified lipid and the predominant menaquinone was Q10. Therefore, the combined chemotaxonomic, phenotypic and phylogenetic data indicated that the strain was considered to represent a novel species of the genus Pseudaestuariivita and the name Pseudaestuariivita rosea sp. nov. was proposed for strain H15T (MCCC 1K04420T = KCTC 82505T).

Williamsia soli sp. nov., an actinobacterium isolated from soil at a thermal power plant in Yantai, China.

Strain C17T, a novel strain belonging to the phylum Actinobacteria, was isolated from a thermal power plant in Yantai, Shandong Province, China. Cells of strain C17T were Gram stain positive, aerobic, pink, non-motile and round with neat edges, showing optimum growth at 28 °C. Phylogenetically, strain C17T was a member of the class Actinobacteria, order Mycobacteriales, family Gordoniaceae. Phylogenetic analysis based on 16S rRNA gene sequence comparisons revealed that the related strains were Williamsia faeni JCM 17784 T and Williamsia limnetica KCTC 19981 T with pairwise sequence similarity of 98.5% for both strains. According to the draft genome sequence, the DNA G + C content was 64.7%. The average amino acid identity (AAI), average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between genome sequences of strain C17T and the closest type strain W. faeni JCM 17784 T were 77.5, 77.9, and 20.7%, respectively. Predominant fatty acids were C16:0 (31.7%) and C18:1ω9c (26.8%). The major menaquinone was MK-9. The diagnostic phospholipids were phosphatidylethanolamine (PE), diphosphatidylglycerol (DPG), and phosphatidylinositol (PI). Therefore, the combined phenotypic, chemotaxonomic and phylogenetic data indicated that strain C17T was considered to represent a novel species of the genus Williamsia. Williamsia soli sp. nov. was proposed for strain C17T (= KCTC 49567 T = MCCC 1K04355T).

Echinicola salinicaeni sp. nov., a novel bacterium isolated from saltern mud.

A novel gram-negative, aerobic, pink, motile, gliding, rod-shaped bacterium, designated P51T, was isolated from saline silt samples in Yantai, China. It was able to grow at 4-42 °C (optimum 33 °C), pH 4.0-9.0 (optimum 7.0), and in 0-11.0% NaCl (optimum 4.0%, w/v). It grew at 4 °C, which was lower than the minimum temperature for related strains. The genome consisted of 4111 genes with a total length of 5 139 782 bp. The 16S rRNA gene sequence analysis indicated that strain P51T was a member of the genus Echinicola and most closely related to 'Echinicola shivajiensis'. A genome analysis identified genes encoding proteins associated with carbon source utilisation, and the carotenoid biosynthesis and ß-lactam resistance pathways. Strain P51T shared an average nucleotide identity value below 84.7%, an average amino acid identity value between 70.8 and 89.3%, and a digital DNA-DNA hybridisation identity of between 17.9-28.2% with closely related type strains within the genus Echinicola. The sole menaquinone was MK-7, and the major fatty acids were iso-C15:0, summed feature 3 (C16:1ω7c and/or C16:1ω6c), summed feature 4 (anteiso-C17:1 B and/or iso-C17:1 I), and summed feature 9 (iso-C17:1ω9c and/or 10-methyl C16:0). The polar lipids included one phosphatidylethanolamine, one unidentified aminophospholipid, one unidentified phospholipid, three unidentified aminolipids, and one unknown lipid. The phenotypic, chemotaxonomic, and phylogenetic analyses suggest that strain P51T is a novel species of the genus Echinicola, for which the name Echinicola salinicaeni sp. nov. is proposed. The type strain was P51T (KCTC 82513T = MCCC 1K04413T).

The two-step strategy for enhancing the specific activity and thermostability of alginate lyase AlyG2 with mechanism for improved thermostability.

To overcome the trade-off challenge encountered in the engineering of alginate lyase AlyG2 from Seonamhaeicola algicola Gy8T and to expand its potential industrial applications, we devised a two-step strategy encompassing activity enhancement followed by thermal stability engineering. To enhance the specific activity of efficient AlyG2, we strategically substituted residues with bulky steric hindrance proximal to the active pocket with glycine or alanine. This led to the generation of three promising positive mutants, with particular emphasis on the T91S mutant, exhibiting a 1.91-fold specific activity compared to the wild type. To mitigate the poor thermal stability of T91S, mutants with negative ΔΔG values in the thermal flexibility region were screened out. Notably, the S72Ya mutant not only displayed 17.96 % further increase in specific activity but also exhibited improved stability compared to T91S, manifesting as a remarkable 30.97 % increase in relative activity following a 1-hour incubation at 42 °C. Furthermore, enhanced kinetic stability was observed. To gain deeper insights into the mechanism underlying the enhanced thermostability of the S72Ya mutant, we conducted molecular dynamics simulations, principal component analysis (PCA), dynamic cross-correlation map (DCCM), and free energy landscape (FEL) analysis. The results unveiled a reduction in the flexibility of the surface loop, a stronger correlation dynamic and a narrower motion subspace in S72Ya system, along with the formation of more stable hydrogen bonds. Collectively, our findings suggest amino acids substitutions resulting in smaller side chains proximate to the active site can positively impact enzyme activity, while reducing the flexibility of surface loops emerges as a pivotal factor in conferring thermal stability. These insights offer valuable guidance and a framework for the engineering of other enzyme types.