Computational identification of key residues regulating fluorescence emission in a red/green cyanobacteriochrome.

Cyanobacteriochromes (CBCRs) are linear tetrapyrrole bilin-binding photoreceptors of cyanobacteria that exhibit high spectral diversity, gaining attention in optogenetics and bioimaging applications. Several engineering studies on CBCRs were attempted, especially for designing near-infrared (NIR) fluorescent proteins with longer fluorescence wavelengths. However, despite continuous efforts, a key component regulating fluorescence emission property in CBCRs is still poorly understood. As a model system, we focused on red/green CBCR Slr1393g3, from the unicellular cyanobacterium Synechocystis sp. PCC 6803 to engineer Pr to get far-red light-emitting property. Energy profiling and pairwise structural comparison of Slr1393g3 variants effectively reveal the mutations that are critical to the fluorescence changes. H497 seems to play a key role in stabilizing the chromophore environment, especially the α3 helix, while H495, T499, and Q502 are potential key residues determining fluorescence emission peak wavelength. We also found that mutations of α2 and α4 helical regions are closely related to the chromophore binding stability and likely affect fluorescence properties. Taken together, our computational analysis suggests that the fluorescence of Slr1393g3 is mainly controlled by the stabilization of the chromophore binding pocket. The predicted key residues potentially regulating the fluorescence emission property of a red/green CBCR will be advantageous for designing improved NIR fluorescent protein when combined with in vitro molecular evolution approaches.

Domain-wise dissection of thermal stability enhancement in multidomain proteins.

Stability is critical for the proper functioning of all proteins. Optimization of protein thermostability is a key step in the development of industrial enzymes and biologics. Herein, we demonstrate that multidomain proteins can be stabilized significantly using domain-based engineering followed by the recombination of the optimized domains. Domain-level analysis of designed protein variants with similar structures but different thermal profiles showed that the independent enhancement of the thermostability of a constituent domain improves the overall stability of the whole multidomain protein. The crystal structure and AlphaFold-predicted model of the designed proteins via domain-recombination provided a molecular explanation for domain-based stepwise stabilization. Our study suggests that domain-based modular engineering can minimize the sequence space for calculations in computational design and experimental errors, thereby offering useful guidance for multidomain protein engineering.

Parasphingopyxis marina sp. nov. isolated from coastal seawater.

A Gram-stain-negative, aerobic, yellow-pigmented and non-motile rod-shaped bacterium, designated as GrpM-11T, was isolated from coastal seawater collected from the East Sea, Republic of Korea. Strain GrpM-11T could grow at 10-40 °C (optimum, 35 °C), at pH 5.5-9.5 (optimum, pH 7.0) and in the presence of 0-8 % (w/v) NaCl (optimum, 3-4 %). Cells hydrolysed aesculin, gelatin and casein, but could not reduce nitrate to nitrite. The 16S rRNA gene sequence analysis showed that this strain formed a distinct phylogenic lineage with Parasphingopyxis algicola ATAX6-5T (96.2 % sequence identity) and Parasphingopyxis lamellibrachiae DSM 26725T (96.2 % identity) and belonged to the genus Parasphingopyxis. The predominant isoprenoid quinone was ubiquinone-10. The polar lipid profile of strain GrpM-11T consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, sphingoglycolipid and three unknown glycolipids. Cellular fatty acid analysis indicated that summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c; 42.8 %), C16 : 0 (19.0 %), C18 : 1 ω7c 11-methyl (13.3 %) and C18 : 1 ω7c (8.0 %) were the major fatty acids. The DNA G+C content of strain GrpM-11T was 63.7 mol%. Through whole genome sequence comparisons, the digital DNA-DNA hybridization and average nucleotide identity values between strain GrpM-11T and two species of the genus Parasphingopyxis were revealed to be in the ranges of 19.0-22.0 % and 76.3-79.7 %, respectively. Based on the results of polyphasic analysis, strain GrpM-11T represents a novel species of the genus Parasphingopyxis, for which the name Parasphingopyxis marina sp. nov. is proposed. The type strain is GrpM-11T (KCCM 43343T=JCM 34665T).

Pelagicola marinus sp. nov. isolated from deep-sea water.

A Gram-stain-negative, strictly aerobic, non-motile, rod-shaped bacterial strain, designated strain DSW4-44T, was isolated from deep-sea water from the East Sea of Korea. The optimum temperature and pH for growth were 25 °C and pH 7.5. DSW4-44T grew with 1.0-6.0 % NaCl, having optimum growth at 3-4 %. Phylogenetic analyses based on 16S rRNA gene sequences indicated that DSW4-44T represented a member of the class Alphaproteobacteria and was most closely related to Pelagicola litoralis CL-ES2T and Pelagicola litorisediminis D1-W8T, with similarities of 96.5 and 95.6 %, respectively. The major fatty acid was summed feature 8 (comprising C18 : 1ω7c and/or C18 : 1ω6c). The polar lipids of DSW4-44T were phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, an unidentified aminolipid, two unidentified lipids and three unidentified phospholipids. The isoprenoid quinone was Q-10. The DNA G+C content of DSW4-44T was 54.3 mol%. On the basis of evidence from a polyphasic study, strain DSW4-44T represent a novel species of the genus Pelagicola, for which the name Pelagicola marinus sp. nov. is proposed. The type strain is DSW4-44T (=KCTC 62762T=KCCM 43261T=JCM 33637T).

Zhongshania marina sp. nov., isolated from deep-sea water.

A Gram-stain-negative, aerobic, catalase-positive, oxidase-positive, motile by a single polar flagellum, rod-shaped strain, designated DSW25-10T, was isolated from the deep-sea water of the East Sea, Republic of Korea. Strain DSW25-10Tgrew at 4-35 °C (optimum, 20-35 °C), at pH 5.5-9.0 (optimum, pH 7.0) and in the presence of 0-6.0 % NaCl (optimum, 0.5-2 %), and could assimilate valerate, but not assimilate d-mannitol and 3-hydroxy-butyrate. Comparative 16S rRNA gene sequence analysis showed that strain DSW25-10T belongs to the genus Zhongshania in the family Spongiibacteraceae and is most closely related to Zhongshania guokunii ZS6-22T, Zhongshania borealis CL-AS9T, Zhongshania aliphaticivorans SM-2T and Zhongshani antarctica ZS5-23T with a similarities of 97.1, 97.0, 96.7 and 96.6 %, respectively. The G+C content of genomic DNA was 49.1 mol% and the major respiratory quinone was ubiquinone-8. Phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol were identified as the major cellular polar lipids. The predominant cellular fatty acids in strain DSW25-10T were summed feature 3 (comprising C16 : 1ω7c and/or C16 : 1ω6c), summed feature 8 (comprising C18 : 1ω7c and/or C18 : 1ω6c), C16 : 0 and C17 : 1ω8c. The DNA-DNA relatedness values between strain DSW25-10T and related strains were clearly lower than 70 %. On the basis of evidence from a polyphasic analysis, strain DSW25-10T is proposed to represent a novel species, Zhongshania marina sp. nov. The type strain is DSW25-10T (=KCCM 43273T=JCM 17372T).

Pseudoxanthomonas putridarboris sp. nov. isolated from rotten tree.

A Gram-staining-negative, rod-shaped, motile bacterium, designated WD12T, was isolated from a rotten tree at Chungbuk National University, South Korea. WD12T grew optimally at 30-37 °C and pH 7.0-7.5 and could assimilate arbutin and potassium-5-ketogluconate. The major cellular fatty acid were iso-C16 : 0, C16 : 0, cyclo C17 : 0, iso-C15 : 0, summed features 3 (comprising C16 : 1ω7c/iso-C15 : 0 2-OH) and anteiso-C15 : 0. The major polar lipids consisted of phosphatidylethanolamine, diphosphatidylglycerol and phosphatidylglycerol. The major respiratory quinone was ubiquinone-8 (Q-8). The G+C content of the genomic DNA was 69.1 %. The results of phylogenetic and comparative analysis based on the 16S rRNA gene sequence indicated that WD12T formed a tight phylogenetic lineage with Pseudoxanthomonas mexicana AMX 26BT and Pseudoxanthomonas japonensis 12-3T of the the genus Pseudoxanthomonas in the family Xanthomonadaceae. Sequence similarity to other members of the genus Pseudoxanthomonasranged from 98.6 % (P. mexicana AMX 26BT) to 95.1 % (Pseudoxanthomonas taiwanensis CB-226T). DNA-DNA relatedness between WD12T and eight type strains of species of the genus Pseudoxanthomonasshowing more than 97 % 16S rRNA sequence similarity were 6±0-26±1 %. On the basis of the evidence from this polyphasic study, WD12T represents a novel species of the genus Pseudoxanthomonas, for which the name Pseudoxanthomonas putridarboris sp. nov. is proposed. The type strain is WD12T (=KACC 15045T=LMG 25968T).