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      Hanstruepera marina sp. nov. and Hanstruepera flava sp. nov., two novel species in the family Flavobacteriaceae isolated by a modified in situ cultivation technique from marine sediment

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          Abstract

          A modified in situ cultivation technique was developed and applied to resource mining of uncultured microbes from marine sediments of Meishan Island in the East China Sea. Two novel strains NBU2968 T and NBU2984 T were isolated by this method but not standard Petri dish, which indicated the modified technique was more effective compared to conventional approaches for isolating uncultured microbes and could be popularized and applied to other aquatic environments. The two novel strains were identified by the polyphasic taxonomic approach. Cells of both strains were observed to be Gram-staining-negative, rod-shaped, nonmotile, aerobic, and yellow-pigmented. Catalase and oxidase activities and hydrolysis of Tweens 40, 60, and 80 of two novel strains were positive. Methyl red reaction, H 2S production, and hydrolysis of Tween 20 were negative. According to 16S rRNA gene sequence analysis, two novel strains shared the highest similarities (96.4–97.7%) to the species with a validated name in the genus Hanstruepera, while shared lower sequence similarities (<95.6%) to all other genera. Phylogenetic analysis revealed that strains NBU2968 T and NBU2984 T were affiliated with the genus Hanstruepera. ANI and dDDH values between the two novel strains and Hanstruepera species were 77.4–78.3% and 20.4–20.9%, respectively, which were below the thresholds for species delineation. The 16S rRNA gene sequence similarity, ANI, and dDDH values between the two novel strains were 99.3, 88.9, and 36.3%, respectively, indicating that the two strains represent different species. The genomes of NBU2968 T and NBU2984 T were 3.28 Mbp with a G+C content of 34.2% and 3.09 Mbp with a G+C content of 34.4%, respectively. The only respiratory quinone was menaquinone-6 (MK-6). The major cellular fatty acids were iso-C 15:0, iso-C 15:1G, and iso-C 17:0 3-OH. The major polar lipids of the two strains were phosphatidylethanolamine, unidentified amino lipids, and unidentified lipids. Based on the above polyphasic characteristics, strains NBU2968 T and NBU2984 T represent two novel species within the genus Hanstruepera, for which the names Hanstruepera marina sp. nov. and Hanstruepera flava sp. nov. are proposed. The type strains are NBU2968 T (= MCCC 1K06392 T= KCTC 82913 T) and NBU2984 T (= MCCC 1K07472 T= KCTC 92511 T), respectively.

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          MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets.

          We present the latest version of the Molecular Evolutionary Genetics Analysis (Mega) software, which contains many sophisticated methods and tools for phylogenomics and phylomedicine. In this major upgrade, Mega has been optimized for use on 64-bit computing systems for analyzing larger datasets. Researchers can now explore and analyze tens of thousands of sequences in Mega The new version also provides an advanced wizard for building timetrees and includes a new functionality to automatically predict gene duplication events in gene family trees. The 64-bit Mega is made available in two interfaces: graphical and command line. The graphical user interface (GUI) is a native Microsoft Windows application that can also be used on Mac OS X. The command line Mega is available as native applications for Windows, Linux, and Mac OS X. They are intended for use in high-throughput and scripted analysis. Both versions are available from www.megasoftware.net free of charge.
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            The neighbor-joining method: a new method for reconstructing phylogenetic trees.

            N Saitou, M Nei (1987)
            A new method called the neighbor-joining method is proposed for reconstructing phylogenetic trees from evolutionary distance data. The principle of this method is to find pairs of operational taxonomic units (OTUs [= neighbors]) that minimize the total branch length at each stage of clustering of OTUs starting with a starlike tree. The branch lengths as well as the topology of a parsimonious tree can quickly be obtained by using this method. Using computer simulation, we studied the efficiency of this method in obtaining the correct unrooted tree in comparison with that of five other tree-making methods: the unweighted pair group method of analysis, Farris's method, Sattath and Tversky's method, Li's method, and Tateno et al.'s modified Farris method. The new, neighbor-joining method and Sattath and Tversky's method are shown to be generally better than the other methods.
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              A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences.

              Some simple formulae were obtained which enable us to estimate evolutionary distances in terms of the number of nucleotide substitutions (and, also, the evolutionary rates when the divergence times are known). In comparing a pair of nucleotide sequences, we distinguish two types of differences; if homologous sites are occupied by different nucleotide bases but both are purines or both pyrimidines, the difference is called type I (or "transition" type), while, if one of the two is a purine and the other is a pyrimidine, the difference is called type II (or "transversion" type). Letting P and Q be respectively the fractions of nucleotide sites showing type I and type II differences between two sequences compared, then the evolutionary distance per site is K = -(1/2) ln [(1-2P-Q) square root of 1-2Q]. The evolutionary rate per year is then given by k = K/(2T), where T is the time since the divergence of the two sequences. If only the third codon positions are compared, the synonymous component of the evolutionary base substitutions per site is estimated by K'S = -(1/2) ln (1-2P-Q). Also, formulae for standard errors were obtained. Some examples were worked out using reported globin sequences to show that synonymous substitutions occur at much higher rates than amino acid-altering substitutions in evolution.
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                Author and article information

                Contributors
                Journal
                Front Microbiol
                Front Microbiol
                Front. Microbiol.
                Frontiers in Microbiology
                Frontiers Media S.A.
                1664-302X
                22 July 2022
                2022
                : 13
                : 957397
                Affiliations
                Li Dak Sum Yip Yio Chin Kenneth Li Marine Biopharmaceutical Research Center, College of Food and Pharmaceutical Sciences, Ningbo University , Ningbo, China
                Author notes

                Edited by: Ming Ma, Peking University, China

                Reviewed by: Peng Zhang, Tobacco Research Institute, Chinese Academy of Agricultural Sciences (CAAS), China; Zong-Jun Du, Shandong University, China

                *Correspondence: Weiyan Zhang zhangweiyan13@ 123456126.com

                This article was submitted to Aquatic Microbiology, a section of the journal Frontiers in Microbiology

                Article
                10.3389/fmicb.2022.957397
                9355603
                35935200
                0b356e98-5e49-4adb-b14a-16544fbc7825
                Copyright © 2022 Ding, Liu, Yang, Guo, Ding, Jung and Zhang.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                History
                : 31 May 2022
                : 27 June 2022
                Page count
                Figures: 2, Tables: 4, Equations: 0, References: 40, Pages: 12, Words: 6943
                Funding
                Funded by: Natural Science Foundation of Zhejiang Province, doi 10.13039/501100004731;
                Funded by: National Natural Science Foundation of China, doi 10.13039/501100001809;
                Categories
                Microbiology
                Original Research

                Microbiology & Virology
                in situ cultivation,modified ichip,hanstruepera,polyphasic taxonomy,marine sediment

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