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Altererythrobacter Rhizovicinus Sp. Nov., Isolated from Rhizosphere Soil of Haloxylon Ammodendron

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TAXONOMIC DESCRIPTION Li et al., Int. J. Syst. Evol. Microbiol. 2020;70:680–686 DOI 10.1099/ijsem.0.003817 Altererythrobacter rhizovicinus sp. nov., isolated from rhizosphere soil of Haloxylon ammodendron Hui- Ping Li, Dan Yao, Kun- Zhong Shao, Qing- Qing Han, Jing- Yi Gou, Qi Zhao* and Jin- Lin Zhang* Abstract A salt- tolerant, Gram- negative, rod- shaped and yellow-pigmented bacterium, designated strain AY-3R T, was isolated from rhizosphere soil of a desert xerophyte, Haloxylon ammodendron, sampled at Badain Jaran Desert, Alxa region, Inner Mongolia, PR China. Growth of this strain was observed at 20–42 °C (optimum, 28–30 °C), at pH 6.0–9.0 (optimum, pH 6.0–7.0) and at 0–8 % (w/v) NaCl (optimum, 3 %). Results of phylogenetic analysis based on 16S rRNA gene sequences showed that strain AY- 3RT was a member of the genus Altererythrobacter, with the highest similarity to Altererythrobacter aerophilus Ery1T (97.6 %), followed T by Altererythrobacter xinjiangensis S3-63 (96.9 %). The predominant fatty acids (>10.0 %) were C18 : 1ω7c, C17 : 1ω6c and summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c). The major polar lipids were diphosphatidylglycerol, phosphatidylcholine, phosphatidyle- thanolamine, phosphatidylglycerol, sphingoglycolipid and one unknown polar lipid. The predominant respiratory quinone was ubiquinone-10. The G+C content of the genomic DNA of strain AY-3R T was 66.3 mol%. On the basis of the data from this poly- phasic taxonomic study, strain AY- 3RT represents a novel species of the genus Altererythrobacter, named Altererythrobacter rhizovicinus sp. nov. (=MCCC 1K03572T=KCTC 72280T). The family Erythrobacteraceae belonged to the order of Sphin- and ubiquinone-10, respectively [6]. The predominant polar gomonadales, the class of Alphaproteobacteria in the phylum lipids are diphosphatidylglycerol (DPG), phosphatidyletha- of Proteobacteria [1, 2]. At the time of writing, the family nolamine (PE), phosphatidylglycerol (PG) and sphingogly- Erythrobacteraceae contained genera with validly published colipid (SGL) [20]. The DNA G+C content is in the range of names, and the genus Altererythrobacter is one of them [2]. 52.0–69.0 mol%. The aim of this study was to describe a novel Subsequently, characteristics of the genus Altererythrobacter strain in the genus Altererythrobacter based on a polyphasic were revised by Xue et al. [3, 4]. The type species of this genus approach. is Altererythrobacter epoxidivorans, isolated from cold- seep T sediment [2]. Until now, 42 species have been identified in Strain AY- 3R was isolated from the rhizosphere of a desert the genus Altererythrobacter ( www. bacterio. net/- allnamesac. xerophyte, Haloxylon ammodendron, collected at Badain html). Most of them were isolated from marine environments Jaran Desert, Alxa region, Inner Mongolia, PR China (39° 23′ [2, 5–14]. The remaining species were isolated from terrestrial 7″ N, 102° 47′ 40″ E). The soil sample was serially diluted with environments, such as desert sand [4, 15, 16], permafrost sterile 0.9 % NaCl (w/v) solution, then dilutions were spread [17], mountain soil [18], rhizosphere soil [19] and forest soil on Reasoner’s 2A (R2A) agar (Difco) and incubated at 25 °C [20]. Most cells of members of the genus Altererythrobacter aerobically for 7 days. Single colonies were picked and further are Gram- reaction- negative, aerobic, motile or non- motile purified. Finally, the purified isolate was preserved as a glyc- and generally rod- shaped cells, which form yellow colonies. erol suspension (20 %, v/v) at −80 °C. Two closest type strains, T The dominant fatty acid and respiratory quinone are 18C : 1ω7c Altererythrobacer aerophilus Ery1 and Altererythrobacter Author affiliations: 1State Key Laboratory of Grassland Agro- ecosystems; Key Laboratory of Grassland Livestock Industry Innovation, Ministry of Agriculture and Rural Affairs; Engineering Research Center of Grassland Industry, Ministry of Education; College of Pastoral Agriculture Science and Technology, Lanzhou University, Lanzhou 730020, Gansu Province, PR China. *Correspondence: Qi Zhao, qzhao@ lzu. edu. cn; Jin- Lin Zhang, jlzhang@ lzu. edu. cn Keywords: Altererythrobacter rhizovicinus sp. nov.; Phylogenetic analysis; Polyphasic taxonomic. Abbreviations: ANI, average nucleotide identity; DDH, DNA–DNA hybridization; DPG, diphosphatidylglycerol; GGDC, Genome- to- Genome Distance Calculator; GL, glycolipid; ISP 2, yeast extract–malt extract; LB, Luria–Bertani; MA, marine agar; NA, nutrient agar; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, hosphatidylglycerol; PYG, peptone–yeast–glucose; R2A, Reasoner’s 2A; SGL, sphingoglycolipid; TSA, tryptone soya agar; TY, tryptone–yeast. The GenBank/EMBL/DDBJ accession number for 16S rRNA gene sequence of strain AY-3R T is MH611372. The GenBank accession number for the whole genome sequence of strain AY- 3RT is SDPV00000000. Four supplementary figures are available with the online version of this article. 003817 © 2020 The Authors 680 Li et al., Int. J. Syst. Evol. Microbiol. 2020;70:680–686 xinjiangensis CCTCC AB 207166T, were obtained from Genome- to- Genome Distance Calculator 2.1 (GGDC) Xu’s lab and the China Centre for Type Culture Collection (http:// ggdc. dsmz. de/ distcalc2. php). (CCTCC) and used as reference strains for phenotypic tests The draft genome of strain AY-3R T was 3 007 728 bp long and and fatty acid analysis. A. aerophilus Ery1T and A. xinjian- comprised three scaffolds with an N50 value 1 680 395 bp. gensis CCTCC AB 207166T were cultured on marine agar The genome coverage was 200×. A total of 2764 proteins, 2216 (MA; Difco) and 0.3×MA (Difco) at 30 °C, respectively. three rRNAs and 46 tRNAs were predicted. The complete Phylogenetic analysis using the 16S rRNA gene sequence was 16S rRNA gene sequence was 1477 bp long and contained carried out as depicted by Zhao et al. [16]. Genomic DNA of the 16S rRNA gene sequence (1457 bp) derived from the isolate was extracted by Bacterial Genomic DNA Extrac- PCR amplification. The DNA G+C content of AY-3R T was tion kit (TianGen Biotech). The 16S rRNA gene was amplified 66.3 mol%, which fell into the range reported for members by PCR using forward primer 27F (5′- AGAGTTTGATC- of the genus Altererythrobacter (52.0–69.0 mol%). The ANI CTGGCTCAG-3′) and reverse primer 1492R (5′-GGTTAC- value between strain AY- 3RT and the reference strains were CTTGTTACGACTT-3′) [21]. Purification, cloning and 88.2 and 74.0 %, respectively. These values were significantly sequencing of the 16S rRNA gene were respectively executed lower than the threshold of the species boundaries (94–96 %) using a PCR purification kit (Sangon Biotech), the pMD 19 T [32]. DDH values between strain AY-3R T and its reference Vector Cloning kit (Takara) and the Sanger method (Beijing strains, A. aerophilus Ery1T and A. xinjiangensis CCTCC AB augct dna- syn Biotechnology). Then, the almost-complete 207166T, were 34.9 and 19.8 %, respectively. The critical value 16S rRNA gene sequence was compiled with the program of genomic species identification is less than 70 % [33]. These dnaman (version 8.0, Lynnon Biosoft) [22]. The EzTaxon- e data obviously indicated that strain AY- 3RT represented a server ( www. ezbiocloud. net/ apps; [23]) was used to calcu- novel species of the genus Altererythrobacter. late levels of sequence similarity between strain AY- 3RT and Growth was assessed on R2A agar, tryptone soya agar (TSA), related strains available in GenBank ( www. ncbi. nlm. nih. Luria–Bertani agar (LB agar), MA, tryptone–yeast agar (TY gov/ genbank/; [24]). Evolutionary distances were calculated agar), peptone–yeast–glucose agar (PYG agar), yeast extract– using Kimura’s two- parameter model [25] and phylogenetic malt extract agar (ISP 2 agar) and nutrient agar (NA). Cell trees were reconstructed using the neighbour- joining [22], morphology was examined by transmission electron micros- maximum- likelihood [26] and maximum- parsimony [27] copy (JEM 1230, jeol) with cells from the early exponential methods with the mega 7.0 program [28]. In each case, growth phase on MA. Gram- staining was carried out on MA bootstrap analysis of 1000 replicates was performed to assess according to the manufacturer’s instructions [34]. Motility the confidence levels of the branches [29]. was examined in semi-solid medium. Growth at various On the basis of phylogenetic analysis, comparison of the temperatures (4, 10, 15, 20, 25, 28, 30, 35, 40 and 42 °C), pH 16S rRNA gene sequence of strain AY- 3RT revealed the 5.0–10.0 (at intervals of 0.5 pH units) by using the following highest similarity to A. aerophilus Ery1T (97.6 %), followed biological buffer systems: 100 mM citric acid/sodium citrate T by A. xinjiangensis S3-63 (96.9 %), and other recognized (pH 5.0–6.0), 100 mM Na2HPO4/NaH2PO4 (pH 6.5–8.0), members of the genus Altererythrobacter (<96.5 %). The three 100 mM Tris/100 mM HCl (pH 8.5) and 100 mM NaHCO3/ T species living trees showed that strain AY- 3R fell within the Na2CO3 (pH 9.0–10.5) [35, 36]. Tolerance to 0–9 % (w/v) NaCl cluster comprising the Altererythrobacter species (Figs 1, S1 (at intervals of 0.5 %) was determined on MA for 15 days at and S2, available in the online version of this article). Strain 28 °C. Oxidase activity was determined by 1 % (w/v) AY- 3RT formed a distinct genetic lineage with A. aerophilus N,N,N′,N′,-tetramethyl-p - phenylenediamine dihydrochlo- Ery1T based on results from the neighbour- joining (Fig. 1), ride (Sigma), and catalase activity was determined by bubble maximum- likelihood methods (Fig. S1). Thus, these data production with 3 % (v/v) H2O2. Oxidation fermentation, highlighted the view that strain AY- 3RT was affiliated with the Tween 80, degradation of DNA, casein, starch and cellulase genus Altererythrobacter and did not belong to any recognized were tested on MA according to standard methods [37]. species of the genus Altererythrobacter. Cellular pigments were extracted with acetone/methanol (7 : 2, v/v) from cultures grown on MA and were measured The draft genome of strain AY- 3RT was determined using with a spectrophotometer in the wavelength range from paired- end reads with the Illumina HiSeq- PE150 platform 200 to 1100 nm (UV- 6100S, Mapada Instruments) [12].
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    COMMUNITY ANALYSIS OF SOUTHERN APPALACHIAN FENS, AND CHARACTERIZATION AND ISOLATION OF A NOVEL BACTERIUM AND ORDER A Thesis by AUSTIN B. HARBISON Submitted to the Graduate School at Appalachian State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE MAY 2016 Department of BIOLOGY COMMUNITY ANALYSIS OF SOUTHERN APPALACHIAN FENS, AND CHARACTERIZATION AND ISOLATION OF A NOVEL BACTERIUM AND ORDER A Thesis by AUSTIN B. HARBISON MAY 2016 APPROVED BY: Suzanna L Bräuer, Ph.D. Chairperson, Thesis Committee Maryam Ahmed, Ph.D. Member, Thesis Committee Leslie Sargent Jones, Ph.D. Member, Thesis Committee Zack Murrell, Ph.D. Chairperson, Department of Biology Max C. Poole, Ph.D. Dean, Cratis D. Williams School of Graduate Studies Copyright by Austin B. Harbison 2016 All Rights Reserved Abstract COMMUNITY ANALYSIS OF SOUTHERN APPALACHIAN FENS, AND CHARACTERIZATION AND ISOLATION OF A NOVEL BACTERIUM AND ORDER Austin B. Harbison B.S., Appalachian State University M.S., Appalachian State University Chairperson: Suzanna L. Bräuer Peatlands of all latitudes play an integral role in global climate change by serving as a carbon sink and a primary source of atmospheric methane; however, the microbial ecology of mid-latitude peatlands is vastly understudied. Herein, next generation Illumina amplicon sequencing of small subunit rRNA genes was utilized to elucidate the microbial communities in three southern Appalachian peatlands. In contrast to northern peatlands, Proteobacteria dominated over Acidobacteria in all three sites. Members of the Proteobacteria, including Alphaproteobacteria are known to utilize simple sugars and methane among other substrates. However, results described here and in previous studies, indicate that bacteria of the candidate order, Ellin 329, may also be involved in poly- and di-saccharide hydrolysis.
  • Contents Topic 1. Introduction to Microbiology. the Subject and Tasks

    Contents Topic 1. Introduction to Microbiology. the Subject and Tasks

    Contents Topic 1. Introduction to microbiology. The subject and tasks of microbiology. A short historical essay………………………………………………………………5 Topic 2. Systematics and nomenclature of microorganisms……………………. 10 Topic 3. General characteristics of prokaryotic cells. Gram’s method ………...45 Topic 4. Principles of health protection and safety rules in the microbiological laboratory. Design, equipment, and working regimen of a microbiological laboratory………………………………………………………………………….162 Topic 5. Physiology of bacteria, fungi, viruses, mycoplasmas, rickettsia……...185 TOPIC 1. INTRODUCTION TO MICROBIOLOGY. THE SUBJECT AND TASKS OF MICROBIOLOGY. A SHORT HISTORICAL ESSAY. Contents 1. Subject, tasks and achievements of modern microbiology. 2. The role of microorganisms in human life. 3. Differentiation of microbiology in the industry. 4. Communication of microbiology with other sciences. 5. Periods in the development of microbiology. 6. The contribution of domestic scientists in the development of microbiology. 7. The value of microbiology in the system of training veterinarians. 8. Methods of studying microorganisms. Microbiology is a science, which study most shallow living creatures - microorganisms. Before inventing of microscope humanity was in dark about their existence. But during the centuries people could make use of processes vital activity of microbes for its needs. They could prepare a koumiss, alcohol, wine, vinegar, bread, and other products. During many centuries the nature of fermentations remained incomprehensible. Microbiology learns morphology, physiology, genetics and microorganisms systematization, their ecology and the other life forms. Specific Classes of Microorganisms Algae Protozoa Fungi (yeasts and molds) Bacteria Rickettsiae Viruses Prions The Microorganisms are extraordinarily widely spread in nature. They literally ubiquitous forward us from birth to our death. Daily, hourly we eat up thousands and thousands of microbes together with air, water, food.