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Downloaded Halomonas Elongata: High-Afnity Betaine Transport System and Choline- from NCBI Database Rekadwad et al. BMC Res Notes (2021) 14:296 https://doi.org/10.1186/s13104-021-05689-3 BMC Research Notes RESEARCH NOTE Open Access The diversity of unique 1,4,5,6-Tetrahydro- 2-methyl-4-pyrimidinecarboxylic acid coding common genes and Universal stress protein in Ectoine TRAP cluster (UspA) in 32 Halomonas species Bhagwan Narayan Rekadwad1* , Wen‑Jun Li2 and P. D. Rekha1 Abstract Objectives: To decipher the diversity of unique ectoine‑coding housekeeping genes in the genus Halomonas. Results: In Halomonas, 1,4,5,6‑Tetrahydro‑2‑methyl‑4‑pyrimidinecarboxylic acid has a crucial role as a stress‑tolerant chaperone, a compatible solute, a cell membrane stabilizer, and a reduction in cell damage under stressful conditions. Apart from the current 16S rRNA biomarker, it serves as a blueprint for identifying Halomonas species. Halomonas elongata 1H9 was found to have 11 ectoine‑coding genes. The presence of a superfamily of conserved ectoine‑ coding among members of the genus Halomonas was discovered after genome annotations of 93 Halomonas spp. As a result of the inclusion of 11 single copy ectoine coding genes in 32 Halomonas spp., genome‑wide evaluations of ectoine coding genes indicate that 32 Halomonas spp. have a very strong association with H. elongata 1H9, which has been proven evidence‑based approach to elucidate phylogenetic relatedness of ectoine‑coding child taxa in the genus Halomonas. Total 32 Halomonas species have a single copy number of 11 distinct ectoine‑coding genes that help Halomonas spp., produce ectoine under stressful conditions. Furthermore, the existence of the Universal stress protein (UspA) gene suggests that Halomonas species developed directly from primitive bacteria, highlighting its role during the progression of microbial evolution. Keywords: Ectoine, Life under extreme conditions, Saline environments, Single‑copy genes, Ancient bacteria and Archaea, Bioactive compounds Introduction function termed as ‘ectoine’ in general [6]. Te moder- 1,4,5,6-Tetrahydro-2-methyl-4-pyrimidinecarboxylic acid ately halophilic members of family Halomonadaceae (C6H10N2O2, molecular weight 142.16) is a natural pig- displays osmoadaptation facilitated by pigments such as ment produced within the cytoplasm of salt-loving bac- betaine and ectoine [3] and hydroxyectoine [12]. Fam- teria (e.g. genus Ectothiorhodospira; Halomonas). Tis ily Halomonadaceae possess total 18 child taxa. Of pigment helps bacterium to perform osmoregulatory these, names of 14 child taxa are validly published with their correct name. Other16 child taxa have their validly *Correspondence: [email protected] published name including synonyms under the Interna- 1 Yenepoya Research Centre, Yenepoya (Deemed to be University), tional Code of Nomenclature of Prokaryotes (ICNP). On University Road, Deralakatte, Mangalore, Karnataka 575018, India similar note, currently Genus Halomonas represented Full list of author information is available at the end of the article by 114 type strains with 112 candidates having validity © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Rekadwad et al. BMC Res Notes (2021) 14:296 Page 2 of 8 published name and correct name and 10 candidates Radar chart with synonyms. Also, three species have orthographic Halomonas spp., possesses multiple quantitative vari- misspelled variants, and 18 invalidated species were not ables (species in particular) i.e. variable genome length/ validated by ICNP [7]. Description of all Halomonas spe- data points for visualization. Radar chart makes the way cies is given on LPSN portal managed by Leibniz Insti- easy to compare the intra-species variable length to see tute DSMZ-German Collection of Microorganisms and similar values and fnd high or low scoring within outliers Cell Cultures GmbH, Germany. Halomonas species are in the genus. known producer of biotechnologically important ectoine. Being suspended in the cytoplasm, ectoine and hydrox- RAST genome analysis yectoine coded by Halomonas species has benefts to cell. It performs various activities in cell such as stress Complete genome sequences of Ectothiorhodospira tolerant chaperones, as a compatible solute, stabilize of haloalkaliphila ATCC 51935 (CP007268), H. elongata cell membrane and reduce cell damage [10]. Moreover, 1H9 (NC_014532), Halorhodospira halochloris DSM ectoine and hydro-ectoines are high-value chemicals 1059 (AP017372) and Halorhodospira halophila SL1 and exploited for cosmetics, immune protection, sta- (CP000544) analyses done using RAST v2.0 (https:// rast. bilization of antibodies, anti-infammatory and tissue nmpdr. org/) [11]. RAST server is a SEED-based National protective agent, for co-production of bioplastic polyhy- Microbial Pathogen Database Resource (NMPDR), droxybutyrate [8], as a skin aging and protectant agent prokaryotic genome annotation service, to predict sys- against harsh environments viz. radiation and extreme tem coverage, subsystem category distribution and sub- temperatures. Whole cell and macromolecule under hos- system feature count [2]. tile conditions were protected by intracellular ectoine from freezing, drying, high salinity, heat stress, oxygen Identifcation of protein families and single copy genes radicals, radiation and denaturing agents [10]. Various Protein families and single-copy genes in 93 Halomonas applications of ectione produced by Halomonas species spp., were identifed using PATRIC 3.6.9 (https:// www. refect presence of diverse gene profles and other con- patri cbrc. org/). PLfams within the genus were computed served genes in their genomes. It is therefore important with MCL infation = 3.0 to obtain higher sequence simi- to evaluate indicative signatures genes that codes ectoine larity and better specifcity for intra-genus/species close and governs vital biological function under extreme envi- comparisons. ronmental conditions among the genus Halomonas. Present study is a blue print of ectoine coding genes identifed from H. elongata. Genome annotations of Selecting single copy number genes existing Halomonas spp., have uncovered existence of PLfams of 1,4,5,6-Tetrahydro-2-methyl-4-pyrimidi- some common genes that codes ectoine (s) among mem- necarboxylic acid coding genes among 93 Halomonas bers of the genus Halomonas. Tus, genome-wide eval- spp., were extracted. Common genes coded by Halo- uations of ectoine coding genes were assessed. We also monas species were selected for analysis. Te topology analyzed highly close 32 Halomonas spp., with Halo- of the phylogenetic tree generated using concatenated monas elongata 1H9, which has phylogenetic related sequences was compared with the topology of 16S rRNA ectoine coding child taxa inferred using identifed single based Halomonas spp., child taxa tree. copy genes. Phylogeny reconstruction and topology analysis Te evolutionary history of one hundred twenty- Main text eight16S rRNA and 33 Halomonas single-copy genes Methods were inferred using standalone tool MEGA X with 1000 128 type strains 16S rRNA genes and 94 Halomonas spp., bootstrap analysis followed by best scoring ML, NJ and genomes ME tree. Te Jukes-Cantor method and are in the units One hundred twenty-eight 16S rRNA genes of type of the number of base substitutions per site. Te clos- strains and 94 complete genomes and reference est child taxa of biotechnological important ectoine sequences of Halomonas spp., were obtained from LPSN producing H. elongata 1H9 were deciphered. It helps and NCBI genome database deposited during 2006 to for phylogenetic analysis and topology comparison to 2020. delineate nearest species and 1,4,5,6-Tetrahydro-2-me- thyl-4-pyrimidinecarboxylic acid gene coding species. Rekadwad et al. BMC Res Notes (2021) 14:296 Page 3 of 8 Results TBZ202, DX6, 9-2 and MC28) possessed by species Phylogenetic analysis of 16S rRNA genes in the genus were more or less similar kind of representative species Halomonas similar to concatenated sequence of 32 Halomonas spe- H. elongata 1H9 is a bacterium that prefers saline envi- cies (Fig. 1). It was observed that of the 93 annotated ronment and known for 1,4,5,6-Tetrahydro-2-methyl- genome sequences, 31 + 1 (32) species have 11 ectoine 4-pyrimidinecarboxylic acid (ectoine) producer under coding genes (DoeA-DoeC-DoeX-EctC-EctD-EutB- extreme environmental condition. EutC-TeaA-TeaB-TeaC-UspA) as single copy number RAST genome analysis of the H. elongata 1H9 shows genes (Additional fle 5: Figure S5; Table 1). Heatmap that various subsystem feature consists of various path- of 11 ectoine
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