Description of Acinetobacter Pollinis Sp. Nov

Taxonomic Description 1 Acinetobacter pollinis sp. nov., Acinetobacter baretiae 2 sp. nov., and Acinetobacter rathckeae sp. nov., 3 isolated from floral nectar and honeybees 4 Author names 5 Sergio Álvarez-Pérez1,2,†, Lydia J. Baker3,†, Megan M. Morris4, Kaoru Tsuji5, Vivianna A. Sanchez3, 6 Tadashi Fukami6, Rachel L. Vannette7, Bart Lievens1, Tory A. Hendry3,* 7 8 ORCID IDs 9 Sergio Álvarez-Pérez: http://orcid.org/0000-0002-6587-8995 10 Lydia J. Baker: https://orcid.org/0000-0002-1453-421X 11 Megan M. Morris: https://orcid.org/0000-0002-7024-8234 12 Kaoru Tsuji: https://orcid.org/0000-0001-5020-5184 13 Vivianna A. Sanchez: https://orcid.org/0000-0002-8935-7991 14 Tadashi Fukami: http://orcid.org/0000-0001-5654-4785 15 Rachel L. Vannette: https://orcid.org/0000-0002-0447-3468 16 Bart Lievens: https://orcid.org/0000-0002-7698-6641 17 Tory A. Hendry: https://orcid.org/0000-0002-8001-1783 18 19 Affiliations 20 1Laboratory for Process Microbial Ecology and Bioinspirational Management (PME&BIM), 21 Department of Microbial and Molecular Systems, KU Leuven, Willem De Croylaan 46, B-3001 Leuven, 22 Belgium. 23 2Department of Animal Health, Complutense University of Madrid, 28040 Madrid, Spain. 24 3Department of Microbiology, Cornell University, Ithaca, NY 14853, USA. 25 4Physical and Life Sciences Directorate, Lawrence Livermore National Laboratory,7000 East Avenue, 26 Livermore, CA 94550, USA. 27 5Center for Ecological Research, Kyoto University Hirano 2, Otsu, Shiga 520-2113, Japan. 28 6Department of Biology, Stanford University, Stanford, CA 94305, USA. 29 7Department of Entomology and Nematology, University of California Davis, Davis, CA 95616, USA. 30 31 † These authors contributed equally to this work. 32 33 Corresponding author 34 * Tory A. Hendry ([email protected]) 35 Keyword 36 Acinetobacter, floral nectar, honeybee. 37 Repositories: 38 The GenBank/ENA/DDBJ accession numbers for the partial nucleotide sequences of the 16S rRNA 39 gene are indicated in Table S1 and the accession numbers for partial rpoB gene sequences are 40 indicated in Table S2. The whole genome shotgun projects have been deposited at 41 DDBJ/ENA/GenBank under the accession numbers VTDL00000000-VTDT00000000. 42 43 ABSTRACT 44 A detailed evaluation of eight bacterial isolates from floral nectar and animal visitors to flowers 45 shows evidence that they represent three novel species in the genus Acinetobacter. Phylogenomic 46 analysis shows the closest relatives of these new isolates are A. apis, A. boissieri, and A. nectaris, 47 previously described species associated with floral nectar and bees, but high genome-wide sequence 48 divergence defines these isolates as novel species. Pairwise comparisons of the average nucleotide 49 identity (ANI) of the new isolates compared to known species is extremely low (<83%), thus 50 confirming that these samples are representative of three novel Acinetobacter species, for which the 51 names Acinetobacter pollinis sp. nov., Acinetobacter baretiae sp. nov., and Acinetobacter rathckeae 52 sp. nov. are proposed. The respective type strains are SCC477T (= TSD-214T = LMG 31655T), B10AT (= 53 TSD-213T = LMG 31702T), and EC24T (= TSD-215T = LMG 31703T). 54 55 The genus Acinetobacter (Gammaproteobacteria) is a physiologically and metabolically diverse 56 group of bacteria currently including 65 validly published and correct names, plus several other 57 tentative designations and effectively but not validly published species names 58 (https://lpsn.dsmz.de/genus/acinetobacter, last accessed on March 5, 2021). Acinetobacter species 59 are ubiquitous in natural and human-associated environments, and a substantial proportion of them 60 are associated with animal and plant hosts [e.g. 1–6]. 2 61 Detailed analysis of a collection of 14 Acinetobacter strains obtained from the floral nectar of 62 Mediterranean wild plants in southern Spain led to the description of two new species, namely A. 63 nectaris and A. boissieri [7]. Soon thereafter, Kim et al. [8] isolated from the intestinal tract of a 64 honey bee a bacterial strain which clustered with the A. nectaris-A. boissieri clade in both 16S rRNA 65 and rpoB gene trees, but was nevertheless identified and described as a new species with the name 66 Acinetobacter apis. However, the diversity of acinetobacters associated with flowering plants and 67 their natural visitors remains mostly unknown, even when the genus Acinetobacter seems to rank 68 among the main bacterial inhabitants of the floral nectar of angiosperms [9–15], and the mouth and 69 digestive tract of flower-visiting hummingbirds [15] and bumblebees [16]. In this study, we explored 70 the phylogenomic affiliation and physiology of a collection of eight bacterial isolates representing 71 three new Acinetobacter species associated with floral nectar and animal visitors to flowers. 72 73 Isolation and Ecology 74 The eight isolates investigated in this study are listed in Table 1. Six of these isolates were obtained 75 from nectar samples of three plant species (namely Diplacus (Mimulus) aurantiacus, Epilobium 76 canum, and Scrophularia californica, two isolates from each species) collected at different locations 77 in California, USA. The other two isolates were retrieved from the mouth and gut of honey bees 78 (Apis mellifera) sampled on the Stanford University campus (Stanford, CA, USA). Nectar samples 79 were diluted in 500 µL of saline solution (0.85% w/v NaCl, Merck) and a 25-µL aliquot of each was 80 streaked on tryptone soy agar (TSA, Oxoid) [9]. Immediately after capture, honey bees were kept 81 individually in sterile containers and anaesthetized by placing them inside a polystyrene box with ice 82 for 10-15 min, after which they were allowed to feed on sugar water (20% w/v sucrose, Sigma- 83 Aldrich) and then dissected to extract their gut. Honey bee guts were immediately ground inside a 84 microtube containing 1 mL of saline solution using a disposable pellet pestle. Aliquots of the 85 remaining sugar water and homogenized gut samples (2 μL and 10 μL, respectively) were streaked 86 on TSA. All cultures were incubated at 25 °C for 7 days, and a colony of each phenotypically distinct 87 microbial type was picked and separately subcultured on TSA to obtain axenic cultures. All isolates 88 were stored at -20 °C in lysogeny broth (LB, Difco) containing 25% glycerol (Sigma-Aldrich). 89 90 16S ribosomal RNA and rpoB gene sequencing 91 Genomic DNA was extracted from the eight isolates using the Qiagen Blood and Tissue Kit (Qiagen, 92 Hilden, Germany), according to the manufacturer’s protocol for bacterial samples. 16S rRNA gene 3 93 amplification was performed using the New England Biolabs (Ipswich, MA, USA) standard Taq 94 polymerase kit and primers 27F and 1494R [17], with the following thermocycling conditions: initial 95 denaturation at 95 °C for 3 minutes, 35 amplification cycles of 95 °C for 30 sec, 55 °C for 30 sec, and 96 72 °C for 60 s, and a final extension at 72 °C for 10 min. PCR cleanup was performed using the 97 QIAquick PCR purification kit (Qiagen) and the amplicons were sent to Cornell University Institute of 98 Biotechnology Resource Center Genomics Facility for Sanger sequencing. Forward and reverse 99 sequences were trimmed for quality, joined using GeneStudio, and trimmed to the same start and 100 stop site and were >1149 bp in length (GenBank accessions MN701871 – MN701875, MN701877, 101 MN701878 and MN709041). Sequence comparisons using BLAST showed that the studied isolates 102 had the highest sequence identity with members of genus Acinetobacter (Table S1). 103 PCR amplification and sequence analysis of two variable regions (zones 1 and 2, 861 bp in total) of 104 the rpoB gene, which encodes the β subunit of RNA polymerase, was performed following the 105 procedures described in Álvarez-Pérez et al. [7]. Sequence comparisons using BLAST identified A. 106 nectaris and A. boissieri as the closest relatives of all studied isolates (with four isolates most closely 107 matching each species), but sequence similarity was ≥93.6% in all cases (Table S2). 108 109 Genome Features and Phylogenomic Analysis 110 Nextera skim libraries were prepared using genomic DNA and run on a 2×250 paired end Rapid Run 111 HiSEQ 2500 platform at the Cornell University Institute of Biotechnology Resource Center Genomics 112 Facility. Genomic sequencing and annotation resulted in high quality bacterial genomes that were 113 submitted to GenBank (VTDL00000000- VTDT00000000) (Table 2). Genomes were assembled using 114 Discovar de novo (version 52488) and authenticated through comparison of the 16S rRNA gene 115 sequence isolated by BLAST from genomic assembles to the Sanger-sequenced 16S rRNA gene from 116 the extracted DNA of each culture. These 16S rRNA gene sequences were 99.5-99.8% similar, 117 confirming that the genomic assemblies represented the isolated cultures. The N50 for the 118 assembled contigs were between 66 and 302, as calculated by the program stats.sh provided by the 119 Joint Genomics Institute (JGI, Walnut Creek, CA). The average coverage depth of the assembled 120 Acinetobacter genomes was 200× or greater as estimated by BBMap [18]. Genome completeness 121 was estimated by checkM [19]. All assemblies were at least 98% complete, and the majority of 122 genomes were more than 99% complete (Table 2). Using OrthoANIu [20], genome size of the isolates 123 was found to vary between 2.59 and 2.75 Mbp and GC content between 36.6 and 39.3% (Table 2). 4 124 Genetic similarity between the isolates investigated in this study and previously documented 125 Acinetobacter species was evaluated using phylogenomic analysis. A phylogenomic tree was 126 constructed by concatenating the DNA of single-copy protein-coding genes shared by the bacterial 127 isolates and those Acinetobacter species identified as being reference or representative sequences 128 in PATRIC (Table S3). PhyloPhLan3 identified 261 shared proteins from RAST annotations [21]; 129 shared protein sequences concatenated and aligned by PhyloPHLan were used to construct a 130 phylogenomic tree using IQ-TREE [22].

Load more