ABSTRACT
Pseudomonas sp. strain RGM 3321 is a phyllosphere endophyte from Fragaria chiloensis subsp. chiloensis f. patagonica that harbors genes associated with plant growth promotion pathways, as well as genes typically found in plant pathogens.
ANNOUNCEMENT
Fragaria chiloensis subsp. chiloensis f. patagonica is the wild form of the Chilean strawberry, which grows in the mountains and in coastal environments (1). In January 2021, a sample of this species was removed from forest soil in the Yungay Precordillera, Ñuble, Chile (−37.05905, −71.64625). The phyllosphere was surface sterilized by submerging the tissues in 70% ethanol (1 min), 1.5% NaClO (3 min), and 96% ethanol (1 min) and finally rinsed three times. The tissues were subsequently ground in physiological solution, and volumes of 100 μL of serial dilutions were inoculated on King's B (KB) agar medium supplemented with 25 μg/mL nystatin and 50 μg/mL cycloheximide. Plates were incubated at 25°C for 48 h. A UV-fluorescent colony was streaked on KB agar and incubated under the same conditions. This step was repeated twice to obtain axenic cultures. The isolate was deposited in the Chilean Collection of Microbial Genetic Resources (CChRGM), under the code RGM 3321. Strain RGM 3321 grown in yeast extract-malt extract-dextrose broth supplemented with 1% l-tryptophan produced 106.47 μg/mL indole acetic acid (IAA) and grown on NBRIP agar displayed a phosphate solubilization index of 2.4, suggesting potential plant growth-promoting traits (2–4).
Two genomic DNA libraries were constructed using the Nextera XT library preparation kit (Illumina, USA) and sequenced on an Illumina HiSeq/NovaSeq platform using a 250-bp paired-end protocol at MicrobesNG (UK). Whole-genome sequencing reads were adapter trimmed using Trimmomatic v0.30 with a sliding window quality cutoff value of Q15 (5, 6). De novo assembly was performed using SPAdes v3.7 (7). Contigs were annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) v6.0 (8). The assembled genome has a size of 6,380,675 nucleotides (nt), distributed in 121 contigs (the largest contig was 609,627 nt), with mean coverage of 82.91×, an N50 value of 171,869 nt, and a G+C content of 58.41%; 5,669 genes and 56 tRNAs were predicted from the annotation. On the EzBioCloud webserver (9), RGM 3321 shared the greatest 16S rRNA gene similarity with the phytopathogenic species of the Pseudomonas syringae group (10), i.e., Pseudomonas KCTC 12500T (99.93%) and Pseudomonas congelans DSM 14939T (99.86%) and Pseudomonas cerasi 58T (99.86%).
BLAST searches were performed using SequenceServer v2.0.0 (11). Strain RGM 3321 harbors one copy of a gene for 1-aminocyclopropane-1-carboxylate (ACC) deaminase (acdS), which is involved in the degradation of ACC, a precursor of ethylene in plants (12, 13). The strain contains genes for a quinoprotein glucose dehydrogenase (gcd) and the pyrroloquinoline quinone (pqq) operon (14), suggesting a mechanism for phosphate solubilization in soil via gluconic acid synthesis (15, 16). The identification of iaaM and iaaH genes suggests production of IAA via the indole-3-acetamide (IAM) and/or indole-3-acetonitrile (IAN) pathways (17, 18). Genes encoding a nitrile hydratase (nthAB) were also found, suggesting the transformation of IAN into an IAM intermediate in the IAN pathway (19; Table 1).
TABLE 1.
Genome sequence features of Pseudomonas sp. strain RGM 3321
| Trait and protein name (gene name) or BGC | RGM 3321 protein code | Identity to reference protein (%) | UniProt accession no. for reference protein | Strain encoding the BGC | Similarity to reference BGC (%)a |
|---|---|---|---|---|---|
| Trait and protein name | |||||
| Phosphate solubilization | |||||
| Quinoprotein glucose dehydrogenase (gcd) | RGM3321_17085 | 69.27 | A0A0B6F0P5 | ||
| Coenzyme PQQ synthesis protein A (pqqA) | RGM3321_09145 | 95.83 | Q3K5R0 | ||
| Coenzyme PQQ synthesis protein B (pqqB) | RGM3321_09140 | 91.42 | C3K348 | ||
| Pyrroloquinoline-quinone synthase (pqqC) | RGM3321_09135 | 92.83 | Q88QV6 | ||
| PqqA binding protein (pqqD) | RGM3321_09130 | 68.97 | Q4K4U9 | ||
| PqqA peptide cyclase (pqqE) | RGM3321_09125 | 89.10 | Q4K4U8 | ||
| Coenzyme PQQ synthesis protein F (pqqF) | RGM3321_09150 | 44.84 | P55174 | ||
| IAA production | |||||
| Tryptophan 2-monooxygenase (iaaM); IAM pathway | RGM3321_01470 | 94.61 | P06617 | ||
| Indoleacetamide hydrolase (iaaH); IAM pathway | RGM3321_01475 | 93.02 | P06618 | ||
| Indole-3-pyruvate decarboxylase (ipdC); indole-3-pyruvate pathway | RGM3321_13685 | 26.95 | A0A5E6Q147 | ||
| Nitrilase (nit); IAN pathway | RGM3321_22075 | 28.52 | K9NKH3 | ||
| Aldehyde dehydrogenase family protein (aldA); IAN pathway | RGM3321_15565 | 94.16 | Q88BC5 | ||
| Aldehyde dehydrogenase family protein (aldB); IAN pathway | RGM3321_11565 | 97.57 | Q88BC5 | ||
| Aldehyde dehydrogenase family protein (aldB); IAN pathway | RGM3321_06435 | 45.50 | Q88BC5 | ||
| ACC deaminase activity | |||||
| ACC deaminase (acdS) | RGM3321_08860 | 88.76 | Q51813 | ||
| Leucine-responsive regulatory protein (acdR) | RGM3321_08855 | 80.47 | K9NP20 | ||
| T3SS | |||||
| Hypersensitivity response secretion protein HrpJ (hrpJ) | RGM3321_26170 | 98.77 | Q05395 | ||
| Lipoprotein (hrcJ) | RGM3321_26075 | 90.30 | G3XDC8 | ||
| Type III secretion protein HrcR (hrcR) | RGM3321_26130 | 93.00 | Q887B8 | ||
| Type III secretion protein HrcS (hrcS) | RGM3321_26125 | 94.32 | G3XDB8 | ||
| Type III secretion protein HrcT (hrcT) | RGM3321_26120 | 84.09 | G3XDD0 | ||
| Type III secretion protein HrcU (hrcU) | RGM3321_26115 | 85.52 | Q887B9 | ||
| Hypersensitivity response secretion protein HrpI (hrpI) | RGM3321_26165 | 98.99 | P35655 | ||
| T3SS ATPase SctN (sctN) | RGM3321_26155 | 99.33 | Q52371 | ||
| V-type ATP synthase subunit E (hrpE) | RGM3321_26085 | 76.17 | Q887C4 | ||
| Type III secretion protein HrcQb (hrcQb) | RGM3321_26135 | 93.98 | Q60235 | ||
| T3SS regulator | |||||
| RNA polymerase sigma factor HrpL (hrpL) | RGM3321_26175 | 95.70 | P37929 | ||
| Hrp pilus protein HrpA1 (hrpA) | RGM3321_26060 | 99.07 | Q52420 | ||
| T3SS key effector | |||||
| Type III effector HopAA1 (hopAA1) | RGM3321_26015 | 74.74 | G3XDB9 | ||
| Type III effector AvrE1 (avrE1) | RGM3321_26040 | 67.83 | Q887C9 | ||
| Type III effector HopM1 (hopM1) | RGM3321_26030 | 93.87 | Q4ZX82 | ||
| BGC | |||||
| Pyoverdine | Pseudomonas protegens Pf-5 | 22 | |||
| Syringafactin | Pseudomonas syringae pv. tomato DC3000 | 66 | |||
| Syringolin A | Pseudomonas syringae pv. syringae B301 D-R | 100 | |||
| Syringomycin | Pseudomonas syringae pv. syringae B728a | 100 |
Similarity of the BGC found in the genome of RGM 3321 to the reference BGC used by antiSMASH.
Fourteen biosynthetic gene clusters (BGCs) were predicted from the RGM 3321 genome using antiSMASH 6.0 (20), four of which, including the pyoverdine (21), syringomycin (22), syringolin A (23), and syringafactin (24) BGCs, are present in plant-pathogenic bacteria (Table 1). In addition, RGM 3321 contains a type III secretion system (T3SS) gene and additional auxiliary genes (25), including an homolog of the RNA polymerase sigma factor (hrpL), which is a master regulator of the T3SS that interacts with a conserved hrp box motif and promotes the expression of effectors and other virulence factors (26), including HopAA1, which specifically enhances the epiphytic bacterial survival/growth in plants (27).
The draft genome of Pseudomonas sp. strain RGM 3321 expands our knowledge about the bacterial diversity of Chilean wild plants, revealing plant growth-promoting genes and additional open reading frames associated with plant virulence factors. All tools were run with default parameters unless otherwise specified.
Data availability.
This whole-genome shotgun project has been deposited at DDBJ/ENA/GenBank under the accession number JALHBH000000000. The version described in this paper is version JALHBH000000000.1. The raw data are available under SRA accession numbers SRR18554678, SRR18554679, and SRR18554680. All project data are available under BioProject accession number PRJNA820724.
ACKNOWLEDGMENTS
We acknowledge financial support from the FONDECYT INICIACIÓN (project 11191074) from the Chilean National Agency for Research and Development (ANID) to J.F.C. and from ANID to the FONDEQUIP Program (grant EQM200205) for funding a platform of equipment for preservation of microbial genetic resources.
Contributor Information
Jean Franco Castro, Email: jean.castro@inia.cl.
David A. Baltrus, University of Arizona
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
This whole-genome shotgun project has been deposited at DDBJ/ENA/GenBank under the accession number JALHBH000000000. The version described in this paper is version JALHBH000000000.1. The raw data are available under SRA accession numbers SRR18554678, SRR18554679, and SRR18554680. All project data are available under BioProject accession number PRJNA820724.