ABSTRACT
Pseudomonas sp. strains MWU12-2029, MWU12-3088, and MWU12-3091 were isolated from wild and cultivated cranberry bog soils in southeastern Massachusetts. The three isolates are closely related to Pseudomonas kribbensis, a not validly published member of the P. fluorescens group, and contain three putative insecticidal protein genes, including the toxin complex A gene (tcaC).
ANNOUNCEMENT
Pseudomonas spp. comprise a considerable component of bacteria isolated from cultivated and wild cranberry bogs during a multiyear culture-dependent survey conducted at the Cape Cod National Seashore (MWU12-2029) (42.070624N, 70.210548W) and the University of Massachusetts State Bog (MWU12-3088 and MWU12-3091) (41.766767N, 70.66842W) (1–8). Although presumably an important constituent of these wetland microbiomes, little is known about how these organisms affect the dynamics of the bog ecosystem, including their effects on insects. As an initial foray into understanding the cranberry bog soil microbiome, 5 cm by 5 cm soil samples were taken from cultivated and wild bogs in July 2012 for isolating and characterizing bacterial populations. Approximately 1 g from soil cores was vortexed in sterile water, and the rinsate was plated onto King’s medium B (KMB) agar supplemented with 50 μg mL−1 each of cycloheximide and ampicillin and incubated at 26°C for 48 h. Fluorescent colonies were purified three times on KMB and stored at −80°C in 34% glycerol. Isolates from frozen storage were recovered on KMB agar, and populations were inoculated into KMB broth cultures grown overnight for genomic DNA (gDNA) isolation. All kits described below were used according to the manufacturers’ instructions. gDNA was extracted with a DNeasy blood and tissue kit (Qiagen, USA), and Kapa Biosystems Hyperplus library preparation kits (catalog number KK8514; Roche, USA) were used to generate Illumina-compatible genomic DNA libraries: DNA was enzymatically sheared to ~500 bp, end repaired, and A tailed; Illumina-compatible adapters with unique indexes (catalog number 00989130v2; IDT, Coralville, IA) were then ligated to each sample; and adapter-ligated molecules were cleaned using Kapa pure beads (catalog number KK8002) and amplified with Kapa Hifi enzyme (catalog number KK2502). Library fragment sizes were determined on an Agilent TapeStation system and quantified by quantitative PCR (qPCR) (Kapa library quantification kit, catalog number KK4835) on a QuantStudio 5 system (Thermo Fisher, USA). Samples were multiplex pooled and sequenced on an Illumina MiSeq platform in a 2-by-250 flow cell. The software was set to default settings except as indicated below. Raw reads were assembled with Unicycler v0.4.8 (9) and polished with Pilon v1.23 (10) within the PATRIC v3.6.12 comprehensive genome analysis pipeline, except for the trim setting, which was set to “true” (11). The comprehensive analysis pipeline includes quality control and trimming by QUAST v5.0.2 (12) and Trim Galore v0.4.0 (13) and annotation by RASTtk v1.073 (14), supplemented with antiSMASH v6.0 (15) for the recognition of secondary metabolite gene clusters. Using the Type (Strain) Genome Server (TYGS), isolates were placed with high confidence within the genus Pseudomonas (16). All three isolates were most closely related to Pseudomonas kribbensis 46-2T (GenBank accession number CP029608) but fell below the 70% digital DNA-DNA hybridization (dDDHd4) (TYGS v342) (16–18) or 95 to 96% average nucleotide identity by BLAST analysis (ANIb) (JSpeciesWS v3.9.5) (19, 20) cutoff to be included in that species (Table 1).
TABLE 1.
Genomic data summary
| Isolate | BioSample accession no. | GenBank accession no. | SRA accession no. | Genome size (bp) | No. of contigs | N50 (bp) | G+C content (%) | Mean read length (bp) | No. of reads | Coverage (×) | dDDHd4/ANIb with P. kribbensis 46-2T (%) |
|---|---|---|---|---|---|---|---|---|---|---|---|
| MWU12-2029 | SAMN26814158 | JALJDY000000000 | SRR18644892 | 6,318,446 | 38 | 583,019 | 60.68 | 233.75 | 3,403,188 | 126 | 65.1/95.48 |
| MWU12-3088 | SAMN26803934 | JALJEA000000000 | SRR18645887 | 6,305,590 | 37 | 392,904 | 60.67 | 229.72 | 3,774,982 | 138 | 65.6/95.65 |
| MWU12-3091 | SAMN26896969 | JALJET000000000 | SRR18508983 | 6,305,411 | 38 | 392,833 | 60.67 | 224.57 | 3,190,686 | 114 | 65.6/95.64 |
MWU12-2029, MWU12-3088, and MWU12-3091 all contain presumptive genes for insecticidal toxin complex A (tcaC) (21) and two additional putative insecticidal toxins that are widespread among members of the Pseudomonas fluorescens group.
Data availability.
The whole-genome shotgun sequencing project has been deposited in the DDBJ/EMBL/GenBank database under BioProject accession number PRJNA691338 with the accession numbers in Table 1. The versions described in this paper are GenBank accession numbers JALJDY000000000.1 (MWU12-2029), JALJEA000000000.1 (MWU12-3088), and JALJET000000000.1 (MWU12-3091). RASTtk annotations are available under an open license at Zenodo (see https://zenodo.org/record/6413129#.YwaiEUfMKUk, https://zenodo.org/record/6413119#.Ywah6kfMKUk, and https://zenodo.org/record/6413231#.Ywain0fMKUk).
ACKNOWLEDGMENTS
This research was supported by the Office of Research and Sponsored Programs, the College of Graduate Studies, and the Biomedical Sciences Program, Midwestern University.
We appreciate and acknowledge Alisha Harrison for the isolation of genomic DNA. Library construction and Illumina sequencing were performed at the Arizona State University Genomics Core Facility.
This work fulfills a course requirement for Special Topics in Bacterial Genomics for A.M. and A.P.
Contributor Information
Scott Soby, Email: ssobyx@midwestern.edu.
Catherine Putonti, Loyola University Chicago.
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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
The whole-genome shotgun sequencing project has been deposited in the DDBJ/EMBL/GenBank database under BioProject accession number PRJNA691338 with the accession numbers in Table 1. The versions described in this paper are GenBank accession numbers JALJDY000000000.1 (MWU12-2029), JALJEA000000000.1 (MWU12-3088), and JALJET000000000.1 (MWU12-3091). RASTtk annotations are available under an open license at Zenodo (see https://zenodo.org/record/6413129#.YwaiEUfMKUk, https://zenodo.org/record/6413119#.Ywah6kfMKUk, and https://zenodo.org/record/6413231#.Ywain0fMKUk).