ABSTRACT
The genome sequences of Escherichia coli O157:H7 strain 380-94, which was isolated from a 1994 dry-cured salami outbreak, and a stronger Congo red-binding variant, RV06, were determined using long-read sequencing technology and de novo assembly. Both strains possessed one chromosome and one plasmid. Strain RV06 possessed a 4,769-bp deletion in the rcs region.
ANNOUNCEMENT
Shiga toxin-producing Escherichia coli (STEC) O157:H7 strain 380-94 is a clinical isolate from the dry-cured salami outbreak in the western United States in 1994 (1, 2). This strain possesses increased acid resistance (reported as strain 30-2C4 in reference 3), greater thermotolerance (4), and better survival in processed salami at different pH values, water activities, and temperatures (5). Additionally, acid adaptation enhanced its survival against heat and at the moderately acidic pH in salami (6). Previously, we reported laboratory-derived red variants (RVs) of strain 380-94 with enhanced Congo red (CR)-binding and biofilm-forming properties (7). Several RV strains carry mutations in the rcs (regulator of capsule synthesis) operon, including strain RV06, which carries a large deletion beyond the rcs primer-targeted region (7). The E. coli rcs operon encodes an unconventional two-component system that regulates capsule, flagellum, and fimbria synthesis, as well as acid and envelope stress responses (8). Mutations in rcsB resulted in enhanced biofilm formation, curli production, CR binding, and survival in nutrient-limited environments, while some reports indicated an increase in sensitivity to acidic conditions (7, 9, 10).
Genomic DNA (gDNA) was isolated from overnight (~16-h) cultures grown in LB broth at 37°C, with shaking at 180 rpm, using the Monarch high-molecular-weight (HMW) DNA extraction kit for tissue (New England Biolabs, Ipswich, MA). The gDNA was sheared with a g-TUBE (Covaris Inc., Woburn, MA) and underwent size selection for 20-kb fragments using the BluePippin kit (Sage Science Inc., Beverly, MA). The sequencing library was prepared using the SMRTbell library preparation kit (Pacific Biosciences, Menlo Park, CA) and sequenced on a Sequel IIe system at the University of Delaware Sequencing Center (Newark, DE). The sequencing runs generated 1,279,063,590 bases and 1,209,817,474 bases, with average read lengths of 11,796 bases and 12,668 bases, for strains 380-94 and RV06, respectively. Default parameters were used for all software analyses. Single-molecule real-time (SMRT) Link v10.1 was used for quality control and filtering. Flye v2.9.1 (11) was used for de novo assembly of the filtered reads. Only the first one-half of the reads (633,494,813 bases) were used for the de novo assembly of strain 380-94. Genome assembly with Flye resulted in one circular chromosome and one circular plasmid for each strain. The NCBI Prokaryotic Genome Annotation Pipeline (PGAP) v6.2 (12) was used for genome annotation. Details of the genomes and sequence data are presented in Table 1.
TABLE 1.
Genome information for E. coli O157:H7 strains 380-94 and RV06
|
E. coli O157:H7 strain and genomic element |
Length (bp) |
GC content (%) |
Coverage (×) |
Total no. of sequencing reads |
Total no. of coding sequences |
Total no. of RNAs |
GenBank accession no. |
|---|---|---|---|---|---|---|---|
| 380-94 | 54,212 | 5,379 | 131 | ||||
| Chromosome | 5,442,784 | 50.53 | 109 | CP104309 | |||
| Plasmid | 92,704 | 47.60 | 155 | CP104310 | |||
| RV06 | 95,498 | 5,379 | 131 | ||||
| Chromosome | 5,439,697 | 50.53 | 209 | CP103864 | |||
| Plasmid | 92,705 | 47.60 | 398 | CP103865 |
The 6,388-bp annotated rcsCBD region of strain 380-94 (nucleotide positions 1615428 to 1621815) was used for pairwise alignment against strain RV06 with NCBI BLAST+ v2.13.0 (https://blast.ncbi.nlm.nih.gov) (13) to identify deletion junctions. The 4,769-bp deletion in RV06 spans the 3′ end of rcsC, the entire rcsB, and the 3′ end of rcsD. Study of RV06 may shed additional light on the role of the rcs genes in the stress response.
Data availability.
The sequence reads for RV06 and 380-94 were deposited under SRA accession numbers SRX17303818 and SRX17304264, respectively. The BioSample accession numbers for RV06 and 380-94 are SAMN30166758 and SAMN30166759, respectively, under BioProject accession number PRJNA294158. The complete genome sequences were deposited in GenBank under accession numbers CP103864 and CP103865 (RV06 chromosome and pRV06-1 plasmid, respectively) and CP104309 and CP104310 (380-94 chromosome and p380-94 plasmid, respectively).
ACKNOWLEDGMENTS
This research was supported by the U.S. Department of Agriculture (USDA), Agricultural Research Service (ARS), Food Safety Program (in-house project 8072-42000-094-00D). This research used resources provided by the SCINet project and the AI Center of Excellence of the USDA, ARS (ARS project 0500-00093-001-00-D).
Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the USDA. The opinions in this paper are those of the authors and should not be construed to represent any official USDA or U.S. Government determination or policy. USDA is an equal opportunity employer.
Contributor Information
Chin-Yi Chen, Email: chin-yi.chen@usda.gov.
David Rasko, University of Maryland School of Medicine.
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Associated Data
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Data Availability Statement
The sequence reads for RV06 and 380-94 were deposited under SRA accession numbers SRX17303818 and SRX17304264, respectively. The BioSample accession numbers for RV06 and 380-94 are SAMN30166758 and SAMN30166759, respectively, under BioProject accession number PRJNA294158. The complete genome sequences were deposited in GenBank under accession numbers CP103864 and CP103865 (RV06 chromosome and pRV06-1 plasmid, respectively) and CP104309 and CP104310 (380-94 chromosome and p380-94 plasmid, respectively).