Whole-genome sequence of Enterobacter hormaechei, isolate jjbc recovered from the gut of Plutella xylostella feeding on cabbage

TL;DR

This paper reports the whole-genome sequence of a gut bacterium found in a cabbage-feeding pest insect.

Contribution

The study provides a new whole-genome sequence of a gut symbiont from a major agricultural pest.

Findings

The genome of Enterobacter hormaechei was sequenced using both long and short read technologies.

The bacterium is a dominant gut symbiont in Plutella xylostella, a pest known for antimicrobial resistance.

The isolate may serve as a model for studying resistance evolution in gut microbes.

Abstract

We present the whole-genome sequence of Enterobacter hormaechei (previously Enterobacter cloacae) obtained from long and short reads. It is a dominant gut symbiont of the notorious crop pest Plutella xylostella, highly prevalent in lepidopteran midguts and a useful model for the evolution of resistance to antimicrobials.

Figures

(A) Whole-genome phylogeny generated using the TYGS server confirming isolate jjbc belongs to the species E. hormaecehei and clusters with isolates from environmental samples, shrimps, and insect guts. Colored squares show five species clusters and seven sub-species clusters and the relative genome size and protein count of different genomes. Numbers above the branches represent branch support values inferred from 100 pseudo-bootstrap replicates based on Genome BLAST Distance Phylogeny under the algorithm distance formula (18). (B) Top 20 Gene Ontology terms showing the number of genes associated with each function for the categories of molecular function, biological process, and cellular component.

Tables

TABLE 1. Summary of the genome assembly statistics for E. hormaechei isolate jjbc a

Replicon	Length (bp)	GC content (%)	Topology	GenBank accession
Chromosome	4,854,432	55.3	Circular	CP136935
Plasmid 1	87,769	50.5	Circular	CP136936
Plasmid 2	82,397	53.2	Circular	CP136937
Plasmid 3	64,969	52.4	Circular	CP136938
Plasmid 4	53,743	50.8	Circular	CP136939

Full text

ANNOUNCEMENT

Enterobacter symbionts isolated from lepidopterans encode genes with functions such as digestion of plant cell wall carbohydrates, detoxification of plant allelochemicals, and the breakdown of insecticides (1 – 4), capabilities that are consistent with a niche as symbionts

of herbivores. The ease of use of Enterobacter hormaechei in gnotobiotic insect rearing systems and the possession of an AmpC beta-lactamase also mean that this bacterium is a valuable model for antimicrobial resistance in the Enterobactericeae (5).

E. hormaechei isolate jjbc was isolated by homogenizing Plutella xylostella larvae feeding on Chinese cabbage, Brassica pekinensis, at the Department of Zoology, University of Oxford. Homogenates were plated on Luria-Bertani (LB) agar and incubated at 30°C (6). After streaking clones, bacteria were grown in LB broth overnight at 30°C. DNA extraction from these cultures and sequencing were done at MicrobesNG (Birmingham, UK). Briefly, 40 µL of bacterial cell suspension was lysed in 120 µL of TE buffer-containing lysozyme, metapolyzyme, and RNase A. Proteinase K (final concentration 0.1 mg/mL) and SDS [final concentration 0.5% (vol/vol)] were added and incubated for 5 min at 65°C. Genomic DNA was purified using an equal volume of SPRI beads and resuspended in Elution buffer (10 mM Tris-HCl, pH 8.0). Genomic DNA libraries were prepared using the Nextera XT Library Prep Kit (Illumina, San Diego, USA) with these modifications: input DNA was increased twofold, and PCR elongation time was increased to 45 seconds. For the short reads, libraries were sequenced on a lllumina NovaSeq 6000 (Illumina, San Diego, USA) using a 250 bp paired-end protocol. For the long reads, DNA libraries were prepared using the Oxford Nanopore SQK-LSK109 and Native Barcoding EXP-NBD104/114 kits (ONT, UK) with 400–500 ng High Molecular Weight DNA, fragmented without size selection. Barcoded samples were pooled and loaded in a Library prepFLO-MIN111 (R10.3) flow cell, in a GridION (ONT, UK).

We obtained 1,172,223 Illumina reads and 13,666 ONT reads. The read quality was verified using FastQC (v. 0.12.1) (7) and NanoPlot (v. 1.42.0) (8), respectively. Genome assembly was performed using the fully automated hybrid assembly pipeline Unicycler (v. 0.5.0) (9). Default settings were used for all software (10). Manual intervention was required to resolve the following issues in the automatic assembly: (i) erroneous removal of genuine sequences during graph cleaning of the short-read assembly and (ii) collapse of inter-plasmidic repeats (11). The correction of these assembly errors resulted in a fully resolved genome consisting of one circular chromosome and four circular plasmids (Table 1), with a BUSCO completeness score of C:98.8%(S:98.6%,D:0.2%),F:0.2%,M:1.0%,n:440 (12) and a mean read depth of 110×. The closest matching species confirmed using the Type (Strain) Genome Server (TYGS) (13) is E. hormaechei (Fig. 1A). This genome was annotated using PGAP v.6.6 (14) and contains 4,951 predicted genes. Functional annotation shows multiple genes associated with carbohydrate metabolism (Fig. 1B). Using ResFinder 4.4.2 server (15), we identified two antimicrobial resistance genes: fosA for Fosfomycin resistance (96.71% alignment and 100% coverage) and blaACT-16, an AmpC beta-lactamase (99.74% alignment and 100% coverage) common in Enterobacter spp. (16).

**TABLE 1: Summary of the genome assembly statistics for E. hormaechei isolate jjbc

a**

(A) Whole-genome phylogeny generated using the TYGS server confirming isolate jjbc belongs to the species E. hormaecehei and clusters with isolates from environmental samples, shrimps, and insect guts. Colored squares show five species clusters and seven sub-species clusters and the relative genome size and protein count of different genomes. Numbers above the branches represent branch support values inferred from 100 pseudo-bootstrap replicates based on Genome BLAST Distance Phylogeny under the algorithm distance formula (18). (B) Top 20 Gene Ontology terms showing the number of genes associated with each function for the categories of molecular function, biological process, and cellular component.