Complete genome sequence of a carbon tetrachloride-degrading bacterium, Pseudomonas sp. strain Stari2

TL;DR

This paper presents the full genome sequence of a Pseudomonas strain that can break down carbon tetrachloride in the presence of oxygen.

Contribution

The study provides the complete genome sequence of a novel carbon tetrachloride-degrading Pseudomonas strain.

Findings

The genome is a single circular sequence of 6,310,573 bp with 5,680 coding sequences.

The strain has a GC content of 60.3% and 94.06% average nucleotide identity to its closest species.

Abstract

This article reports on the complete genome of Pseudomonas sp. strain Stari2, which was shown to have the ability to degrade carbon tetrachloride (CCl4) in aerobic conditions. A single circular sequence of 6,310,573 bp, a GC content of 60.3%, and 5,680 coding sequences were obtained. In silico analysis revealed an average nucleotide identity of 94.06% to its closest species.

Tables

TABLE 1. CDSs potentially involved in dehalogenation in strain Stari2

Locus tag	Putative homolog	Substrate	Reference
NH234_02775	dhaA	R-X	(20)
NH234_21655	l-dex	C2HO2RX	(21)
NH234_03495	ctrA	CHCl3, 1,1,1-TCA	(22)
NH234_20080	cfrA	CHCl3	(23)
NH234_09950	hadL	C2HO2RX	(24)
NH234_00545	dcmA	CH2Cl2	(25)
NH234_03490	pceA	PCE, hexachloroethane	(26)
NH234_15595	tceA	TCE, cis-DCE, 1,1-DCE	(27)
NH234_14700	cyp108	CT and many others	(28)
NH234_07415	tcbC	1,2-Dichlorobenzene	(29)
NH234_14015	cmuA	Chloromethane	(30)
NH234_19170	vcrA	Chloroethene	(31)
NH234_20830	dehH1	Chloroacetate	(32)
NH234_22340	trmA	CHCl3	(33)
NH234_00830	mmoC	R-X	(34)

Full text

ANNOUNCEMENT

CCl_4_ (CT) is a priority environmental contaminant due to its high toxicity, which poses significant risks to human health and ecosystems through the generation of free radicals via oxidative pathways (1). While CT biodegradation has been extensively studied under anaerobic conditions primarily through cometabolic reduction (2–4), aerobic degradation remains less explored. Our previous work isolated a bacterium capable of aerobic CT degradation from a microbial consortium collected in Iwate Prefecture, Japan (5).

Isolated bacteria were grown in brain heart infusion medium with 5 mM CT at 30°C for 48 h, and high-molecular weight (HMW) DNA was extracted using the Promega Wizard HMW DNA Extraction Kit following the manufacturer’s protocol. DNA was sequenced by Bioengineering Lab Co., Ltd. (Kanagawa, Japan) using two platforms: DNBSEQ (MGI, Shenzhen, China) for short reads and GridION (Oxford Nanopore Technologies, Oxford, UK) for long reads. Default parameters were used for all software, unless otherwise specified.

For short read sequencing, DNA concentration was measured using Synergy LX and QuantiFluor, and quality was confirmed via electrophoresis using the 5200 Fragment Analyzer System with the Agilent HS Genomic DNA 50 kb Kit. Library preparation utilized the MGIEasy FS DNA Library Prep Set and the MGIEasy DNA Adapters-96 Kit. Sequencing was performed under 2 × 200 bp conditions on the DNBSEQ-G400 platform. For long-read sequencing, the Ligation Sequencing Kit from Oxford Nanopore Technologies was used, and sequencing was performed on the GridION platform with an R9.4.1 flow cell. Guppy (Ver. 4.0.11) was used for real-time base calling and barcode sorting.

Adapter sequences from short reads were removed using Cutadapt (Ver. 2.7) (6), and read sampling was performed with SeqKit (Ver. 0.11.0) (7). Low-quality bases were removed using Sickle (version 1.33)(8). Long read adapter sequences were removed with Porechop (Ver. 0.2.3), and reads shorter than 1,000 bases were discarded using Filtlong (Ver. 0.2.0). Hybrid assembly of high-quality short and long reads was conducted using Unicycler (Ver. 0.4.7) (9). Assembly results were confirmed with Bandage (Ver. 0.8.1) (10), and genome data integrity was checked with CheckM (Ver. 1.1.2) (11). Gene prediction was carried out using PGAP (Ver. 6.1) (12).

The DNBSEQ and GridION sequencing generated 5,998,880 and 223,095 high-quality paired reads (N50 3,836 bp), respectively (paired raw reads 20,716,044 and 1,113,507 respectively). The resulting assembly consists of one circular chromosome with an N50 value of 6,310,573 bp, 60.3% GC content, and a coverage of 875×. Average nucleotide identity analysis (13) showed 94.06% similarity to Pseudomonas kribbensis strain 46-2 (type strain, GenBank accession CP029608 [14]), which is lower than the 95% threshold used to distinguish species (15).

In silico analysis of this complete genome using Prokka, BlastKoala, GhostKoala, KofamKoala, and EggNogMapper (16–19) indicated that strain Stari2 has complete pathways for carbon metabolism, partial pathways for nitrogen metabolism, and complete pathways for sulfur metabolism and cofactor biosynthesis. Fifteen CDSs potentially involved in dehalogenation were identified, including four associated with aerobic dehalogenation (Table 1). While enzymes for chloroalkane and chloroalkene degradation were detected, none were specific to carbon tetrachloride (CCl₄).