Draft Genome Sequence of Pseudomonas sp. Strain CES, Containing the Entire Alkylxanthine Gene Cluster for Caffeine Breakdown

Pseudomonas strain CES was isolated from caffeine-enriched soil and found to possess the N-demethylation pathway for caffeine breakdown. We report the nucleotide sequence of the draft genome with 5,827,822 bp, 62.6% G+C content, and 5,427 protein-coding regions.

ABSTRACT

Pseudomonas strain CES was isolated from caffeine-enriched soil and found to possess the N-demethylation pathway for caffeine breakdown. We report the nucleotide sequence of the draft genome with 5,827,822 bp, 62.6% G+C content, and 5,427 protein-coding regions.

ANNOUNCEMENT

Various bacteria present in the soil, in water, or in the alimentary canal of insects have been shown to catabolize caffeine (1,3,7-trimethylxanthine) using N-demethylation or C-8-oxidative pathways (1–4). The bacterium Pseudomonas sp. strain CES was isolated from coffee-enriched soil through a caffeine-enrichment procedure as described previously (5, 6). Prior to isolation, coffee grounds were amended to the soil for 10 days. The strain was initially characterized as a Pseudomonas sp. based on 16S rRNA sequencing and fatty acid methyl ester (FAME) analysis (5) and can grow on up to 8 g/liter caffeine, which is over three times higher than the concentration used for most caffeine-degrading bacteria (2, 3).

Here, we report the draft genome sequence of Pseudomonas sp. CES, which uses the N-demethylation pathway for caffeine breakdown (6). Genomic DNA extraction and library preparation were performed at the Beijing Genomics Institute (BGI). DNA was extracted from CES cells using the BGI standard operating procedure based on the cetyltrimethylammonium bromide procedure (7), and 200- to 400-bp fragments were recovered after fragmentation by sonication. Fragments were end repaired, ligated to adaptors, amplified by PCR, heat denatured, and circularized with splint oligonucleotides. This final library was sequenced using next-generation sequencing on the Illumina HiSeq BGISEQ-500 platform, resulting in 177,377,152 total reads (88,688,576 paired-end reads) of 90 to 100 bp. Low-quality reads (50% Q < 20) and adapter sequences were identified and removed using SOAPnuke (8), and cleaned reads were assembled de novo using the IDBA-UD assembly program (9) using the parameters mink 33 and maxk 59. This analysis resulted in 3,143,938 paired-end reads and 28,542,398 single-end reads assembled into 198 contigs. Reads were mapped back to the assembled contigs for validation, and the N₅₀ contig length was 71,749 nucleotides. The 198 contigs of 5,827,822 bp have an overall base composition of 62.6% G+C; the average coverage per base position was 553×. The assembled 198 contigs were run through Prodigal version 2.5.3 GeneFinder (10) and annotated using BLASTP against the RefSeq database of bacterial proteins downloaded from NCBI. The programs tRNAscan-SE version 1.4 (11) and RNAmmer version 1.2 (12) were used to predict regions encoding tRNAs and rRNAs. Default parameters were used for all software unless otherwise specified.

The chromosome has 5,427 protein-coding regions, 16S and 23S rRNA genes, and 29 tRNA genes. The putative N-demethylase genes ndmABCDE, required for N-demethylation of caffeine to xanthine, were identified in the genomic sequence with a BLAST search of the encoded protein using BLASTP (13) against NCBI’s nonredundant protein database. The caffeine-degrading gene cluster in CES is similar to the alkylxanthine (Alx) gene clusters in Pseudomonas putida CBB5 and Pseudomonas sp. NCIM 5235 (Table 1); Clustal Omega analysis of the three cluster nucleotide sequences revealed that the CES cluster has identities of 71.92% and 91.65% to the clusters from CBB5 and NCIM 5235, respectively. The CES Alx gene cluster contains putative transcriptional regulators cafT and cafR, a putative caffeine permease, cafP, and homologs to Escherichia coli frmAB genes, which encode enzymes for formaldehyde metabolism (14–16), but differs from that of CBB5 due to the presence of a vicinal oxygen chelate (VOC) family protein upstream of the ndmA gene. This VOC gene was found in strain NCIM 5235, which can also survive at high levels of caffeine (17) and may be one reason that CES can grow above 2.5 g/liter caffeine. The CES draft genome could help elucidate the molecular evolution of Pseudomonas species and may reveal additional genes or regulatory mechanisms to enable bacteria to grow at higher caffeine concentrations.

TABLE 1.

Genes in the Alx gene cluster for Pseudomonas sp. CES, Pseudomonas putida CBB5, and Pseudomonas sp. NCIM 5235

Gene	CES GenBank accession no.	CBB5		NCIM 5235		E. coli MG1655
		GenBank accession no.	Nucleotide identity (%)	GenBank accession no.	Nucleotide identity (%)	GenBank accession no.	Nucleotide identity (%)
ndmA	KAF4561170	KKX58007	90.31	AWV66915	99.72
ndmB	KAF4561167	KKX58004	90.14	AWV66912	98.03
ndmC	KAF4561164	KKX58001	87.68	AWV66909	98.59
ndmD	KAF4561163	KKX58000	80.78	AWV66908	96.26
ndmE	KAF4561162	KKX57999	84.68	AWV66906	98.20
cafR	KAF4561168	KKX58005	90.45	AWV66913	100
cafTa	KAF4561173	KKX58009	64.13
cafP	KAF4561166	KKX58003	87.25	AWV66911	96.42
frmA	KAF4561172	KKX58008	92.7	AWV66917	99.19	AAC73459	73.17
frmB	KAF4561161	KKX57998	75.35	AWV66907	96.84	AAC73458	48.01
VOCb	KAF4561171			AWV66916	99.21

^aThe NCIM 5235 gene cluster sequencing did not extend to include cafT.

^bThe CBB5 gene cluster does not contain a VOC gene.

Data availability.

This genome was deposited in GenBank under the BioProject number PRJNA613486, BioSample accession number SAMN14432513, and SRA accession number SRP259409. This whole-genome shotgun project was deposited at DDBJ/ENA/GenBank under the accession number JAAXLL000000000. The version described in this paper is version JAAXLL010000000.