Permanent Draft Genome Sequence of Sulfoquinovose-Degrading Pseudomonas Putida Strain SQ1 Ann-Katrin Felux1,2, Paolo Franchini1,3 and David Schleheck1,2*

Erschienen in: Standards in Genomic Sciences ; 10 (2015). - 42 Felux et al. Standards in Genomic Sciences (2015) 10:42 DOI 10.1186/s40793-015-0033-x SHORT GENOME REPORT Open Access Permanent draft genome sequence of sulfoquinovose-degrading Pseudomonas putida strain SQ1 Ann-Katrin Felux1,2, Paolo Franchini1,3 and David Schleheck1,2* Abstract Pseudomonas putida SQ1 was isolated for its ability to utilize the plant sugar sulfoquinovose (6-deoxy-6-sulfoglucose) for growth, in order to define its SQ-degradation pathway and the enzymes and genes involved. Here we describe the features of the organism, together with its draft genome sequence and annotation. The draft genome comprises 5,328,888 bp and is predicted to encode 5,824 protein-coding genes; the overall G + C content is 61.58 %. The genome annotation is being used for identification of proteins that might be involved in SQ degradation by peptide fingerprinting-mass spectrometry. Keywords: Pseudomonas putida SQ1, aerobic, Gram-negative, Pseudomonadaceae, plant sulfolipid, organosulfonate, sulfoquinovose biodegradation Introduction sediment of pre-Alpine Lake Constance, Germany [13]. Pseudomonas putida strain SQ1 belongs to the family of SQ is the polar headgroup of the plant sulfolipid sulfo- Pseudomonadaceae in the class of Gammaproteobac- quinovosyl diacylglycerol, which is present in the teria. The genus Pseudomonas was first described by photosynthetic membranes of all higher plants, mosses, Migula (in the year 1894 [1]) and the species Pseudo- ferns and algae and most photosynthetic bacteria [14]. monas putida by Trevisan (in 1889 [2]). P. putida strain SQ is one of the most abundant organosulfur com- KT2440 was the first strain whose genome had been se- pounds in the biosphere, following glutathione, cyst- quenced (in 2002 [3]), and it is the most well-studied P. eine, and methionine, and the global production of SQ putida strain thus far [4]. Currently, there are more than is estimated at 10 gigatons (1010 tons) per year [15]. 30 genome sequences of P. putida strains available (e.g., Hence, the complete degradation of SQ concomitant 12 complete and 24 draft genomes in NCBI; January with a recycling of the bound sulfur in form of inor- 2015), including the complete genome sequence of type ganic sulfate is an important process of the carbon and strain NBRC 14164T [5]. P. putida species are highly sulfur cycle, e.g. in soils. abundant in water, soil and in the rhizosphere [6, 7], can Until today only one bacterial degradation pathway for be plant-beneficial [8], and are extensively studied for SQ has been identified, ‘sulfoglycolysis’ in Escherichia their capabilities to degrade a broad range of substrates, coli K-12 [16]. In this pathway, SQ is catabolized in ana- especially aromatic compounds [9–12]. logy to glucose-6-phosphate via an adapted Embden- P. putida strain SQ1 was isolated for its ability to Meyerhof-Parnas (glycolysis) pathway, involving four utilize the sulfonated plant sugar sulfoquinovose (6-deoxy- newly identified enzymes and genes, and four newly 6-sulfoglucose) as a sole source of carbon and energy identified metabolites. The pathway yields dihydroxy- for growth, and was enriched from a sample of littoral acetone phosphate (DHAP), which drives energy metab- olism and growth of E. coli, and sulfolactaldehyde, which is reduced to dihydroxypropanesulfonate and excreted * Correspondence: [email protected] Pseudomonas 1Department of Biology, University of Konstanz, Konstanz, Germany [16]. For species, it is well-known that 2Konstanz Research School Chemical Biology, University of Konstanz, these bacteria lack the key enzyme for glycolysis, Konstanz, Germany Full list of author information is available at the end of the article © 2015 Felux et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Konstanzer Online-Publikations-System (KOPS) URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-0-297588 Felux et al. Standards in Genomic Sciences (2015) 10:42 Page 2 of 6 phosphofructokinase, but that the alternative Entner- Based on its 16S rRNA gene sequence, strain SQ1 is a Doudoroff pathway is operative, i.e., an oxidative entry member of the genus and species Pseudomonas putida, into glucose-6-phosphate catabolism via a dehydrogen- which is placed in the family Pseudomonadaceae within ase enzyme. We detected a SQ-dehydrogenase activity in the order Pseudomonadales of Gammaproteobacteria,as crude extract of SQ-grown P. putida SQ1 cells, and we illustrated by a phylogenetic tree shown in Fig. 2. Cur- therefore suspect that a ‘Sulfo-Entner-Doudoroff’-type of rently, 1,732 genome sequences of member of the order pathway might be operative in P. putida SQ1 for catab- Pseudomonadales of Gammaproteobacteria, and 707 olism of SQ, but not sulfoglycolysis. genome sequences within the family Pseudomonadaceae A draft genome sequence of strain SQ1 has been have been established (IMG JGI, January 2015). established and annotated in the IMG pipeline, and the annotation has been transferred to a proteomics (Mas- Genome sequencing information cot) database for peptide fingerprinting-mass spectrom- Genome project history etry: in our present (unpublished) work, the database is The DNA sample was submitted to GATC Biotech used to identify enzymes and genes that are specifically (Konstanz, Germany) in December 2012 where the induced during growth with SQ, e.g. in comparison to whole-genome shotgun sequencing phase was completed cells grown with glucose, by two-dimensional protein gel in April 2013; the whole-genome shotgun sequencing electrophoresis. Here, we present a summary classifica- was performed by GATC using the Illumina HiSeq2000 tion and a set of features for Pseudomonas putida strain platform and a 100-bp paired-end library. After the SQ1, together with the description of the shotgun gen- mapping and de-novo assembly of the unmapped reads, omic sequencing and annotation. which was done at the Genomics Center of the Univer- sity of Konstanz, the draft genome sequence was uploaded into the IMG Pipeline for annotation and pre- Organism Information sented for public access on December 2014. The draft Classification and features genome annotation is available at IMG under the IMG P. putida SQ1 is a rod-shaped (Fig. 1), motile, Gram- submission ID 14279, and was also deposited in Gen- negative bacterium that grows aerobically in complex bank under the accession number JTCJ00000000. Table 2 medium (e.g. LB-medium), or prototrophically in mineral- presents the project information and its association with salts medium with a single carbon source (e.g., succinate, MIGS version 2.0 compliance [17]. glucose, SQ). Strain SQ1 grows overnight on LB-agar plates and forms beige-whitish, smooth colonies (Table 1). Pseudomonas putida SQ1 has been deposited in the Leib- Growth conditions and genomic DNA preparation niz Institute DSMZ-German Collection of Microorganisms Genomic DNA was extracted from an overnight culture and Cell Cultures under reference number DSM 100120. of P. putida SQ1 grown at 30 °C in LB medium (500-ml Fig. 1 Scanning electron micrographs of Pseudomonas putida SQ1. Cells derived from a liquid culture (LB medium) Felux et al. Standards in Genomic Sciences (2015) 10:42 Page 3 of 6 Table 1 Classification and general features of Pseudomonas putida SQ1 [32] MIGS ID Property Term Evidence codea Current Domain Bacteria TAS [33] classification Phylum Proteobacteria TAS [34] Class Gammaproteobacteria TAS [34, 35] Order Pseudomonadales TAS [36, 37] Family Pseudomonadaceae TAS [38, 39] Genus Pseudomonas TAS [1, 38–40] Species putida TAS [1, 2] Fig. 2 Phylogenetic tree based on the 16S rRNA gene sequence Strain SQ1 TAS [13] of P. putida SQ1, and sequences of other strains of the species P. Gram stain Negative TAS [13] putida, P. aeruginosa and P. fluorescens. The sequences were aligned with the CLUSTAL W program and the tree was built with the Cell shape Rod-shaped TAS [13] neighbor-joining algorithm integrated in the MEGA 6.0 program [31]. Motility Motile TAS [13] The phylogenetic tree was tested with 1000 bootstrap replicates; Sporulation Non-sporulating TAS [13] bootstrap values are shown at each node. The scale bar represents a 0.005 % nucleotide sequence divergence Temperature Mesophile TAS [13] range Optimum 30 °C TAS [13] using the Illumina HiSeq2000 platform and a 100-bp temperature paired-end library, which resulted in 23,816,201 se- pH range; Not tested; 7.2 TAS [13] quenced reads (1.85 × 109 total bases). The trimming, Optimum mapping, as well as the de novo assembly of the un- Carbon Succinate, glucose, IDA,TAS [13] source sulfoquinovose mapped raw reads, was performed at the Genomics Cen- ter of the University of Konstanz, Germany. First, the Energy Chemoorganotroph IDA,TAS [13] source remaining adapters were removed and reads were trimmed by quality in CLC Genomics Workbench v6.5 MIGS-6 Habitat Aerobic habitat TAS [13] (CLC bio, Aarhus, Denmark). In the next step, Bowtie MIGS-22 Oxygen Aerobic TAS [13] requirement v2.2.3 [18] was used to align the filtered reads against the genome of the closest relative, P. putida strain MIGS-15 Biotic Free-living NAS relationship W619, to which 21,943,994 reads matched. These mapped reads were assembled with a reference-guided MIGS-14 Pathogenicity Potentially pathogenic, Risk group 2 (classification approach using the Columbus module implemented in according to German TRBA) Velvet v1.2.10 [19]. Velvet was then used to de novo as- MIGS-4 Geographic Isolated from littoral TAS [13] semble also all unmatched 1,872,207 reads (8.5 % of total location sediment of Lake Constance, reads).

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