Ridl et al. Standards in Genomic Sciences (2018) 13:3 DOI 10.1186/s40793-017-0306-7 EXTENDED GENOME REPORT Open Access Complete genome sequence of Pseudomonas alcaliphila JAB1 (=DSM 26533), a versatile degrader of organic pollutants Jakub Ridl1†, Jachym Suman2†, Serena Fraraccio2, Miluse Hradilova1, Michal Strejcek2, Tomas Cajthaml3, Andrea Zubrova2, Tomas Macek2, Hynek Strnad1* and Ondrej Uhlik2* Abstract In this study, following its isolation from contaminated soil, the genomic sequence of Pseudomonas alcaliphila strain JAB1 (=DSM 26533), a biphenyl-degrading bacterium, is reported and analyzed in relation to its extensive degradative capabilities. The P. alcaliphila JAB1 genome (GenBank accession no. CP016162) consists of a single 5.34 Mbp-long chromosome with a GC content of 62.5%. Gene function was assigned to 3816 of the 4908 predicted genes. The genome harbors a bph gene cluster, permitting degradation of biphenyl and many congeners of polychlorinated biphenyls (PCBs), a ben gene cluster, enabling benzoate and its derivatives to be degraded, and phe gene cluster, which permits phenol degradation. In addition, P. alcaliphila JAB1 is capable of cometabolically degrading cis-1,2-dichloroethylene (cDCE) when grown on phenol. The strain carries both catechol and protocatechuate branches of the β-ketoadipate pathway, which is used to funnel the pollutants to the central metabolism. Furthermore, we propose that clustering of MALDI-TOF MS spectra with closest phylogenetic relatives should be used when taxonomically classifying the isolated bacterium; this, together with 16S rRNA gene sequence and chemotaxonomic data analyses, enables more precise identification of the culture at the species level. Keywords: Pseudomonas alcaliphila JAB1, Pseudomonadaceae, Genome, Dioxygenase, Monooxygenase, Biodegradation, Bioremediation, Aromatic compounds, Biphenyl, Polychlorinated biphenyls (PCBs), Chlorobenzoic acids (CBAs), cis-1,2-dichloroethylene (cDCE), Phenol, bph genes, ben genes, phe genes, MALDI- TOF MS Introduction of industrial and agricultural origin. Many of these com- Over recent decades, significant quantities of potentially pounds have been found to have toxic, mutagenic and car- harmful chemicals have been released into the environ- cinogenic effects on living organisms. Removal of these ment, creating countless numbers of contaminated sites. xenobiotics usually involves physical and chemical pro- Major contaminants include halogenated and nitrated cesses, such as landfill, excavation and incineration, which alicyclic, aliphatic, aromatic and polyaromatic compounds are expensive and difficult to execute. An alternative approach, bioremediation, uses ubiquitous plant-microbe * Correspondence: [email protected]; [email protected] interactions to degrade xenobiotics [1]. Bacteria and fungi †Equal contributors are natural recyclers capable of funneling toxic organic 1 Department of Genomics and Bioinformatics, Institute of Molecular compounds to central metabolism intermediates [2], Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic thereby creating harmless products [3] such as carbon 2Department of Biochemistry and Microbiology, Faculty of Food and dioxide and water. In addition to the enormous phylo- Biochemical Technology, University of Chemistry and Technology, Prague, genetic diversity of microorganisms, the richness of Czech Republic Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. 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. Ridl et al. Standards in Genomic Sciences (2018) 13:3 Page 2 of 10 their metabolic activities promotes the degradation of species P. alcaliphila, strain JAB1, whose extensive bio- pollutants and xenobiotics in different environments. degradation capabilities are highlighted. Members of the genus Pseudomonas [4, 5], one of the most diverse bacterial genera, inhabit several environ- Organism information mental niches and have been studied in relation to hu- Classification and features man and plant pathogenicity, antibiotic resistance, plant P. alcaliphila was described as a facultatively psychrophilic growth promotion, plant-derived organic matter degrad- alkaliphilic species isolated from seawater off the coast of ation and bioremediation [6]. Pseudomonads, which are Rumoi, Hokkaido, Japan. The characteristics of this species metabolically highly versatile, contain both abundant are as follows: alkaliphile, incapable of growth at >40 °C, and unique metabolic pathways [7], which, most import- catalase- and oxidase-positive and also capable of reducing antly, catabolize a broad range of substrates. Many of nitrate to nitrite and of hydrolyzing casein and gelatin [21]. these substrates are pollutants, including aliphatic and Further physiological features are listed in Table 1. The aromatic petroleum hydrocarbons [8–12], BTEX [13, JAB1 cells are monotrichous rods as shown in Fig. 1. 14], phenolic compounds ranging from phenol via The JAB1 strain was originally misidentified as P. methylphenols and nitrophenols to chlorophenols [15], pseudoalcaligenes [22]. The consensus 16S rRNA gene benzoate, CBAs and toluates [16], biphenyl and PCBs sequence, compiled from four 16S rRNA gene copies [17–19], chlorinated aliphatics [20] and many others. In contained in the JAB1 genome, had 99.93% similarity to this study, we present the first complete genome of the those of P. alcaliphila AL 15-21T [21], P. chengduensis Table 1 Classification and general features of P. alcaliphila JAB1 MIGS ID Property Term Evidence codea Classification Domain Bacteria TAS [55] Phylum Proteobacteria TAS [56, 57] Class Gammaproteobacteria TAS [58, 59] Order Pseudomonadales TAS [5, 60] Family Pseudomonadaceae TAS [61] Genus Pseudomonas TAS [4, 5] Species Pseudomonas alcaliphila TAS [21] Strain JAB1 (Accession no. DSM 26533) Gram stain Negative TAS [21] Cell shape Rod-shaped IDA, TAS [21] Motility Motile IDA, TAS [21] Sporulation Non-sporulating TAS [21] Temperature range Mesophile IDA Optimum temperature 28–37 °C IDA pH range; Optimum Not tested; Neutral TAS [21] Carbon source Biphenyl, phenol, other organic substrates IDA MIGS-6 Habitat Soil TAS [22] MIGS-6.3 Salinity Up to 7% NaCl (w/v) TAS [21] MIGS-22 Oxygen requirement Aerobic TAS [22] MIGS-15 Biotic relationship Free-living TAS [22] MIGS-14 Pathogenicity Non-pathogen NAS MIGS-4 Geographic location Czech Republic TAS [22] MIGS-5 Sample collection 2000 NAS MIGS-4.1 Latitude 50°1′52″N NAS MIGS-4.2 Longitude 16°35′55″E NAS MIGS-4.4 Altitude 420 m NAS IDA Inferred from Direct Assay, TAS Traceable Author Statement (i.e., a direct report exists in the literature), NAS Non-traceable Author Statement (i.e., not directly observed for the living, isolated sample, but based on a generally accepted property for the species, or anecdotal evidence) aEvidence codes Ridl et al. Standards in Genomic Sciences (2018) 13:3 Page 3 of 10 Fig. 1 Transmission electron photomicrograph of P. alcaliphila JAB1 MBRT [23] and P. oleovorans subsp. lubricantis RS1T PCBs Delor 103 and incubated for 48 h. The content of [24]. Additional closest matches included P. toyotomien- individual PCB congeners present in the microcosms as sis HT-3T (99.86% similarity) and P. mendocina CH50T well as CBA accumulation were determined using GC-MS (99.24% similarity). A phylogenetic tree indicates closest (450-GC, 240-MS ion trap detector, Varian, Walnut relatedness of the JAB1 strain to P. alcaliphila AL 15- Creek, CA). PCBs were analyzed in ethyl acetate extracts 21T (Fig. 2). In addition, the JAB1 strain was unable to according to the method described by Čvančarová M. grow at 41 °C, which is a typical feature of P. alcaliphila et al. [25]. CBAs were analyzed in the extracts using GC- but not of other closely related pseudomonads [23]. MS after methylation with diazomethane according to our Furthermore, whole-cell MALDI-TOF MS analysis, per- previously published protocol [26]. The respective chem- formed following the methodology described elsewhere ical standards for the analytes were obtained from Merck [12], indicated that JAB1 spectra clustered with those of (Darmstadt, Germany), Supelco (Steinheim, Germany), P. alcaliphila AL 15-21T (Fig. 3). The results of MALDI- TCI Europe (Zwijndrecht, Belgium) and AccuStandard TOF MS profiling thus further confirmed the identity of (New Haven, USA). the JAB1 strain as P. alcaliphila. Therefore, we propose Metabolic activity of the JAB1 strain resulted in the that MALDI-TOF MS analysis be performed of the depletion of various mono-, di-, tri- and tetra-chlorinated isolate and its closest phylogenetic relatives when biphenyls as shown in Fig. 4. At the same time, 2-CBA, taxonomically classifying the isolated bacterium. In 3-CBA, 4-CBA, 2,3-diCBA, 2,4-diCBA and 2,5-diCBA for- addition to 16S rRNA gene sequence and chemotaxo- mation (data not shown) was observed over the course of nomic data analysis, MALDI-TOF MS can provide the coincubation period; these are common biodeg- additional