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Characterizing Proteases in an Antarctic Janthinobacterium Sp • Article • Advances in Polar Science doi: 10.13679/j.advps.2015.1.00088 March 2015 Vol. 26 No. 1: 88-95 Characterizing proteases in an Antarctic Janthinobacterium sp. isolate: Evidence of a protease horizontal gene transfer event Cecilia Martinez-Rosales1,2☆, Juan José Marizcurrena1☆, Andrés Iriarte3,4,5☆, Natalia Fullana1, Héctor Musto5 & Susana Castro-Sowinski1,2* 1 Sección Bioquímica y Biología Molecular, Facultad de Ciencias, Universidad de la República (UdelaR), Igua 4225, 11400, Montevideo, Uruguay; 2 Microbiología Molecular, Instituto de Investigaciones Biológicas Clemente Estable (IIBCE). Av Italia 3318, 11600, Montevideo, Uruguay; 3 Dpto de Bioquímica y Genómica Microbiana and Dpto de Genómica (IIBCE); 4 Dpto de Desarrollo Biotecnológico, Instituto de Higiene, Facultad de Medicina, UdelaR; 5 Laboratorio de Organización y Evolución del Genoma, Facultad de Ciencias, UdelaR Received 5 August 2014; accepted 6 January 2015 Abstract We report the isolation of a cold-adapted bacterium belonging to the genus Janthinobacterium (named AU11), from a water sample collected in Lake Uruguay (King George Island, South Shetlands). AU11 (growth between 4°C and 30°C) produces a single cold-active extracellular protease (ExPAU11), differentially expressed at low temperature. ExPAU11 was identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS) as an alkaline metallo-protease (70% coverage with an extracellular protease of Janthinobacterium sp. PI12), and by protease-inhibitor screening identified as a serine-protease. To the best of our knowledge this is the first experimental evidence of a cold-active extracellular protease produced by Janthinobacterium. Furthermore, we identified a serine-protease gene (named JSP8A) showing 60% identity (98% ☆ query coverage) to subtilisin peptidases belonging to the S8 family (S8A subfamily) of many cyanobacteria. A phylogenetic analysis of the JSP8A protease, along with related bacterial protein sequences, confirms that JSP8A clusters with S8A subtilisin sequences from different cyanobacteria, and is clearly separated from S8A bacterial sequences of other phyla (including its own). An analysis of the genomic organization around JSP8A suggests that this protease gene was acquired in an event that duplicated a racemase gene involved in transforming L- to D-amino acids. Our results suggest that AU11 probably acquired this subtilisin-like protease gene by horizontal gene transfer (HGT) from a cyanobacterium. We discuss the relevance of a bacterial protease-HGT in the Antarctic environment in light of this hypothesis. Keywords Antarctic, cold-active protease, horizontal gene transfer, Janthinobacterium, subtilisin Citation: Martinez-Rosales C, Marizcurrena J J, Iriarte A, et al. Characterizing proteases in an Antarctic Janthinobacterium sp. isolate: Evidence of a protease horizontal gene transfer event. Adv Polar Sci, 2015, 26:88-95, doi: 10.13679/j.advps.2015.1.00088 1 Introduction negative Betaproteobacteria (order Burkholderiales, family Oxalobacteraceae), commonly found in soil and water, and [1] Bacteria belonging to the genus Janthinobacterium are Gram- in a variety of foods where they cause food spoilage . Two species (J. agaricidamnosum and J. lividum) and many strains * Corresponding author (email: [email protected]) have been described. Janthinobacterium strains have been ☆ Martinez-Rosales C, Marizcurrena J J and Iriarte A equally contributed to this work. journal.polar.org.cn Characterizing proteases in an Antarctic Janthinobacterium sp. isolate 89 isolated from temperate environments, but many Arctic and yeast extract, 10 g·L-1 NaCl) and MM (5% LB and 5% skim Antarctic cold-tolerant isolates have also been reported[2-5]. milk) liquid medium, as described by Martinez-Rosales and The production of extracellular hydrolytic enzymes Castro-Sowinski[17]. Proteolytic activity was determined in by isolates of this genus has been reported, e.g. production the cell-free supernatant of MM grown cells, using azocasein of chitinase[6], β-agarase[7], and chitosanase[8]. However, the as a substrate[18], when clarification of the medium (milk production of extracellular proteases by Janthinobacterium coagulation) was evident. Cell-free supernatant was obtained strains is quite rare[1], in spite of reports to the contrary by after centrifugation and filtration (0.45 μm Millipore filters) Dainty et al.[9], Bach et al.[10], and Tomova et al.[11]. Proteases of grown culture medium. One unit of enzyme activity (U) are ubiquitous endo- and exo-peptidases that catalyze the was defined as the amount of cell-free supernatant required hydrolytic breakdown of proteins into peptides or amino to increase absorbance by one unit at 340 nm under the assay acids. They are classified into families based on significant conditions. similarity in amino acid sequence, with each family identified by a letter representing the catalytic type (A, aspartic; C, 2.3 Zymography and serine protease inhibitor effects cysteine; G, glutamic; M, metallo; N, asparagine; S, serine; T, on extracellular protease activity threonine; U, unknown), and subfamilies based on evidence of very ancient evolutionary divergence. Protease profiles were analyzed using zymogram gels with Proteases have many industrial applications, and gelatin as the copolymerized substrate and 8% acrylamide have been recognized as a key step in the degradation and as the resolving gel (5% for the stacking gel). Cell-free utilization of proteinaceous polymers by bacteria[12-14]. supernatant samples were obtained after clarification of Major interest exists in finding novel proteolytic enzymes the MM medium at 4°C. In parallel, zymograms were with new properties, such as high performance at low- run with copolymerized gels supplemented with 2 mM temperature, because of potential industrial applications. phenylmethilesulfonyl fluoride (PMSF; serine protease Many investigators have isolated proteolytic bacteria from inhibitor). Methods were as described by Martinez-Rosales extremely cold environments and the proteases have been and Castro-Sowinski[17]. characterized[15-17]. For an interesting review of proteases from psychrotrophs, see Kasana[14]. 2.4 Extracellular protease identification via proteomics We reported previously the isolation of Pseudomonas and Flavobacterium strains that produce extracellular cold- Cell-free supernatant of MM grown cells was used to identify active proteases, from water samples collected at Fildes the extracellular protease by matrix-assisted laser desorption/ Peninsula (King George Island, South Shetlands, Antarctica)[17]. ionization time-of-flight mass spectrometry (MALDI-ToF Our current work aims to analyze the occurrence of serine- MS). The cell-free supernatant was analyzed by sodium proteases in the cold-tolerant Janthinobacterium sp. AU11 dodecyl sulfate polyacrylamide gel electrophoresis (SDS- isolate. Serine proteases are among the most important PAGE) (12% acrylamide for the resolving gel and 5% for enzymes with industrial applications. During this work we the stacking gel), and then the proteins were visualized by found evidence that AU11 most likely acquired a serine- Coomassie staining. In-gel digestion and MALDI-ToF MS protease gene by horizontal gene transfer (HGT) from of the prominent band was done as described by Piñeyro et [19] a cyanobacterium. Given this hypothesis, we discuss al. in the Analytical Biochemistry and Proteomics Unit of the relevance of a protease-HGT event in the Antarctic the Institute Pasteur (Montevideo, Uruguay). The protein was environment. identified by database searching at NCBI (National Center for Biotechnology Information; http://www.ncbi.nlm.nih. gov[20]) with peptide m/z values (mass divided by charge) 2 Materials and methods using the MASCOT software (www.matrixscience.com[21]). 2.1 Isolation and identification of an extracellular 2.5 Amplification of protease genes protease-producing bacterium Genomic DNA was extracted using the phenol-chloroform An extracellular protease-producing bacterium was isolated [22] from a water sample collected from Lake Uruguay (near the procedure described by Sambrook et al. and used as Uruguayan Antarctic Scientific Base; 62°11′4″S, 58°51′7″W; template for the Polymerase Chain Reaction (PCR). For protease gene amplification the consensus-degenerate King George Island, South Shetlands), and identified by [23] sequencing a 1 500 bp 16S rDNA fragment as described by hybrid primers designed by Acevedo et al. (B2F: 5′GGC Martinez-Rosales and Castro-Sowinski[17]. CAC GGC ACC CAY GTB GCS GG 3′ and B2R: 5′CGT GAG GGG TGG CCA TRS WDG T 3′) were used. Then, 2.2 Growth rate and protease production specific primers (PROTJF: 5′GGT TTG ATC CTG CCC ATC TTT GC 3′ and PROTJR: 5′CAG TTG CTG TTG The ability of AU11 to grow and produce extracellular GGC ATG GGA G 3′) were designed and used to amplify proteases at different temperatures (4°C, 18°C, and 30°C) was the protease gene fragment. The entire coding sequence analyzed in Luria-Bertani (LB; 10 g·L-1 tryptone, 5 g·L-1 (CDS) and genomic context was obtained by inverse PCR 90 Martinez-Rosales C, et al. Adv Polar Sci March(2015) Vol. 26 No. 1 (iPCR) using the outward primers OUT1 (5′GC AAA GAT accession numbers were assigned: JN416568 and JN416569 GGG CAG GAT CAA ACC 3′) and OUT2 (5′TCC CAT for the 16S rDNA and the protease JSP8A gene, respectively. GCC CAA CAG CAA CTG 3′) (both primers are the reverse complement of specific primers PROTJF and PROTJR). 3 Results Purified PCR
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