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Sulfolobus Acidocaldarius

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Sulfolobus Acidocaldarius ORIGINAL RESEARCH Molecular analysis of the UV-inducible pili operon from Sulfolobus acidocaldarius Marleen van Wolferen1, Małgorzata Ajon2, Arnold J. M. Driessen2 & Sonja-Verena Albers1 1Molecular Biology of Archaea, Max Planck Institute for terrestrial Microbiology, Karl-von-Frisch-Strasse 10, 35043, Marburg, Germany 2Department of Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 7, NL-9747 AG Groningen, The Netherlands Keywords Abstract Archaea, conjugation, DNA exchange, type IV pili. Upon ultraviolet (UV) stress, hyperthermophilic Sulfolobus species show a highly induced transcription of a gene cluster responsible for pili biogenesis: Correspondence the UV-inducible pili operon (ups operon). This operon is involved in UV- Sonja-Verena Albers, Molecular Biology of induced pili assembly, cellular aggregation, and subsequent DNA exchange Archaea, Max Planck Institute for terrestrial between cells. As the system increases the fitness of Sulfolobus cells after UV Microbiology, Karl-von-Frisch-Strasse 10, light exposure, we assume that transfer of DNA takes place in order to repair 35043 Marburg, Germany. Tel: +496421178426; Fax: +496421178429; UV-induced DNA damages via homologous recombination. Here, we studied E-mail: [email protected] all genes present in the ups cluster via gene deletion analysis with a focus on UpsX, a protein that shows no identifiable functional domains. UspX does not Funding Information seem to be structurally essential for UV-induced pili formation and cellular This study was supported by Max Planck aggregation, but appears to be important for efficient DNA transfer. In addi- Society, DFG, NWO. tion, we could show that pilin subunits UpsA and UpsB probably both function as major pilin subunits in the ups pili. Received: 19 June 2013; Revised: 21 July 2013; Accepted: 29 July 2013 MicrobiologyOpen 2013; 2(6): 928–937 doi: 10.1002/mbo3.128 Introduction stress (Ajon et al. 2011). Transfer of DNA therefore prob- ably plays a role in repair of double-strand breaks (DSBs) Upon ultraviolet (UV) stress Sulfolobus species show a caused by UV radiation as was proposed in Ajon et al. high upregulation of a gene cluster encoding proteins (2011). Because the aggregation was also shown to be responsible for formation of type IV pili (T4P) (Fro¨ls inducible by the DNA strand-break-inducing agent bleo- et al. 2007; Go¨tz et al. 2007): the ups operon (UV-induc- mycin, the first trigger for pili formation and subsequent ible pili operon of Sulfolobus) (Fro¨ls et al. 2008). Indeed, aggregation is thought to be the sensing of DSBs in the when analyzed by transmission electron microscopy DNA (Fro¨ls et al. 2008). The mechanism behind this pro- (TEM), a large number of pili could be observed on the cess remains unknown. DNA exchange mechanisms surface of UV-stressed Sulfolobus cells from different spe- among hyperthermophiles for repair of DNA have been cies (Ajon et al. 2011). An additional phenotypic charac- described in more detail by van Wolferen et al. (2013). teristic of UV-stressed Sulfolobus cells is the formation of In other organisms, T4P were also shown to mediate large cellular aggregates (Fro¨ls et al. 2008), which was DNA transfer (Filloux 2010). Different studies showed shown to be mediated by ups pili in a species-specific that the pili facilitate the uptake of extracellular DNA in manner (Ajon et al. 2011). Moreover, Sulfolobus cells can competent Gram-positive and Gram-negative bacteria. exchange chromosomal DNA in a pili-dependent manner The exact role of T4P in competence is still not well which was shown to increase cellular fitness under UV understood, but their presence is essential for successful 928 ª 2013 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. M. van Wolferen et al. Analysis of the S. acidocaldarius ups Operon DNA uptake (reviewed in Kruger€ and Stingl 2011). It has was supplemented with 1.5% gelrite. Plates were incubated been suggested that DNA is brought close to the cell for 5–6 days at 78°C. E. coli–competent cells DH5a and surface by binding to the pili that subsequently retract. A ER1821 (NEB, Frankfurt am Main, Germany), used for, recent study for the first time reported the binding of a respectively, cloning and methylation of plasmid DNA, pilin subunit (minor pilin ComP) of competent Neisseria were grown in Lysogeny broth medium (10 g/L tryptone; meningitidis to specific sequences of self-DNA suggesting 5 g/L yeast extract; 10 g/L NaCl) at 37°C supplemented that pili indeed bring DNA to the cell surface (Cehovin with the appropriate antibiotics. Growth of cells was moni- et al. 2013). Besides having a role in competence, two tored by optical density measurements at 600 nm. examples are known in which T4P are involved in a conju- gative system. First, the IncI1 conjugative plasmid R64 in Ultraviolet treatment, aggregation assays Escherichia coli that carries the pil genes (Kim and Komano 1997; Yoshida et al. 1999; Komano et al. 2000), and sec- Ultraviolet light treatment was performed as described in ond, PAPI-1 DNA which conjugates between Pseudomonas Fro¨ls et al. (2008). Ten mL culture (OD600 0.2–0.3) was species and also comprises pil genes. The latter is located treated with a UV dose of 75 J/m2 (254 nm, UV crosslin- on a pathogenicity island that has been obtained via hori- ker; Spectroline, Westbury, NY) in a plastic petri dish. zontal gene transfer (Carter et al. 2010). A role of T4P in Subsequently cultures were incubated at 78°C for 3 h. cellular chromosomal exchange and subsequent repair of Samples taken at different time points were analyzed with damaged DNA has never been shown before. Moreover, phase contrast microscopy, survival rate assays, and elec- unlike the described bacterial T4P, the archaeal ups genes tron microscopy. To quantify aggregated cells after induc- are not present on a conjugative plasmid or acquired via tion with UV, 5 lL of cell culture (diluted to OD 0.2) was horizontal gene transfer. spotted on a microscope slide covered with a thin layer of The UV-induced pili system was initially studied in S. 1% agarose in Brock minimal medium. A coverslip was solfataricus (Fro¨ls et al. 2008; Ajon et al. 2011), but added when the drop had dried. Cells were visualized with because of the availability of genetic tools for manipulat- phase contrast microscopy. Free and aggregated cells (≥3) ing the Sulfolobus acidocaldarius genome (Wagner et al. were counted for at least three fields per strain using 2009, 2012) we switched to the latter organism. There is a ImageJ cell counter (NIH, Bethesda, MD). Percentages of high conservation and functional similarity among all cells in aggregates were subsequently calculated. sequenced Sulfolobus species. The ups operon encodes five proteins: UpsX, a hypothetical protein with no conserved Deleting/tagging genes in S. acidocaldarius regions; UpsE, an ATPase; UpsF an integral membrane protein; and UpsA and UpsB, two putative pilin subunits To construct deletion and gene replacement strains, up- containing class III signal peptides. and downstream flanking areas of the genomic regions of Here, we have analyzed the different roles of the genes interest (~600 bp) were amplified with primers listed in in the ups cluster. The secretion ATPase UpsE and the Table S1. Primers were designed according to the geno- membrane protein UpsF are, as expected, essential for the mic sequence of Sulfolobus acidocaldarius DSM639. Over- formation of ups pili. Interestingly, UpsX is not essential lap polymerase chain reaction (PCR) was performed to for pili formation, but the deletion of its gene resulted in connect the up- and downstream fragments (Wagner decreased DNA exchange suggesting a role in DNA trans- et al. 2012). The PCR product was subsequently cloned fer. Pilin subunits UpsA and B are essential for cellular into pSVA406, carrying an ampicillin resistance gene, aggregation. However, as deletion mutants of either of which resulted in the plasmids summarized in Table 1. the subunits still formed pili, it appears that a mixed The plasmids were methylated in E. coli ER1821-contain- structure is essential for cellular aggregation. ing pM.EsaBC4I (NEB) (Kurosawa and Grogan 2005) and transformed into S. acidocaldarius MW001/MR31 Material and Methods (Wagner et al. 2012). Integrants were selected on plates lacking uracil and grown in 24-well plates for 2 days in the same medium. Subsequently cultures were plated and Culture conditions grown for 5 days on second selection plates containing Sulfolobus acidocaldarius strains MW001, MR31, and uracil and 100 lg/mL 5-fluoroorotic acid to select for JDS183 and derived mutants were grown aerobically at clones in which the plasmid looped out by homologous 78°C in basic Brock medium (Brock et al. 1972), supple- recombination. Obtained colonies were tested by PCR for mented with 0.1% NZ amine AS (Sigma, Munich, successful deletion/replacement of the genes. Correctness Germany), 0.2% dextrin, and 20 lg/mL uracil, and adjusted of strains was confirmed by DNA sequencing. Strains that to pH 3.5 with sulfuric acid. For solid media the medium were made during this study are listed in Table 2. ª 2013 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd. 929 Analysis of the S. acidocaldarius ups Operon M. van Wolferen et al. Table 1. Plasmids used during this study. Table 2. Strains used during this study. Plasmid Description Source/Reference Source/ Strain Background strain Genotype Reference pMA08 Deletion plasmid for This study DupsX+promoter MR31 S. acidocaldarius DpyrE Reilly and region (Dsaci1493, (wt1) DSM639 (Dbp 154–171) Grogan À40 bp) (2001) pSVA406 Backbone of deletion (Wagner et al.
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