Diversity of Sinorhizobium (Ensifer) Meliloti Bacteriophages in the Rhizosphere of Medicago Marina: Myoviruses, Filamentous and N4-Like Podovirus
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fmicb-11-00022 January 22, 2020 Time: 17:45 # 1 ORIGINAL RESEARCH published: 24 January 2020 doi: 10.3389/fmicb.2020.00022 Diversity of Sinorhizobium (Ensifer) meliloti Bacteriophages in the Rhizosphere of Medicago marina: Myoviruses, Filamentous and N4-Like Podovirus María Teresa Cubo1*, Cynthia Alías-Villegas1, Eduardo Balsanelli2, Dany Mesa2, Emanuel de Souza2 and María Rosario Espuny1 1 Departamento de Microbiología, Facultad de Biología, Universidad de Sevilla, Seville, Spain, 2 Department of Biochemistry and Molecular Biology, Universidade Federal do Paraná, Curitiba, Brazil Using different Sinorhizobium meliloti strains as hosts, we isolated eight new virulent phages from the rhizosphere of the coastal legume Medicago marina. Half of the isolated phages showed a very narrow host range while the other half exhibited a wider host range within the strains tested. Electron microscopy studies showed that Edited by: Helene Sanfacon, phages M_ort18, M_sf1.2, and M_sf3.33 belonged to the Myoviridae family with feature Agriculture and Agri-Food Canada long, contractile tails and icosaedral head. Phages I_sf3.21 and I_sf3.10T appeared (AAFC), Canada to have filamentous shape and produced turbid plaques, which is a characteristic of Reviewed by: Michael Francis Hynes, phages from the Inoviridae family. Phage P_ort11 is a member of the Podoviridae, with University of Calgary, Canada an icosahedral head and a short tail and was selected for further characterization and Ahmed Askora, genome sequencing. P_ort11 contained linear, double-stranded DNA with a length of Zagazig University, Egypt 75239 bp and 103 putative open reading frames. BLASTP analysis revealed strong *Correspondence: María Teresa Cubo similarities to Escherichia phage N4 and other N4-like phages. This is the first report of [email protected] filamentous and N4-like phages that infect S. meliloti. Specialty section: Keywords: bacteriophages, P_ort11, Podoviridae, N4-like phage, Sinorhizobium/Ensifer meliloti, filamentous This article was submitted to phage, Medicago marina Virology, a section of the journal Frontiers in Microbiology INTRODUCTION Received: 31 October 2019 Accepted: 07 January 2020 Sinorhizobium (Ensifer) meliloti is a soil bacterium able to induce the formation of root nodules on Published: 24 January 2020 Medicago, Melilotus and Trigonella legumes, where these bacteria fix atmospheric nitrogen (Béna Citation: et al., 2005; Willems, 2006; Gibson et al., 2008; Zhang et al., 2012). The symbiotic relationship Cubo MT, Alías-Villegas C, established between bacteria and legumes is important for sustainable agriculture because the Balsanelli E, Mesa D, de Souza E and conversion of dinitrogen to ammonia improves the overall productivity of crops without the need Espuny MR (2020) Diversity of adding nitrogenous fertilizers (Pérez-Montaño et al., 2014). In addition, some recent studies of Sinorhizobium (Ensifer) meliloti have shown the use of S. meliloti-Medicago for bioremediation purposes on soils contaminated with Bacteriophages in the Rhizosphere of Medicago marina: Myoviruses, heavy metals (Fan et al., 2011; Alías-Villegas et al., 2015). Filamentous and N4-Like Podovirus. The most abundant and genetically diverse entities on Earth are viruses of bacteria 31 Front. Microbiol. 11:22. (bacteriophages), with the global count estimated to be greater than 10 (Hendrix, 2002; Clokie doi: 10.3389/fmicb.2020.00022 et al., 2011). It is well-known that bacterial species can be infected by several different phages, Frontiers in Microbiology| www.frontiersin.org 1 January 2020| Volume 11| Article 22 fmicb-11-00022 January 22, 2020 Time: 17:45 # 2 Cubo et al. Diversity of Sinorhizobium meliloti Bacteriophages which can serve as agents of gene mobilization across bacterial (Barnet, 1972) with some modifications. Soil samples (5 g) were population (Canchaya et al., 2003). Moreover, the reports of the mixed with 10 ml of TY and incubated at 28◦C for 24 h. Soil genome sequence from a large number of bacteria have shown particles were removed by centrifugation (3,000 g, 15 min, two that many of them harbor prophages as well as genes from phages times) and finally the samples were filter sterilized. Two different (Casjens, 2003; Decewicz et al., 2017). In the decade of the 1970s sets of 8 overnight S. meliloti strains cultures (100 ml of each and 1980s of the 20th century some papers about morphological bacteria in 1 ml of TY) were mixed with 4 ml of soil-sample description of several S. meliloti phages (sinorhizobiophages) and incubated at 28◦C (180 rpm) for 3 days. Bacterial cells were were published (Krsmanovic-Simic and Werquin, 1973; Werquin then removed by centrifugation, and the supernatant was filtered et al., 1988). Initially, interest in sinorhizobiophages was based through 0.45 mm Millipore filter. Four ml of this solution was on their use as molecular genetic tools due to their transducing used to inoculate a fresh set of bacteria as above indicated, finally ability, as well as their use for phage typing of indigenous rhizobia yielding a solution presumably containing phages. The presence (Kowalski, 1967; Casadesús and Olivares, 1979; Sik et al., 1980; of phages was tested using the double agar overlay plaque assay Finan et al., 1984; Bromfield et al., 1986; Kankila and Lindström, (Kropinski et al., 2009). Briefly, 100 ml of phage solution were 1994). New genome sequences and structural analysis have added to 200 ml of middle log-phage S. meliloti strains, the provided a broader and deeper knowledge about the diversity of mixture was incubated 5 min at room temperature and then S. meliloti phages (Ganyu et al., 2005; Schulmeister et al., 2009; mixed with soft agar to form a lawn on TY solid medium. Deak et al., 2010; Brewer et al., 2014; Dziewit et al., 2014; Stroupe Plates were incubated at 28◦C until plaques became visible. Single et al., 2014; Crockett et al., 2015; Hodson et al., 2015; Johnson plaques were picked with a sterile toothpick, and replated three et al., 2015, 2017; Schouten et al., 2015; Decewicz et al., 2017). times in order to ensure the purity of the phage stocks. It is clear that phages play major roles in the ecological High-titer phage lysates were obtained by infecting 5-ml balance of microbial life. In the context of the symbiotic N2- cultures of S. meliloti strains in TY medium at an optical density fixation bacteria, phages that infect rhizobia may have a great at 600 nm (OD600) of 0.3 with 500 ml of phage solution and impact on the population dynamics of these bacteria in the incubating at 28◦C (180 rpm) for 24–48 h. Cell debris were rhizosphere environment by altering, for instance, the relative removed by centrifugation, and the resulting supernatants were numbers of resistant and/or susceptible rhizobial strains in soil filtered twice through a 0.45 mm Millipore filter. Phage stocks (Mendum et al., 2001; Sharma et al., 2002; Malek et al., 2009). were stored at 4◦C. Preliminary studies on Medicago marina from two polluted areas of south-western Spain allowed the isolation and characterization Electron Microscopy of several new S. meliloti strains that showed tolerance to different For transmission electron microscopy, aliquots of high-titer stresses including salinity, alkalinity and heavy metals (Alías- lysates (20 ml) were absorbed onto 300-mesh copper grids with Villegas et al., 2015). In order to obtain a full understanding of collodion support films for 3 min and left to dry for 24 h. the biology of those sinorhizobia strains and their relationship Then the samples were negatively stained with 2% (w/v) uranyl to the plant, we studied phages from the rhizosphere sand- acetate for 10 min. Phages were examined by transmission soil of M. marina. In this work we identified eight novel electron microscopy in Centro de Investigación, Tecnología e bacteriophages using different S. meliloti strains as trapping host Innovación (CITIUS, University of Seville) using a Zeiss Libra and the rhizosphere harbors a broad diversity of phages not 120 TEM at 80 kV. yet shown before. Host Range Determination Host range of the isolated phages was tested as described MATERIALS AND METHODS previously (Kankila and Lindström, 1994), with some modifications. Briefly, a total of 43 rhizobial strains were Bacterial Strains, Media, and Growth tested for infection with the eight phages isolated in this work Conditions (Table 1). Top agar overlays containing 3.5 ml of 0.4% (wt/v) TY and 200 ml of an overnight culture of the bacterial strain The bacterial strains used in this work are listed in Table 1. Most to be tested were poured onto a solid TY plate and allowed to of the S. meliloti strains were isolated from nodules of M. marina set. The lysates used had a phage titer of ca 1 × 109 pfu (plaque in the south-west region of Spain, as previously reported (Alías- forming units) ml−1 except the lysates from phages S_sf3.10C Villegas et al., 2015). All bacterial strains were routinely grown and I_sf3.10T, which reached titers of ca 1 × 105 pfu and 1 × 107 at 28◦C on tryptone-yeast (TY) medium (Beringer, 1974). When pfu ml−1, respectively. A 10 ml sample of three serial dilutions, appropriate, gentamicin was used at a final concentration of from undiluted to 100-times-diluted, of the lysate of phages was 10 mg ml−1. spotted onto each bacterial overlay, and the plates were incubated 2 days at 28◦C. After incubation, each plate was scored for lysis. Bacteriophage Isolation, Purification, Assays were performed in triplicate. and Propagation Bacteriophages were isolated from M. marina-rhizosphere sand P_ort11-Phage Adsorption samples from Odiel river marshes (Huelva, Spain, 37◦ 100 Phage adsorption experiments were carried out as described 28.500 N, 6◦ 550 51.500 W) using a multiple-enrichment protocol previously (Hyman and Abedon, 2009), with some modifications. Frontiers in Microbiology| www.frontiersin.org 2 January 2020| Volume 11| Article 22 fmicb-11-00022 January 22, 2020 Time: 17:45 # 3 Cubo et al.