Central JSM Microbiology Research Article Corresponding author Simone Cappello, Istituto per l’Ambiente Marino Costiero (IAMC) – CNR of Messina, Spianata San Raineri, Study of Bacterial 86 - 98122 Messina, Italy; Tel: +39-090-6015421; Fax: +39- 090-669003, Email: Submitted: 28 October 2014 Communities in Mussel Mytilus Accepted: 09 January 2015 Published: 12 January 2015 galloprovincialis (Bivalvia: Copyright © 2015 Cappello et al. Mytilidae) by a Combination OPEN ACCESS Keywords of 16s Crdna and 16s Rdna • Mytilus galloprovincialis • Microbial community Sequencing • Symbiont Simone Cappello1*, Anna Volta1,2, Santina Santisi1,3, Lucrezia Genovese1, Giulia Maricchiolo1, Martina Bonsignore1 and Michail M. Yakimov1 1Istituto per l’Ambiente Marino Costiero (IAMC) – CNR of Messina, Italy 2Department of Industrial and Mechanical Engineering, University of Catania, Italy 3PhD School of “Biology and Cellular Biotechnology” of University of Messina, Italy Abstract In this study has been analyzed the genetic potential (rDNA) versus expression (crDNA) of microbial populations associated to gills of living mussel Mytilus galloprovincialis (Bivalvia: Mytilidae) in natural environment. Data obtained (16S rDNA/crDNA clones libraries) showed as sequences mainly related to Bacteroides/ Chlorobi, Firmicutes and Gamma-Proteobacteria groups are specific in live mussels. It is presumed that further studies of microbial population structure with culture-independent methods will demonstrate the active interactions (symbiosis) between filter-feeding organisms and gill-associated bacteria. ABBREVIATIONS GC-MS: Gas Chromatography-Mass Spectrometry; DAPI: 4’, carbon,(e.g. sponges, chelating mussels, iron, transforming corals…) may molecular be involved, nitrogen, at least, nitrate in 6-Diamidino-2- Phenylindole 2hcl; CFU: Cultivable Heterotrophic three different typology: (i) nutrient acquisition, (i.e., cycling Bacteria; PAST: Paleontological Staatistics or polyphosphate into bio-available form for organisms) [6- INTRODUCTION production8]; (ii) processing of special of metabolicsecondary wastemetabolites, by eliminating such as protectiveit via the Mussels are ecologically important members of the coastal antibioticsbiotransformation and other of biologicallyseveral toxic active metabolites compounds [9]; [10-12]. and (iii) In and estuarine communities. These bivalve molluscs have the feeders represent one of the most complex and reliable topic and may potentially impact the planktonic structure in a body forany understandingcase, the relationship the biodiversity between bacteriaof bacterial and benthiccommunities filter ability to filter large volumes of waters for their food requirements in marine environment and have functionally important systems [1] and, in general, they may provide a useful nutrient evolutionary and ecological implications. of water. Bacteria are an important component of planktonic bacterial proteins can be digested and assimilated into mussel proteinssource for [2]. the filter-feeding organisms (mussels); indeed, as attribution of in situ microbe-mediated processes to the In this optic the first challenge is to overcome the complicated mussels and bacteria responsible organism(s) improving knowledge on dynamics and Different studies showed the relation between the characteristic of these active interactions. As previously reported studiesfunctional based activity on 16S together rDNA could with not identification indicate activity of ecologically nor prove [1,3,4] but very little is known about the thesignificant viability strains. [13] or evenAlthough the functional many authors role of haveeach phylogeneticargued that [5] the interactions between bacteria and marine organisms Cite this article: Cappello S, Volta A, Santisi S, Genovese L, Maricchiolo G, et al. (2015) Study of Bacterial Communities in Mussel Mytilus galloprovincialis (Bivalvia: Mytilidae) by a Combination of 16s Crdna and 16s Rdna Sequencing. JSM Microbiology 3(1): 1016. Cappello et al. (2015) Email: Central group, the rapid developments in reverse transcription-PCR and improvement in RT enzymes have provided opportunities for evaluation of active communities inferred from analysis of toreaction, cDNA RNAusing templates First-Strand were cDNAtreated Synthesis with DNase SuperScript I (Invitrogen)TM II crDNA and mRNA, demonstrating that uncultured bacteria could according to the manufacturer’s instructions. RNA was converted be active in situ. 14. Possible Reverse Transcriptase (Life Technologies, Carlsbad, CA, USA) RT reaction mixtures (20 µl) as previously reported DNA contamination of RNA templates was routinely monitored The aim of the present study was to perform the culture- of active dominant bacterial groups associated to gills of by PCR amplification of aliquots of RNA that were not reverse independent 16S crDNA phylogenetic survey of the identification reactions. Primers used for PCR included the above reverse mussels Mytilus galloprovincialis in marine transcribed; no contaminating DNA was detected in any of these (Bivalvia: Mytilidae) Bacteria primer (Uni_1492R) and 16S crDNA forward domain-specific consortiaenvironments; inhabiting in the in same living time mussels. we have analyzed the genetic potential (rDNA) versus expression (crDNA) of microbial , Bac27_F (5’-AGAGTTTGATCCTGGCTCAG-3’) [14]. PCR MATERIALS AND METHODS (initial denaturation: 5 min hot-start at 95°C; 1 min at 94°C, 1 min at 50°C, 2 min at 72°C, 30 cycles; extension: 10 min at 72°C) was Sampling collection performed with GeneAmp 5700 (PE Applied Biosystems, Foster Living Mytilus galloprovincialis City, CA, USA), using a 50 μl (total volume) mixture containing 1x solution Q (Qiagen, Hilden, Germany), 1x Qiagen reaction buffer, specimens were harvested in 1 µM of each forwardTaq and reverse primer, 10µM dNTPs (Gibco, September 2012 to the Lake Faro (38°15′59.95N; 15°38′19.56″E),2 and Invitrogen Co., Carlsbad, CA), 2.0 ml (50-100 ng) of template and located on the north-eastern tip of Sicily (Messina, Italy ;). Lake 2.016S U Clone of Qiagen libraries Polymerase (Qiagen). Faro is a meromictic lake with a surface area of 0.263 km reaches a maximum depth of ∼30 m at its centre. GC-MS analysis was used to reveal the presence of chemicals in the lake’s water After gel electrophoresis of PCR products, purified amplicons transported(data not shown). to the Mussels laboratory were in collecteda cool box by and hand used from for boats further to fromrepresenting the transformed 16S crDNA E. sequencescoli DH10 were cloned into the pGEM a depth of 30 cm. After collection all samples were immediately T-easy Vector II (Promega, Madison, Wis., USA), and inserts β colonies. After blue/white analysis. The organisms were cleaned under running tap water 16screening, mM NH randomlySO ; 5 Mpicked colonies were suspended in PCR with a stiff brush and aseptically shucked with a sterilized knife. lysis solution4 A4 without Proteinase K (67 mM Tris–Cl (pH2 8.8); looselyAfter dissection, associated each microbes. gill samples were further washed four μ β-mercaptoethanol; 6.7 mM MgCl ; 6.7 times with 0.2 µm-filtered autoclaved seawater to remove any μM EDTA (pH 8.0) and heated at 95°C for 5 min. The lysate Bacterial Total Count (DAPI Count) (∼0.2 ng DNA) was used as the template for PCR amplification To determine the bacterial abundance in gills from freshly with primers M13F (5′-GACGTTGTAAAACGACGGCCAG-3) and M13R (5′-GAGGAAACAGCTATGACCATG-3). PCR products were sequenced using Macrogen Service (Macrocen,et al. Korea). collected mussels, samples of gills tissue were thawed and The analysis of the sequences (1400 to determinebp of average the numberlength) subsequently lightly ground in 10% formaldehyde in sterile-1 was performed as previously described estimated (Yakimov total diversity., 2009). seawater using a ground-glass tissue grinder. After appropriate Rarefaction analysis was performed dilution, aliquots of the homogenates were stained with 1 mg l of unique OTUs as a proportion of the of DAPI (Sigma-Aldrich S.r.L., Milan, Italy) for 5 min [3]. Densities Calculations were performed using the freeware program of gills-associated bacteria were determined by epifluorescence PAleontological STatistics (PAST) version 1.32 (http://folk.uio. counting of fluorescing bodies of appropriate-1. size (~1 µm). Three no/ohammer/past).RESULTS AND DISCUSSION separate homogenates were prepared from each sample. Results wereHeterotrophic expressed as Cultivable number of cell Bacteria gr (CFU) affect microbial communities in terms of both abundance and biodiversity.As previously Measures reported of estimations[3], mussel offiltration the total activity bacteria can For determination of cultivable heterotrophic bacteria (CFU) present in the gills of M. galloprovincialis samples of gills homogenates (as previously described) were values(DAPI ofcount) 2.2 × and10 of heterotrophic cultivable6 CFU gbacteria-1. Data of (CFU) total serially diluted whit sterile seawater. Different dilutions were 7 showed, respectively, estimated by spreading 100 µl onto Marine Agar 2216 medium cells g-1 and 7.2 × 10 (Difco S.p.a., Milan,-1 Italy) and incubated at 20 ± 1°C for 7 days. bacteria (DAPI) count highlighted that microbial abundance Results were expressed as colony forming units (CFU) in grams was significantly higher in the gills of mussels than in natural ofDNA/RNA gill (CFU gr Extraction). totalenvironment bacterial (data concentration not shown). detected These inresults the mussels are in accordance is directly with data obtained