tRNA genes were found in Piscirickettsia salmonis 16S^23S rDNAspacer region (ITS)

Andres Casanova, Johanna Obreque C., Ana Mar|a Sandino G., Matilde Jashes *Laboratorio de Virolog|a, Departamento de Biolog|a, Facultad de Qu|mica y Biolog|a, Universidad de Santiago de Chile, Alameda 3363, Santiago-Chile,

Casilla 40 Correo 33, Santiago-Chile, Chile

Received 16 November 2000; received in revised form 5 January 2001; accepted 6 January 2001

Abstract

Piscirickettsia salmonis is the etiological agent of Salmonid Rickettsial Septicemia, a disease affecting salmon aquaculture industry. Weanalyzed the 16S^23S rDNA spacer region (internal transcribed spacer, ITS) of Chilean P. salmonis isolates LF-89 and EM-90. Two mainITS amplification products were obtained by PCR using L1 and G1 primers, differing from that described where only one ITS region wasfound. By Southern blot, it was established that these two amplification products contained sequences related to P. salmonis ITS. Sequenceanalysis confirmed that P. salmonis had two ITS regions: ITS A and ITS B. In both isolates, the smaller (ITS B) corresponded to ITSsequences previously described for each one, and the larger (ITS A) were almost the same as their respective ITS B sequences interrupted byan insert which contained two tRNAs genes: tRNA-Ile and tRNA-Ala. ß 2001 Federation of European Microbiological Societies.Published by Elsevier Science B.V. All rights reserved.

Keywords: 16S^23S rDNA spacer region (ITS); tRNA gene; Polymerase chain reaction; Piscirickettsia salmonis

1. Introduction

Piscirickettsia salmonis is an obligate intracellularGram-negative bacterium. It is the causative agent of Sal-monid Rickettsial Septicemia (SRS), or piscirickettsiosis,the most important disease that a¡ects salmonid maricul-ture in Chile. It was isolated for the ¢rst time in ourcountry [1,2] and also reported in Canada, Norway, andIreland [3^5]. Initially, it was classi¢ed as a Rickettsiales-like organism, constituting the ¢rst time a Rickettsia-likebacterium was isolated from ¢sh [6,7]. Through 16S rRNAgene analysis of P. salmonis type strain LF-89, isolated insouthern Chile, a new genus and species were proposed:P. salmonis [8]. At present, there is a great interest todetermine genetic di¡erences among P. salmonis isolates.

It is now established that the sequence of the 16S rRNAgene is a powerful method for determining phylogeneticrelationships, commonly used to establish inter- and intra-generic relationships [9]. Nevertheless, the spacer region

between the 16S and 23S genes (internal transcribedspacer, ITS) is useful to di¡erentiate species or even strainsof prokaryotes. This region exhibits a great deal of se-quence and length variation [10,11]. It is shown that with-in a single genome there are frequently multiple rRNA lociand the spacer regions found within these loci also showvariations in length and sequence. This diversity is due inpart to variations in number and type of tRNA genes theymay contain. Therefore, in the last few years a lot of in-formation about ITS sequences of prokaryotic organismshas become available [10^14].

Recently the ITS of six P. salmonis isolates was studied.Because only one ITS was obtained by PCR it was sug-gested that the P. salmonis genome has a single rRNAoperon. Also, no tRNA genes were found in the ITS se-quences [15]. Nevertheless, we analyzed the ITS of 12Chilean P. salmonis isolates and a di¡erent result was ob-tained. By PAGE analysis of ITS ampli¢ed using G1 andL1 primers a characteristic electrophoretic pattern wasobtained, di¡erent to other bacteria including ¢sh patho-gens. We also observed that in all of them, more than oneproduct was obtained suggesting more than one ITS re-gion may exist in its genome (manuscript submitted). Todetermine if this is true, the 16S^23S rDNA spacer regionof two Chilean P. salmonis isolates, the ATCC strain LF-

0378-1097 / 01 / $20.00 ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.PII: S 0 3 7 8 - 1 0 9 7 ( 0 1 ) 0 0 0 2 9 - 5

* Corresponding author. Present address: Departamento de CienciasBiologicas, Facultad de Qu|mica y Biolog|a, Universidad de Santiago deChile, Alameda 3363, Santiago-Chile, Casilla 40 Correo 33, Santiago-Chile, Chile. Tel. : +56 (2) 6810185; Fax: +56 (2) 6812108;E-mail : mjashes@lauca.usach.cl

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89 and strain EM-90, were studied. In this report the elec-trophoretic data, Southern blot analysis and the sequencesof ITS ampli¢cation products are discussed.

2. Materials and methods

2.1. Bacteria culture and DNA isolation

P. salmonis LF-89 (LF-89 ATCC VR 1361), and theEM-90 isolates were used [7,15]. EM-90 isolated previ-ously from Atlantic salmon (Salmo salar) was providedby Mr. Erwin Landskron from Marine Harvest, a salmonExport Company located in Puerto Montt, Chile.

P. salmonis was grown at 15³C in the chinook salmonembryo cell line CHSE-214 until 90% of the cell sheet waslysed (approximately 14 days). 0.2 mg ml31 of proteinaseK and 125 Wl of a disruption solution (280 mM Tris, 80mM EDTA, 45 mM SDS, 685 mM NaCl, 1% L-mercap-toethanol) were added to 500-Wl aliquots of an infectedcells suspension. Then it was incubated for 1 h at 50³Cand DNA was extracted by the phenol^chloroform meth-od [16].

2.2. ITS ampli¢cation

The P. salmonis ITS was ampli¢ed through PCR usingthe universal primers L1 and G1, as described previously[11]. A 5-Wl aliquot of DNA suspension was added to 45 Wlof reaction mixture containing PCR bu¡er (20 mM Tris^HCl (pH 8.4), 50 mM KCl), 1 mM MgCl2, 200 WM eachof dATP, dGTP, dCTP, dTTP; 0.473 WM (2.5 ng Wl31) ofG1 and L1 primers and 2.5 U Taq DNA polymerase (Gib-co). The mixture was denatured at 94³C for 5 min andampli¢ed by 25 cycles at 94³C for 1 min, 55³C for 7 minand 72³C for 2 min. The ¢nal cycle was followed by 7 minat 72³C. PCR ampli¢cation was performed with a DNAthermal cycler (Biometra UNO-Thermoblock1). Thelength and number of ampli¢ed DNA fragments were an-alyzed by 7% PAGE with Tris^glycine bu¡er [17] andafterwards silver nitrate-stained [18].

2.3. ITS analysis by Southern blot

Brie£y, the ITS of P. salmonis was submitted to 7%PAGE with a Tris^glycine running bu¡er. Electrotransferto a nylon membrane was performed, and subsequentlyhybridization with the radioactive probe [19]. Oligonucleo-tide used as a probe was synthesized based on theP. salmonis LF-89 ITS sequence available in the database (GenBank access U36943) (from base 74 to 119; 5P-CAA TGT GGT AAA AAG TAT AAG TAA AGA TTCCTT GAT TAA TTT AGG G-3P). Approximately 50 ngwas labelled with [K32P]dATP 1 WCi Wl31 using the primersDNA labelling system (Gibco random primers kit, Rock-

ville, MD, USA) and then it was puri¢ed by SephadexG-50.

2.4. Cloning and sequencing of ITS ampli¢cation products

The PCR products were puri¢ed from a 1.5% agarosegel using the QIAquick gel extraction kit (Qiagen, Valen-cia, CA, USA). The puri¢ed products were cloned into apGEM-T easy vector using the manufacturer's protocol(Promega, Madison, WI, USA). The plasmid DNA wasextracted by an alkaline lysis method, as described [16],and then it was analyzed by PCR to con¢rm the insertionof DNA fragments. Sequencing was carried out on anautomated sequencer (ABI Perkin Elmer 377, Norwalk,CT, USA) in the Laboratorio de Oligopeptidos S.A., San-tiago, Chile. Each sequence was con¢rmed from three sep-arate clones, obtained from a di¡erent ampli¢cation reac-tion. They were sequenced in both forward and reversedirections.

3. Results

3.1. P. salmonis ITS ampli¢cation by PCR

The primers L1 and G1 have complementary sequencesof 16S and 23S rRNA genes and amplify the spacer regionlocated in between them. PAGE analysis of the ampli¢ca-tion products of P. salmonis LF-89 ITS region showed thepresence of two products: A and B (Fig. 1A, lane 1).Similar products were observed with the EM-90 isolate.Nevertheless, it was possible to distinguish di¡erences inthe electrophoretic migration of the products betweenboth isolates (Fig. 1A, lane 2).

Fig. 1. Analysis of P. salmonis ITS ampli¢cation products by PAGEand Southern blot. PCR ampli¢cation products of P. salmonis LF-89and P. salmonis EM-90 isolates obtained with the universal primers L1and G1 (lanes 1 and 2, respectively) were submitted to PAGE. A: Theelectrophoresis was performed at 100 V for 5 h. The polyacrylamide gelwas silver nitrate-stained and dried. B: Autoradiograph of ITS ampli¢-cation products after electrotransfer to a nylon membrane and subse-quent hybridization with the [K32P]dATP-ITS probe (as described inSection 2). Gibco 123 molecular mass marker in bp.

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3.2. Southern blot analysis

A Southern blot analysis was performed, using a radio-active probe based on the published P. salmonis LF-89ITS sequence (GenBank access U36943). A and B ampli-¢cation products of P. salmonis LF-89 and EM-90 strainshybridized with this probe (Fig. 1B, lanes 1 and 2). ITS ofbacteria from di¡erent genera and species were used ascontrol, but none of these hybridized (data not shown).This group included salmon pathogens such as Renibacte-rium salmoninarum, Yersinia ruckeri, and also Thiomicro-spira crunogena, Leucotrix mucor, which are phylogeneti-cally related to P. salmonis.

3.3. ITS nucleotide sequences analysis

ITS ampli¢cation products of P. salmonis LF-89 andEM-90 isolates, were cloned and sequenced. Fig. 2 showsthe sequence alignment of A and B products of P. salmo-nis LF-89 and EM-90. The ITS sequence of product Bcorresponded to the ITS sequence described previouslyfor each one (GenBank access U36943 and U36944). Inboth isolates, the ITS A sequence was almost the same as

their respective ITS B sequence, but it was interrupted byan insert of around 300 bp. BLAST analysis determinedthat they contained two rRNAs genes. These sequenceswere 100% homologous to those that encode tRNA-Alaand tRNA-Ile (shown by boldface letters in Fig. 2).

4. Discussion

When we analyzed the 16S^23S rDNA spacer region ofP. salmonis obtained by PCR using G1 and L1 primers, adi¡erence was found from that described previously[15,20], i.e. two major ampli¢cation products were ob-tained instead of one. By Southern blot analysis it wasestablished that both products contained sequences relatedto P. salmonis ITS (Fig. 1B). Through sequencing we es-tablished that ITS B of LF-89 and EM-90, correspondedto ITS previously described for these isolates (Fig. 2)(GenBank access U36943 and U36944). ITS A sequences,in both isolates, were almost the same as their respectiveITS B sequences, but they were interrupted by an insertthat contained two tRNA genes: tRNA-Ala and tRNA-Ile(GenBank access AF205384 and AF205385). This contra-

Fig. 2. Sequence alignment of the P. salmonis LF-89 and EM-90 A and B ampli¢cation products. The regions corresponding to 16S and 23S genes areshown by small letters and the spacer region 16S^23S (ITS) by capital letters. The tRNA-Ala and tRNA-Ile sequences are written in boldface capitals.Nucleotides identical to the P. salmonis LF-89 sequence are indicated by dots, and insertion^deletions by dashes.

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dictory result with respect to those obtained previously[15,20] can be explained by analyzing the experimentaldesign of these studies. Marshall et al. [20] ampli¢ed theITS using primers complementary to the available P. sal-monis ITS sequence (corresponding to our ITS B). Whenanalyzing the ITS sequences obtained in our study (ITS Aand B) we veri¢ed that sequences complementary to Mar-shall's primers were present only in ITS B but not in ITSA. In the latter, these were interrupted by tRNA sequencesavoiding the ITS A product ampli¢cation. On the otherhand, the primers used in Mauel's study [15] had comple-mentary sequences to 16S and 23S rDNA but unlike L1and G1 primers they amplify a larger region of bothrRNA genes. When we performed a PCR with Mauel'sprimers only one major ampli¢ed product was observed.In addition, a larger sized product appeared only as a lightsignal.

Our results agree with those observed in other bacteriain that they frequently had multiple rrn loci and theirspacer regions have a signi¢cant degree of variation[11,12,21]. At least 50% of the bacteria analyzed havemultiple copies of the rRNA operon per genome: 13 outof 22 cases of Gram-negative bacteria and 11 out of 20Gram-positive bacteria. The variation among the spacerregions of rRNA operon is due in part to the presenceof tRNA genes. Moreover, for the ITS sequenced it wasdetermined that the majority of the spacer regions ofGram-negative bacteria also contained tRNA-Ala andtRNA-Ile while others contain only tRNA-Glu. The ma-jority of ITS of Gram-positive bacteria do not have tRNAgenes, but if they do, they correspond to the tRNA-Ala ortRNA-Ile or both [9].

This report shows that in the P. salmonis genome thereare two 16S^23S rRNA spacer regions. Thus, more thanone rRNA operon may exist. It would be very interestingto determine if ITS of P. salmonis isolated from othercountries have similar characteristics, and also if thereare tRNA-Ala and tRNA-Ile genes in their 16S^23SrDNA spacer region. This would contribute to the under-standing of the relationship between P. salmonis isolatesfrom di¡erent geographic origins.

Acknowledgements

This study was supported by Grants #1980634 fromFONDECYT and from FONDAP program: Oceanogra|ay Biolog|a Marina, Subprograma de peces. We thank Ma-rine Harvest for providing the isolate P. salmonis EM-90.

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