26 t. toyama, k. kita-tsukamoto and h. wakabayashi

魚病研究 Fish Pathology,31(1),25-31,1996.3

Identification of Flexibacter maritimus, Flavobacterium branchiophilum

and Cytophaga columnaris by PCR Targeted 16S Ribosomal DNA

Takashi Toyama*1,3, Kumiko Kita-Tsukamoto*2

and Hisatsugu Wakabayashi*1,4

*1 Department of Fisheries, University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113, Japan *2 Ocean Research Institute , University of Tokyo, Minamidai 1-15-1,

Nakano-ku, Tokyo 164, Japan

(Received November 8, 1995)

On the basis of the 16S rRNA sequence data analysis among the closely related species, the specific primers for Flexibacter maritimus, Flavobacterium branchiophilum, and Cytophaga columnaris were constructed. The specificity in amplifying the 16S rDNA of each species was confirmed by using selected strains of related bacteria and principal fish pathogenic bacteria. A primer pair of MAR 1 and MAR2 could differentiate F. maritimus from other species. The PCR performed with a pair of primers specific to F. branchiophilum failed to amplify the 16S rDNA. However, the PCR with a pair of a specific primer BRA1 and a universal primer 1500R succeeded in specifically amplifying 16S rDNA from F. branchio

philum. The Nucleotide Sequence Database (GenBank) contains two different 16S rRNA sequence data for C. columnaris. On the basis of these data, we produced two primer pairs. A pair of COL1 and 1500R amplified the 16S rDNA from 5 of 7 strains of C. columnaris, and a pair of COLa and COLb worked for the rest of the strains. In addition, these two groups within C. columnaris were supported by PCR-RFLP.

Key words : Flexibacter maritimus, Flavobacterium branchiophilum, Cytophaga columnaris, 16S ribosomal DNA, PCR

Flexibacter maritimus, Flavobacterium branchio

philum, Cytophaga columnaris (syn. Flexibacter columnaris) and Cytophaga psychrophila (syn. Flexibacter psychrophilus) are known as principal fish

pathogens belonging to the Flavobacterium-Cytophaga group. Nakagawa (1993) revealed the phylo

genetically close relationship between these fish pathogens by 16S rRNA sequencing. In a previous paper (Toyama et al., 1994), we described a primer pair for PCR amplification of small-subunit 16S rRNA gene (rDNA) of C. psychrophila. The

primers were designed on the basis of the 16S rRNA sequence comparison between the closely related species. It was confirmed that the PCR products were species-specific and not conserved even between relatively closely related species, such as C. colunmaris, F. branchiophilum and F. maritimus. The present study expands on the use of PCR amplification of

16S rDNA for identification of these three fish

pathogens.

Materials and Methods

Primers According to the phylogenetic tree for Flavobacterium-Cytophaga group based on 16S rRNA com

parison (Nakagawa, 1993), twenty-four species belonging to cluster 9 through 12, including F. maritimus, F branchiophilum and C. columnaris, were selected for comparison of the 16S rRNA sequence data. The 16S rRNA sequence data of these bacteria were obtained by accessing the Nucleotide Sequence Database (GenBank). The data were examined with multiple alignment analysis (CLUSTAL V) (Higgis et al., 1992), the specific regions for each bacterial species identified, and the specificity was confirmed by FASTA (Lipman and Pearson., 1985). The

primers were synthesized by Sawady Technology Co., Tokyo, Japan, and the primer pairs were designated as MAR1-MAR2 for F maritimus, BRA1

*3 Present address: JICA Kanagawa International Fisheries

Training Centre. 5-25-1 Nagai, Yokosuka, Kanagawa 238

-03, Japan. *4 Author to whom correspondence should be addressed .

26 T. Toyama, K. Kita-Tsukamoto and H. Wakabayashi

BRA2 for F. branchiophilum, and COL 1 -COL2 and

COLa-COLb for C. columnaris. The COL 1 -COL2

primer pair and the COLa-COLb primer pair were

based on the 16S rRNA sequence of C. columnaris

NCIMB2248 and ATCC43622, respectively, because

the Nucleotide Sequence Database gave us two dif

ferent 16S rRNA sequences of these strains. The

general applicable primers designated 20F and

1500R, which were conserved in 16S rDNA genes of

most bacteria, were also employed (Weisburg et al.,

1991). The primer sequences and the positions cor

responding to those on the Escherichia coli 16S

rRNA numbers are listed in Table 1.

Bacterial strains

The bacterial strains used for the PCR are listed in

Table 2. These were some of the species included in

the cluster 9 through 12 and other principal fish

pathogens. All strains of the genus Cytophaga or

Flavobacterium were grown on TYES broth consist

ing of 0.4% tryptone, 0.04% yeast extract, 0.05%

MgSO4 7H2O, 0.05% CaCl2 10H2O and pH 7.2 (Holt

et al., 1993). Flexibacter maritimus strains were

cultured on "marine TYES" broth containing 60%

aged sea water. The other bacterial strains were

grown in brain heart infusion broth (Nissui, Tokyo).

The bacteria were cultured at 25•Ž except for C.

psychrophila, which was grown at 18•Ž. Cells were

harvested at approximately early stationary phase by

centrifugation, washed with sterile distilled water,

suspended in distilled water to give a cell concentra

tion of 30 mg of wet weight per ml, and stored at

-20•Ž.

PCR amplification

Forty microliters of the cell suspension was mixed

with 10 ,ul of proteinase K solution (1 mg/m/) and 50

,u1 of K buffer (40 mm Tris-HCl, 0.2% Nonidet p-40,

0.2 mm EDTA, 1% Tween 20, pH 8.0). The mixture

was heated at 60•Ž for 20 min, followed by at 100•Ž

for 5 min, then cooled rapidly on ice, and centrifuged

at 8,000 rpm for 5 min. Amplification was performed

in 50ƒÊl of reaction mixture containing 1.5 mm

MgCl2, 0.5 nmol of each dNTP (the nucleotide tri

phosphate of adenine, guanine, cytosine and thy

mine), 10 pmol of each primer, 2.5 units of Taq

DNA polymerase and 5 ill of the cell lysate as the

template DNA. Samples were subjected to 30 or 35

cycles of amplification in a DNA thermal cyder

(Mini Cyder TM; MJ Research, Inc., Massachu

setts, USA). A preheating cycle at 94•Ž for 2 min

was included. The amplification cycles used for

denaturation, primer annealing to the template, and

primer extension were as follows: 94•Ž for 2 min,

45•Ž for 1.5 min, and 72•Ž for 2 min for each

primer pair. After amplification, 10 ,ul of the PCR

products were mixed with 5ƒÊl of tracking dye solu

tion (2% SDS, 25% Ficoll, 0.125% bromphenol

blue, 40 mm Tris-HCl, 5 mm EDTA), submitted to

electrophoresis in a 1.0% agarose gel, stained with

ethidium bromide, and photographed at 302 nm UV

transillumination.

Restriction enzyme digestion

Seven strains of C. columaris listed in Table 2 were

used for restriction enzyme digestion of the 16S

rDNA. For each strain, 4ƒÊl of PCR products

amplified by the universal 20F-1500R primer pair

was digested with 20 units of the restriction enzyme

HaeIII (5'-GG •« CC-3') or Hhal (5'-•«C-3')

according to Hiraishi et al. (1995). Ten microliter of

the digested solution was mixed with 8ƒÊl of tracking

Table 1. The primer sequences and their positions corresponding to the numbering of Escherichia coli

Identification of F. maritimus by PCR 27

Table 2. Bacterial strains used for the PCR

Abbreviations: Fx., Flexibacter; F., Flavobacterium; C., Cytophaga; A., Aeromonas; E., Eschricha; Ed., Edwardsiella; P., Pasteurella; Ps., Pseudomonas; V., Vibrio. Y., Yersinia;

dye solution, submitted to electrophoresis in a 2.0% agarose gel, and visualized by staining with ethidium

bromide.

Results

PCR-amplification Although the sequences of the primers BRA1 and

BRA2 were based on the 16S rRNA sequence of F. branchiophilum ATCC35035, attempts at PCR am

plification of the 16S rDNA gene of the strain by using the primer pair were unsuccessful. Attempts at amplification of the 16S rDNA gene of C. columnaris NCIMB 2248 by using the COL1 -COL2 primer pair

also were without success. Hence, combinations of each of these primers and the universal primers, 20F and 1500R, were tested. As shown in Fig. 1, the

BRA 1-1500R primer pair and the COL 1-1500R were capable of amplifying F. branchiophilum ATCC 35035 and C. columnaris NCIMB2248, respectively.

To test the utility of each primer pairs for identification of the bacterial species, several strains of the targeted species as well as several strains of other species were used for the PCR with each of the

primer pairs. The MAR1-MAR2 primer pair amplified the 16S rDNA from all of 6 strains of F maritimus, but none of 17 strains of other species (Fig.

2). The BRA 1-1500R primer pair amplified the 16S


rDNA from all of 6 strains of F. branchiophilum, butnone of 18 strains of other species (Fig. 3). TheCOLa-COLb primer pair was capable of amplifyingonly 2 of 7 strains of C. columnaris, EK28 andFPC74 (Fig. 4). Although these primers were basedon the 16S rRNA sequence of C. columnaris ATCC-43622, this strain was not used for the PCR amplifi-cation because we did not have it. The COL1 and1500R primer pair amplified the 16S rDNA from therest of C. columnaris strains, NCIMB2248, EPC54,FPC77, FPC666 and FPC492 (Fig. 5). Both of the

primer pairs reacted on some bacteria other than C.columnaris, too. However, the size of their PCR

products was different from that of C. columnaris(Figs. 4 and 5).

Restriction enzyme digestionDigestion pattern of 16S rDNA from 7 strains of

C. columnaris with HaeIII or HhaI is shown in Fig.6. The PCR-restriction fragment length polymor-

phism (RFLP) patterns of the digested 16S rDNA

from C. columnaris differentiated strains EK28 and

FPC74 from the other strains. It was consistent with

the results of PCR amplification by using the COLa-

COLb, and COL1-1500R primer pairs.

(A) (B)Fig. 1. Confirmation of the validity of the primers for

PCR amplification. (A) F branchiophila ATCC-35035 was amplified with the primer pairs,BRA1-BRA2 (lane 1), BRA1-1500R (lane 2),20F-BRA2 (lane 3), and 20F-1500R (lane 4).Left-most lane (M); molecular weight markers.

(B) C. columnaris NCIMB2248 was amplifiedwith the primer pairs, COL1-COL2 (lane 1),COL1-1500R (lane 2), 20F-COL2 (lane 3), and20F-1500R (lane 4). Left-most lane (M);molecular weight markers.

Fig. 2. Agarose gel electrophoresis of PCR productsfrom selected bacterial strains with a primer pairof MAR1 and MAR2. Lanes 1 through 6: Fmaritmus NCIMB2154, NCIMB2153, FPC386,FPC394, FPC454 and JIP32/91(6); Lane 7: C.

psychrophila NCIMB1947; Lane 8: F. branchio-philum ATCC35036; Lane 9: C. columnarisEK28; Lane 10: F. aquatile IAM12316; Lane 11:C. aquatilis IAM12365; Lane 12: C. flevensis;Lane 13: F. indologenes FPC954; Lane 14: F.breve ATCC14234; Lane 15: E. coli K12; Lane16: E. tarda EPC801; Lane 17: A. hydrophilaFPC868; Lane 18: A. salmonicida; Lane 19: P.

piscicida FPC851; Lane 20: Y. ruckeri FPC442;Lane 21: P. anguilliseptica NCIMB 1950; Lane22: P. fluorescens FPC348; and Lane 23: V.anguillarum FPC346. Left-most lane (M);molecular weight markers.


Discussion

It was theorized that the MAR1-MAR2 and

BRA1-1500R were specific for the detection of F.

maritimus and F. branchiophilum, respectively. It is

uncertain why the primers BRA2 and COL2 did not

work. We are discussing these primers.

The COLa-COLb and COLI-1500R primer pairs

distinguished two groups among C. columnaris

strains, and there was no cross-reaction between the

groups. One group consisted of EK28 and FPC74,and another group was composed of NCIMB2248,

FPC54, FPC77, FPC666 and FPC492. The group-

ing was supported by the restriction enzyme diges-

tion analysis of the 16S rDNA from these strains of

Fig. 3. Agarose gel electrophoresis of PCR productsfrom selected bacterial strains with a primer pairof BRA1 and 1500R. Lanes 1 through 6: F.braniophilum ATCC35035, TW1, ATCC35036,FDL 1, FDL3 and FL15; Lane 7: C. psychro-

phila NCIMB1974; Lane 8 and 9: C. columnarisNCIMB2248 and EK28; Lane 10: F. maritimusNCIMB2154; Lane 11: F. aquatile IAM12316;Lane 12: C. aquatilis IAM12365; Lane 13: C.

flevensis; Lane 14: F. indologenes FPC954; Lane15: F. breve ATCC14234; lane 16: E. coli K12;Lane 17: E. tarda EPC801; Lane 18: A. hydro-

phila FPC868; Lane 19: A. salmonicida; Lane20: P. piscicida FPC851; Lane 21: Y ruckeriFPC442; Lane 22: P. anguilliseptica NCIMB-1950; Lane 23: P. fluorescens FPC348; and Lane24: V. anguillarum FPC346. Left-most lane

(M); molecular weight markers.

Fig. 4. Agarose gel electrophoresis of PCR productsfrom bacterial strains and a primer pair ofCOLa and COLb. Lanes 1 through 7: C.columnaris EK28, FPC74, NCIMB2248, FPC54,FPC77, FPC666 and FPC492; Lane 8: C. psy-chrophila NCIMB1947; Lane 9: F branchio-

philum; Lane 10: F. maritimus NCIMB2154;Lane 11: F. aquatile IAM12316; Lane 12: C.aquatilis IAM12365; Lane 13: C. flevensis; Lane14: F indologenes FPC954; Lane 15: F breveATCC14234; Lane 16: E. coli K12; Lane 17: E.tarda EPC801; Lane 18: A. hydrophila FPC868;Lane 19: A. salmonicida NCIMB2020; Lane 20:P. piscicida FPC851; Lane 21: Y. ruckeriFPC442; Lane 22: P. anguilliseptica NCIMB-1950; Lane 23: P. fluorescens FPC348; and Lane24: V. anguillarum FPC346. Left-most lane



C. columnaris. Therefore, it appears that there isintra-species variation among C. columnaris strainson the 16S rRNA level.

According to Bernardet and Grimont (1989), thestrains EK28 and NCIMB2248 which were sepa-rated to different 16S rRNA group had the relativevalue of 79% in the DNA-DNA hybridization test,and so it was formally admitted that these two strain

belonged to the same species of C. columnaris. Fur-ther studies should be performed on the phylogenetic

variations within C. columnaris strains.

Acknowledgments

We greatly thank Dr. Y. Nakagawa for generously

supplying the 16S rRNA sequence data of his Ph. D.dissertation. We are also grateful to Dr. K. Ohwada,Dr. K. Kogure, Dr. M. Nishimura, and the members

in Marine Microbiology Division, Ocean ResearchInstitute, University of Tokyo, for their advice and

encouragement. We express our appreciation to Dr.R. Holt and Dr. M. Mauel, Department of Micro-

biology, Oregon State University, who kindly review-ed the manuscript. This work was supported by a

grant from the Ministry of Education, Science andCulture, grant No. 07556047.

References

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(A) (B)

Fig. 5. Agarose gel electrophoresis of PCR productsfrom selected bacterial strains with a primer pairof COL1 and 1500R. Lanes 1 through 7: C.columnaris EK28, FPC74, NCIMB2248, FPC54,FPC77, FPC666 and FPC492; Lane 8: C. psy-chrophila NCIMB1947; Lane 9: F. branchio-

philum; Lane 10: F. maritimus NCIMB2154;Lane 11: F. aquatile IAM12316; Lane 12: C.aquatilis IAM12365; Lane 13: C. flevensis; Lane14: F. indologenes FPC954; Lane 15: F breveATCC14234; Lane 16: E. coli K12; Lane 17: E.tarda EPC801; Lane 18: A. hydrophila FPC868;Lane 19: A. salmonicida NCIMB2020; Lane 20:P. piscicida FPC851; lane 21: Y. ruckeriFPC442; Lane 22: P. anguilliseptica NCIMB-1950; Lane 23: P. fluorescens FPC348; and Lane24: V. anguillarum FPC346. Left-most lane


Fig. 6. PCR-RFLP patterns of 16S rDNA from C.columnaris digested with HaeIII (A) and Hhal

(B). Lane 1: EK28; Lane 2: FPC74; Lane 3:NCIMB2248; Lane 4: FPC54; Lane 5: FPC77;Lane 6: FPC666; and Lane 7: FPC492. Left-most lane (M); molecular weight markers.


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26 t. toyama, k. kita-tsukamoto and h. wakabayashi

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