Microbiol. Immunol. Vol. 36 (2), 161-167, 1992

Organization

of the Ribosomal

RNA Genes in Treponema

phagedenis and Treponema pallidum Masahito FUKUNAGA,*4 Noriko OKUZAKO,1 Ichiji MIFUCHI,1 Yoshiko 1

ARIMITSU,2

and

Masaaki

SEKI3

Faculty of Pharmacyand PharmaceuticalScience,Universityof Fukuyama, Fukuyama, Hiroshima 729-02, Japan, 2Departmentof Applied Immunology , National Institute of Health, Shinagawa-ku, Tokyo141, Japan, and 3Japan LyophilizationLaboratories, Kiyose, Tokyo 180-04, Japan (Accepted for publication, November 9, 1991)

Abstract The genomic DNA fragment which contains ribosomal RNA (rRNA) genes for Treponemaphagedeniswas cloned into bacteriophage vector lambda EMBL3. A restriction map of the fragment was constructed and the organization of the rRNA genes was determined. The fragment contained at least one copy of the 16S, 23S and 5S sequences and the genes are arranged in the order 16S-23S-5S . Southern hybridization using radiolabeled rRNA gene probes to genomic DNA from T . phagedenis strain Reiter and T. pallidum strain Nichols showed that these organisms have two radioactive fragments which hybridize to the probes in their genome . These results suggest that both pathogenic and non-pathogenic strains of Treponemamay carry at least two sets of rRNA genes on their chromosomes.

We are interested in studying the organization and regulation of the genes coding for the ribosomal RNAs (rRNA) of spirochetes. Our interest in rRNA genes in spirochetes has arisen because the organization of rRNA genes for leptospires is unique (5). In addition, our finding of a functional promoter immediately preceding one operon comprising only the 5S rRNA gene gave further evidence that the other rRNA genes of the strains of leptospires are organized in separate transcription units. By the transcriptional analysis of the 5' regions of the 16S and 23S rRNA genes for Leptospirainterrogans,primary transcripts were detected and the sequence resembling a promoter was seen at upstream from the 5'-terminus of the gene in each case. Organization and transcription of genes for rRNA have been highly conserved among procaryotes because the synthesis of rRNA is an essential process in the growth of any organism (13, 14). In most eubacterial species so far examined, the sequences for the three rRNAs are closely linked in gene sets, in the order 16S-23S5S (11). Some exceptions to this rule have been reported . In Thermusthermophilus (21), 16S and 23S rRNA genes are separated; in Mycoplasmagallisepticum(2), the 16S gene is separated from the other genes; and in another Mycoplasmastrain (20), the 161

162

M.

FUKUNAGA

ET AL

5S rRNA gene is separated from the other genes. However, there is no reported sequence like that of the Leptospira genome. Spirochetes are thin and spiral shaped and constitute one of the few groups correctly identified by their common characteristics. Spirochetes include treponemes, leptospires, borrelias. In this paper we describe the organization and the cloning of the rRNA genes of Treponema phagedenis as the first step towards a detailed study of their structure and expression. MATERIALS

AND

METHODS

Bacterial strains and media. Treponema phagedenis strain Reiter was cultured anaerobically for five days at 37 C in modified Brewer's medium (16) supplemented

with calf serum at 10% (v/v). Bacterial cells were collected by centrifugation, washed three times with saline-EDTA buffer (0.15 M NaCl, 0.1 M sodium EDTA, pH 8.0) and stored at -80 C. The virulent Treponemapallidum strain Nichols was cultured in rabbit testes and the testes were aseptically removed, minced and dipped in phosphate-buffered saline (pH 7.2) to remove loose cellular debris. The bacterial cells were purified by differential centrifugation and stored at -80 C. Escherichia coli strains HB101 and NM539 were grown in Luria broth either in the liquid form or

with

were

2%

used

agar

added

to isolate

(12).

L

plates

supplemented

with

100 ƒÊg/ml

of

ampicillin

E. coli transformants.

Preparationof DNA, and genomic-and plaque-hybridization. Total cellular DNA was extracted by the method described in a previous paper (4, 5). The hybridization probes were also prepared as described previously (5). DNAs were cloned by the standard method of Sambrook et al (17) by using plasmid vector pUC18 or bacteriophage lambda EMBL3 arm DNA. DNA fragments in agarose gels or the phage plaques on the plates were transferred and hybridized to the probes. Experimental conditions were as described in our previous papers (4, 5) and by Southern (18). Ordered deletion mutants were generated by digestion with exonuclease III. Total cellular RNA of T. phagedenisstrain Reiter was extracted by the method described in a previous paper (7). All other experimental conditions were as described previously (7). Probes used in this study. The DNA fragment generated with BanII digestion of the 16S rRNA gene for Leptospirainterrogansstrain Moulton (nucleotide numbers 220 to 590, 9) was used as a 16S gene probe (probe A in the Fig. 1). The fragments including the 5' part of the 23S rRNA gene for the same organism (SmaI-PmaCI, nucleotide numbers 34 to 451, probe B, 6) and the sequence generated with BamHIBg/II digestion (nucleotide numbers 1587 to 2839, probe C, 6) were used as a 23S gene probe. The DNA fragment including the entire sequence of the 5S rRNA gene from strain Moulton (EcoRI-HincII, probe D in Fig. 1, 7) was used as a 5S gene Probe.

Each

DNA

fragment

was

labeled

by

using [ƒ¿-32P]dCTP,

222

TBq/mmol

(NEN Research Products) ; and random primer labeling kit (Takara Biochemicals). Ribosomal RNA (a mixture of 16S, 23S, and 5S rRNA) was partially hydrolyzed as

rRNA

GENE

ORGANIZATION

IN

TREPONEMA

163

described by Suzuki et al (19) and labeled with T4-polynucleotide [ƒÁ-32P]

ATP

and

used

as

an

RNA

kinase and

probe.

RESULTS

Cloningof rRNA GeneSetsfor Treponema phagedenis Strain Reiter, Linkage and Order In our preliminary work, we showed that one of the rRNA gene sets in T. phagedenisstrain Reiter is located on the 13-kb BamHI fragment. Therefore, genomic DNA of strain Reiter was cleaved with BamHI and a lambda EMBL3 gene bank was constructed by using BamHI arms, and recombinant bacteriophages containing the rRNA genes were selected by plaque hybridization. One of these clones was selected, and physical maps were constructed by digestion, partial digestion, and double digestion of the DNA fragment with each enzyme. A restriction map for the BamHI fragment is shown in Fig. 1. Hybridization analysis according to Southern (18) was used to map the organization of the rRNA genes in this BamHI fragment. Four different DNA fragments (probes A, B, C and D, shown in Fig. 1) were radiolabeled and employed as probes against restriction digests of recombinant phage DNA. The results of one such experiment are shown in Fig. 2. The DNA was digested by several enzymes indicated in the figure in combination with BamHI or Sail and the location of the 23S rRNA gene was determined by using probes B and C. Hybridization of probe C to Xhol digest of recombinant phage DNA yielded small (0.8-kb) and large fragments. Larger fragment was separated into radioactive 4.5-kb fragment and phage arm in double digests with BamHI or Sail enzyme. The largest radioactive fragment in Xhol digest is composed of both phage arms. The location of the 16S rRNA gene was also determined by the same procedure and the results are shown in Fig. 1. For determination of the 5S rRNA gene locus, the

Fig.

1.

Phage

Physical DNA

map containing

of the recombinant the

BamHI

phage fragment

DNA was

and digested

the figure, electrophoresed, transferred and hybridized fragment sizes were estimated by using the size marker gene was determined and is indicated in this experiment are also indicated. text.

location with

of ribosomal each

to an rRNA DNA. The

enzyme

RNA

genes.

indicated

in

gene probe. Restriction location of each rRNA

in the figure. The leptospiral rRNA gene probes used The other experimental conditions are described in the

164

M.

FUKUNAGA

ET Al

Fig. 2. Patterns of hybridization of the 23S rRNA gene probe to restriction endonucleasedigested recombinant phage DNA. Recombinant phage DNA was digested with each restriction enzyme indicated in the figure, electrophoresed on agarose gels, transferred to a nylon membrane (Zeta probe membrane) by the alkaline Southern blotting method (4), and hybridized to a 23S rRNA gene probe (probe (1 in Fig. 1). Restriction fragment sizes were estimated by using the size marker DNA.

3.0-kb HindIII fragment was ligated into pUC18 DNA, and competent cells of E. coli K-12 strain HB101 were transformed. A bacterial clone harboring the recombinant plasmid DNA and another clone which contained the same HindIII fragment but in the opposite direction were isolated. Ordered deletion mutants for both recombinant plasmid DNAs were generated by digestion with exonuclease I I I. The location of the 5S rRNA gene was determined by hybridization of these ordered deletion mutant DNAs with probe D.

Copy Number of rRNA Genes in Treponema pallidum Southern hybridization was used to determine

and T . phagedenis the copy number of the rRNA

genes in T. pallidum and T. phagedenis. rRNAs were prepared from strain Reiter, radiolabeled and employed as a probe against restriction digests of genomic DNA from T. pallidum strain Nichols and T. phagedenis strain Reiter. The results of one such experiment are shown in Fig. 3, which shows the Southern hybridization patterns of the genomic DNA digests of strain Nichols with the rRNA probes. The restric-

rRNA

GENE

ORGANIZATION

IN

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TREPONEMA

Fig. 3. Patterns of hybridization of the rRNA probe to restriction endonuclease-digested treponemal genomic DNA. Genomic DNA of T. pallidum strain Nichols was digested with the enzymes indicated in the figure and electrophoresed in 1% agarose gel. The rRNA probe prepared from T. phagedenis strain Reiter was nicked and labeled by using [32P]ATP and T4-polynucleotide kinase. Hybridization and the other experimental conditions were as described in a previous paper (7). Table

1.

Sizes

of restriction hybridized

endonuclease-digested with

the rRNA

DNA

fragments

probe

tion digests of genomic DNA from strain Nichols yielded two radioactive bands. The leptospiral DNA probes (A, B or D) revealed identical hybridization profiles (data not shown). These results indicate that the sequences for the three rRNA genes are close to each other. The molecular sizes of hybridization fragments in

166

M.

kilobases were calculated a molecular size marker

FUKUNAGA

ET AL

by using HindIII-KpnI digests of lambda phage and all the results are summarized in Table

DNA as 1. Both

pathogenic and non-pathogenic strains of treponema produced two radioactive bands in each restriction endonuclease digestion. The sizes of the fragments varied with the strain, but these results suggest that there are at least two gene sets for rRNAs in both strains used in this experiments. DISCUSSION

The location, linkage and number of rRNA genes in leptospires were determined previously (4, 5, 7, 8). We have found that the leptospiral genome has two genes for 16S rRNA, two genes for 23S rRNA, and two 5S rRNA genes for saprophytic leptospires but one gene for parasitic leptospires (7, 8). Furthermore, there is no linkage at all among those rRNA genes. Contrary T. phagedenis

to our expectation, as we demonstrated here, strain Reiter are arranged in gene sets, probably

the rRNA genes in all in the order 16S-

23S-5S. In one of these gene sets, the sequences corresponding to the three rRNAs are all contained in 5.3-kb, implying that the three rRNA genes are closely linked. The other gene sets, including the genes of pathogenic treponema, may also contain at least one copy of the 16S, 23S and 5S gene sequence. The copy number of these gene sets in treponema found in the eubacteria.

is in the lower

number

of the

range

of the

copy

number

The spirochetes have a unique and complex morphology and at present the group is divided into two families (1, 10). The family Leptospiraceaeis composed of two genera, Leptospiraand Leptonema. The other family, Spirochaetaceae,is composed of four genera : Spirochaeta,Treponema,Borrelia and Cristispira. There are many differences between the two families regarding their habitat, energy-yielding metabolism, and DNA composition (1, 15). Phylogenetic studies using 16S rRNA oligonucleotide cataloging have demonstrated that there are some taxonomic distinctions between leptospires and treponemes (3, 15, 22, 23). The results reported here, therefore, confirm the phylogenetic situation of treponemes in the evolution of the spirochetes. In carry gene sion

this two

study, rRNA

arrangement and regulation

expression

we found

that

gene

in

sets

both their

parasitic

and

chromosomes

in one gene set for strain Reiter. of gene sets will provide insight

non-parasitic and

we

also

treponema

strains

determined

rRNA

Further studies on the expresinto the mechanisms of gene

in treponemes.

This work was supported in part by Grant from the Ministry of Education, Science and Culture, Japan. We thank Y. Ohshiro, H. Ueda and K. Hirata for their technical assistance. We also thank T. Yamada for invaluable advice. REFERENCES

1)

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10)

11) 12) 13) 14) 15) 16) 17) 18) 19) 20)

21) 22) 23)

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