Gene, 9X (1991) 123-127

123

Elsevicr

GENE

03919

Cloning and characterization Escherichia coli

of the Mycobacterium

leprue putative ribosomal

(Recombinant

RNA;

primer extension;

DNA;

16s ribosomal

mycobacteria;

bacilli;

RNA promoter in

leprosy)

Shlomo Sela” and Josephine E. Clark-Curtiss”*b Departments

of ‘I Biology

and ‘Molecular Microbiology. Washington University, St. Louis, MO 63130 (U.S.A.)

Received by W.M. Holmes: 7 June Accepted: 2X October 1990

1990

SUMMARY

The putative promoter region of the 16s ribosomal RNA-encoding gene (rRNA) of Mycobacterium leprue was cloned and characterized in Escherichiu coli. A 932-bp Hue111 restriction fragment, containing the 5’ end of the 16s rRNA gene and flanking upstream region, was cloned in front of a promoterless reporter gene in the shuttle vector, pMH109, to generate the plasmid, pYA 110 1. This clone exhibits promoter activity both in Gram (E. coli) and Gram + (Bacillus subtilis) bacteria. Sequence analysis and primer extension experiments with mRNA derived from the M. leprue clone were used to determine the structure and the location of the promoter, as well as the transcription start point in E. coli. The promoter region contains sequences that resemble the -35 and -10 consensus sequences found in many bacteria. A region located 34 bp distal to the promoter is a putative rRNA processing signal, based on sequence homology with processing signals involved in the maturation of the rRNA precursor in B. subtilis and several Mycoplusmu species.

INTRODUCTION

M. leprue, the etiologic agent of leprosy, is still one of the least understood eubacteria. Studies on the biochemistry and genetics of this organism are inhibited by the inability to grow it in vitro. A correlation between the growth rate of mycobacteria and the number of sets of rRNA genes they possess has been observed (Bercovier et al., 1986). Fast-growing mycobacteria were shown to contain two sets of rRNA genes

Correspondence to: Dr. S. Sela, University,

Campus

box

Tel. (314)726-7323;

No.

Department 1137,

of Biology,

St. Louis,

MO

Washington

63130

whereas slow-growing mycobacteria contain only one set. We have recently demonstrated that M. leprue also contains only a single set of rRNA genes (Sela et al., 1989) and thus fits into the slow-growers group. However, one should note that even among the slow-growing mycobacteria there is a large variability in the growth rates of different species. Therefore, growth rate cannot be due solely to the number of the rRNA genes. To begin to study the regulation of M. leprue rRNA synthesis and its correlation to the growth rate of this organism, we have cloned and characterized the putative promoter of the 16s rRNA gene from M. leprae.

(U.S.A.)

Fax (314)726-4432. EXPERIMENTAL

Abbreviations:

Ap, ampicillin;

B., Bacillus; bp, base pair(s);

acetyltransferase; cat, gene encoding CAT; Cm, chloramphenicol; kb, kilobase( LB, Luria-Bertani (medium); M., Mycobacterium; nt, nucleotide(s);

oligo, oligodeoxyribonucleotide;

ribosomal RNA; rRNA, gene encoding transcription start point(s).

0378-l

I lY~Y1.‘$03.50

0

199 I Elsevicr

R, resistance/resistant; rRNA;

Science

rRNA,

ss, single strand(ed);

Publishers

AND DISCUSSION

CAT, Cm

B.V. (Biomedical

fsp,

Division)

(a) Cloning of the 5’ region of Mycohacterium leprae rRNA gene The rRNA gene cluster in M. leprue was previously identitied and cloned (Sela et al., 1989). However, that clone

124 lacked a small portion

of the beginning

(5’ end) of the /ftS

rRNA gene as well as the upstream region. Therefore, the initial step in this study was to clone the promoter region. We screened a cosmid M. leprae library (Clark-Curtiss ct al., 1985) with a DNA probe from another mycobacterium, M. sn~~egmrrtis, which contains the 5’ part of the IfiS rRIVA gene (Bercovier et al., 1959). This resulted in the identification of several cosmid clones. One of these clones,

from

pYA 1100 &m

dlgest

ww.ww \

pYAllO0, was further analyzed by Southern-blot hybridization of &I-digested pYA 1 100 DNA with the IW. snre:UZ&S DNA probe and revealed a 4%kb P.~tf fragment identical to a M. ieprcte chromosomal fragment, previously shown to contain the 5’ end of the Zf?S rRNA gene and the upstream DNA flanking region (Sela et al.. 1989). (b) Detecting

promoter activity in Escherichiu

Tronsfarmotion of --E. colt ond selection on chioromohenicol Dlotes

coli

To find the specific promoter region within the 4.X-kb PstI fragment of pYA 1100, the fr~~gment was digested with frequent cutter HueIII. and the resulting fragments were subcloned into a SmuI site of the promoter-cloning shuttle vector, pMHl09 (Fig. 1). This vector contains a Gram ’ (~r~~~~!~~~~cc~.~ aweus) cat gene including its ribosomcbinding site. and can replicate both in E. co/i and R. suhtih hosts (Hudson and Stewart, 1986). Transformed E. co/i cells were initially selected on LB rncdiut~l cont~~inin~ 5 klg Cm/ml. and later grown on media containing 30 pg Cmiml. Several clones which displayed resistance to Cm contained a I-kb insert. One of the CmK clones, pYAl101, was partially restriction-lllapped and used for further experiments. A 700-bp XhrrI fragment (Fig. 1; note that the 5’ X/WI site is dcrivcd from the vector), was rccloned into the X/X/I site of pMH 109. This clone, designated pYA 1 107, conferred the Cm” phenotype on E. coli cells. thereby narrowing the location of the potential promoter. Plasmid pYA 1107 also conferred the Cm” phenotype on B. .suhtifis 16XT ’ cells grown on LB plates c~~~~taining 5 pg Cm/ml. However, B. suhtilis with pYAllO7 was unable to grow on medium supplemented with 30 pg Cm/ml, as in case of E. coli. If we assume that the different sensitivity to Cm reflects the expression of cut, a possible explanation would be a variation in the copy number of pYAllO7 in the two bacteria as was reported for other plasmids derived from the same shuttle vector (Hudson and Stewart, 1986). (c) Structural

Smo I digesr

/

analysis

The M. leprue fragment cloned in pYAl101 was sequenced (Fig. 2). Analysis of the sequence revealed a promoter-like sequence which is close to the canonical -10 and -35 regions found in many bacteria (Harley and Reynolds, 1987). Although the - 10 region appeared to dii’fer from the expected consensus TATAAT by change of the first two nt (TA --f AT), the mycobacterial promoter fused to the cat gene on plasmid pMHl09 functioned reasonably well in

Reclonrng of the XboI-XboI fragment into pMHIO9 Tronsformotlon of --E. co11 ond selection

on chloramphenlcol

plates

n pYAl107

Fig. I. Construction (Hudson

of plasmids

and Stewart,

gene lacking its own promoter, plasmid

contains

enables

replication

replication tetracycline Bg, &!II;

in B.

pYAl 101 and

1986) in a 7.4-kb plasmid,

two origins

preceded

pY41 107. pMHl09 which contains

a

cat

by a multiple cloning site. The

of replication,

one (designated

in E. co/i, and a second (designated

pBR ori)

PUB ori) enables

suhG/is.Plasmid pMHIO9 bears t\vo selective markers.

resistance E, EcoRl;

(Tc) and kanamycin H, flindlll;

resistance

(Km). B, Bcr,nHI;

P, PvuII; S. &~znl; Ss, Ssrf; X, XhctI.

E. di. This is evident by the ability of cells harboring this fusion to grow in medium suppleI~lented with 30 /rg Cm:ml. The -35 region differed from the consensus sequence only in the last nt (T instead of A). However, it is interesting to note that it is identical to the -35 region of the second promoter (PZ) in rrn operons of E. cnli (Jinks-Robertson and Nomura, 1987). The distance between the two consensus hexamcrs in M. lepme is 16 nt, similar to the situation in E. cwfi rm promoters. This is slightly shorter than the 17 nt consensus spacer present in many other penes (Harle! and Reynolds, 1987). An oligo derived from an nt sequence distal to the putativc promoter (PI in Fig. 2) was used as a primer to map the rs/) of the 16s rRNA gene in E. co/r’by primer extension analysis. Extension ofthe primer annealed to RNA purified from E. cofi cells harboring plasmid pYA 1 101 and plasmid pYA1 107 demonstrated a specific transcript whose 5’ end mapped to a G residue 8 bp distal to the -10 region. No specific transcript was seen in cells containing the vector

125

G ATC

-691

I23

-631 -571 GATTCCGCTTTGTCGAAGAGATGATCCGGCTGGGTGCCGATGCCCGCACGGATGGGCACC ATGCCGTTGTGCGGGGTCTTCCACAACTCTCTCGAGCGCGCCGGTCTGGTGTTCGGATATCC

-511

P-2 -451 GTGCTGGTGCCGGTTTGGTACTCGCGGGACTTGTCGCTGATGGAGACACCGAGGTCTACG -391 -33:

-35

-10

-271 AGGCATTAAGATGTTATAGTCAACCCGGGA~CTCTGCTGGATCTG~

‘I -211 TGGCTGGGTTGCCGAAGCGGGGGMGTAAGCTTGMGTGTTGTTTTGAG~CTC~TA~ -151 TGTTTGGTTTTGTTGTTGTTGATTTTTTGACTACTACATCTAGCATTCCTCGTGTGTGTAGGT

P-l -91

GTAGTTTATTATGTTATTTATAGATGCCAGTTTTGGTGTC~GTCAGGTATCTCTAG~

-31

1 16s , TTGlUVLATTTCGTCTAGTTATTGATGGAGTTTTTTGT~GGAGAGTTTGATCCTGGCTCAG

30

GACGAACGCTGGCGGCGTGCTTAACACATGCXAGTCGAACTC

90 150 210 TGTGGTGGAAAGCTTTTTGCGGTGCAGGATGG

Fig. 2. The nt sequence

of the 5’ region

of the coding

M. leprue 16.9 rRNA gene and its upstream of the putative

5’ end (nt + 1) of the mature

based on the sequence (Suzuki

alignment

et al., 1988). Putative

(nt -205)

is indicated

extension

experiments

performed

with

3Zf + I-

; Promega,

technique

(Sanger

et al. (1984).

GenBank-Los

Alamos

K710 Los Alamos,

templates

Sequencing from

WI), employing

Laboratory

NM 87545 (U.S.A.)

plasmids

were pGEM

kit (US Biochemical were done according

are deposited

Group

under

similar to

reactions

the chain-termination

analyses

The nt sequences

National

--

fsp

used for the primer

The sequence

(derived

OH). Computer

to Devereaux

are boxed. The

and the primers

et al., 1977) and the Sequenase

Cleveland,

1 is

and is

with M. bovis BCG 16s rRNA sequence

signal is overlined.

Madison,

of the

16s rRNA molecule

(PI, P2) are underlined.

ss DNA

strand

region. Position

-35 and -10 regions

by a triangle

the B. subfilis processing

Corporation,

flanking

with the

T-10, Mail stop MS

accession

No. M58020. Fig. 3. Primer extension

pMH 109 (Fig. 3). Primer extension with primer 2 (P2 in Fig. 2) did not reveal any transcript that derived from the mycobacterial DNA (data not shown). The distance between the tsp and the - 10 region, 8 bp, is similar to that of rRNA operons found in other bacterial species (Brosius et al., 1981; Gafny et al., 1988; Hartmann and Erdmann, 1989; Hyman et al., 1988; Taschke and Herrmann, 1988). In many bacterial species, tandem promoters preceding the 16s rRNA gene have been found (see for example: Jink-Robertson and Nomura, 1987; Stewart and Bott, 1983). In several cases, only a single promoter is observed (see for example: Hyman et al., 1988; Hartmann and Erdmann, 1989). Based on primer extension analyses and sequencing data, only a single promoter was identified near

T and C represent fragment) PI-extension boring pYAllO1

using

primer

reactions,

plasmids

analysis

sequencing PI

of the pYAllO7

(see Fig. 2). Lanes l-3

done with RNA purified

pMHl09

and pYAl107,

polyacrylamide-6

of the M. leprae promoter. reactions

(rRNA-less respectively.

control Denaturing

Lanes G, A, insert

represent

(XbaI primer

from E. co/i cells harwith the vector

itself),

gel consisted

of 6%

M urea.

the 5’ terminus of the M. leprue 16s rRNA gene. However, it is possible that another promoter(s) might exist that is not recognized by the E. coli transcriptional apparatus. Processing of the rRNA molecules into the mature RNA transcripts in E. coli has been suggested to involve the generation of stem-and-loop structures between complementary sequences flanking the mature transcript. These structures serve as processing signals for specific RNases (King and Schlessinger, 1987). A similar mechanism was

126 Mycobacterirm

lep-a’ Bacillus slrbtilis MIJCO$~~I cnpricohm Mycoplnsmn pnerlmonine

GAAGTGTTGTTTGAGAACTCAA

-100 CCATGCTCTTGATGCCCCGTTGTCGGGGGGCGTGGCCGTT

GAAATGATCTTTGAAAACTAAA

I I III I III II I III1 -100 TGTGTAGGTGTAGTTTATTATGTTATTTATAGATGCCAGTTTTGGTGTCT

ATCACGATCTTTGAAAACTAAA AAAATGTTCTTTCAAAACTGGA

Fig.4. Comparisonof the

conserved 16s rRNA processing signal sequcncesfound in B.subtilis (Loughneyetal..1983),i2~~~plnsn~~1 wpricolw? (Gafnyetal., 1988; Taschkeand Hermann,198X), andMJCO/)[~1.~1~~~1p~zezlnlolii~le (Hyman etal., 1988)~theM. leprcrr promoter:leader scquence.Thenumberofthe firstntin eachsequence is 337,-118.P37, and -178,respectively (numbersareaccording totheoriginal papers).

suggested to exist in B. subtilis. However, the sequences involved in the stem structures were different from those reported in E. coIi(Loughney et al., 1983). Recently, several researchers (Gafny et al., 1988; Taschke and Herrmann. 1988) have demonstrated the existence of processing sequences in mycoplasma homologous to those in B. .subtili.v. Sequence analysis of the M. leprue promoter region revealed a region of 22 bp (- 178 to - 157; Fig. 2) that demonstrated striking similarity to the processing signals located in the 5’ end of the 16s RNA genes of B. subtilis. M~roplr.sm~r pneumorlirre and Mlcoplasmu capricolum (Fig. 4). The similarity values are 77?,,, 59”” and 68%, respectively. Our data suggest that M. Ieprue might possess rRNA processing mechanisms similar to those found in B. subtilis and mycoplasma. This would support the suggestion that the M. leprue rRNA genes are organized in an operon structure which is transcribed to form a precursor that is later processed to the different mature rRNA molecules. Since B. subtilis and M~wplasma belong to one branch and M. leprue belongs to the other branch of of the Gram + phylum (Woese, 1987), the conservation of the putative processing signal sequence might be a common feature of this phylum. (d) Sequence of the Mycobacterium leprae 16s rRNA gene Our sequence data also contain some information regarding the primary structure of the 16s rRNA gene. Other groups have sequenced regions in the 3’ end of the actual 16s rRNA molecule (Estrada-G. et al., 1988), and the 16s rRNA molecule excluding part of the 5’ end (Smida et al., 1988). The sequence data presented here for the 5’ end of the 16s rRNA gene contain a 25-bp overlap with the latter RNA sequence (unpublished data, kindly provided by E. Stackebrandt). This permits us to deduce the entire gene sequence. Comparison of the 241 bp of the 5’ end of the 16s r-RNA gene demonstrated homology with sequences of several bacteria (Fig. 5). The similarity values ranged from 94”; to the reported M. bovis BCG sequence (Suzuki et al.. 1988). to 7O”,, and 632, to rRNA sequences from B. suhtilis and E. coli, respectively (Stewart and Bott, 19X3; Brosius et al., 198 1). These results suggested a relatively close relationship between the two mycobacterial species, supporting data by

-50 GGATATTT&AAATACCTT+GGCTCCCTT+TCCAAAGGGAGTGTTTGGG+ I I I I II I I II I I I II -50 TGTCAGGTATCTCTAGAAATTGAAAATTTCGTCTAGTTATTGATGGAGTT 1 TTTGTTTGGAGAGTTTGATCCTGGCTCAGGACGAACGCTGGCGGCGTGC~ IIlIIIIlIIIIIIIlllIllIlIIIIIIIIIIIllllIIlIIIIIIIII 1 TTTGTTTGGAGAGTTTGATCCTGGCTCAGGACGAACGCTGGCGGCGTGCT 51 TAACACATG&AGTCGAA&GAAAGGTCT&........:..TTCGGAGA IIIIIllIIIIIIIIllIIlIlIIllIIII II /III 51 TAACACATGCAAGTCGAACGGAAAGGTCTCTAAAAAATCTTTTTTAGAGA 89 TACTCGAGTGGCGAACGGGTGAGTAACACGTGGGTGATCT lIIIlIIIIIIIlIIIIIIIIIIIIIlIIlllIIl IIIIIIIIIl//lI 101 TACTCGAGTGGCGAACGGGTGAGTAACACGTGGGTAATCTGCCCTGCACT 139 TC.GGGATAAGCCTGGGAAACTGGGTCTAATACCGGATAGGACCACGGG~ II lIIIIIlII IIIlllllllIllllIIlIIIIIIIIIIIl I I 151 TCAGGGATAAGCTTGGGAAACTGGGTCTAATACCGGATAGGACTTCAAGG 188 TGCATGTCT+GTGGTGGAA;GCGCTTTAGCGGTGTGGGA+GA IlIIIIIIIlIlIIIIIIIII III IIIIII IIIII 201 CGCATGTCTTGTGGTGGAAAGC.TTTTTGCGGTGCAGGATGG Fig. 5.ComparisonbetweentheM. leprue sequence (lowerlines; see Fig.3)and M. bovis BCG sequence (upperlines; Suzukieta]., 1988).

others (Smida et al., 1988), who compared other segments of 16s rRNA molecules from several mycobacteria. In spite of the high degree of sequence similarity of the 16s rRNA genes in the two mycobacteria, the M. leprw gene has a sequence of 12 bp (81-92) which is entirely missing in the M. bovis BCG gene and is part of a unique stem-and-loop structure (nt 77-100). This sequence might be a useful candidate for an M. leprcle species-specific probe. (e) Conclusions Our hypothesis that the identified promoter is an actual M. leprue promoter is supported by the observations of promoter activity in E. coli and in B. subtih, sequence and structure similarity to the consensus promoter, and the existence of nearby sequences homologous to Gram + processing signals. Information on other mycobacterial promoters is limited. Sequence determinations for several mycobacLerial antigens have identified putative promoter sequences (Mehra et al., 19X6; Booth et al., 19X8). In another case, random M. bovis DNA fragments were shown to have promoter activity in E. coli when ligated to a promoterless vector (Sirakova et al., 19X9). Also. observations in our laboratory (Sathish et al., 1990) indicated that a few i,gt 11 : : M. leprue clones specify apparent nonfusion proteins based on molecular size. Thus, it seems that wnc mycobacterial promoters can function in E. co/i. The ultimate characterization of the controlling elements must Lvait until enough intact precursor rRNA can be isolaled from this organism and/or the isolation of the mycobacterial RNA polymerase.

127 cannot

M. ieprue

Since

to choose

another

yet be cultivated

system

in vitro, one has

to study the regulation

of its

genes. According to our study, the 16s rRNA promoter directs transcription both in E. coii and in B. subtilis. However, some regulatory elements might be restricted to the phylum level, as we have suggested in the case of the putative processing signal. This implies that at least some aspects of the regulation of M. kprae rRNA synthesis, might be better approached by studying the promoter fusion in a Gram + background (B. subti&s). The emerging availability of systems for genetic manipulations in cultivable mycobacteria (Snapper et al., 1988; Zainuddin et al., 1989) offers an alternative, promising approach for studying regulation of the rRNA synthesis in M. leprue.

the

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C.B. and Reynolds,

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This work was supported by a postdoctoral feIlowship from the Heiser Program for Research in Leprosy awarded to S.S. and by Public Health Service grant AI-23470 from the National Institute of Allergy and Infectious Diseases awarded to J.E.C.-C. We thank M. Hudson for plasmid pMH109, A. Honeyman for B. subtilis 168T+, E. Stackebrandt for sharing the unpublished sequence data of M. leprue 16s rRNA, H. Bercovier and R. Gafny for discussions and helpful advice and Roy Curtiss III for critically reading the manuscript.

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sequences.

pp. 1358-1385.

ACKNOWLEDGEMENTS

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Dale,

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~~~~~~bu~~erjz~~7 5n7~gn?u~j~ with ~.~~be~j~~~i~~ coli plasmids

selectable

resistance

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carrying

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of a