John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, UK1
Dept of Molecular Microbiology and Centre for Tuberculosis Research, Johns Hopkins School of Hygiene and Public Health, 615 N Wolfe Street, Baltimore, MD 21205, USA2
Author for correspondence: K. F. Chater. Tel: +44 1603 452571. Fax: +44 1603 456844. e-mail: chater{at}bbsrc.ac.uk
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ABSTRACT |
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Keywords: tuberculosis, leprosy, actinomycetes, prokaryotic phylogeny, sporulation in Streptomyces
The GenBank accession numbers for the sequences reported in this paper are: wblA, AJ239085; wblB, AJ239086; wblE, AJ239087; and wblI, AJ239088.
a Present address: Departamento de Microbiología y Parasitología, Facultad de Farmacía, Universidad de Alcalá, 28871-Alcalá de Henares, Spain.
b Present address: Laboratory of Infection Biology, The Rockefeller University, Box 21, 1230 York Ave, New York, NY 10021, USA.
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INTRODUCTION |
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Streptomycetes are developmentally complex (Chater & Losick, 1997 ; Chater, 1998
). They form mycelial colonies in which different physiological functions take place in different locations. Thus, most antibiotic synthesis takes place in the older parts of the substrate mycelium, whilst sporulation takes place in specialized aerial hyphae. Gene regulation and the possibility of development in mycobacteria are less well characterized, but there is some evidence that alterations in cell type may occur during infection by pathogenic mycobacteria (Chatterjee, 1976
; Parrish et al., 1998
).
The prototype of the gene family discussed in this paper, whiB, is a developmental regulatory gene identified and characterized in S. coelicolor as being essential for sporulation of aerial hyphae (Davis & Chater, 1992 ). It encodes an 87 amino acid polypeptide with attributes suggesting that it may be a DNA-binding protein. The sequence of WhiB contains four cysteines a common motif in metal-coordinating DNA-binding proteins such as Zn-binding GAL4 and other zinc finger proteins (Schjerling & Holmberg, 1996
) (including a class of small transcriptional activator proteins encoded by some phages; Julien et al., 1998
), Fe-binding SoxR (Gaudu et al., 1997
) and Hg-binding MerR (OHalloran, 1993
). Secondary structure analysis of WhiB indicated a potential helixloophelix structure from residues 64 to 84 (Davis & Chater, 1992
). The importance of this region was underscored by the fact that the whiB70 mutant, which contains a missense mutation leading to substitution of Leu74 by Pro, had the same phenotype as a whiB deletion mutant (Davis & Chater, 1992
). The whiB genes of Streptomyces aureofaciens (Kormanec et al., 1998
) and Streptoverticillium griseocarneum (Soliveri et al., 1993
) function in a similar manner to whiB of S. coelicolor.
The existence of a mycobacterial gene encoding a WhiB homologue was first suggested by hybridization of a whiB-derived probe to restriction-endonuclease-digested Mycobacterium bovis BCG DNA (Soliveri et al., 1993 ). Subsequently, a whiB-like gene downstream of the groEL operon of M. tuberculosis was detected during database searches (Chater, 1993
). The partial sequencing of the M. leprae genome (Honoré et al., 1993
; Smith et al., 1997
), and the recently completed sequencing of the M. tuberculosis genome (Cole et al., 1998
) have provided an opportunity to scan large amounts of mycobacterial genomic DNA sequence for the presence of ORFs encoding homologues of WhiB. In this paper we describe multiple whiB homologues in mycobacteria (the whm gene family) and the use of this information to reveal their equivalents in Streptomyces (the wbl, for whiB-like, gene family). Remarkably, this substantial gene family appears to be present in most (perhaps all) actinomycetes, yet absent from all other sufficiently characterized organisms.
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METHODS |
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Genetic manipulation.
For E. coli, plasmid preparation and transformation of competent cells were as described by Sambrook et al. (1989) . For S. coelicolor, genomic DNA preparations were as described by Hopwood et al. (1985)
. Standard methods were used for restriction enzyme digestion, ligation, Southern blotting, PCR and radiolabelling of DNA (Sambrook et al., 1989
) unless stated otherwise.
Screening of an S. coelicolor genomic library.
An S. coelicolor genomic library of ordered cosmids (Redenbach et al., 1996 ) was screened by DNA hybridization using different genes as probes (Table 1
). Probes of PCR products were amplified from the following plasmids: whmAMl, pTA577F [containing whmAMl, amplified by PCR from M. leprae cosmid B57, in pCRII (Invitrogen)]; whmBMt, pTA63.3 (containing whmBMt, amplified by PCR from M. tuberculosis genomic DNA, in pCRII); whmCMl, pTA1937 (J. Gomez, unpublished); whmDMs, pBS718 (J. Gomez, unpublished); whmEMt, pJS5001 [whmEMt was amplified by PCR from M. tuberculosis genomic DNA and inserted into the SmaI site of pIJ2926 (Janssen & Bibb, 1993
)]; saf (
wblI), pUL300 (Daza et al., 1990
); whiB, pIJ558 (whiB gene in pUC19; Davis & Chater, 1992
). Probes were [
-32P]dCTP-labelled using an Oligolabelling Kit (Amersham Pharmacia Biotech), following the suppliers instructions. The cosmid library was probed under low-stringency conditions, prehybridization and hybridization being performed at 65 °C in a solution containing 5xSSC, 5xDenhardts solution (Hopwood et al., 1985
), 0·5% (w/v) SDS and 100 µg nonhomologous DNA ml-1. Hybridization overnight was followed by two washes in 2xSSC, 0·1% SDS at 60 °C. After isolation, cosmid DNAs were further digested and a second Southern blot was performed.
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Phylogenetic analysis of whiB-like genes.
Amino acid sequences of the various whm and wbl genes were aligned and analysed using the PROTDIST, FITCH and CONSENSE programs from the PHYLIP software package (Felsenstein, 1988 ). The dendrogram shown in Fig. 4
is the consensus tree of 500 runs of the FITCH program. Branch lengths are proportional to the genetic distance between proteins as assessed by PROTDIST.
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RESULTS |
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In most members of the Whm/Wbl family, a conserved set of cysteine residues is arranged as Cys-X(1422)-Cys-X2-Cys-X5-Cys (see below), but in WhmG (Rv0022c in the M. tuberculosis genome) the spacing of the corresponding residues is Cys-X11-Cys-X3-Cys-X7-Cys. Nonetheless, the other similarities to conserved regions of the Whm/Wbl protein family are striking enough to warrant the designation of Rv0022c as whmG. Further sequence comparisons are made later in this paper.
Multiple whm genes are also present in a rapidly growing, saprophytic mycobacterium, M. smegmatis
M. smegmatis is saprophytic and has a significantly faster generation time than the pathogenic M. tuberculosis (3 vs 1824 h in culture). Southern blotting experiments on M. smegmatis DNA gave unique and strong signals with probes from whmAMl, whmBMt and whmCMl (Fig. 1), whmE (not shown) and whiB (revealing the previously cloned whmDMs; see above). Similar results (but different fragment sizes) were also obtained with DNA of M. bovis BCG (see Fig. 1
).
Multiple whiB-like genes are present in S. coelicolor
The presence of paralogues of whiB in mycobacteria suggested that a similar situation might prevail in Streptomyces spp., and, in particular, in the extensively studied strain S. coelicolor A3(2). This would not only extend (and be informed by) the large amounts of genetic and biological information available for this strain, but might also in future help to evaluate the roles of whiB-like genes in mycobacteria. A firm indication that S. coelicolor has multiple whiB-like genes came from the use of a probe obtained by PCR using S. coelicolor DNA as template. The oligonucleotides designed as primers for PCR were based on two segments conserved among the known whiB-like genes (see Methods). Thus, it was hoped that several whiB-like genes would be represented in the PCR-amplified DNA. Indeed, the PCR-generated probe hybridized fairly strongly at six different positions to Southern blots of chromosomal DNA digests, one of which had the mobility expected for whiB.
Thus encouraged, we used PCR products corresponding to each of five whm genes (whmAwhmE; see Methods) to detect whm orthologues in S. coelicolor DNA. Each of these probes except the whmC probe hybridized strongly to one band in Southern blots of S. coelicolor DNA digests, and each band corresponded to one of the bands obtained in the preliminary experiment. (Of the two other bands, one was later identified as wblH, which is described below, and the other remains unidentified.) The probes were then used to identify hybridizing cosmids in the S. coelicolor library, and after suitable subcloning we sequenced the relevant regions to reveal a whiB-like (wbl) gene in each case. With one exception (the whmD/whiB pair) the genes have been given letters to match their likely equivalent whm genes. Thus, considering a conserved core segment corresponding to WhiB residues 2480, WblA protein was 67% identical to WhmA; WblB was 66% identical to WhmB; no WhmC homologue was found (but see below); WhmD was 87% identical to WhiB; and WblE was 75% identical to WhmE. Furthermore, the ongoing S. coelicolor genome sequencing project (www.sanger.ac.uk/projects/S_coelicolor/) revealed another wbl gene in cosmid St4C6, which was not closely similar to any whm genes and so was termed wblH (at the time of completing this paper, approximately 80% of the genome was available). wblH is identified as St4C6.25 (PID e1490484). The WblH protein is most similar to WhiB (58% identity) and WhmD (59·5% identity). In some cases, the corresponding whm and wbl genes had similar genetic contexts. Thus, whmAMt and wblA both diverge from genes encoding related putative penicillin-binding proteins; whmBMt and wblB both converge on groEL, though wblB is separated from the convergent groEL1 operon by an intervening gene; and whmEMt and wblE both converge on genes encoding related putative membrane sensor kinases. wblH, which has no close whm homologue, converges on a gene encoding a putative exported desaturase.
In summary, S. coelicolor contains close homologues (presumptive orthologues) of at least four of the seven known whm genes of mycobacteria. The seven whm genes of M. tuberculosis are noticeably clustered in less than 1 Mb of the circular 4411529 base pair H37Rv chromosome, between positions 3468401 (whmC) and 27442 (whmG) (position 1, located near the origin of replication, is defined as the first base in the dnaA ORF); the homologues from S. coelicolor are more widely scattered and in a quite different order (Fig. 2), illustrating an absence of large-scale synteny between these diverged actinomycetes.
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The discovery of wblI brought the total number of whiB paralogues so far identified in S. coelicolor to six (including whiB itself).
Evidence of transcription of wbl genes
To find out whether transcription occurred in wbl genes during normal liquid culture (representing vegetative growth), a preparation of RNA isolated late in exponential growth of S. coelicolor A3(2) strain M145 was evaluated by RT-PCR, using primers specific to each wbl gene (Table 2). In each case, a product of the predicted size was readily obtained, whilst control experiments showed no signal (Fig. 3
). Thus, all of the wbl genes are transcribed.
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DISCUSSION |
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It is interesting to note the presence of a whiB-like gene, gp49, in a recently sequenced mycobacterial phage genome (Tm4; Ford et al., 1998 : Figs 4
and 5
), which raises the possibility that comparatively recent phage-mediated horizontal transfer of whiB-like genes might have occurred; however, this could not readily account for the presence of similar sets of several such genes in nearly all actinomycetes. Small transcriptional activator proteins containing four cysteine residues, albeit with quite different spacing (Cys-X2-Cys-X23-Cys-X4-Cys) from the array of conserved cysteines in the WhiB family (and showing no other similarities to WhiB) are specified by several phages of Gram-negative bacteria (Julien et al., 1998
).
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The presence of four conserved cysteine residues suggests that the Whm/Wbl proteins may be sensitive to redox changes, perhaps through a bound metal atom or through direct sensitivity to oxidation via disulphide bond formation, as in the OxyR transcription factor of E. coli (Zheng et al., 1998 ). To date, these possibilities have been difficult to address experimentally because no biochemical activity or cellular target for the activity of any of the proteins has been defined. Nevertheless, the possession of these genes by actinomycetes, and the reliance of actinomycetes on mycothiol in place of glutathione, may be no coincidence; both may perhaps reflect a specialization coinciding with biologically very significant increases in oxygen concentration believed to have occurred in the earths atmosphere around 700800 million years ago, and to have permitted the emergence of fully aerobic bacteria (Ochman & Wilson, 1987
).
In summary, we speculate that the proteins are transcription factors that activate different target genes, possibly sensing redox changes that could originate from environmental change or be generated internally during metabolic shift-down (for example, at the end of vegetative growth or when aerial hyphae switch from growth to sporulation) (Nyström, 1999 ).
Studies of the effects of wbl mutations in the genetically manipulable S. coelicolor A3(2) may help to illuminate the functions of whm genes in pathogenic mycobacteria. A particular hope is that the epidemiologically important, but little understood phenomenon of mycobacterial dormancy within hosts may share regulatory features with the extensively studied process of sporulation in Streptomyces, in which whiB plays a very important part. Intriguingly, it has been demonstrated that M. tuberculosis is capable of achieving a dormant state in vitro following a gradual shift to anaerobic conditions (Lim et al., 1999 ). The presence of whm genes in M. smegmatis, a non-pathogen, suggests that the whm genes do not play a specific role in mycobacterial virulence, although indirect or contributory roles in virulence cannot be ruled out. At least one whm gene (whmDMs) is essential for colony formation by M. smegmatis (J. Gomez & W. R. Bishai, unpublished), whereas a very recent paper (Hutter & Dick, 1999
) reported that a whmB homologue of M. smegmatis did not affect growth or dormancy.
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NOTE ADDED IN PROOF |
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ACKNOWLEDGEMENTS |
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Received 21 July 1999;
revised 15 October 1999;
accepted 27 October 1999.