School of Biological Sciences, University Park, Nottingham University, Nottingham NG7 2RD, UK1
Author for correspondence: C. J. Ingham. Tel: +44 20 7589 5111. Fax: +44 20 7584 2056. e-mail: c.ingham{at}ic.ac.uk
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ABSTRACT |
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Keywords: rifampicin resistance, RNA polymerase, NusG, Bacillus subtilis
a Present address: Department of Biology, Imperial College of Science and Technology, Sir Alexander Fleming Building, Imperial College Road, London SW7 2AZ, UK.
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INTRODUCTION |
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Bacterial transcription can be inhibited by a small number of chemotherapeutic agents, most notably the semi-synthetic antibiotic rifampicin (Wehrli et al., 1968 ). Rifampicin binds to the RNA polymerase and prevents productive initiation of transcription, but does not inhibit transcription after promoter clearance. The effectiveness of rifampicin varies depending on the sigma factor that is directing the initiation of transcription (Wegrzyn et al., 1998
). Rifampicin derivatives are highly effective clinically. However, resistant bacteria arise during treatment due to mutations within the rpoB gene encoding the ß subunit of the RNA polymerase (Jin & Gross, 1988
). Rifr RNA polymerases have been extensively characterized in E. coli; these have altered properties in transcription elongation and/or termination (Jin et al., 1988a
, b
; Jin & Gross, 1991
). In E. coli, Rifr mutations are usually located in the central region of the ß subunit polypeptide within clusters I, II and III (Jin & Gross, 1988
; Severinov et al., 1993
). Rifr mutations can also occur near the N terminus of the ß subunit (Severinov et al., 1994
). In Bacillus subtilis it is known that Rifr mutations map to rpoB (Boor et al., 1995
) but the effect of these mutations on transcription elongation and termination is unknown. Indeed, little is known of the effect of Rifr mutations on transcription elongation and termination in non-enteric bacteria.
B. subtilis and closely related Gram-positive bacteria differ in several aspects of transcription elongation and termination compared to E. coli (Henkin, 2000 ). B. subtilis has a number of distinctive means of regulating Rho-independent termination that are lacking in Gram-negative bacteria, including tryptophan RNA-binding attenuator proteins (TRAP) (Babitzke, 1997
), as well as the T-box antitermination system and the S-box regulon (Grundy & Henkin, 1998
). In addition, both transcription termination factor Rho and transcription elongation factor NusG are essential in E. coli but neither are required for growth by B. subtilis (Quirk et al., 1993
; Ingham et al., 1999
). NusG has also been shown to be inessential in Staphylococcus aureus (Xia et al., 1999
). NusG appears to be a universal transcription factor. A nusG homologue has been found in all eubacteria for which the genome has been sequenced and has been found in the archaea and eukaryotes (Hartzog et al., 1998
). In E. coli, NusG regulates Rho-dependent termination (Sullivan & Gottesman, 1992
) and increases the rate of transcription elongation by decreasing pausing by the RNA polymerase (Burova et al., 1995
). The function of NusG in other bacteria is largely unknown and even in E. coli it is not clear in detail how NusG regulates the RNA polymerase.
In this work, Rifr mutants of B. subtilis were isolated and characterized with respect to the autogenous regulation of the rho and nusG genes. Two Rifr mutants with a highly conserved glutamine residue mutated to a basic residue (Q469K and Q469R) enhanced autogenous regulation of nusG. Q469K and Q469R are the first RNA polymerase mutants identified which show increased sensitivity to regulation by NusG. Autoregulation of nusG was used to isolate a strain expressing a truncated NusG which had lost this regulation. This confirmed that we had identified a genetic system for examining fundamental aspects of transcription elongation/termination in a non-enteric bacterium.
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METHODS |
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Isolation of mutants of B. subtilis in which nusG is not autoregulated.
Strain BSNusG3 was plated on L agar containing 200 µg X-Gal ml-1 with IPTG, erythromycin, lincomycin and rifampicin at the concentrations described previously. Under these growth conditions, induction of NusG with IPTG decreased expression of (nusGlacZ) so that colonies of BSNusG3 grown overnight appeared nearly white (allowing for a degree of orange-brown pigmentation due to growth on rifampicin). Over 2000 unmutagenized colonies were visually screened for derepression of
(nusGlacZ) as indicated by blue colonies despite the presence of IPTG. These mutants were subjected to further analysis by Western blotting for NusG expression and sequencing of the nusG gene from PCR products.
Quantification of transcripts by RNase protection assay and Northern analysis.
RNA was purified from B. subtilis cultures by rapid protoplast formation followed by repeated acid phenol extractions and 2-propanol precipitation (Volker et al., 1994 ). Radiolabelled antisense transcripts for use in RNase protection experiments were generated by in vitro transcription using T7 RNA polymerase from a Maxiscript kit as instructed by the manufacturer (Ambion). NTP concentrations used in transcription reactions were 500 µM unlabelled ATP, CTP and GTP, and 5 µM [
-32P]UTP [400 Ci mmol-1 (14·8 TBq mmol-1)]. The antisense transcripts used were complementary to RNA transcribed within the secEnusG region (Fig. 1a
). The DNA template used for in vitro transcription was degraded using RNase-free DNase I as instructed by the supplier (Ambion) and the radiolabelled RNA (>90% full length) was purified using a spin column-100 (Sigma) before hybridization to B. subtilis RNA. RNase protection assays were performed using an HybSpeed RPA kit (Ambion) using 3 µg B. subtilis RNA or 3 µg of yeast tRNA as a control. Protected RNAs were resolved on 5% acrylamide, 1xTBE, 8 M urea denaturing gels, sized against Century RNA markers (Ambion). Quantification of protected transcripts was by phospor-imaging (Bio-Rad).
Northern analysis of transcripts was by the method of Igo & Losick (1986) using RNA prepared as described above. Probes for hybridization to Northern blots were prepared by random-priming (High Prime; Boehringer).
Western immunoblotting.
Cells were broken by sonication in lysis buffer containing 20 mM Tris (pH 8·0), 2 mM EDTA and complete protease inhibitor cocktail (Boehringer). Soluble protein was quantified using Bradford reagent (Bio-Rad). Protein samples were resolved by SDS-PAGE (8% acrylamide gels) and blotted to PVDF membrane (ICN). Detection of cross-reacting proteins was by Western blotting using a BM chemiluminescent kit (Boehringer) and a polyclonal rabbit antibody raised against purified B. subtilis NusG protein (Ingham et al., 1999 ).
ß-Galactosidase assays.
The ß-galactosidase activity of B. subtilis strains containing lacZ fusions was assessed by the hydrolysis of ONPG using standard methods and calculations (Harwood & Cutting, 1990 ). Strains were grown to an optical density at 600 nm of 0·40·6 in 2TY medium. Where a qualitative assessment of lacZ expression was required, strains were patched on L agar microtitre plates containing 200 µg X-Gal ml-1 with antibiotics and IPTG as appropriate.
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RESULTS |
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NusG inhibits transcription within the secEnusG region
RNase protection assays were used to quantify transcription within the secEnusG intergenic region (Fig. 1). In strain BSNusG1 grown without NusG, three protected RNAs were detected, including a 230 nt RNA full-length RNA, indicating transcription from secE into nusG (Fig. 4
). Two smaller RNAs (120 and 160 nt) were also detected. It is not clear if these truncated transcripts were generated by different initiation, termination or processing of the 230 nt RNA. When NusG was induced in BSNusG1, the 230 nt RNA decreased in abundance more than tenfold (Fig. 4a
, b
). The truncated transcripts were reduced around threefold by NusG expression. In the absence of NusG, the pattern of transcription within this region was similar when comparing strains BSNusG1 (Rifs) and BSNusG3 (Q469K). In strain BSNusG3, induction of NusG to a similar level to BSNusG1 (Fig. 4b
) almost completely eliminated all transcripts detected in the secEnusG region, except for a minor amount of the 120 nt RNA (Fig. 4a
). RNase protection supported the results obtained from the analysis of
(nusGlacZ) expression (Fig. 3a
). The Q469K mutation in the RNA polymerase confers hypersensitivity to NusG in that NusG becomes a more potent inhibitor of nusG transcription in this genetic background.
To understand the transcription regulation in more detail, an antisense RNA corresponding to the first 81 nt of the secE ORF (probe N2; Fig. 1a) was used to protect RNA purified from strain BSNusG3. Transcription of the 5' region of the secE gene was the same in the presence or absence of NusG (Fig. 5a
, b
). Autoregulation of nusG was therefore not due to regulation of any promoter(s) co-transcribing secE and nusG. This suggests that the site(s) of NusG regulation were within either the secE gene, the secEnusG intergenic region or the 5' region of the nusG gene.
Northern analysis of transcription was used to confirm autoregulation and clarify the pattern of transcription within the secEnusG region (Fig. 6). Probing for mRNAs transcribed from the secEnusG into the lacZ reporter gene in strain BSNusG3 detected two transcripts of 3400 and 4000 nt which were no longer detected when NusG was induced with IPTG. In contrast, rho transcription was not significantly regulated by NusG (Fig. 7
). The size of these mRNAs (including the lacZ fusion) suggests that nusG is transcribed from at least two promoters, one upstream of secE and one apparently within the secEnusG intergenic region (Fig. 1
). Neither transcript was detected when NusG was induced with IPTG, which is consistent with the RNase protection data (Fig. 4a
).
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Taken together with RNase protection experiments (Fig. 4a), these data suggest that autoregulation of transcription was mediated by sequences within the region -31 to +272 relative to the start codon of the nusG gene (the latter co-ordinate within the nusG gene was deduced from the 3' limit of the fusion of nusG to lacZ). In this work, we have excluded regulation of the initiation of transcription and Rho-dependent termination as possible explanations. Rho-independent termination and/or the regulation of mRNA stability are therefore the most likely candidates. A search of RNA secondary structure in this region using the GCG program mFold (University of Wisconsin) did not reveal any likely Rho-independent terminators (data not shown). Therefore, if NusG is acting as a Rho-independent termination factor, the mode of action is an unusual one. Termination of transcription within the -31 to +272 region does not explain the down-regulation of all of the transcripts (observed by RNase protection and Northern analysis) of the region -229 to +4 (Figs 4a
, 6
) unless decreased stability and/or processing of terminated transcripts is also a factor. Therefore, NusG may act (directly or indirectly) within the -31 to +272 region with a knock-on effect on the abundance of upstream transcripts within the secEnusG intergenic region, but not further upstream within the 5' sequence of the secE gene.
Effect of Rifr mutants on autoregulation of the rho gene
Despite being inessential (Quirk et al., 1993 ), the B. subtilis rho gene is known to be autoregulated by a similar attenuation mechanism to rho in E. coli. Autoregulation of rho in B. subtilis occurs at the level of transcription, requires sequences downstream of the rho promoter and is mediated by Rho-dependent termination within the mRNA leader and coding region of the rho gene (Ingham et al., 1999
). Given that Rifr mutants in E. coli usually antiterminate or facilitate Rho-dependent termination (Jin et al., 1988b
), it was logical to test the effects of B. subtilis Rifr mutations on the expression of a lacZ transcription fusion to rho in B. subtilis. The Rifr mutants Q469K, Q469R and H482Y were transformed into strain BSRho2 (Fig. 1b
) to create strains BSRho9, BSRho10 and BSRho11, respectively. In each strain Rho was induced with IPTG and Rho-dependent termination was assessed by a lacZ fusion to the rho gene. In all cases, induction of Rho from pSPAC (to a similar level in all strains as judged by Western blotting; data not shown) decreased expression of
(rholacZ). However, the effect of Rho was twofold greater in BSRho9 and BSRho10 and was twofold less in BSRho11 when compared to the Rifs strain with the same lacZ fusion (Fig. 8
). Therefore, the Q469K and Q469R mutations enhanced autogenous regulation of rho and the H482Y was inhibitory. The observed changes in lacZ expression due to Rho induction were alleviated by bicyclomycin (Fig. 8
), confirming that these were dependent on Rho. As autoregulation of Rho is independent of NusG (Ingham et al., 1999
), this phenotype appears to be distinct from nusG hypersensitivity.
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DISCUSSION |
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Unusual features of NusG function in B. subtilis allowed us to screen for Rifr RNA polymerase mutants that were also altered with respect to regulation by NusG; the nusG gene is both inessential for viability and appears autoregulated. Transcription of nusG was dependent on NusG (Figs 4a, 6
) but transcription of secE was not (Fig. 5a
), although the mRNA co-transcribing secE and nusG is regulated in some fashion, possibly by termination immediately downstream of secE. Transcription regulation within the secEnusG region in B. subtilis appears more complex than reported in other bacteria, despite the arrangement of genes around nusG being highly conserved within the eubacteria (Downing et al., 1990
; Joeng et al., 1993
; Puttikhunt et al., 1995
). The upstream secE gene encodes a secretory protein that is essential in both E. coli and B. subtilis (Joeng et al., 1993
). In E. coli, secE and nusG are invariably co-transcribed (Downing et al., 1990
). In Streptomyces coelicolor both genes appear essential but have their own promoters (Puttikhunt et al., 1995
). In E. coli, the intergenic region between secE and nusG is only 1 bp (Downing et al., 1990
); in B. subtilis this region is 180 bp and may contain additional regulatory elements. Sequences sufficient to mediate autoregulation were located within the 5' region of nusG (nt -31 to +272). NusG appears to act in this system after the initiation of transcription, which is consistent with its known properties as a transcription elongation and antitermination factor (Sullivan & Gottesman, 1992
; Li et al., 1993
). The most likely mechanism of action is that NusG enhances Rho-independent termination within the nusG gene but that these terminated mRNAs are unstable or processed, explaining the apparent regulation of transcription within the upstream secEnusG intergenic region. We have, however, been unable to locate a classical Rho-independent terminator within nusG or the secEnusG region. If regulation is due to termination, it is at an unusual site, not an RNA stemloop with poly(U) tail. Alternatively, we cannot exclude the possibility that NusG may be indirectly autoregulated via another factor. However, Rho was not such an intermediary despite targeting Rho-dependent termination being a major function of the E. coli NusG (Sullivan & Gottesman, 1992
; Li et al., 1993
; Burns & Richardson, 1995
).
With the exception of the enteric bacteria, no good genetic systems exist for identifying and characterizing general transcription elongation factors. In part this is due to the lack of analogues of coliphage termination and antitermination systems. N-mediated antitermination has proved valuable in identifying enteric Nus factors (Sullivan et al., 1992
) and HK022 Nun termination has been used to isolate nusG mutations (Burova et al., 1999
). Our attempts to isolate a B. subtilis phage which is sensitive in its lytic cycle to NusG have failed. However, we have shown in this work that a mutation in nusG can be obtained on the basis of the loss of autoregulation. We have therefore identified an approach that may lead to the discovery of novel components of the Gram-positive transcription elongation and termination complex.
In strains of B. subtilis with the Rifr mutations Q469K or Q469R, autoregulation of nusG was enhanced and transcription of this gene became more sensitive to NusG. In E. coli, four Rifr mutations (Severinov et al., 1993 ) have been found at the equivalent glutamine residue (Q513) in the ß subunit of the RNA polymerase. The best characterized of these is rpoB8 (Q513P). The RNA polymerase with the allele rpoB8 exhibits increased Rho-dependent and Rho-independent termination, altered transcription elongation and an increased Km for purine nucleotides (Jin & Gross, 1991
). In addition, both rpoB8 and rpoB101 (Q513L) have altered properties with respect to interaction with the nusA1 allele (Jin et al., 1988a
). Interestingly, the nusG4 mutation in E. coli also suppresses the nusA1 allele to restore
N-mediated antitermination (Sullivan et al., 1992
). Taken together with our data, this suggests that the conserved glutamine Q513/Q469 is a critical residue in the regulation of the RNA polymerase by both NusA and NusG. This is despite these factors having generally opposite but non-competitive effects on the RNA polymerase in elongation and Rho-dependent termination (Burns et al., 1998
). Basic mutations at this residue may cause the RNA polymerase to adopt a conformation that is more sensitive to NusG, possibly mediated via altered contacts with DNA or RNA. Current structural models suggest that direct contacts between NusG and the RNA polymerase at this residue are a less likely explanation (Korzheva et al., 2000
). Targeted mutagenesis of Rifr cluster I in E. coli has yielded the rpoB mutation Q513R (Severinov et al., 1993
). It is not known how Q513R affects transcription elongation and termination, but this work suggests that the mutants containing Q513R and Q513K in E. coli are well worth studying with respect to regulation by NusG. Q513K has been found to affect the copy number of ColE1 plasmids in E. coli but it is not known if NusG is involved in this phenotype (Yang & Polisky, 1999
). Despite the genetics of Nus factors and Rifr RNA polymerases being well developed in E. coli, there have been no reports of RNA polymerases unusually sensitive to NusG. Knowledge of the genetics of NusGRNA polymerase interactions is limited. It is possible that NusG hypersensitive mutants have not been isolated in E. coli because they are lethal, given that the E. coli NusG is essential for growth. An intriguing alternative is that NusG hypersensitive mutants have already been isolated but have not been recognized as such. Purified RNA polymerases from B. subtilis or E. coli with altered sensitivity to NusG should prove useful for future in vitro studies on transcription elongation.
Conclusions
The B. subtilis NusG protein regulates nusG transcription, apparently by a novel Rho- and promoter-independent mechanism. Sequences required for autoregulation are located within the 5' region of nusG or immediately upstream. Autoregulation can be used to isolate mutations within nusG, providing the first approach to studying the genetics of this universal transcription elongation factor outside the enteric bacteria.
Rifr mutations in the ß subunit of the RNA polymerse that alter Q513 to a basic residue render nusG autoregulation hypersensitive to NusG. This is the first evidence that Rifr RNA polymerases can be enhanced in their sensitivity to NusG.
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ACKNOWLEDGEMENTS |
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Received 18 May 2000;
revised 17 August 2000;
accepted 22 August 2000.