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Journal of Bacteriology, August 1999, p. 4617-4627, Vol. 181, No. 15
Departamento de Microbiologia, Escuela
Nacional de Ciencias Biologicas, IPN, Mexico, Distrito Federal 06400, Mexico,1 and Division of Mycobacterial
Research, National Institute for Medical Research, London NW7 1AA,
United Kingdom2
Received 29 January 1999/Accepted 21 May 1999
All mycobacteria studied to date have an rRNA operon, designated
rrnA, located downstream from a single copy of the
murA gene, which encodes an enzyme (EC 2.5.1.7) important
for peptidoglycan synthesis. The rrnA operon has a
promoter, P1(A), located within the coding region of murA,
near the 3' end. Samples of RNA were isolated from Mycobacterium
tuberculosis at different stages of the growth cycle and from
Mycobacterium smegmatis grown under different conditions.
RNase protection assays were used to investigate transcripts of both
murA and rrnA. Transcription of
murA was found to continue into the 16S rRNA gene, as if
murA and rrnA form a hybrid (protein
coding-rRNA coding) operon. During the growth of M. tuberculosis, the hybrid operon contributed approximately 2% to
total pre-rRNA. Analysis of M. smegmatis RNA revealed that the level of murA RNA depended on the growth rate and that
the patterns of expression during the growth cycle were different for
murA and rrnA. M. smegmatis has a
second rRNA operon, rrnB, located downstream from a single
copy of the tyrS gene, encoding tyrosyl-tRNA synthetase.
Transcription of tyrS was found to continue into the 16S
rRNA gene rrnB. The hybrid tyrS-rrnB operon
contributed 0.2 to 0.6% to rrnB transcripts. The pattern
of tyrS expression during the growth cycle matched the
pattern of rrnB expression, reflecting the essential role
of TyrS and rRNA in protein biosynthesis.
All the mycobacteria studied to date
have either one or two rrn operons, designated
rrnA and rrnB. The rrnA operon, which is present in all mycobacteria, is located downstream from the murA gene, which encodes the enzyme
UDP-N-acetylglucosamine 1-carboxyvinyl transferase (EC
2.5.1.7), or MurA (10). This enzyme catalyzes the transfer
of the enol ether from phosphoenolpyruvate to the 3' OH of
UDP-N-acetylglucosamine during the early stages of
peptidoglycan synthesis. Mycobacterium smegmatis, which is
representative of many fast-growing mycobacteria (10), has a
second rrn operon (rrnB), located downstream from
the tyrS gene, encoding the enzyme tyrosyl-tRNA synthetase
(EC 6.1.1.1), or TyrS (8, 15). TyrS is essential for protein
biosynthesis; its role is the attachment of L-tyrosine to
the ribose moiety of the 3'-terminal adenosine residue of tyrosyl-tRNA.
Each of the known rrnA operons has a promoter, P1(A), which
has a transcription start point (tsp) situated within the coding region
of murA or no more than 3 nucleotides downstream from the stop codon (10) (Fig. 1).
Thus, a region of the murA gene near the
3' end is transcribed both as part of the murA gene and as part of the rrnA operon, raising the possibility that
murA and rrnA are transcribed as a single unit.
Similarly, it is possible that tyrS and rrnB are
transcribed as a single unit because of the absence of a transcription
terminator in the 54-bp region downstream from the 3' end of the
tyrS coding region and the tsp for rrnB (8,
15).
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Effects of Growth Conditions on Expression of
Mycobacterial murA and tyrS Genes and
Contributions of Their Transcripts to Precursor rRNA
Synthesis
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
View larger version (17K):
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FIG. 1.
Upstream regions of mycobacterial rrn
operons and constructs used in RNase protection assays. A horizontal
arrow indicates the location of the binding site for the primer
specified; a diagonally bent arrow indicates the location of the
binding site for the primer specified (horizontal section of the arrow)
and the presence of the T7 promoter sequence (angled section); a
horizontal arrow with a circle at the end indicates the binding site
for primer JY15, used in DNA sequencing reactions. An arrow bent at a
right angle denotes the location of a tsp (vertical section) and the
direction of transcription (horizontal section). 
,
murA; 
, tyrS;
, 16S rRNA gene; , T7
promoter; tspT7 or tsp(T7), tsp for the T7 promoter. Mtu,
M. tuberculosis; Msm, M. smegmatis.
(A) rrnA operon of M. tuberculosis. (i) Upstream
region of the rrnA operon. The scheme shows the locations of
murA, tsp's (arrows bent at right angles labelled tsp1 and
tspCL1) for rrnA, binding sites for the primers used to
construct the minigenes, and the binding site for primer JY15. (ii)
Minigene Mtu 1 constructed by amplification of a section of
the upstream region with primers rp5 and rp101. (iii) Minigene
Mtu 2 constructed by amplification of a section of the
upstream region with primers rp5 and rp8. The same alignment and scale
(shown by the bar) are used in panels i to iii. (B) rrnA
operon of M. smegmatis. (i) Upstream region of
rrnA. The scheme shows the locations of murA,
tsp's (arrows labelled tsp1, tsp2, and tspCL1) for rrnB
binding sites for the primers used to construct the minigenes, and the
binding site for primer JY15. (ii) Minigene Msm A1
constructed by amplification with primers rp3 and rp101. (iii) Minigene
Msm A2 constructed by amplification with primers rp3 and
rp7. The same alignment and scale (shown by the bar) are used in panels
i to iii. (C) rrnB operon of M. smegmatis. (i)
Upstream region of rrnB. The scheme shows the locations of
tyrS, the tsp (arrow labelled tsp1) for the rrnB
operon, the binding sites for the primers used to construct the
minigenes, and the binding site for primer JY15. (ii) Minigene
Msm B1 constructed with primers rp9 and rp101. (iii)
Minigene Msm B2 constructed with primers rp9 and rp10. The
same alignment and scale (shown by the bar) are used in panels i to
iii.
In this study, the RNA fraction was isolated from Mycobacterium tuberculosis and M. smegmatis at different stages of growth and from M. smegmatis grown in two different media. RNase protection assays were used to investigate the extents of coordination of the expression of murA with rrnA and of tyrS with rrnB.
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MATERIALS AND METHODS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Materials.
A Sequenase (U.S. Biochemicals) sequencing kit
was supplied by Cambridge Biosciences. [
-35S]dATP was
obtained from Amersham. A GeneClean kit was obtained from Bio 101. Oligonucleotide primers were prepared with an automated DNA synthesizer
(model 370A; Applied Biosystems).
Bacterial strains, media, and vector. M. smegmatis NCTC 8159 (National Collection of Type Cultures) was maintained on Löwenstein-Jensen slopes and grown at 37°C with vigorous shaking in either Lemco broth (3) or Kohn-Harris glucose medium (described below) containing 0.1% Tween 80. Seed cultures of M. smegmatis for inoculation were grown in Lemco broth for 26 h. These cultures were used to inoculate medium at the rate of 20 ml of inoculum/liter. Kohn-Harris glucose medium was based (6) on the medium of Kohn and Harris (13) with glucose (5 g/liter) as the carbon source and trace elements provided at 5 ml/liter as described by Kelly and Clarke (12). M. tuberculosis H37Rv was grown in Dubos medium (7) containing 0.1% Tween 80.
Isolation of RNA.
Cells were collected, resuspended in 1 ml
of guanidinium buffer (6 M guanidinium chloride, 0.1% [vol/vol]
Tween 80, 10 mM EDTA, 1 mM 2-mercaptoethanol), and kept at 
20°C for
15 min. The suspension was added to half the volume of heat-sterilized
glass beads (0.15-mm diameter) contained in a 2-ml screw-cap
microcentrifuge tube. Mycobacteria were ruptured by three pulses of 1 min each on a Mini-BeadBeater device (Biospec Products); debris and
beads were sedimented by centrifugation (10,000 × g
for 3 min), and the cleared lysate was retained. The pellet of beads
and mycobacterial residue was briefly reextracted on the
Mini-BeadBeater (30-s pulse) with 300 µl of fresh guanidinium buffer,
and the resulting extract was pooled with the first. The lysate was
extracted three times with 2 volumes of chloroform-3-methyl-1-butanol
(24:1, vol/vol). The RNA fraction was precipitated by the addition of
ethanol and redissolved in an appropriate volume of
morpholinepropanesulfonic acid (MOPS) buffer, and residual DNA was
removed by digestion with RNase-free DNase. The integrity of the RNA
was checked by electrophoresis through formaldehyde gels.
Construction of minigenes.
Regions upstream from the 5' end
of the 16S rRNA coding region of rrnA of M. tuberculosis and rrnB of M. smegmatis were
amplified by PCR with the primers shown in Table
1 used to amplify the sequences
illustrated in Fig. 1. Appropriate plasmids for PCR amplification for
M. tuberculosis and M. smegmatis were described previously (8). The procedures used resulted in a T7
promoter at the 3' end of the minigene to provide a probe for RNase
protection assays.
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Transcription of minigenes.
Radiolabelled transcripts were
obtained by use of a Riboprobe kit (Promega, Madison, Wis.) according
to the instructions provided. Each reaction mixture contained 50 µCi
of [-32P]CTP; transcription was carried out at 37°C
for 1 h, and the reaction was terminated by heating the sample at
approximately 90°C for 3 min. DNase was added to the cooled reaction
mixture, which was then kept at 37°C for 15 min. The products were
purified by electrophoresis through a 6% (wt/vol) polyacrylamide-8 M
urea sequencing gel. An appropriate band was excised from the gel, and
radiolabelled RNA was recovered after the gel fragment was soaked
overnight in elution buffer (0.5 M ammonium acetate, 1 mM EDTA, 0.2%
[wt/vol] sodium dodecyl sulfate). The eluate was desalted and then
made 0.3 M in sodium acetate, and RNA was precipitated with ethanol.
RNase protection assays. Hybridizations were carried out with either formamide buffer [80% (vol/vol) formamide, 0.2 M sodium acetate, 40 mM piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES) (pH 6.4)] at 50°C for 16 h as described by Sambrook et al. (16) or PES buffer (1 M NaCl, 1 mM EDTA, 25 mM PIPES [pH 6.8]) at 90°C for 70 min (14). Each sample contained radiolabelled probe (1 × 105 to 4 × 105 cpm) and 10 µg of M. tuberculosis RNA or 30 µg of M. smegmatis RNA.
After the hybridization step, samples were treated with RNase A and RNase T1 and then protein was removed by treatment with phenol-chloroform. RNA was precipitated, redissolved, and analyzed by electrophoresis through 6% polyacrylamide-8 M urea gels. The gels were calibrated by use of either HaeIII-digested
X174 DNA molecular size markers (Promega, Madison, Wis.) or the products of
sequencing reactions generated by the methods described below. After
separation by electrophoresis, radioactive products were located by
autoradiography at either approximately 20°C or 70°C with an
intensifying screen.
Sequencing of double-stranded DNA.
DNA sequences were
determined by the dideoxy chain termination procedure with
[-35S]dATP as described by Ji et al. (11)
and with primer JY15.
Measurements of radioactivity. Quantitative measurements of radioactivity were obtained with a PhosphorImager (model 4005; Molecular Dynamics, Chesham, Buckinghamshire, United Kingdom) and the software supplied with the instrument.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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In order to investigate the coordinated expression of murA and rrnA of both M. tuberculosis and M. smegmatis and tyrS and rrnB of M. smegmatis, RNase protection assays were carried out. The constructs used to generate two sets of appropriate radioactive probes are illustrated in Fig. 1. One set was designed to detect the 3' end of M. tuberculosis murA RNA (Fig. 2A) and the 3' ends of murA RNA and tyrS RNA of M. smegmatis (Fig. 3A).
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The data shown in Fig. 2B (for M. tuberculosis murA) and 3C (for M. smegmatis murA and tyrS) provide evidence for large transcriptional units which extend through the protein coding region and into the 16S rRNA coding region. These transcripts comprise two regions, one coding for protein (MurA or TyrS) and the other coding for rRNA. For this reason, these RNA species are considered to be hybrid transcripts derived from hybrid operons. The hybrid operon identified in M. tuberculosis (Fig. 2) comprises murA at the 5' end with the rrnA operon located downstream. The hybrid operons identified in M. smegmatis (Fig. 3) comprise murA at the 5' end with the rrnA operon located downstream and tyrS at the 5' end with the rrnB operon located downstream.
Previously (10), we described two categories of promoters that are present in M. tuberculosis in particular and in mycobacterial rrn operons in general (Fig. 1) and that are dedicated to pre-rRNA synthesis. One category (P1) is located within the coding region of murA, near the 3' end; the second category (PCL1) is located within the hypervariable multiple-promoter region which extends from the 3' end of murA to a conserved sequence motif, CL2, which lies upstream from the 16S rRNA gene and within the leader region of pre-rRNA transcripts (10). PCL1 promoters are associated with another conserved sequence motif, (CL1) (10, 11). A third category of promoter (PmurA), which lies upstream from murA, has now been identified. Previously, we identified a single (P1) promoter of the rrnB operon of M. smegmatis, located between tyrS and the 16S rRNA gene (8, 9). We have now identified a second promoter upstream from tyrS; this promoter is responsible for transcription of the hybrid operon.
M. tuberculosis has a single copy of murA per genome (4). Restriction enzyme digests of M. smegmatis genomic DNA revealed murA and tyrS to be present in single copies per genome (results not shown). Thus, murA, which forms part of a hybrid operon with rrnA, is the sole source of MurA in both M. tuberculosis and M. smegmatis. Similarly, tyrS, which forms part of a hybrid operon with rrnB in M. smegmatis, is the sole source of TyrS in M. smegmatis. We therefore carried out RNase protection studies using the probes described in Fig. 4A, 5A, and 5B in order to study murA RNA and tyrS RNA synthesis under different conditions of mycobacterial growth and to relate their synthesis to pre-rRNA synthesis.
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The RNA fraction comprises mainly (approximately 83%) rRNA
(2) and much lower levels of mRNA. The abundance of a
particular mRNA within the RNA fraction represents a steady-state value
which reflects both the rate of synthesis and the rate of degradation or processing. Comparative values of radioactivity per assay provide a
measure of the relative number of transcripts at the steady state. The
abundance of murA RNA within the RNA fraction was related to
the abundance of transcripts of rrnA originating from tsp
one (tsp1) and designated pre-rRNAA(P1) as shown in Fig. 4
and Table 2 for M. tuberculosis and in Fig. 5A, 5B, and
6A for M. smegmatis.
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The results for M. tuberculosis (Fig. 4B and Table 2) show that murA was expressed at all stages of growth, that is, not only during balanced growth but also during the stationary phase. For each assay, the number of murA transcripts was related first to the number of pre-rRNAA(P1) transcripts. The results obtained for samples r, s, t, and u (Table 2) show that there were 50 to 360 copies of murA RNA per 1,000 copies of pre-rRNAA(P1). Previously, we showed (9) that pre-rRNAA(P1) comprises approximately 20% of pre-rRNA. These estimates suggest that, on average, there were 10 to 70 copies of murA RNA for every 1,000 copies of pre-rRNA. The low level of pre-rRNA in the late log phase (sample v), originating from the P1 and PCL1 promoters and reported earlier (9), accounts for the observed high ratio of murA RNA to pre-rRNAA(P1) shown in Table 2.
The data obtained for M. smegmatis (Fig. 5C) show that murA was expressed during the stationary phase as well as during balanced growth. A comparison of the radioactivities of murA RNA- and pre-rRNAA(P1)-protected fragments revealed that more copies of murA RNA per copy of pre-rRNAA(P1) were found during exponential growth in Lemco broth than during the stationary phase (Fig. 6A, panel i) or when growth conditions were less favorable, as in Kohn-Harris glucose medium (Fig. 6A, panel ii).
Previously, it was shown that the contribution of
pre-rRNAA(P1) to all transcripts
(pre-rRNAA) of rrnA depended on the growth rate; pre-rRNAA(P1) accounted for approximately 1% of
pre-rRNAA during early growth (e.g., Table 3, samples a and
b) in Lemco broth; this value rose to approximately 15% during the
stationary phase (e.g., Table 3, samples
e, f, and g) or in Kohn-Harris glucose medium (9). During
early growth in Lemco broth, the numbers of murA transcripts
were found to exceed the numbers of transcripts of
pre-rRNAA(P1) (Fig. 6A, panel i). On the basis of the
above-mentioned data, we estimate that, during culturing in Lemco
broth, for every 1,000 copies of pre-rRNAA, we detected approximately 15 copies of murA RNA during the early stages
of growth and 60 to 100 copies during the stationary phase. However, the overall pattern of murA RNA synthesis does not reflect
the pattern of pre-rRNAA synthesis. The burst of
pre-rRNAA synthesis [approximately 100-fold that of
pre-rRNAA(P1)] that is known to take place during early
growth in Lemco broth (9) has no counterpart in
murA RNA synthesis. We infer that although the stimuli for murA and rrnA transcription may have features in
common, they are not identical.
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The results show that tyrS RNA was detected at all stages of growth of M. smegmatis (Fig. 5D). When the mycobacterium was grown in Lemco broth, tyrS RNA was found to be most abundant during the early stages of growth and least abundant during the early and late stationary phases. When growth took place in Kohn-Harris glucose medium, the abundance of tyrS RNA was scarcely higher than the value found during the stationary phase in Lemco broth. In general, the profiles of tyrS RNA synthesis were found to correlate with the profiles of transcripts (pre-rRNAB) of rrnB (Fig. 5D); for example, the burst in the synthesis of tyrS RNA noted during early growth in Lemco broth was also seen for the synthesis of pre-rRNAB. The abundance of tyrS RNA transcripts was related to the abundance of transcripts of pre-rRNAB (Fig. 6B); ratios within the range of 1 to 6 copies of tyrS RNA for every 1,000 copies of pre-rRNAB were obtained. The highest ratio of tyrS RNA to pre-rRNAB, which was found during early growth in Lemco broth, is thought to reflect the need of the cells for a higher ratio of TyrS per ribosome during a period of rapid protein biosynthesis.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Here we have shown that the rrn operons of mycobacteria may form parts of larger hybrid operons in which both protein encoding and rRNA encoding DNAs are transcribed as single units. The starting point for the transcription of the hybrid operon of M. tuberculosis is likely to be between the insertion sequences IS1557, which lies immediately upstream of murA (4), and the 5' end of the coding region of murA. No similar map is available for M. smegmatis; therefore it is not certain that murA is the first gene of the putative hybrid operon, and it is not known whether it is preceded by other genes. The RNase protection experiments show that transcription in both species continues beyond the 5' end of the 16S rRNA coding region, suggesting that transcription will continue until an intrinsic terminator downstream from the 5S rRNA gene is reached because of the operation of particular (antitermination) mechanisms that ensure complete transcription of the rrnA operon (5). Initiation of murA transcription from the PmurA promoter ensures that there is one copy of pre-rRNA for each copy of murA RNA derived from that promoter. However, the contribution of the hybrid operon to pre-rRNA synthesis is small (approximately 1 to 4% [Table 2]) because of the presence of additional promoters dedicated to pre-rRNA synthesis and located between the end of the murA gene and the beginning of the 16S rRNA gene (8).
The second rRNA operon of M. smegmatis, rrnB, also may form part of a hybrid operon, in this case including the tyrS gene. In the genome of M. tuberculosis, tyrS is preceded by the Rv1688 gene, which probably codes for 3-methylpurine DNA glycosylase; the two genes appear to be transcribed as a single unit (4), as judged by the size and composition of the sequence separating them. Should a similar arrangement occur in the M. smegmatis genome, then the homologue of the Rv1688 gene would be part of the hybrid operon.
Within the bacterial cell, transcription and translation are coupled so that mRNA is translated as it is being synthesized. If, like mRNA translation, both ribosome assembly and rRNA processing take place as transcription proceeds, then it is likely that only a small proportion of hybrid transcripts will remain intact after transcription is complete. Early pre-rRNA processing events would have the effect of releasing mRNA into the cytosol; a transcriptional terminator also has this effect. Thus, the hybrid operon appears to make economical use of cell resources by using a minimum number of initiation and termination steps.
Our analysis of the pre-rRNA fractions of both M. tuberculosis and M. smegmatis has shown that in normal growth, transcripts originating from promoters dedicated to the expression of rRNA gene sequences represent the majority. The str operon of Escherichia coli provides another example of promoters located within an operon but dedicated to the expression of a particular gene within the operon. This operon (19, 20) comprises four genes in the order rpsL, rpsG, fus, and tufA. In addition to Pstr, the principal promoter for the operon, promoters dedicated to the expression of tufA (encoding elongation factor Tu) are thought to lie within fus (encoding elongation factor G).
Results obtained for E. coli rrn operons help to place our
findings in a wider context. The rrnG operon, which does not
appear to form part of a hybrid operon, is located downstream from
clpB, a gene coding for E. coli heat shock
protein F84.1 (18). An intrinsic terminator for
clpB is thought to be located 31 bp downstream from the 3'
end of the coding region and 83 bp upstream from the 35 box of
promoter P1 of rrnG (17), suggesting that
clpB and rrnG are transcribed independently. In
contrast, transcripts of the open reading frame upstream from the
rrnB operon of E. coli were shown to continue
into the 16S rRNA coding region; this finding was the first evidence
for a hybrid operon (1). The open reading frame, which is
regulated by two promoters (1), encodes glutamate racemase,
an enzyme that catalyzes the synthesis of D-glutamate, an
essential component of bacterial peptidoglycan.
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ACKNOWLEDGMENTS |
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We thank our colleague Andrew Lane for helpful discussions. We thank Simon A. Cox for help in the preparation of the manuscript.
J. A. G.-y-M. received financial support from COFAA and EDD, IPN, Mexico. This work was supported as part of the European Commission Science Research and Development Programme (contract ERBIC 18CT 9720253).
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FOOTNOTES |
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* Corresponding author. Mailing address: Division of Mycobacterial Research, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, United Kingdom. Phone: 44 181 959 3666. Fax: 44 181 906 4477. E-mail: rcox{at}nimr.mrc.ac.uk.
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Journal of Bacteriology, August 1999, p. 4617-4627, Vol. 181, No. 15
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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[Abstract] [Full Text] -
Kumar, A., Bose, M., Brahmachari, V.
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