Biomedical Research, 11937 US Hwy@271, The University of Texas Health Center at Tyler, Tyler, TX-75708-3154, USA1
Department of Medical Microbiology, Barts and the London, Queen Marys School of Medicine and Dentistry, Turner St, Whitechapel, London E1 2AD, UK2
Author for correspondence: Murty V. V. S. Madiraju. Tel: +1 903 877 2877. Fax: +1 903 877 5969. e-mail: murty.madiraju{at}uthct.edu
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ABSTRACT |
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Keywords: gene replacement, regulated expression, mycobacteria, DNA synthesis, filamentation
Abbreviations: oriC; origin of replication; kan, kanamycin; SCO, single crossover; DCO, double crossover; d.t., doubling time(s)
a These authors contributed equally to this work.
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INTRODUCTION |
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The genus Mycobacterium includes fast-growing species, with doubling times (d.t.) of between 2 and 3 h (Mycobacterium smegmatis and Mycobacterium fortuitum) and 10 h (Mycobacterium aviumintracellulare complex), and slow-growing species, with d.t. of 24 h (Mycobacterium tuberculosis and Mycobacterium bovis) (Ratledge, 1976 ). Although M. smegmatis is viewed as a fast-growing member of the genus Mycobacterium, its d.t. is approximately six to seven times slower than that of E. coli. In addition to their growth characteristics, some members of the mycobacteria, e.g. M. tuberculosis, are believed to maintain two physiologically distinct growth states an active, multiplicative state and a dormant, non-replicative state (Manabe & Bishai, 2000
). In the latter state, the bacterium remains metabolically active but is in a state of non-growth for extended periods, only to revive and multiply and cause infection later. The non-replicative persistent state can be induced in vitro by growing the organisms under oxygen-depletion conditions (Dick et al., 1998
; Wayne, 1977
, 1994
). These experiments also suggested that bacteria in the non-replicative, persistent state have completed DNA synthesis but are blocked at the cell-division stage; they also showed that the bacteria exhibited synchronous replication when resuspended in fresh growth media (Wayne, 1977
; Wayne & Hayes, 1996
). The growth characteristics of M. smegmatis cultured under oxygen-depletion conditions are similar to those described for M. tuberculosis (Dick et al., 1998
). The genetic elements responsible for the differences in growth rates and the ability to shift between the two physiologically distinct growth states remain unknown.
The genetic and biochemical aspects of replication initiation in members of the mycobacteria are poorly understood. To date, only the key elements involved in the replication initiation process in members of the mycobacteria, dnaA and oriC, have been identified and characterized (Madiraju et al., 1999 ; Qin et al., 1999
; Rajagopalan et al., 1995
; Salazar et al., 1996
). Based on the results of a plasmid replication assay, we reported that the dnaAdnaN intergenic regions from M. smegmatis, M. avium and M. tuberculosis can function as oriC in each organism (Madiraju et al., 1999
; Qin et al., 1999
; Rajagopalan et al., 1995
) and that point mutations in the designated DnaA boxes of M. smegmatis oriC severely decrease oriC activity (Qin et al., 1997
). These results are different from those reported for E. coli, where single, double or triple mutation combinations in two or three DnaA boxes did not affect plasmid-based oriC replication (Holz et al., 1992
; Langer et al., 1996
). The nucleotide sequences of the oriC region in members of the mycobacteria are approximately 65% similar, and many of the designated DnaA boxes in mycobacterial oriC are positionally conserved (Madiraju et al., 1999
; Qin et al., 1999
), even though mycobacterial oriC genes are species-specific (Madiraju et al., 1999
; Qin et al., 1999
). DnaA of M. tuberculosis has been purified, and its interactions with oriC and adenine nucleotides and the effects of acidic phospholipids on these interactions have been investigated (Yamamoto et al., 2002
). At the deduced amino acid sequence level, mycobacterial DnaA proteins are approximately 82% similar, with differences noted in the amino acids located between residues 100 and 160. The conserved regions are believed to contain the lipid-,ATP- and DNA-binding domains (Messer et al., 2001
; Schaper & Messer, 1997
; Skarstad & Boye, 1994
). While these and other studies suggest a relationship between dnaA and oriC, it is unknown as to whether the dnaA gene is essential in any members of the mycobacteria and, if it is, whether it is needed for replication. Here, we show that the M. smegmatis dnaA gene is an essential replication gene and that cells containing altered levels of DnaA are filamentous and multinucleoidal. The implications of these results for understanding the function of M. smegmatis DnaA are discussed.
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METHODS |
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Construction of a dnaA antisense expression vector.
The M. smegmatis dnaA coding region was amplified by PCR using the oligonucleotide primers MVS1 (5'-GCGGATCCAATGACTGCTGACCCCGAC-3') and MVM72 (5'-CAGGATCCGGTGTGGAGAGATTC-3') and cloned in the antisense orientation into pMV261, an E. coliMycobacterium shuttle vector, under the transcriptional control of the hsp60 promoter (Stover et al., 1993 ). The BamHI restriction sequence is shown in bold. The recombinant plasmid, pMBL9, was electroporated into M. smegmatis and transformants were selected at 30 °C. Antisense expression was induced by shifting growth cultures to higher temperatures, i.e. 37 and 44 °C. One merodiploid expressing dnaA antisense RNA (also referred to as andA), BLM4, was selected and used.
Construction of dnaA expression vectors
dnaA from the native promoter.
A DNA fragment containing the putative dnaA promoter and its coding region from M. smegmatis was amplified by PCR from pMR40 (Rajagopalan et al., 1995 ) using the oligonucleotide primers MVM30 (5'-TTGTTCGGCTGGAAGGTCCGCT-3') and MVM72. The DNA fragment was then released from the TA vector by digestion with NotI and cloned into pMV306, an integration-proficient E. coliMycobacterium shuttle vector (Stover et al., 1993
), to create pMG36 (Fig. 1
). The integration-proficient vector pMV306 carries attP and int genes derived from the mycobacteriophage L5, and transformation of mycobacteria with this plasmid leads to site-specific integration into the chromosomal attB site (Stover et al., 1993
).
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Construction of non-replicating (suicide) gene replacement vectors.
pMR40, carrying the M. smegmatis dnaA region, was digested with BstBI and the large fragment was self-ligated to make a 470 bp unmarked deletion in dnaA. The 3·5 kb dnaA region was then released by digestion with BglII and PstI and cloned into the BamHI/PstI sites of the vector p2NIL (Parish & Stoker, 2000 ) to create pMG24. In the next step, a 6·1 kb PacI marker cassette from pGOAL17 (Parish & Stoker, 2000
) carrying lacZ, sacB and aph was cloned into the PacI site of pMG24 to generate pMG25. This suicide recombination delivery vector was used to disrupt dnaA.
Disruption of dnaA and construction of a conditional expression strain.
The two-step recombination protocol, followed essentially as described, was used to disrupt dnaA of M. smegmatis (Hinds et al., 1999 ; Parish & Stoker, 2000
). In the first step, pMG25 was integrated into the dnaA region by homologous recombination, generating a single crossover (SCO) strain that was blue, resistant to kanamycin (kan) and sensitive to sucrose; this strain was designated as RGM15. In the next step, homologous recombination events were selected to identify double crossovers (DCOs) that were white and sensitive to kan but resistant to sucrose. The integration-proficient pMV306-derivative expressing M. smegmatis dnaA (pMG36) from its native promoter was integrated into the attB site of RGM15 SCO to create the merodiploid strain RGM27. Selection for DCOs was then carried out in the same genetic backgrounds. In some experiments, pMG40 integrated in the wild-type background was used; this strain, which was a dnaA merodiploid expressing dnaA from the ami promoter, was designated as RGM35. Similarly, for the construction of a conditional expression strain, M. smegmatis dnaAMsmttg (pMG40) and dnaAMsmgtg (pMG44), expressed from the ami promoter, were integrated into the SCO strain RGM15 to generate the merodiploids RGM34 and RGM18, respectively. DCOs were then selected for on agar plates containing 0·2% acetamide. DCOs were verified by PCR and Southern analyses (Sambrook et al., 1989
). One strain, RGM36 (derived from RGM34, which was kan-sensitive, sucrose-resistant, white and expressed dnaAMsmttg; Table 1
), was used in all of the experiments described. This strain was referred to as the conditionally complemented dnaA mutant.
Growth and viability experiments.
Unless stated otherwise, all mycobacterial cultures were grown in Middlebrook 7H9 broth supplemented with albumin/glucose/sodium chloride. The conditionally complemented dnaA mutant strain RGM36 was grown in the presence of 0·2% acetamide, washed with acetamide-free medium twice, resuspended in the same medium and then grown for different periods of time, prior to sampling for determinations of DnaA levels by Western blotting and viability by plating, and visualization of cells and nucleoids by microscopy. In the latter case, cells were fixed in 75% ethanol prior to their visualization by either light or fluorescent microscopy (Dziadek et al., 2002a ). The conditionally complemented dnaA mutant strain grown in medium supplemented with acetamide was always used as the control. Growth was followed as the increase in the OD600 value; d.t. was defined as the time required to double the OD600 value. In some experiments, the viability of cultures grown in the absence of acetamide for 6 and 8 d.t. was also determined independently. For determinations of intracellular levels of DnaA, cultures were inoculated to a low cell density (OD600 0·05) and grown for different periods of time. Cultures harvested at an OD600 value of 0·6 after approximately 24 h growth were referred to as being in the exponential phase.
Preparation of cellular lysates and Western blotting.
Preparation of cellular lysates and Western blotting protocols were followed essentially as described (Dziadek et al., 2002a ). Recombinant M. tuberculosis DnaA was used to raise polyclonal antibodies in rabbits. The DnaA antibodies were affinity-purified following the protocols described for M. tuberculosis FtsZ antibodies (Dziadek et al., 2002a
). The extent of cross-reactivity of the M. tuberculosis DnaA antibodies with the M. smegmatis DnaA antigen is weak as compared to that of the M. tuberculosis counterpart. Nitrocellulose blots were probed with 250-fold diluted affinity-purified M. tuberculosis DnaA antibodies. Under the experimental conditions used here, the total amount of soluble protein recovered in the clear cell lysates of stationary phase cultures of M. smegmatis was less as compared to those of exponential phase cultures, consistent with our earlier results with M. smegmatis and M. tuberculosis cultures (Dziadek et al., 2002b
). One reason for the reduced recovery of soluble protein was that the bead-beating-based cell lysis protocols that we used are not optimal for stationary phase cultures, even though these cultures remain viable for the growth period followed. Consequently, DnaA levels were expressed as a percentage of the total soluble protein as determined by using the BCA protein assay kit (Pierce), as opposed to total DnaA molecules per cell. Results are presented as the mean values from three experiments.
Incorporation experiments.
DNA synthesis was followed using [5, 6-3H]uracil essentially as described by Wayne (1977 , 1994
) and Dick et al. (1998)
. Wayne (1994)
showed that only a negligible amount of thymidine is incorporated into mycobacterial DNA, whereas uracil is not only incorporated into RNA but is also methylated and incorporated into mycobacterial DNA. To permit distinction between DNA and RNA synthesis, duplicate samples of incorporation experiments were incubated for 24 h at 37 °C in 0·3 M KOH before filtering.
Microscopy.
Mycobacterial cells were resuspended in buffer A (10 mM Tris/HCl, pH 7·5, 10 mM MgCl2 and 0·02%, v/v, Tween 80), sonicated for 90 s in a Branson waterbath sonicator to break clumps and then examined either by conventional microscopy or under a Nikon TS 100 inverted microscope with a 100xNikon Plan Fluor oil immersion objective with a numerical aperture of 1·3 and working distance of 0·17. The cells were imaged using a Sensicam 12-bit monochromatic CCD camera and SlideBook 3.0 software (3I Imaging). Mycobacterial cells were fixed in 75% ethanol. Prior to staining, ethanol was removed from the cells by a brief centrifugation and the cells were then washed with buffer A. Cells were stained with a combination of ethidium bromide (40 µg ml-1) and mithramycin (180 µg ml-1), incubated on ice for 30 min and then photographed, as described previously (Dziadek et al., 2002a ). The fluorescence images were acquired using a 100 W mercury lamp and a Chroma filter set (excitation wavelength 540565 nm; emission wavelength 560623 nm). All images were optimized using Adobe Photoshop 5.0.
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RESULTS |
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Expression of dnaA antisense RNA results in inhibition of growth
If M. smegmatis dnaA is essential for cell survival, we would expect that conditions that block the production of dnaA would result in cell death. One approach to decreasing the production of a target protein is to express antisense RNA specific to the gene encoding the protein and then evaluate the consequences associated with the blockage of protein production. To test the above possibility, we constructed a strain expressing M. smegmatis dnaA antisense RNA from the heat-inducible M. bovis BCG hsp60 promoter and evaluated the consequences of dnaA antisense RNA expression on cell viability. Even though the hsp60 promoter is constitutively active at 30 °C its activity is elevated at 42 °C (Stover et al., 1991 ). Expression of antisense RNA by shifting growth temperatures, i.e. 2 h at 42 °C followed by an additional 3 h growth at 37 °C, significantly reduced the intracellular levels of DnaA (Fig. 2a
). Analysis of the bands produced after electrophoresis of the cellular extracts from M. smegmatis using Quantity One software (Bio-Rad) indicated a reduction of approximately 65% in the amount of DnaA produced under antisense expression conditions. The viability of the cultures carrying the antisense construct decreased significantly at 37 °C as compared to 30 °C (Fig. 2b
). Taken together, these results indicate that M. smegmatis dnaA is an essential gene for replication and that reductions in the intracellular levels of DnaA decrease viability.
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Conditional expression of dnaA
To gain insights into the roles of dnaA in DNA replication, we constructed a dnaA conditional expression strain by placing the coding region of dnaA under the transcriptional and translational control of the ami promoter region (Parish et al., 1997 ; Triccas et al., 1998
). The M. smegmatis ami promoter is one of the best characterized regulatable promoters in mycobacteria and its promoter activity can be induced by the addition of acetamide to the growth medium (Parish et al., 1997
; Triccas et al., 1998
). Two potential translational start sites for M. smegmatis dnaA have been proposed one starting with GTG (Rajagopalan et al., 1995
) and another starting with TTG, which is located 90 codons upstream of the predicted GTG start codon (Salazar et al., 1996
). The dnaA coding regions starting with the GTG (dnaAMsmgtg) and TTG (dnaAMsmttg) initiation codons were cloned individually into pMV306 (see Methods) to create a translational fusion with the first six amino acids of the product of amiE (Triccas et al., 1998
). The resulting recombinant plasmids were integrated into the RGM15 SCO strain and their sites of integration were verified by PCR; DCOs were selected as described earlier. To enable a continuous supply of DnaA, DCOs were selected on agar plates containing acetamide. The concentration of acetamide used elevated the intracellular levels of DnaA by approximately sixfold (see below), but it was not toxic to the cells as it did not interfere with their growth or viability (see below). Analyses of 10 DCOs obtained with the dnaAMsmttg construct, by PCR (Fig. 4a
) and Southern blotting (Fig. 4b
), confirmed the presence of mutant and wild-type dnaA patterns in a ratio of 4:6. One mutant DCO, designated RGM36, was characterized further. In contrast to the DCOs obtained with the dnaAMsMttg construct, all 80 DCOs obtained with the dnaAMsmgtg construct revealed only the wild-type pattern, indicating that the truncated DnaA protein produced from the dnaAMsmgtg construct was non-functional.
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We also noted that the appearance of colonies of RGM36 and of the dnaA merodiploid (RGM35) on acetamide plates was delayed, when compared to the wild-type control (M. smegmatis). Presumably, this delay was due to cell lengthening and the loss of synchrony between DNA replication and cell-division events (see below). Western analyses confirmed a gradual depletion in the DnaA levels in RGM36 cells that had been grown in the absence of the inducer, acetamide (Fig. 5b). Analyses of the bands produced by Western blotting, using the QUANTITY ONE software (Bio-Rad), indicated that parent RGM36 cells grown in the presence of acetamide had approximately sixfold higher levels of DnaA compared to the wild-type cells, whereas RGM36 cells starved of acetamide had significantly depleted levels of DnaA, i.e. they were reduced by half at 6 d.t. and nearly undetectable at 7 and 8 d.t. (data not shown).
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DnaA-depleted cells recover and multiply following the addition of acetamide
To determine whether cells depleted in DnaA would recover and divide, acetamide was added to RGM36 cells that had been starved of acetamide for 36 h of growth. The viability and cell morphology of these cultures were then examined. The viability of the acetamide-replenished cultures was restored to levels comparable to those of the RGM36 cells grown in the presence of acetamide (data not shown). Importantly, the lengths of the filament cells decreased, and were comparable to those of the RGM36 cells grown in the presence of acetamide (Fig. 8b, d
). Furthermore, the short filaments also contained distinct, well-separated nucleoids (Fig. 8a
, c
). No visible DNA-less cells were detected under these conditions. The appearance of colonies on agar plates was delayed by approximately 2436 h, and the colonies were heterogeneous upon their appearance (data not shown). Presumably, the delayed appearance of the colonies was due to the slow accumulation of DnaA to sufficient levels for subsequent resumption of cell division, prior to the initiation of new rounds of cell-cycle events.
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DISCUSSION |
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The intracellular levels of M. smegmatis DnaA were found to be constant during the exponential and stationary phases of growth (data not shown). In contrast, we found that the intracellular levels of FtsZ (a putative initiator protein of the cell-division process in mycobacteria) were growth-phase dependent, suggesting that FtsZ is required in actively dividing cells. The relatively high and stable intracellular levels of DnaA present in M. smegmatis suggest a continued requirement for this protein in cell metabolism. DnaA is believed to function as a transcriptional regulator of many genes, including itself in E. coli (Messer et al., 1988 ; Messer & Weigel, 1997
). The binding of E. coli DnaA to DnaA boxes located in the promoter regions of dnaA and polA has been shown to result in transcriptional repression (Atlung et al., 1984
, 1985
; Braun et al., 1985
; Braun & Wright, 1986
; Polaczek & Wright, 1990
; Skovgaard et al., 1998
; Smith et al., 1997
). Scanning the putative promoter regions of several genes, including, for example, dnaA, ftsY and ftsQ of M. smegmatis (Institute of Genome Research; http://www.tigr.org/) and M. tuberculosis (Cole et al., 1998
), revealed the presence of several nine nucleotide long sequences with one to two mismatches to the conserved DnaA box sequence TTCA/GGCACA (Madiraju et al., 1999
; Qin et al., 1999
; Rajagopalan et al., 1995
). Thus, the possibility that the fairly constant levels of DnaA present in vivo are needed to bind to the putative DnaA boxes located in the promoter region, for subsequent transcriptional repression of some unknown target genes, cannot be ruled out.
DNA synthesis experiments clearly indicated that depletion of DnaA leads to the inhibition of DNA synthesis (Fig. 6). It is pertinent to note that nearly complete inhibition of DNA synthesis occurred only after acetamide starvation for periods corresponding to 78 d.t. This result is different from those observed with other bacterial dnaA conditional mutants, where blockage of DNA synthesis has been shown to occur after approximately 1 d.t. (Gorbatyuk & Marczynski, 2001
). In the present study, conditional dnaA expression was attained from the acetamide-regulatable ami promoter. Thus, one explanation for our observations is that the ami promoter could be leaky, thereby enabling a slow but continuous accumulation of DnaA. In support of this assumption, we have recently found that the expression of ftsZgfp (the gene for a green fluorescent fusion protein of FtsZ) from the ami promoter in M. smegmatis hosts is leaky under uninduced conditions (J. Dziadek, M.V.V.S. Madiraju & M. Rajagopalan, unpublished data). Thus, elevated levels of DnaA in RGM36 cells grown in the presence of acetamide (Fig. 4b
) combined with a leaky expression of dnaA, if any, could lead to a gradual (but not abrupt) decrease in the intracellular pools of DnaA, leading to a slow and continued synthesis of DNA under acetamide-starved conditions. Alternatively, slow but continuous DNA synthesis under acetamide-starved conditions (Fig. 6
; near normal levels of synthesis up to 4 d.t. and slow but continuous synthesis up to 6 d.t.) could be due to a prolonged replication period. The elevated levels of DnaA in RGM36 parent cells could promote overinitiation at oriC, resulting in multiple replication forks that proceed slowly to bidirectional replication, thereby resulting in an overall increase in DNA content. Thus, a prolonged replication period during overproduction of DnaA could have led to the multinucleoidal state. Detailed determinations of the DNA contents of cells and the origin-to-terminus ratios produced under altered DnaA levels could provide clues as to the changes in the replication period, if any. Another possibility, which is speculative at the moment, is that M. smegmatis contains a hitherto unidentified gene that is paralogous to dnaA and has some residual dnaA activity. These possibilities are not mutually exclusive and further studies are required to address these issues.
The bacterial cell cycle is divided into two parts: a replication cycle, which includes both chromosomal replication and partitioning of DNA, and a division cycle. Both processes are believed to be linked, and defects in either of these processes lead to filamentation of the cell (Cook & Rothfield, 1999 ; Sun & Margolin, 2001
). Consistent with this assumption are reports showing that either overproduction of FtsZ, a putative initiator of cell division (Dziadek et al., 2002a
), inactivation of WhmD, a putative cell-division protein (Gomez & Bishai, 2000
), or thermal inactivation of DnaG primase, required for DNA-chain elongation (Klann et al., 1998
), lead to filamentation in M. smegmatis. Similar to these results, we noted that alterations in DnaA levels, i.e. either increases or decreases, resulted in cell lengthening, although DnaA-depleted cells were much more filamentous than DnaA-overproducing cells (compare Fig. 7j
, l
, n
with f
). Interestingly, the filamentous cells contained several distinct, well-separated nucleoids under overproduction and depletion conditions. The major difference between the two conditions was that filamentation due to depletion of DnaA was associated with a severe loss of cell viability. In contrast, filamentous cells produced after exposure to DnaA-overproduction conditions continued to grow without any decrease in viability (Fig. 5
). These results tend to indicate a loss of coordination between the DNA replication and cell-division events under altered levels of DnaA.
Our results showing the multinucleoidal state in filamentous cells under DnaA-depleted conditions are somewhat unexpected. Cell division is expected to proceed to completion in dnaA mutants, thereby resulting in cells containing a single nucleoid (Cook & Rothfield, 1999 ; Sun & Margolin, 2001
). Obviously, this was not the case with the acetamide-starved cells of M. smegmatis, thus signalling a correlation between the initiation of DNA replication and cell-division events in this organism, and that DnaA may have a role in these two events.
How can we explain the multinucleoidal state of cells under altered DnaA production conditions? The conditionally complemented dnaA mutant cells (RGM36) grown in the presence of acetamide had elevated levels of DnaA (Fig. 5b) and were multinucleoidal, although these cells continued to grow without any reduction in their viability. Presumably, cell lengthening and the multinucleoidal state of the DnaA-overproducing cells are due to induction of an SOS-like response. It is conceivable that either the activity or the level(s) of protein(s) responsible for cell division in M. smegmatis could be sensitive to the intracellular levels of DnaA, and this might be one way by which M. smegmatis tightly coordinates its DNA replication with its cell division. DnaA could exert such an effect directly either by binding to the presumptive DnaA boxes located in the promoter regions of target genes or by interacting with the proteins, or indirectly by recruiting other protein(s). It is worth noting that the filamentous and multinucleoidal state observed under DnaA-overproduction conditions is reminiscent of the B. subtilis yabA mutant phenotype (Noirot-Gros et al., 2002
). The yabA gene product has been implicated in the negative regulation of B. subtilis replication. Thus, the possibility that alterations in the activity and/or the levels of the putative negative regulators of M. smegmatis DNA replication under elevated DnaA production conditions remains open.
Similarly, growth in the absence of acetamide, which led to a gradual depletion in the levels of DnaA and DNA synthesis (Figs 5 and 6
), also resulted in the multinucleoid state. In this scenario, the residual levels of DnaA in acetamide-starved cells may be sufficient to trigger new rounds of replication that proceed to completion, thereby resulting in the multinucleoidal state, but not sufficient to coordinate DNA replication with cell-division events. This could lead to a blockage in cell division, again signalling a linkage between DNA replication and cell-division events. Our results showing the polynucleoidal state of merodiploid cells expressing antisense dnaA RNA are somewhat surprising (Fig. 9
). With a non-DnaA-overproducing cell population we thought that induction of dnaA antisense RNA would ultimately produce elongated cells with a single nucleoid, but this was not the case (Fig. 9d
). Presumably, constitutive levels of dnaA RNA expression under uninduced conditions were sufficient to affect the septation process, thereby resulting in the multinucleoidal state. Currently, our studies are limited by the number of available regulatory promoters known to affect gene expression in mycobacteria; hence, we are unable to ascertain the effect(s) of dnaA expression on septation at this time. The expression of dnaA antisense RNA from tight, inducible promoters that eliminate background expression could produce a starting population of cells that contain a single nucleoid. It should be noted that antisense expression under induced conditions, although effective in reducing intracellular DnaA levels (Fig. 2a
), is not totally leak-proof. This could in turn lead to a slow but steady accumulation of DnaA; hence, an increase in the cell length under induced conditions (42 °C for 2 h followed by continued growth at 37 °C) comparable to that observed with the acetamide-starved cells was not observed (Fig. 7i
, m
, n
).
It is pertinent to note that the overproduction of B. subtilis DnaA has been shown to induce cell lengthening but, unlike the results reported in the present study, this lengthening resulted in inhibition of cell growth (Ogura et al., 2001 ). Increasing the intracellular levels of DnaN in the DnaA-overproducing cells restored them to a wild-type phenotype. In our experiments, approximately sixfold DnaA overproduction was attained under acetamide growth conditions (Fig. 5b
). It is not known whether we can attain more than sixfold DnaA overproduction in cells and, if so, whether this would result in growth inhibition. Further characterization of the roles of DnaA in DNA replication and cell-cycle events will enable us to understand why and at what stage DnaA-overproducing cells are arrested in cell division and how these replication processes are related to those of B. subtilis and E. coli.
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ACKNOWLEDGEMENTS |
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Received 27 June 2002;
revised 30 July 2002;
accepted 27 August 2002.