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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Mol Microbiol. Author manuscript; available in PMC 2010 January 1.
Published in final edited form as:
PMCID: PMC2733369
NIHMSID: NIHMS118579

Synchronous replication initiation in novel Mycobacterium tuberculosis dnaA cold-sensitive mutants

Abstract

The genetic aspects of oriC replication initiation in Mycobacterium tuberculosis are largely unknown. A two-step genetic screen was utilized for isolating M. tuberculosis dnaA cold-sensitive (cos) mutants. First, a resident plasmid expressing functional dnaA integrated at the attB locus in dnaA null background was exchanged with an incoming plasmid bearing a mutagenized dnaA gene. Next, the mutants that were defective for growth at 30°C, a nonpermissive temperature, but resumed growth and DNA synthesis when shifted to 37°C, a permissive temperature, were subsequently selected. Nucleotide sequencing analysis located mutations to different regions of the dnaA gene. Modulation of the growth temperatures led to synchronized DNA synthesis. The dnaA expression under synchronized DNA replication conditions continued to increase during the replication period, but decreased thereafter reflecting autoregulation. The dnaAcos mutants at 30°C were elongated suggesting that they may possibly be blocked during the cell division. The DnaA115 protein is defective in its ability to interact with ATP at 30°C, but not at 37°C. Our results suggest that the optimal cell cycle progression and replication initiation in M. tuberculosis requires that the dnaA promoter remains active during the replication period and that the DnaA protein is able to interact with ATP.

Introduction

Mycobacterium tuberculosis, the causative agent of tuberculosis, is responsible for approximately 1.7 million deaths each year. Tuberculosis infections are also the leading cause of morbidity and mortality in AIDS patients. M. tuberculosis is a slow grower with an average doubling time of 24 h. Its efficiency as an infectious agent relies on its ability to shift between two physiologically distinct growth states upon infection including an active replicative state with an increase in the bacterial burden and a latent or quiescent state characterized by a limited bacterial turnover. It is believed that the bacterium in the latter state remains metabolically active, but in a state of non-replicative persistence (NRP) (Wayne and Hayes, 1996). The NRP bacteria reactivate and resume active replication under conditions that are favourable for their growth, and multiply to cause an infection. DNA replication, which is a critical aspect of the cell cycle, is essential for cell duplication. Although the M. tuberculosis genome sequence was determined nearly a decade ago (Cole et al., 1998), the genetic aspects of the regulation of DNA replication in this medically important pathogen are largely unknown.

DNA replication is believed to be initiated when the DnaA protein recognizes and binds to repeats of specific sequences consisting of nine nucleotides, called DnaA boxes, located in the origin of replication (oriC) (Bramhill and Kornberg, 1988a, 1988b). Binding of the ATP-bound form of DnaA to these DnaA-boxes triggers a cascade of events that result in the melting of the oriC DNA region, the assembly of additional proteins of the oriC-DnaA complex, and ultimately the unwinding and synthesis of DNA. Extensive genetic and biochemical studies dealing with dnaA and oriC have revealed that E. coli employs multiple mechanisms to prevent multiple initiations at oriC such that the “once-per-cell-cycle initiation at any given time” rule is maintained (Skarstad et al., 1986; Skarstad et al., 1988). These mechanisms include: (i) sequestration of the dnaA promoter so that de novo synthesis of DnaA is prevented (Campbell and Kleckner, 1990; Theisen et al., 1993); (ii) sequestration of the DnaA protein away from the oriC toward newly replicated chromosomal elements, which provides a sink for DnaA (Kitagawa et al., 1996; Kitagawa et al., 1998; Morigen et al., 2001); (iii) inactivation of DnaA by stimulating its intrinsic ATPase activity by both the DnaN and Hda proteins via a process called regulatory inactivation of DnaA (RIDA) (Katayama et al., 1998; Kurokawa et al., 1999); and finally (iv) sequestration of oriC by SeqA, which shows high affinity for hemimethylated DNA and binds GATC sites that are abundantly present in the oriC region (Baker, 1994; Lu et al., 1994; Slater et al., 1995). All of these mechanisms lead to either modulation of DnaA activity and or levels of DnaA protein in addition to the reduction of oriC availability.

Earlier studies on DnaA mediated oriC replication in M. tuberculosis have revealed that both DnaATB and oriC are essential and that DnaATB is a DnaA-box binding protein (Dziadek et al., 2002; Madiraju et al., 2006; Qin et al., 1999; Yamamoto et al., 2002a; Zawilak et al., 2004). The DnaATB protein exhibits weak ATPase activity (Yamamoto et al., 2002a; Yamamoto et al., 2008) and uses its intrinsic ATPase activity to rapidly oligomerize at oriC (Madiraju et al., 2006). The M. tuberculosis strain producing DnaA that is defective for ATPase activity, but is proficient for binding to ATP, is inviable (Madiraju et al., 2006). It is unknown how oriC replication mediated by DnaA is regulated in M. tuberculosis and whether or not oriC replication in M. tuberculosis involves any or all of the aforementioned regulatory mechanisms. The availability of well-defined and genetically characterized M. tuberculosis dnaA mutants will facilitate the dissection of the initiation process and will help us to begin understanding the regulatory mechanisms operating in this organism. To begin addressing this issue, we have developed a simple and robust protocol for isolating several novel alleles of M. tuberculosis dnaA strains that demonstrate a cold-sensitive (cos) phenotype. Characterization of these mutants revealed that DNA replication initiation can be synchronized by modulating the growth temperatures and that optimal cell cycle progression requires that dnaA expression remain high during the DNA synthesis period.

Results

M. tuberculosis dnaAcos mutants

A two-step genetic screen in a dnaA strain background was developed for isolating M. tuberculosis dnaAcos mutants. The dnaA strain is a chromosomal null for dnaA but contains an L5 phage based vector carrying a functional copy of dnaA integrated at attB locus (attB::dnaA) (Madiraju et al., 2006). The dnaA at attB locus in this strain was located approximately 2.7 Mbp relative to its native location. Thus, the dnaA gene in this strain is expected to be replicated with a significant delay relative to oriC, since it is close to the end of the replication period.

In the first step, the dnaA strain was electrotransformed with a plasmid DNA library expressing mutant dnaA genes. Since resident mycobacterial L5-based integrating vectors can be very efficiently excised with an incoming plasmid carrying a different antibiotic marker (Pashley and Parish, 2003), this step could result in the replacement of the integrated plasmid carrying a functional copy of dnaA with that of the incoming plasmid expressing the mutant copy. In the second step, the dnaA transformants that were viable at 37°C (permissive temperature), but not at 30°C (nonpermissive temperature), were selected by replica patching and were characterized further. Using this protocol, several viable transformants were obtained and 7 out of the 3300 analyzed were found to be consistently cold sensitive (cos) (Fig. 1A). These cos transformants recovered readily when shifted to the permissive temperature (Fig. 1B). Since the coding region of the dnaA gene and not its promoter was PCR mutagenized in our protocol, we would expect that the cos phenotype could be due to an altered activity of the DnaA protein, but not due to altered expression levels of dnaA. DnaA protein autoregulates its expression (Braun et al., 1985). Thus, it is possible that mutations in the dnaA coding region affect its ability to regulate its own expression and therefore DnaA levels. Evaluation of the dnaA transcript and the translated product could provide additional insights into these issues.

Figure 1
Characterization of dnaAcos strains: (A): Replica patching of M. tuberculosis dnaAcos strains at 37 and 30 °C. Agar plates were incubated at respective temperatures and visualized after 2 weeks for 37°C and 4 weeks for 30°C. Note ...

Nucleotide sequence analysis identified that the mutations occurred in different regions of the dnaA gene (Fig. 1C). With the exception of dnaA231, all of the strains contained multiple mutations. The dnaA231 strain contained a single point mutation at codon 375 and the affected residue is part of a region that is believed to be important for its ATPase activity. Strains dnaA108, dnaA115, dnaA132, dnaA183 and dnaA255 carried an identical set of mutations at codons 73, 151 and 215, However, strain dnaA255 also possessed another mutation at codon 468. Interestingly, codons 215 and 468 are in regions believed to be important for the binding of ATP and DNA, respectively (Erzberger et al., 2002; Roth and Messer, 1995, 1998; Schaper and Messer, 1995). Finally, strain dnaA245 possessed three mutations, but all of these are at different sites (Fig. 1C).

Characterization of the dnaAcos mutants

The dnaAcos mutants showed reduced viability (Fig. 1D) and filamentation at the nonpermissive temperature, but recovered when shifted to the permissive temperature (Fig. 1E shows the phenotype of the representative strain dnaA115). Measurement of the incorporation of radiolabeled uracil into alkali stable DNA revealed that dnaAcos mutants were as proficient as the wildtype strain in synthesizing DNA at 37°C (Fig. 2A), but were defective at 30°C (Fig. 2B). DNA synthesis in dnaAcos cultures at 30°C showed a small initial increase and reached a plateau between 16h (dnaA115) to 34h (dnaA231 and dnaA245). The observed gradual plateau is possibly due to a heterogeneous population of cells completing ongoing rounds of DNA synthesis that were unable to initiate new rounds of DNA synthesis. In contrast, DNA synthesis in wildtype cells continued to increase at 30°C (Fig. 2B). These data suggest that the dnaAcos mutants are defective for reinitiation at 30°C. It should be noted that the wildtype strain in these and other experiments described below refers to the parent strain wherein the dnaA gene is located at its native location. Hence the wildtype strain does not constitute an identical control to dnaAcos cells that contained a functional copy of the dnaA gene at the attB site. However, the location of the dnaA gene is not expected to affect the outcome of the experiments presented in this study.

Figure 2
DNA synthesis profiles of the dnaAcos cultures at 37°C and 30°C. Actively growing cultures of M. tuberculosis wildtype and dnaAcos strains were diluted and pre-labelled with 3H-uracil (0.5 microCurie/mL) and grown to O.D600=0.6. The pre-labelled ...

DNA synthesis in dnaAcos mutants is aligned

The DNA synthesis patterns of dnaAcos mutants at the nonpermissive temperature (Fig. 2) combined with the reversal of the cold-sensitive phenotype upon a temperature shift (Fig. 1D and E) suggested a possibility that DNA replication in these mutants can be aligned by modulating the growth temperatures. Accordingly, experiments as shown in Fig. 3A were performed. In these experiments, two identical sets of cultures actively growing at 37°C were transferred to 30°C (marked as I in Fig. 3A) and incubated for 26h prior to shifting the cultures back to 37°C allowing for DNA synthesis (Fig. 3A, marked as ‘II’). The 26h incubation period at 37°C corresponds to approximately one doubling time for the culture. Preliminary experiments established that dnaAcos cultures growing at 30°C were able to resume DNA synthesis with a 2h lag period when the cultures were returned to 37°C (not shown). Therefore, 2h after incubation at 37°C, one set of cultures was returned to 30°C (see Fig. 3A, marked as III) and the other remained at 37°C. DNA synthesis was followed at both temperatures.

Figure 3
Synchronized DNA replication in M. tuberculosis dnaAcos cultures. (A) Experimental plan. Two identical sets of M. tuberculosis cultures, one indicated by dotted line and the other by solid line, were pre-labelled with 3H-uracil (0.5 μCi/mL). Both ...

As can be seen, cultures that were returned to 30°C after the 2h incubation period at 37°C continued DNA synthesis for approximately 11h where it reached a plateau (see Fig. 3B, time point at II is marked as ‘0’). This portion of the curve is referred to as the DNA synthesis period, “C”. An additional burst of DNA synthesis occurred only when a second temperature shift experiment was performed by transferring the culture to 37°C for 2h and then transferring these cultures back to 30°C (see Fig. 3B, marked as IV and V, respectively at 26 and 28 h). Again, DNA synthesis continued for 11h and reached a plateau thereafter. Each burst of DNA synthesis led to a doubling in the amount of radioactivity indicating that bacteria were able to complete one round of DNA synthesis, but failed to initiate new rounds at 30°C. These results suggest that the C period at 30°C is approximately 11h in length. In contrast, the wildtype strain continued to synthesize DNA in a linear fashion without requiring temperature shifts.

The dnaAcos cultures maintained at 37°C showed a similar trend with the exception that the new burst of DNA synthesis occurred after roughly 24h (Fig. 3C time point at II marked as ‘0’). As in the situation observed at 30°C, DNA synthesis continued for approximately 10h, reached a plateau and underwent a burst at about 24h, which again continued for a period of about 10h (Fig. 3C). Here, as observed previously, the radioactivity was doubled in each cycle. The plateau period between the two bursts of DNA synthesis at 37°C may include ‘D’ in addition to any of the ‘B’ periods. The latter period corresponds to the time after cell division that precedes the initiation of a new round of DNA replication. Evaluation of the cell number determinations at various periods during the cell cycle could enable us to determine the ‘D’ and ‘B’ periods precisely. As can be seen, the wildtype cells continued DNA synthesis that proceeded in a linear manner. Analysis of the DNA synthesis data revealed a rate of ~ 3030 nt/min at 30°C and 3333 nt/min at 37°C (see Fig. 3 legend for details).

In the aforementioned experiments, cultures were incubated for 26h at 30°C prior to the temperature shift. Identical results, with respect to DNA synthesis (see above) and dnaA expression (see below), were also obtained when the incubation period of the cultures at 30°C was continued for 30h (data not shown). Incubation periods longer than 30h were not performed since these cultures became filamentous and clumpy. From the above results we may conclude that modulation of the growth temperatures of dnaAcos mutants lead to synchronized replication initiation in M. tuberculosis.

Rifampicin, an inhibitor of RNA polymerase and DNA transcription, has been shown to inhibit initiation of new rounds of DNA synthesis (Lark, 1972; Messer, 1972). Accordingly, we evaluated if the addition of rifampicin at the time of temperature shift interferes with oriC initiation and subsequent DNA synthesis at 37°C. The addition of rifampicin allowed the initial round of DNA synthesis to proceed but prevented a second burst of DNA synthesis (supplementary Figure S1). Since dnaA transcription is active even at 30°C (see below), we interpret the data to mean that the necessary DnaA potential sufficient for the initiation of one round of DNA replication may have been accumulated during incubation at 30°C. However, addition of rifampicin at the time of temperature shift prevented new rounds of dnaA transcription and hence prevented the second burst of initiation and subsequent DNA synthesis.

dnaA expression under synchronized replication initiation conditions

De novo expression of dnaA is believed to be one of the factors that are important for the initiation of new rounds of DNA synthesis. Hence, the kinetics of dnaA expression under synchronized replication initiation conditions (see Fig. 3) was examined. RNA samples at various periods after temperature shift, in addition to samples taken prior to the shift, were prepared and dnaA expression relative to 16S rRNA was evaluated (Fig. 4). Since the dnaA expression profiles at both 30 and 37°C were identical, the results obtained under only one condition are shown. The following observations were evident: (1) dnaA expression in the wildtype and dnaAcos strains was reduced at 30°C when compared to expression at 37°C. It is not evident why dnaA expression levels decreased at 30°C, however one possibility is that the affinity of DnaA to the DnaA boxes at 30°C is higher than at 37°C resulting in tighter repression of dnaA promoter. (2) dnaA expression levels in the wildtype strain returned to normal levels upon performing a temperature shift that was maintained thereafter, although a small reduction was noted at 16h. The 16h time point corresponds to the post-replication period and since increased levels of dnaA are not expected to be required, the dnaA expression was repressed.

Figure 4
Expression levels of the dnaA and the mtrA genes by quantitative real-time (QRT) PCR: RNA was isolated from wild type M. tuberculosis and dnaAcos strains growing at 37°C (marked as I) prior to temperature shift, 26h after incubation at 30°C ...

(3) dnaA expression in strains of dnaAcos mutants show two distinct features including an elevated expression level during the C period (8h) ín addition to a rapid reduction thereafter. It should be noted that the dnaAcos gene located at the attB locus is within close proximity to the midpoint of the genome [~2. 7 Mbp of 4. 4 Mbp]. Accordingly, one would expect that duplication of dnaA gene would occur toward the end of the C period at approximately 9h. Presumably, the increased expression of dnaA during the C period is not be due to gene duplication, but rather due to an increased requirement of dnaA. To address this question, we examined the expression profile of the wildtype dnaA gene located at attB under identical growth conditions (supplementary Figure S2). The attB::dnaA expression profile was similar to that of the strain that has the dnaA gene located at its native locus next to oriC, again confirming that the increased expression of dnaA during the C period in synchronized dnaAcos mutants is not due to gene duplication or relocation of the gene, but rather due to a requirement for increased dnaA expression (see below and discussion). We propose that the rapid reduction in dnaA expression following dnaA gene duplication (approximately 9h) is due to the autoregulatory activity of DnaA, the sequestration of its promoter by hitherto unidentified regulatory proteins (see discussion), or some combination of both mechanisms.

Addition of chloramphenicol, a protein synthesis inhibitor, at the time of the temperature shift, did not interfere with dnaA expression (supplementary Figure S3). We also examined the expression pattern of the essential mtrA response regulator under these conditions. Earlier studies had indicated that the dnaA promoter is the MtrA response regulator target (Fol et al., 2006). Interestingly, our results demonstrated that the mtrA expression remained relatively similar at all growth periods indicating that similar expression levels of mtrA are required at all periods of the cell cycle (Fig. 4B).

Intracellular levels of DnaAcos remain stable

We considered the possibility that the cold-sensitive phenotype of the dnaAcos mutants is due to alterations in the levels or possibly in the activity of the DnaA protein. To obtain insights into this issue, we examined the status of DnaA by immunoblotting in dnaAcos mutants growing at 30°C and 37°C and compared these results to analogous assays performed with the wildtype strain. SigA was used as an internal control (Fig. 5A, see panels I and ii). Quantitation of the band intensities confirmed that the relative ratios of DnaA:SigA were similar at both temperatures and in all strains (Fig. 5B). Interestingly, the DnaA:SigA ratios of dnaAcos strains were lower than that observed of the wild type strain. Although the reason for this is not evident, nonetheless, these results support a notion that both the cold-sensitive phenotype (Fig. 1) and the inability to initiate new rounds of DNA synthesis (Fig. 3) at the nonpermissive temperature is not due to altered levels of DnaA.

Figure 5
DnaA protein levels in dnaAcos strains: (A): Actively growing wildtype and dnaAcos cultures at 37°C were shifted to 30°C and incubated for 48h. Samples at both temperatures were removed, clear cellular lysates were prepared and the DnaA ...

The DnaA115 protein is defective for binding to ATP at 30°C

Initial interactions of DnaA with ATP are critical for the formation of a productive oriC-DnaA initiation complex that is competent for replication initiation (Bramhill and Kornberg, 1988a). Hence, we considered the possibility that the cos phenotype of the dnaAcos mutants is due to an altered DnaA activity. This is not an unreasonable assumption since some of the dnaAcos mutants, for example dnaA108, dnaA115, dnaA132, dnaA183 and dnaA255, had mutations in the conserved GxGxxGKT motif that is critical for the binding of ATP (Walker et al., 1982). It is important to note that the E. coli dnaA46 temperature sensitive (ts) mutant, which is reversible in its thermostability, is defective for the binding of ATP (Hwang and Kaguni, 1988a, 1988b). Hence, in order to test this prediction, we overproduced the mutant DnaA as his-fusion proteins and attempted to purify the proteins using nickel affinity columns. Notably, we were able to purify DnaA115 but not DnaA231 and DnaA255 due to observed aggregation problems. The ability of DnaA115 to bind ATP at 30 and 37°C was examined using filter binding assays (Fig. 6). The wildtype DnaA protein bound ATP at both 37 and 30°C with similar affinities (Kd ~146 nM). In contrast, DnaA115 was proficient for the binding of ATP at 37°C (Kd ~6.6 μM), but was completely defective at 30°C (Fig. 6). DnaA115 showed a sigmoidal binding tendency at 37°C. Mutations at other locations in DnaA115 may have affected the overall ability of this mutant protein to interact with ATP.

Figure 6
Binding of Wild type DnaA and DnaA115 proteins to ATP. Nitrocellulose filter binding assay was used to measure the binding of wildtype DnaA (A) or DnaA115 (B) to radiolabelled ATP. Concentrations of ATP used are shown. The amount of ATP bound (nmoles) ...

dnaA115 growing at the nonpermissive temperature is blocked at cell division

The dnaA expression patterns of dnaAcos and the altered interactions of DnaA115 with ATP at 30°C suggest that de novo synthesis of DnaA that is competent to interact with ATP, is necessary for cell cycle progression. We have shown that dnaAcos mutants at the nonpermissive temperature were not proficient for the initiation of new rounds of DNA synthesis (Fig. 3) presumably due to defective interactions between DnaA and ATP (Fig. 6). These cells, however, would be expected to be proficient for cell division. Thus, continuous growth at the nonpermissive temperature could lead to filamentous cells with single nucleoids. To test this prediction we visualized the cell morphology by using brightfield microscopy (Fig. 7, panels marked as ‘I’) and visualized the nucleoids by using fluorescence microscopy following staining with a mixture of ethidium bromide and mithramycinA (Fig. 7, panels marked as ‘ii’). The wildtype cells growing at 30°C and 37°C and the dnaAcos cells growing at 37°C were nearly similar in size with one to two nucleoids per cell (Fig. 7). In contrast, the dnaA115 cells growing at 30°C were elongated and blocked at cell division (Fig. 7). The DNA staining pattern indicated that the nucleoids in the dnaA115 cells at 30°C were diffuse, as though they contained more than two nucleoids per filamentous cell. Certainly, more sensitive methods such as flow-cytometry and microarray techniques are required to determine the total number of origin copies per cell. Continued incubation of dnaA115 at 30°C increased the cell length although the majority of these cells contained diffuse nucleoids with nucleoid-free zones (see supplementary Figure S4). Together, these results indicate that the optimal cell cycle progression in M. tuberculosis requires a DnaA protein that is competent to bind and possibly hydrolyze ATP in order to catalyze other activities.

Figure 7
Visualization of the cell and nucleoid morphology of the wildtype and the dnaA115 strains. M. tuberculosis cultures growing at 37°C and 30°C were removed, stained with ethidium bromide and mithramycinA mixture and visualized by bright-field ...

Discussion

M. tuberculosis dnaAcos versus the E. coli dnaA conditional lethal mutants

DnaA, a member of AAA+ family ATPases, is a highly conserved protein in eubacteria (reviewed in (Erzberger et al., 2002)), and is recognized to contain several functional domains that are implicated in oligomerization, association with DnaB, ATP, polar lipids and DNA (Messer, 2002; Schaper and Messer, 1997). The conditional lethal M. tuberculosis dnaAcos mutants that we have isolated possesed mutations distributed in all these regions of the DnaA protein (Fig. 1C) and importantly, the affected residues are conserved in eubacteria (see supplementary Figure S5).

While we obtained a single point mutation in the conserved box VII of the Walker type B ATPase motif, RELEGA (Neuwald et al., 1999), we did not obtain a single point mutation in the ATP-binding and DNA-binding motifs (see Fig. 1C, compare dnaA231 with dnaA108, dnaA115, dnaA132, dnaA183 and dnaA255). Failure to obtain a single point mutation in the ATP- or DNA-binding regions suggest that mutations in this region could result in a defective protein that renders the cell inviable and that mutations located in other regions may suppress this phenotype. For the sake of comparision, classic E. coli dnaA mutants, including dnaA46 and dnaA5, that bear mutations in the ATP-binding pocket carry a secondary mutation elsewhere in the gene (Hansen et al., 1992). An alternate possibility, although unlikely, is that our mutation search is not exhaustive.

The M. tuberculosis dnaAcos mutants, unlike the E. coli dnaA46 (ts), show a ‘delayed replication defect’ (slow stop) at the non-permissive temperature, but recover readily at the permissive temperature (Abe and Tomizawa, 1971; Evans et al., 1979). DNA synthesis in dnaA46 strains can be aligned if cells are allowed to accumulate excess initiation potential at 42°C prior to returning to the permissive temperature. E. coli dnaAcos is a spontaneous revertant of the dnaA46 (ts) mutation, and carries two intragenic suppressor mutations (Braun et al., 1987). This mutant shows excessive reinitiations (Kellenberger-Gujer et al., 1978), presumably due to defective interactions with ATP (Simmons and Kaguni, 2003). Our results suggest that the M. tuberculosis dnaA115 and possibly other dnaAcos mutants are comparable to the E. coli reversible type of mutants, such as dnaA46.

We showed that dnaA115 carries three mutations (Fig. 1C). However, it is unknown which of these mutations is responsible for the observed phenotypes such as cold sensitivity, defective binding to ATP, and an ability to exhibit synchronous bursts of DNA synthesis at the permissive temperature following incubation at the non-permissive temperature. Evaluation of the contribution of these mutations individually and together in various combinations will provide valuable clues to the contribution of the amino acid residues to the replication initiation process in M. tuberculosis. Our future studies will address these important yet unresolved questions.

Synchronized DNA replication and initiation once-per-cell cycle

Results presented in this study reveal (Figs. 3 and and4)4) several features associated with oriC replication in M. tuberculosis. First, initiation of DNA replication in M. tuberculosis, as in E. coli, requires de novo DnaA synthesis. This claim is substantiated by our findings that the addition of rifampin enabled the completion of on-going rounds of DNA synthesis, but did not allow for the start of new rounds of DNA synthesis (Fig. S2). Second, although dnaA transcription and translation occur at both 37 and 30 °C, only DnaA competent to interact with ATP was found to be proficient for initiation (Figs. 3 and and4).4). The inability of DnaA115 to bind ATP at 30°C explains why dnaAcos is not proficient for the second-round of reinitiation at 30°C (Fig. 3).

Third, optimal cell cycle progression and viability require that the dnaA promoter remains active during the entire cell cycle, with elevated activity during the C period and reduced activity immediately thereafter. The dnaA gene in the dnaAcos mutants is expected to duplicate just prior to the end of the C period. Thus, reduction in the dnaA expression levels following dnaA gene duplication, therefore at the end of the C period, could either be due to an autoregulation event or due to the sequestration of the dnaA promoter (see below). Since dnaAcos cells at the nonpermissive temperature are filamentous and possibly blocked at cell division (Fig. 7), we predict that elevated dnaA expression during the C period provides the necessary pools of active DnaA required for regulated transcription of genes involved in cell division and other processes. This is not an unreasonable assumption since the transcriptional regulatory role for DnaA has been well recognized (Messer and Weigel, 1997). M. tuberculosis DnaA, like its E. coli counterpart, recognizes and binds to DnaA-boxes (Dziadek et al., 2002; Madiraju et al., 2006). Our bioinformatics analysis identified the DnaA-box like sequences in the putative promoters of genes involved in cell division and other metabolic pathways such as lipid biosynthesis and nucleotide biosynthesis (not shown). Presumably, the interactions of DnaA with the target promoter sequences results in the temporal expression of genes involved in septation and cell cycle progression. These results substantiate our earlier report showing that overproduction or conditional depletion of DnaA in M. smegmatis produces filamentous cells blocked at cell division (Greendyke et al., 2002).

Finally, the nucleotide (ATP) bound state of DnaA is important for cell cycle progression and the initiation of DNA synthesis. This is because even though transcription and translation of M. tuberculosis dnaA115 proceed at 37 and 30°C, new rounds of DNA synthesis were observed only at 37°C. M. tuberculosis DnaA binds ATP and ADP with relatively high affinity (Yamamoto et al., 2002a; Yamamoto et al., 2002b), but only DnaA protein competent to bind and hydrolyze ATP promotes the rapid oligomerization required for the initiation of replication at oriC (Madiraju et al., 2006). Since DnaA115 protein is defective for binding to ATP at 30°C (Fig. 6), this could explain why the dnaA115 strain failed to reinitiate DNA synthesis at the non-permissive temperature (Fig. 3).

The observed 10h C period in synchronized population of M. tuberculosis is longer than we expected, but is consistent with an earlier report describing the DNA synthesis time in M. tuberculosis (Hiriyanna and Ramakrishnan, 1986). The C period in E. coli is also long when grown in chemostats and in media containing succinate as opposed to glucose, although the reasons for this are unknown (Bipatnath et al., 1998). The long C period in M. tuberculosis with a tD=24h is not due to the presence of single rrn operon in its genome because M. marinum with tD=3h has also one rrn operon (Helguera-Repetto et al., 2004). It is possible that DnaA could regulate the intracellular levels of nucleotide pools, hence the overall rate of DNA synthesis. Alternatively, lagging strand DNA synthesis could be slow in M. tuberculosis. Clearly, further studies are required to understand this issue. It is pertinent to note that the slow growth rate of M. tuberculosis is also correlated to the presence of large number of insertion sequences in its genome (Cox, 2004).

The M. tuberculosis dnaA promoter contains several DnaA-box like sequences (Qin et al., 1999). The binding of DnaA to these DnaA boxes immediately after gene duplication could lead to regulated expression of dnaA, similar to the reported autoregulatory activity of E. coli DnaA (Braun et al., 1985). We have recently shown that the MtrA response regulator promotes dnaA transcription by binding to the dnaA promoter in a phosphorylation-dependent manner (Fol et al., 2006). Thus, another possibility is that intracellular ratio of the phosphorylated to the non-phosphorylated MtrA response regulators dictates the outcome of dnaA expression. A consequence would be the regulation of dnaA expression.

DNA synthesis under synchronized replication conditions reveal that hyper-initiation following reinitiation does not occur in M. tuberculosis (Fig. 3C). This raises the question as to how ‘initiation once-per-cell cycle’ is achieved in this organism. Studies with E. coli reveal that DnaA mediated oriC replication is initiated when a threshold level of DnaA within a cell is reached (Lobner-Olesen et al., 1989; Skarstad et al., 1986; Skarstad et al., 1989), and that a sequestration of both the dnaA promoter and the oriC regions are required for achieving the controlled once-per-cell-cycle initiation [reviewed in (Riber and Lobner-Olesen, 2005)].

The M. tuberculosis oriC does not contain GATC methylation sequences and the genome does not encode for the SeqA-like protein. Our earlier data show that the active species for initiating replication at oriC is the DnaA protein that is capable of binding and hydrolyzing ATP (Madiraju et al., 2006). Hence, in order to explain how oriC initiation in M. tuberculosis is regulated, we propose that DnaA-box like sequences present in the promoters of several genes including the dnaA gene provide a sink for DnaA-ATP pools remaining after initiation thereby minimizing the active DnaA threshold available for reinitiation. We have recently shown that the intrinsic ATPase activity of DnaA promotes rapid oligomerization of DnaA-ATP complexes on oriC (Madiraju et al., 2006). This process may result in the conversion of DnaA-ATP complexes to DnaA-ADP complexes (Madiraju et al., 2006). Thus, another possibility is that a tight association of DnaA-ADP complexes with oriC following its rapid oligomerization prevents the reinitiation from oriC (Madiraju et al., 2006). This situation may be analogous to that of the RIDA mechanism (Katayama et al., 1998). M. tuberculosis DnaA is membrane associated (Yamamoto et al., 2008) and acidic phospholipids promote dissociation of nucleotides from DnaA and possibly restore levels of active DnaA (Yamamoto et al., 2002a; Yamamoto et al., 2008). Finally, in addition to DnaA, other hitherto unidentified regulatory proteins may target both the dnaA promoter and the oriC in order to regulate hyper-initiation. One such regulator could be MtrA (Fol et al., 2006). Further characterization of dnaAcos mutants could provide valuable insights into the regulation of oriC replication process in M. tuberculosis.

Finally, investigations in DNA replication regulation and cell cycle progression in M. tuberculosis remain relatively unexplored as compared to those in other organisms. Larry Wayne developed a hypoxia-based culture system where actively growing M. tuberculosis are subject to a gradual hypoxic state over a period of two to three weeks in sealed, air-tight tubes in order to convert the population to a non-replicative persistent state. Hypoxic M. tuberculosis cells are shown to catalyze synchronous replication upon resuspension of cultures in fresh oxygenated media (Wayne, 1977; Wayne and Hayes, 1996). These are time consuming experiments, and the status of the dnaA promoter activity following reactivation and cell cycle progression have not been investigated. The simple growth temperature shifts for aligning DNA synthesis with the dnaA mutants that we generated should prove useful in investigating the regulatory mechanisms involved in M. tuberculosis DNA replication and cell cycle.

Materials and Methods

Bacterial strains and culture conditions

The E. coli strain Top-10 (Stratagene) and M. tuberculosis strain H37Ra was used in all of the experiments presented herein. Middlebrook 7H9 broth supplemented with oleic acid, albumin, dextrose and sodium chloride was used to culture M. tuberculosis. Transformants were selected on media supplemented with agar. In order to select for transformants, hygromycin was used at either 50 μg ml−1 for M. tuberculosis or 100 μg ml−1 for E. coli. In addition, kanamycin was used at either 10 μg ml−1 for M. tuberculosis or 50 μg/ml for E. coli to select transformants.

Protocol for creating the dnaAcos mutants

Our protocol for constructing the M. tuberculosis dnaA mutants involved several steps. In the first step, we created the PCR mutagenized plasmid dnaA library wherein the mutated dnaA coding region was cloned downstream of its native promoter. The native dnaA promoter was amplified using primers 5′CCATCGATCGCCGGTTGTTCGGCTGGAAGGTC-3′ (MVM372) and 5′TTGCAAGCTTCATATGCGTATCTCCCTGGTTCTC-3′ (MVM408), with ClaI, HindIII, and NdeI sites, respectively, underlined. The PCR fragments that were amplified were cloned into a kanamycin-resistant pMV306 vector digested with the same enzymes. Second, the dnaA coding region was amplified using primers 5′-CGCCTGCAGCATATGGCCGATGACCCCG GTTC-3′ (Q58) and 5′-TGCTCTAGACTACTAGCGCTTGGAGCGCTGAC-3′ (MVM379) with NdeI and XbaI sites, respectively, in the presence of 1.5 mM MgCl2 and 0.5 mM MnCl2, Taq DNA polymerase and genomic DNA as the template. PCR amplification under these conditions is shown to generate 2 to 3 mutations per 1.5-kb of DNA generated (Guo et al., 1999; Zhou et al., 2003). The PCR amplified fragments were then digested with NdeI and XbaI, cloned downstream of the pMV306 plasmid bearing the dnaA promoter, transformed into E. coli Top10 (Invitrogen Inc.) and was selected for kanamycin resistance. All transformants were pooled, the plasmid DNA was purified and electrotransformed into M. tuberculosis dnaA strain carrying the integrated copy of dnaA (expressing from its native promoter) at the attB site (Madiraju et al., 2006). Mycobacterial plasmids integrated at the attB site can be excised with an incoming target plasmid carrying an alternate antibiotic marker (Pashley and Parish, 2003). Next, all kanamycin resistant and viable transformants were replica patched, incubated at both 37°C and 30°C. Those colonies that grew at 37°C but not at 30°C were identified, subsequently colony purified and are designated as dnaA cold sensitive mutants (dnaAcos).

Mapping and nucleotide sequencing

Genomic DNA was prepared from dnaAcos strains grown at the permissive temperature (37°C), the dnaA coding region was amplified with proof reading DeepVent DNA polymerase and sequenced to map mutations.

DnaA overproduction and purification

Purification of the recombinant wildtype M. tuberculosis DnaA from E. coli BL21(DE3)/pLysS cells on nickel affinity columns was performed as described (Madiraju et al., 2006; Yamamoto et al., 2002a). The coding region of dnaAcos115 was amplified, cloned downstream of the T7 promoter in a pET19b vector previously digested with NdeI and BamHI enzymes. The recombinant mutagenic protein was purified using the same protocol as described for the wild-type DnaA protein.

Filter binding experiments

A nitrocellulose filter binding assay for detecting the binding of ATP to DnaA protein was performed similarly to that described previously (Yamamoto et al., 2002a). Reactions were carried out in 50μl of buffer containing 50 mM Tris-acetate (pH 8.2), 0.05 mM magnesium acetate, 0.3 mM EDTA, 5 mM 2-mercaptoethanol, 10 mM ammonium sulphate, 20% glycerol, and 0.005% (v/v) Tween-20 was incubated with DnaA and [α-32P]-labelled ATP. DnaA was also incubated with ATP at the indicated temperature, samples were filtered, washed, and the amount of bound radioactivity was measured by using scintillation counting.

RNA extraction and quantitative real-time (QRT) PCR

Extraction of total RNA from broth-grown cultures of M. tuberculosis were performed at different times (see Fig. 3A) and at different temperatures as described previously (Chauhan et al., 2006b; Fol et al., 2006). DNA contamination was removed by treatment with DNaseI (Ambion). Approximately 50–100 ng of total RNA was reverse-transcribed using 100 nM of RT16S3 with RTdnaA primers and Superscript II reverse transcriptase (Invitrogen). Target cDNA from the control and the experimental sets were amplified in separate reaction tubes. Real-time PCR (Taqman chemistry) was performed in a Bio-Rad I Cycler using the Taq DNA polymerase (NEB), Taqman probes (Biosearch Technologies), and reverse and forward primers (see Table 1). The calculated threshold cycle (Ct) value for each gene of interest was normalized to the Ct value for the 16S and the fold-expression was calculated using the formula: fold change = 2-Δ(ΔCt). No reverse transcriptase (RT) reactions were included as negative controls. Expression data are the average from three independent RNA preparations, each reverse-transcribed and quantified by real-time PCR in triplicate. Real-time PCR conditions include initial denaturation at 95°C for 3 min; followed by 40 cycles of denaturation at 95°C for 30 sec, annealing and extension at 60°C for 1 min.

Table 1
Primers for RT and QRT-PCR.

DNA synthesis: Incorporation of 3H Uracil

A quantitative measurement of DNA synthesis with M. tuberculosis cultures was performed as described previously (Greendyke et al., 2002) by following Wayne's protocols (Wayne, 1977). To increase the specific activity, all cultures were pre-labelled by growing with uracil (0.5 μCi/mL) for several generations until the OD600 reached approximately 0.6. The pre-labelled cultures were then diluted into fresh 7H9 Middlebrook broth containing 3H-uracil, grown at 37°C for 24h (~ 1 generation). One half of the sample was shifted to 30°C and the other half of the sample was continued at 37°C for 26h. DNA synthesis at both temperatures was then followed at the indicated time periods. In some experiments, the cultures grown at 30°C for 26h were returned to 37°C for 2h. At this point these cultures were either incubated at 37°C or were shifted back to 30°C. Samples consisting of 0.5 ml aliquots were collected for monitoring DNA synthesis at the time points indicated. After incubation with 0.2N KOH at 37°C for 24h, paraformaldehyde was added to a final concentration of 4% and incubated at 37°C for 1h. The samples were then filtered through a 0.45 micron pore size filter, washed twice with phosphate buffered saline followed by 70% ethanol. The membranes were dried and radioactivity counted in a scintillation counter.

Microscopy

M. tuberculosis wildtype and dnaAcos strains were grown under various conditions, fixed in 4% paraformaldehyde and visualized using microscopy as previously described (Chauhan et al., 2006a; Chauhan et al., 2006b). Nucleoids were visualized by staining with a mixture of ethidium bromide (40 μg ml-1) and mithramycin A (180 μg ml-1) for 30 min on ice. Prior to staining, cells were exposed to 2 % (v/v) toluene for 2 min. A Nikon Eclipse 600 microscope with a 100 × Nikon Plan flour oil immersion objective, a numerical aperture (NA) of 1.3, a 100 W mercury lamp, and a Chroma filter set (excitation from 540 to 565 nm and emission from 560 to 623 nm) was used for microscopy. Images were acquired using a Photometrics Coolsnap ES camera and Metamorph 6.2 imaging software (Universal Imaging Corporation).

Immunoblotting

Preparation of cellular lysates and immunoblotting using anti-DnaA and anti-SigA antibodies were performed essentially as previously described (Chauhan et al., 2006a; Chauhan et al., 2006b). The SigA protein was used to normalize for protein amounts loaded in each lane when comparing the DnaA levels. Anti-sigma 70 antibodies were obtained from Neoclone Biotechnology (Madison, WI) and used as recommended. Immunoblots were processed with the ECF Western blotting kit from GE Healthcare and scanned on a Bio-Rad Molecular Imager (FX), and DnaA and SigA levels were determined with the volume analysis function of the QuantityOne software.

Supplementary Material

Supp figs

Figure S1: Pre-labeled actively growing cultures of M. tuberculosis at 37°C were shifted to 30°C for 30 h and then returned to 37°C. Rifampicin at a final concentration of 200 μg/ml was added and incubation was continued at 37°C. Samples at indicated time periods were collected and the DNA synthesis was measured as described under materials and methods section. (A) Wild type and (B) dnaA115.

Figure S2:attB::dnaAwt expression levels by quantitative real-time (QRT) PCR: Actively growing cultures of M. tuberculosis RGM85 strain carrying the wildtype dnaA gene at the attB locus was grown at 37°C for 26h. The culture was then shifted to 30°C for another 30h without dilution and returned to 37°C. RNA was extracted from cultures at different time points as indicated in Figure 3A and the dnaA levels were measured by QRT-PCR using Taqman chemistry (Probes and oligos used are listed in Table 1). The expression levels of dnaA were normalized relative to 16S mRNA levels. Relative DNA expression levels as fold change with respect to “0” time point is depicted.

Figure S3: Effect of chloramphenicol on the dnaA expression levels: Actively growing culture of the M. tuberculosis dnaA115 was shifted to 30°C for 30h and then returned to 37°C. Chloramphenicol at a final concentration of 20 μg/ml was added. At indicated time periods, samples were removed and processed for RNA extraction and the evaluation of the dnaA expression levels as described under Figure 4. The expression levels of dnaA were normalized relative to 16S rRNA.

Figure S4: Cell morphology and nucleoid localization in the dnaA115 and the wildtype M. tuberculosis. Actively growing M. tuberculosis cultures were shifted to 30°C, grown for 30, 48 and 72 h and stained with a mixture of ethidium bromide and Mithramycin A. Cell morphology and nucleoids were visualized by bright-field and fluorescent microscopy, respectively.

Figure S5: Alignment of DnaA proteins. The deduced DnaA amino acid sequences of M. tuberculosis and E. coli were aligned and compared. The N-terminal region of the DnaA protein containing domains 1 and 2 responsible for oligomerization and DnaB interaction activities, respectively is shown without any shading. The domain 3 responsible for ATP-binding and hydrolysis and lipid interaction is shown with dark green shading whereas the C-terminal region containing domain 4 responsible for DNA binding is shown as light yellow box. As can be noted, majority of the mutations in the M. tuberculosis DnaA were mapped to the conserved residues of the ATP-binding and DNA-binding domains. For clarity, the affected residues are boxed and strains carrying those mutations are listed beneath. Number of mutants with a particular mutation is shown in the parenthesis.

Acknowledgments

We thank members of our laboratory for helpful discussions and Dr. Dorota Stankowska for artwork. This work is supported in part by grants from the National Institutes of Health AI41406 (MM) and AI48417 (MR). The authors thank the anonymous reviewers for their helpful and critical comments.

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