Propionate represses the dnaA gene via the methylcitrate pathway-regulating transcription factor, PrpR, in Mycobacterium tuberculosis

During infection of macrophages, Mycobacterium tuberculosis, the pathogen that causes tuberculosis, utilizes fatty acids as a major carbon source. However, little is known about the coordination of the central carbon metabolism of M. tuberculosis with its chromosomal replication, particularly during infection. A recently characterized transcription factor called PrpR is known to directly regulate the genes involved in fatty acid catabolism by M. tuberculosis. Here, we report for the first time that PrpR also regulates the dnaA gene, which encodes the DnaA initiator protein responsible for initiating chromosomal replication. Using cell-free systems and intact cells, we demonstrated an interaction between PrpR and the dnaA promoter region. Moreover, real-time quantitative reverse-transcription PCR analysis revealed that PrpR acts as a transcriptional repressor of dnaA when propionate (a product of odd-chain-length fatty acid catabolism) was used as the sole carbon source. We hypothesize that PrpR may be an important element of the complex regulatory system(s) required for tubercle bacilli to survive within macrophages, presumably coordinating the catabolism of host-derived fatty acids with chromosomal replication.


Introduction
Faithful transmission of genetic material to daughter cells requires the precise regulation of chromosomal replication and its coordination with the cell cycle. Chromosomal replication in all three domains of life is mainly regulated at the initiation step. In bacteria, it is initiated through cooperative binding of the initiator protein, DnaA, to multiple DnaA boxes (9-mers) within the oriC region, which leads to the unwinding of DNA (reviewed in Kaguni 2006;Leonard and Grimwade 2011;Ozaki and Katayama 2009). The activity and availability of both key elements of replication initiation, DnaA and oriC, are tightly regulated to ensure that replication is initiated only once per cell cycle. Several factors that regulate replication initiation have been identified in both Gram-negative (Escherichia coli, Caulobacter crescentus) and Gram-positive (Bacillus subtilis, Streptomyces coelicolor) bacteria Katayama et al. 2010;Wolański et al. 2012). A few regulatory systems, such as the inactivation of DnaA-ATP by ATP hydrolysis, appear to be used universally by all bacteria. In contrast, others systems appear to be specific for particular bacteria, such as the CtrA, GcrA and CcrM proteins, which temporarily and spatially coordinate replication initiation with cell differentiation and cell cycle progression in C. crescentus (Collier 2012).
Surprisingly, little is known about the regulation of DNA replication in response to various environmental conditions and factors (Wang and Levin 2009). Recent studies have demonstrated that there is a direct link between central carbon metabolism (CCM) and the initiation and elongation stages of DNA replication in E. coli (Maciąg et al. 2011(Maciąg et al. , 2012 and B. subtilis (Jannière et al. 2007). These discoveries indicate the existence of a global correlation between metabolic status and the key cell cycle processes leading to bacterial proliferation (e.g., replication). In this light, it is important to investigate the relationship between CCM and replication in intracellular pathogens such as Mycobacterium tuberculosis that utilize compounds ''scavenged'' from the host.
The success of M. tuberculosis as the causative agent of TB lies mostly in its ability to maintain a dormant, non-replicating state for extended periods under unfavorable conditions (reviewed in Gengenbacher and Kaufmann 2012). During the infection of macrophages, M. tuberculosis is exposed to nutrient limitation and thus must re-route its carbon metabolism from sugars to fatty acids and cholesterol (see McKinney et al. 2000;Munoz-Elias and McKinney 2006;Shi et al. 2010). The CCM of M. tuberculosis is known to be a key determinant of its pathogenicity (Rhee et al. 2011), but little is known about the coordination of CCM with replication during the transition to dormancy.
In the present work, we show for the first time that PrpR, a transcription factor that regulates genes encoding enzymes responsible for fatty acid catabolism (Masiewicz et al. 2012), represses dnaA expression during M. tuberculosis growth on propionate as a sole carbon source.

Materials and methods
DNA manipulations, bacterial strains, culture conditions, and protein purification DNA manipulations were carried out using standard protocols (Sambrook et al. 1989). Enzymes were supplied by Fermentas and Promega; [c-32 P]ATP radioisotope was purchased from Hartmann Analytic; and oligonucleotides were synthesized by Genomed (Poland). The utilized bacterial strains and oligonucleotides, as well as their relevant characteristics, are given in Table 1. M. tuberculosis strain H37Rv and its derivatives were cultured aerobically at 37°C in Middlebrook 7H9 broth (Difco, Detroit, MI.) or on 7H10 agar plates supplemented with 10 % OADC (oleic acid-albumindextrose-catalase) and 25 lg/ml kanamycin (when required). For RNA extraction and gene expression measurements, M. tuberculosis strains were cultivated at 37°C either in 7H9 ? OADC medium or in M9 minimal salts medium (Sambrook et al. 1989), containing 2 mM MgSO 4 and 0.1 mM CaCl 2 , with glucose, sodium acetate or sodium propionate (0.5 % each) as a sole carbon source. The fusion protein, 6HisPrpR, was purified using affinity chromatography (HIS-Select HF resin), as described previously by Masiewicz et al. (2012).
The samples were then centrifuged (16,0009g, 15 min, 4°C) and the protein concentrations in the supernatants were determined using the Bradford assay (Bradford 1976). For ''fishing'' experiments, 5 mg of total protein extract (final volume, 1 ml) was incubated with 10 pmol of DNA-immobilized Dynabeads with constant gentle mixing for 1 h at 25°C. The Dynabeads were then washed and eluted with PBS supplemented with increasing NaCl concentrations. The eluates were resolved by 10 % SDS-PAGE, and the gels were stained with Coomassie brilliant blue. Visible protein band was excised from the gel and analyzed by mass spectrometry.

Whole-cell immunoprecipitation assay
The immunoprecipitation assay was performed as described previously Masiewicz et al. 2012). Briefly, M. tuberculosis H37Rv wild type and prpR-deletion strains were cultivated in 7H9 ? OADC broth at 37°C to OD 600 = 0.9 and treated with glutaraldehyde (1 %) for 5 min, and the cross-linked nucleoprotein complexes were immunoprecipitated with anti-PrpR antibodies, as described previously (Masiewicz et al. 2012). Cells treated in parallel without cross-linking served as negative controls. The immunoprecipitated DNA was PCR amplified using primers flanking the dnaA and prpR promoter regions. Two independent experiments of immunoprecipitation assays were carried out.

Results and discussion
PrpR interacts weakly with the dnaA promoter region To identify dnaA promoter (pdnaA)-and oriC regionbinding protein(s) that might control the initiation of replication in M. tuberculosis, we performed streptavidin affinity chromatography using biotinylated DNA fragment (Fig. 1b) containing either the dnaA promoter region (645 bp, Fig. 1a) or the oriC region (557 bp, data not shown) as bait against cell extracts prepared from M. tuberculosis H37Rv cultured to an OD 600 of 0.9 on 7H9 ? OADC medium. We identified a putative dnaA promoter region-binding protein of *60 kDa (Fig. 1b) but no binding protein was identified for the oriC region (presumably due to the fact that frequently the interactions between proteins and oriC are transient). The protein was identified by mass spectrometry (47 % protein sequence coverage) as corresponding to Rv1129c (PrpR), a recently described transcriptional factor that is directly involved in regulating the methylcitrate and glyoxylate pathways (Masiewicz et al. 2012 (Masiewicz et al. 2012). In the present work, using in silico search we identified only one putative PrpR binding sequence in the dnaA promoter; this sequence differs from the high affinity binding sequence by two nucleotides (TTTtCAAc). Interestingly, the identified sequence is located immediately downstream the transcription start site (transcription from the p2dnaA promoter, see Fig. 1a) (Salazar et al. 2003;Li et al. 2010). To verify that PrpR was able to bind the dnaA promoter region in vitro, we performed an EMSA. This binding was confirmed, although the purified PrpR protein exhibited a lower affinity toward the dnaA promoter region than toward a DNA fragment containing the prefect palindrome (e.g., its own promoter, pprpR) (Fig. 2a). A single nucleoprotein PrpR-pdnaA complex was visible in reactions containing 200 nM protein, whereas incubation with higher protein concentrations (500, 1,000 nM) resulted in the formation of two nucleoprotein complexes probably as a result of PrpR dimer binding (PrpR forms stable dimers and possibly trimers, Masiewicz et al. 2012). It has to be noted that the mtrA promoter region, which served as a negative control, was not bound by the PrpR protein (even at highest concentration). Surface plasmon resonance (SPR) analysis confirmed the weak interaction between PrpR and the dnaA promoter. The dissociation of PrpR from the dnaA promoter was much faster than that from the prpR promoter region, suggesting that the complexes between PrpR the dnaA promoter are not stable (data not shown).
To determine whether the PrpR regulator binds the dnaA gene promoter region in M. tuberculosis, we performed glutaraldehyde cross-linking of proteins to DNA in intact cells, followed by selective immunoprecipitation of the protein-DNA complexes with anti PrpR antibodies, as described previously (Masiewicz et al. 2012). Both PrpR-pdnaA and PrpR-pprpR (positive control) complexes were detected, whereas no signal (or faint background signal) was observed  (Salazar et al. 2003;Li et al. 2010) and PrpR binding site are indicated by bend arrow and gray square, respectively. Primers MtrpmH_Fwor, pmtdnaA_Fw2, and MtrpmH_Rv were used to amplify a 645-bp intergenic region for affinity chromatography or a 270-bp dnaA promoter fragment for EMSA analysis (Table 1). B Streptavidin affinity chromatography using a biotinylated rpmH-dnaA intergenic region as bait. The PCR amplified rpmH-dnaA region (10 pmol) was immobilized on Dynabeads and incubated with protein extracts (5 mg) prepared from M. tuberculosis H37Rv grown on 7H9 ? OADC medium (OD 600 = 0.9). The Dynabeads were then washed and eluted with PBS buffer supplemented with increasing concentrations of NaCl. The eluted proteins were resolved by 10 % SDS-PAGE, and visible bands were excised for analysis by mass spectrometry from a prpR-deletion strain or PrpR-pmtrA, which served as negative controls (Fig. 2b).
Collectively, these results indicate that PrpR binds the dnaA promoter region relatively weakly in vitro and within intact M. tuberculosis cells. This suggests that, in addition to the regulation of fatty acid catabolism, PrpR might play a role in regulating the expression of the dnaA gene, which is responsible for the initiation of chromosomal replication.

PrpR represses transcription of dnaA during growth on propionate
We previously demonstrated that the prpR gene is most highly expressed during the growth of M. tuberculosis on propionate, a degradation product of odd-chain-length fatty acids and cholesterol (Masiewicz et al. 2012). The growth of M. tuberculosis on medium containing propioniate as the sole carbon source was impaired in contrast to growth on glucose (Masiewicz et al. 2012). Under such conditions, one would expect that the high expression of PrpR might influence the expression of the dnaA gene. To test this hypothesis, we performed qRT-PCR analysis of dnaA expression using RNA isolated from the wild type and prpR-deleted M. tuberculosis strains growing on M9 minimal medium containing glucose, acetate or propionate as a sole source of carbon. In this experiment, we measured the activity of both p1 and p2 dnaA promoters (using primers complementary to dnaA gene, Table 1). The expression level of dnaA in the DprpR strain growing on propionate was four-fold higher than that in the wild type strain, suggesting that PrpR may act as a repressor of dnaA gene expression (Fig. 3). Notably, we did not observe any significant difference in dnaA expression when these strains were cultivated on minimal medium containing glucose or acetate (Fig. 3). It must be mentioned that in the M.  Table 1) was incubated with a non-specific competitor and increasing amounts of 6HisPrpR, and the nucleoprotein complexes were analyzed on a 4 % polyacrylamide gel; prpDR (containing the perfect PrpR biding site) and the mtrA promoter region served as positive and negative controls, respectively. Arrows along horizontally orientated bars inserted below each figure indicate number of PrpR boxes in corresponding DNA fragment. Digits above arrows indicate number of nucleotide mismatches from the consensus (TTTGCAAA) within each PrpR box. B In vivo immunoprecipitation: PrpR-DNA complexes cross-linked with glutaraldehyde were immunoprecipitated with anti-6HisPrpR polyclonal antibodies (sample 1). PCR was carried out with the following primer pairs: p1129_Fw and p1129_Rv (pprpDR, positive control); pmtdnaA_Fw2 and MtrpmH_Rv (pdnaA); and pmtrA_Fw and pmtrA_Rv (pmtrA, negative control). A second negative control (2) consisted of extracted DNA subjected to immunoprecipitation without cross-linking. Positives controls (?) were also performed using templates obtained from strains subjected only to cross-linking (3) or total DNA extracted from the cells (4) tuberculosis DprpR complemented strain the repression of dnaA gene on propionate was not completely restored ( Fig. 3) (despite the fact that the expression level of prpR gene was not altered compared to the wild type strain, data not shown). However, the complementing copy of the prpR gene was introduced on the M. tuberculosis chromosome together with its promoter region containing strong (TTTGCAAA) PrpR boxes (in addition to the PrpR binding sites present in the native locus-a common prpD and prpR promoter region), which presumably titrated PrpR protein away from the dnaA promoter region what could result in higher that in the wild type dnaA expression. It is noteworthy that the expression of prpD gene (activated by PrpR) in the M. tuberculosis DprpR complemented strain did also not reach the level measured in the wild type strain (Masiewicz et al. 2012).
The relatively low affinity of PrpR towards the dnaA promoter is presumably compensated by the elevated level of the protein in M. tuberculosis during growth on propionate. It is worth mentioning that M. tuberculosis utilizes fatty acids and cholesterol as a major carbon source in host macrophages during infection (Gengenbacher and Kaufmann 2012;Rhee et al. 2011). Thus, the data presented here and in our earlier study (Masiewicz et al. 2012) suggest that PrpR might play a dual role in media containing fatty acids and cholesterol as the sole carbon source, acting both as an activator of genes involved in fatty acid catabolism including its own gene and as a repressor of the dnaA gene (Fig. 4). It is worth mentioning that besides PrpR there are also DnaA and MtrA proteins that may be directly involved in negative regulation of dnaA gene expression in Mycobacterium by binding dnaA promoter region (Salazar et al. 2003;Fol et al. 2006;Nguyen et al. 2010). It is noteworthy to point out that the MtrA is supposed also to negatively influence DnaA oligomerization along oriC and thus chromosome replication by binding DnaA boxes within oriC (Rajagoplan et al. 2010).
Tubercle bacilli can survive for decades in humans or hypoxic-and nutrient-depleted media (Hett and Rubin 2008). However, surprisingly little is known Fig. 3 PrpR represses dnaA gene expression during growth on propionate. Total RNA was extracted from cultures grown in M9 minimal medium containing glucose, acetate or propionate (0.5 %) as the carbon source. PCR was carried out with primers RT_MtdnaA_Fw and RT_MtdnaA_Rv. The mRNA levels of dnaA were normalized with respect to that of the constitutively expressed housekeeping gene, sigA. Means were calculated from three independent experiments and three determinations per experiment. The error bars indicate standard deviations of triplicate samples. Statistical significance was calculated by the Student's t test Fig. 4 PrpR coordinates fatty acid catabolism with the initiation of chromosomal replication. In M. tuberculosis, PrpR activates the expression of genes involved in the methylcitrate and glyoxylate cycles, and also inhibits dnaA gene expression. Perpendicular lines indicate negative regulation. Adapted from Masiewicz et al. (2012) about the regulatory mechanisms responsible for the ability of these bacilli to enter and exit dormancy. Based on the novel findings presented herein and on our earlier study (Masiewicz et al. 2012) we hypothesize that PrpR could be an important element of the complex regulatory system(s) required for the persistence of TB within macrophages, controlling both the catabolism of host-derived fatty acids and the initiation of chromosomal replication (Fig. 4).