Replication of the bacterial chromosome initiates at a single origin of replication that is called oriC. This occurs via the concerted action of numerous proteins, including DnaA, which acts as an initiator. The origin sequences vary across species, but all bacterial oriCs contain the information necessary to guide assembly of the DnaA protein complex at oriC, triggering the unwinding of DNA and the beginning of replication. The requisite information is encoded in the unique arrangement of specific sequences called DnaA boxes, which form a framework for DnaA binding and assembly. Other crucial sequences of bacterial origin include DNA unwinding element (DUE, which designates the site at which oriC melts under the influence of DnaA) and binding sites for additional proteins that positively or negatively regulate the initiation process. In this review, we summarize our current knowledge and understanding of the information encoded in bacterial origins of chromosomal replication, particularly in the context of replication initiation and its regulation. We show that oriC encoded instructions allow not only for initiation but also for precise regulation of replication initiation and coordination of chromosomal replication with the cell cycle (also in response to environmental signals). We focus on E. coli, and then expand our discussion to include several other microorganisms in which additional regulatory proteins have been recently shown to be involved in coordinating replication initiation to other cellular processes (e.g., Bacillus, Caulobacter, Helicobacter, Mycobacterium, and Streptomyces). We discuss diversity of bacterial oriC regions with the main focus on roles of individual DNA recognition sequences at oriC in binding the initiator and regulatory proteins as well as the overall impact of these proteins on the formation of initiation complex.

Mycobacterium tuberculosis, the pathogen that causes tuberculosis, presumably utilizes fatty acids as a major carbon source during infection within the host. Metabolism of even-chain-length fatty acids yields acetyl-CoA, whereas metabolism of odd-chain-length fatty acids additionally yields propionyl-CoA. Utilization of these compounds by tubercle bacilli requires functional glyoxylate and methylcitrate cycles, respectively. Enzymes involved in both pathways are essential for M. tuberculosis viability and persistence during growth on fatty acids. However, little is known about regulatory factors responsible for adjusting the expression of genes encoding these enzymes to particular growth conditions. Here, we characterized the novel role of PrpR as a transcription factor that is directly involved in regulating genes encoding the key enzymes of methylcitrate (methylcitrate dehydratase [PrpD] and methylcitrate synthase [PrpC]) and glyoxylate (isocitrate lyase [Icl1]) cycles. Using cell-free systems and intact cells, we demonstrated an interaction of PrpR protein with prpDC and icl1 promoter regions and identified a consensus sequence recognized by PrpR. Moreover, we showed that an M. tuberculosis prpR-deletion strain exhibits impaired growth in vitro on propionate as the sole carbon source. Real-time quantitative reverse transcription-polymerase chain reaction confirmed that PrpR acts as a transcriptional activator of prpDC and icl1 genes when propionate is the main carbon source. Similar results were also obtained for a non-pathogenic Mycobacterium smegmatis strain. Additionally, we found that ramB, a prpR paralog that controls the glyoxylate cycle, is negatively regulated by PrpR. Our data demonstrate that PrpR is essential for the utilization of odd-chain-length fatty acids by tubercle bacilli. Since PrpR also acts as a ramB repressor, our findings suggest that it plays a key role in regulating expression of enzymes involved in both glyoxylate and methylcitrate pathways.

In bacteria, chromosomal DNA must be efficiently compacted to fit inside the small cell compartment while remaining available for the proteins involved in replication, segregation, and transcription. Among the nucleoid-associated proteins (NAPs) responsible for maintaining this highly organized and yet dynamic chromosome structure, the HU protein is one of the most conserved and highly abundant. HupB, a homologue of HU, was recently identified in mycobacteria. This intriguing mycobacterial NAP is composed of two domains: an N-terminal domain that resembles bacterial HU, and a long and distinctive C-terminal domain that contains several PAKK/KAAK motifs, which are characteristic of the H1/H5 family of eukaryotic histones. In this study, we analyzed the in vivo binding of HupB on the chromosome scale. By using PALM (photoactivated localization microscopy) and ChIP-Seq (chromatin immunoprecipitation followed by deep sequencing), we observed that the C-terminal domain is indispensable for the association of HupB with the nucleoid. Strikingly, the in vivo binding of HupB displayed a bias from the origin ( oriC ) to the terminus ( ter ) of the mycobacterial chromosome (numbers of binding sites decreased toward ter ). We hypothesized that this binding mode reflects a role for HupB in organizing newly replicated oriC regions. Thus, HupB may be involved in coordinating replication with chromosome segregation. IMPORTANCE We currently know little about the organization of the mycobacterial chromosome and its dynamics during the cell cycle. Among the mycobacterial nucleoid-associated proteins (NAPs) responsible for chromosome organization and dynamics, HupB is one of the most intriguing. It contains a long and distinctive C-terminal domain that harbors several PAKK/KAAK motifs, which are characteristic of the eukaryotic histone H1/H5 proteins. The HupB protein is also known to be crucial for the survival of tubercle bacilli during infection. Here, we provide in vivo experimental evidence showing that the C-terminal domain of HupB is crucial for its DNA binding. Our results suggest that HupB may be involved in organizing newly replicated regions and could help coordinate chromosome replication with segregation. Given that tuberculosis (TB) remains a serious worldwide health problem (10.4 million new TB cases were diagnosed in 2015, according to WHO) and new multidrug-resistant Mycobacterium tuberculosis strains are continually emerging, further studies of the biological function of HupB are needed to determine if this protein could be a prospect for novel antimicrobial drug development.

It has recently been demonstrated that bacterial chromosomes are highly organized, with specific positioning of the replication initiation region. Moreover, the positioning of the replication machinery (replisome) has been shown to be variable and dependent on species-specific cell cycle features. Here, we analyzed replisome positions in Mycobacterium smegmatis , a slow-growing bacterium that exhibits characteristic asymmetric polar cell extension. Time-lapse fluorescence microscopy analyses revealed that the replisome is slightly off-center in mycobacterial cells, a feature that is likely correlated with the asymmetric growth of Mycobacterium cell poles. Estimates of the timing of chromosome replication in relation to the cell cycle, as well as cell division and chromosome segregation events, revealed that chromosomal origin-of-replication ( oriC ) regions segregate soon after the start of replication. Moreover, our data demonstrate that organization of the chromosome by ParB determines the replisome choreography. IMPORTANCE Despite significant progress in elucidating the basic processes of bacterial chromosome replication and segregation, understanding of chromosome dynamics during the mycobacterial cell cycle remains incomplete. Here, we provide in vivo experimental evidence that replisomes in Mycobacterium smegmatis are highly dynamic, frequently splitting into two distinct replication forks. However, unlike in Escherichia coli , the forks do not segregate toward opposite cell poles but remain in relatively close proximity. In addition, we show that replication cycles do not overlap. Finally, our data suggest that ParB participates in the positioning of newly born replisomes in M. smegmatis cells. The present results broaden our understanding of chromosome segregation in slow-growing bacteria. In view of the complexity of the mycobacterial cell cycle, especially for pathogenic representatives of the genus, understanding the mechanisms and factors that affect chromosome dynamics will facilitate the identification of novel antimicrobial factors.

AdpA is a key regulator of morphological differentiation in Streptomyces . In contrast to Streptomyces griseus , relatively little is known about AdpA protein functions in Streptomyces coelicolor . Here, we report for the first time the translation accumulation profile of the S. coelicolor adpA ( adpA _Sc ) gene; the level of S. coelicolor AdpA (AdpA _Sc ) increased, reaching a maximum in the early stage of aerial mycelium formation (after 36 h), and remained relatively stable for the next several hours (48 to 60 h), and then the signal intensity decreased considerably. AdpA _Sc specifically binds the adpA _Sc promoter region in vitro and in vivo , suggesting that its expression is autoregulated; surprisingly, in contrast to S. griseus , the protein presumably acts as a transcriptional activator. We also demonstrate a direct influence of AdpA _Sc on the expression of several genes whose products play key roles in the differentiation of S. coelicolor : STI, a protease inhibitor; RamR, an atypical response regulator that itself activates expression of the genes for a small modified peptide that is required for aerial growth; and ClpP1, an ATP-dependent protease. The diverse influence of AdpA _Sc protein on the expression of the analyzed genes presumably results mainly from different affinities of AdpA _Sc protein to individual promoters.

The primary role of the bacterial protein DnaA is to initiate chromosomal replication. The DnaA protein binds to DNA at the origin of chromosomal replication (oriC) and assembles into a filament that unwinds double-stranded DNA. Through interaction with various other proteins, DnaA also controls the frequency and/or timing of chromosomal replication at the initiation step. Escherichia coli DnaA also recruits DnaB helicase, which is present in unwound single-stranded DNA and in turn recruits other protein machinery for replication. Additionally, DnaA regulates the expression of certain genes in E. coli and a few other species. Acting as a multifunctional factor, DnaA is composed of four domains that have distinct, mutually dependent roles. For example, C-terminal domain IV interacts with double-stranded DnaA boxes. Domain III drives ATP-dependent oligomerization, allowing the protein to form a filament that unwinds DNA and subsequently binds to and stabilizes single-stranded DNA in the initial replication bubble; this domain also interacts with multiple proteins that control oligomerization. Domain II constitutes a flexible linker between C-terminal domains III–IV and N-terminal domain I, which mediates intermolecular interactions between DnaA and binds to other proteins that affect DnaA activity and/or formation of the initiation complex. Of these four domains, the role of the N-terminus (domains I–II) in the assembly of the initiation complex is the least understood and appears to be the most species-dependent region of the protein. Thus, in this review, we focus on the function of the N-terminus of DnaA in orisome formation and the regulation of its activity in the initiation complex in different bacteria.