Dagmara Jakimowicz

Bacterial replication initiator DnaA. Rules for DnaA binding and rolesof DnaA in origin unwinding and helicase loading

We review the processes leading to the structural modifications required for the initiation of replication in Escherichia coli, the conversion of the initial complex to the open complex, loading of helicase, and the assembly of two replication forks. Rules for the binding of DnaA to its binding sites are derived, and the properties of ATP-DnaA are described. We provide new data on cooperative interaction and dimerization of DnaA proteins of E. coli, Streptomyces and Thermus thermophilus, and on the stoichiometry of DnaA-oriC complexes of E. coli.

Cell division and DNA segregation in Streptomyces: how to build a septum in the middle of nowhere?

Streptomycetes are antibiotic‐producing filamentous microorganisms that have a mycelial life style. In many ways streptomycetes are the odd ones out in terms of cell division. While the basic components of the cell division machinery are similar to those found in rod‐shaped bacteria such as Escherichia coli and Bacillus subtilis, many aspects of the control of cell division and its co‐ordination with chromosome segregation are remarkably different. The rather astonishing fact that cell division is not essential for growth makes these bacteria unique. The fundamental difference between the cross‐walls produced during normal growth and sporulation septa formed in aerial hyphae, and the role of the divisome in their formation are discussed. We then take a closer look at the way septum site localization is regulated in the long and multinucleoid Streptomyces hyphae, with particular focus on actinomycete‐specific proteins and the role of nucleoid segregation and condensation.

ParA of Mycobacterium smegmatis co‐ordinates chromosome segregation with the cell cycle and interacts with the polar growth determinant DivIVA

Mycobacteria are among the clinically most important pathogens, but still not much is known about the mechanisms of their cell cycle control. Previous studies suggested that the genes encoding ParA and ParB (ATPase and DNA binding protein, respectively, required for active chromosome segregation) may be essential in Mycobacterium tuberculosis. Further research has demonstrated that a Mycobacterium smegmatis parB deletion mutant was viable but exhibited a chromosome segregation defect. Here, we address the question if ParA is required for the growth of M. smegmatis, and which cell cycle processes it affects. Our data show that parA may be deleted, but its deletion leads to growth inhibition and severe disturbances of chromosome segregation and septum positioning. Similar defects are also caused by ParA overproduction. EGFP–ParA localizes as pole‐associated complexes connected with a patch of fluorescence accompanying two ParB complexes. Observed aberrations in the number and positioning of ParB complexes in the parA deletion mutant indicate that ParA is required for the proper localization of the ParB complexes. Furthermore, it is shown that ParA colocalizes and interacts with the polar growth determinant Wag31 (DivIVA homologue). Our results demonstrate that mycobacterial ParA mediates chromosome segregation and co‐ordinates it with cell division and elongation.

Developmental Control of a parAB Promoter Leads to Formation of Sporulation-Associated ParB Complexes in Streptomyces coelicolor

The Streptomyces coelicolor partitioning protein ParB binds to numerous parS sites in the oriC-proximal part of the linear chromosome. ParB binding results in the formation of large complexes, which behave differentially during the complex life cycle (D. Jakimowicz, B. Gust, J. Zakrzewska-Czerwinska, and K. F. Chater, J. Bacteriol. 187:3572-3580, 2005). Here we have analyzed the transcriptional regulation that underpins this developmentally specific behavior. Analysis of promoter mutations showed that the irregularly spaced complexes present in vegetative hyphae are dependent on the constitutive parABp(1) promoter, while sporulation-specific induction of the promoter parABp(2) is required for the assembly of arrays of ParB complexes in aerial hyphae and thus is necessary for efficient chromosome segregation. Expression from parABp(2) depended absolutely on two sporulation regulatory genes, whiA and whiB, and partially on two others, whiH and whiI, all four of which are needed for sporulation septation. Because of this pattern of dependence, we investigated the transcription of these four whi genes in whiA and whiB mutants, revealing significant regulatory interplay between whiA and whiB. A strain in which sporulation septation (but not vegetative septation) was blocked by mutation of a sporulation-specific promoter of ftsZ showed close to wild-type induction of parABp(2) and formed fairly regular ParB-enhanced green fluorescent protein foci in aerial hyphae, ruling out strong morphological coupling or checkpoint regulation between septation and DNA partitioning during sporulation. A model for developmental regulation of parABp(2) expression is presented.

Developmental-Stage-Specific Assembly of ParB Complexes in Streptomyces coelicolor Hyphae

In Streptomyces coelicolor ParB is required for accurate chromosome partitioning during sporulation. Using a functional ParB-enhanced green fluorescent protein fusion, we observed bright tip-associated foci and other weaker, irregular foci in S. coelicolor vegetative hyphae. In contrast, in aerial hyphae regularly spaced bright foci accompanied sporulation-associated chromosome condensation and septation.

The level of AdpA directly affects expression of developmental genes in Streptomyces coelicolor

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.

Dynamic interplay of ParA with the polarity protein, Scy, coordinates the growth with chromosome segregation in Streptomyces coelicolor.

Prior to bacterial cell division, the ATP-dependent polymerization of the cytoskeletal protein, ParA, positions the newly replicated origin-proximal region of the chromosome by interacting with ParB complexes assembled on parS sites located close to the origin. During the formation of unigenomic spores from multi-genomic aerial hyphae compartments of Streptomyces coelicolor, ParA is developmentally triggered to form filaments along the hyphae; this promotes the accurate and synchronized segregation of tens of chromosomes into prespore compartments. Here, we show that in addition to being a segregation protein, ParA also interacts with the polarity protein, Scy, which is a component of the tip-organizing centre that controls tip growth. Scy recruits ParA to the hyphal tips and regulates ParA polymerization. These results are supported by the phenotype of a strain with a mutant form of ParA that uncouples ParA polymerization from Scy. We suggest that the ParA–Scy interaction coordinates the transition from hyphal elongation to sporulation.

Cluster of DnaA Boxes Involved in Regulation of Streptomyces Chromosome Replication: from In Silico to In Vivo Studies

In Streptomyces coelicolor, replication is initiated by the DnaA protein in the centrally located oriC region and proceeds bidirectionally until the replication forks reach the ends of the linear chromosome. We identified three clusters of DnaA boxes (H69, H24, and D78) which are in a relatively short segment of the chromosome centered on the oriC region. Of the clusters analyzed, D78 exhibited the highest affinity for the DnaA protein; the affinity of DnaA for the D78 cluster was about eightfold higher than the affinity for oriC. The high-affinity DnaA boxes appear to be involved in the control of chromosome replication. Deletion of D78 resulted in more frequent chromosome replication (an elevated ratio of origins to chromosome ends was observed) and activated aerial mycelium formation, leading to earlier colony maturation. In contrast, extra copies of D78 (delivered on a plasmid) caused slow colony growth, presumably because of a reduction in the frequency of initiation of chromosome replication. This suggests that the number of high-affinity DnaA boxes is relatively constant in hyphal compartments and that deletion of D78 therefore permits an increased copy number of either the chromosomal origin region or a plasmid harboring the D78 cluster. This system conceivably influences the timing of decisions to initiate aerial mycelial formation and sporulation.

Choreography of the Mycobacterium Replication Machinery during the Cell Cycle

ABSTRACT 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. 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, key regulator for morphological differentiation regulates bacterial chromosome replication

AdpA, one of the most pleiotropic transcription regulators in bacteria, controls expression of several dozen genes during Streptomyces differentiation. Here, we report a novel function for the AdpA protein: inhibitor of chromosome replication at the initiation stage. AdpA specifically recognizes the 5′ region of the Streptomyces coelicolor replication origin (oriC). Our in vitro results show that binding of AdpA protein decreased access of initiator protein (DnaA) to the oriC region. We also found that mutation of AdpA-binding sequences increased the accessibility of oriC to DnaA, which led to more frequent replication and acceleration of Streptomyces differentiation (at the stage of aerial hyphae formation). Moreover, we also provide evidence that AdpA and DnaA proteins compete for oriC binding in an ATP-dependent manner, with low ATP levels causing preferential binding of AdpA, and high ATP levels causing dissociation of AdpA and association of DnaA. This would be consistent with a role for ATP levels in determining when aerial hyphae emerge.