Bacterial chromosomes (though not Escherichia coli and some other c-proteobacterial chromosomes) contain parS sequences and parAB genes encoding partitioning proteins, i.e. ParA (ATPase) and ParB (DNA-binding proteins) that are components of the segregation machinery. Here, mycobacterial parABS elements were characterized for the first time. parAB genes are not essential in Mycobacterium smegmatis; however, elimination or overexpression of ParB protein causes growth inhibition. Deletion of parB also leads to a rather severe chromosome segregation defect: up to 10 % of the cells were anucleate. Mycobacterial ParB protein uses three oriC-proximal parS sequences as targets to organize the origin region into a compact nucleoprotein complex. Formation of such a complex involves ParB-ParB interactions and is assisted by ParA protein. INTRODUCTIONTuberculosis (TB) is the world's most common disease caused by an infectious organism, Mycobacterium tuberculosis (DeAngelis & Flanagin, 2005; and see http:// www.who.int/topics/tuberculosis/en/). Due to the spread of multi-drug-resistant strains of M. tuberculosis and a synergy with HIV, the TB epidemic is growing and becoming more dangerous in both developing and industrialized countries. A unique feature of M. tuberculosis is its ability to maintain a dormant, non-replicating state for extended periods of time under unfavourable conditions. The mechanism (or mechanisms) by which this bacterium shifts to a dormant state and reverts to active growth is not well understood (Hampshire et al., 2004;Gó mez & Smith, 2000;Wayne & Hayes, 1996: Wayne & Sohaskey, 2001. Knowledge regarding the steps of the mycobacterial cell cycle (replication, chromosome segregation and cell division) seems to be critical for understanding the mechanisms that are responsible for the transition from an active to a non-replicative persistent state (and vice versa) of pathogenic mycobacteria, particularly M. tuberculosis. While initiation of chromosome replication Qin et al., 1999;Rajagopalan et al., 1995;Zawilak et al., 2004) and cell division (FtsZ ring formation) (Chauhan et al., 2006; Dziadek et al., 2002Dziadek et al., , 2003Huang et al., 2007;Rajagopalan et al., 2005) are relatively well studied in mycobacteria, nothing is known about the segregation of chromosomes in these bacteria.Bacterial chromosome segregation has been recently found to be an active and complex process closely coupled with replication (see Bartosik & Jagura-Burdzy, 2005;Errington et al., 2005;Hayes & Barilla, 2006; Leonard et al., 2005 for reviews). In bacteria studied to date, the newly synthesized origin (oriC) regions undergo a symmetric or asymmetric segregation process; two copies of the duplicated oriC regions migrate from the cell centre toward opposite cell poles, i.e. to the 1/4 and 3/4 positions (Escherichia coli, Bacillus subtilis, Vibro cholerae chromosome II), or one copy of the newly synthesized origins remains at the pole while the other copy migrates to the opposite pole Abbreviations: EMSA, electrophoretic mo...

Bacterial chromosome replication is mediated by single initiator protein, DnaA, that interacts specifically with multiple DnaA boxes located within the origin (oriC). We compared the architecture of the DnaA-origin complexes of evolutionarily distantly related eubacteria: two Gram-negative organisms, Escherichia coli and Helicobacter pylori, and two Gram-positive organisms, Mycobacterium tuberculosis and Streptomyces coelicolor. Their origins vary in size (from approx. 200 to 1000 bp) and number of DnaA boxes (from 5 to 19). The results indicate that: (i) different DnaA proteins exhibit various affinities toward single DnaA boxes, (ii) spatial arrangement of two DnaA boxes is crucial for the H. pylori and S. coelicolor DnaA proteins, but not for E. coli and M. tuberculosis proteins, and (iii) the oriC regions are optimally adjusted to their cognate DnaA proteins. The primary functions of multiple DnaA boxes are to determine the positioning and order of assembly of the DnaA molecules. Gradual transition from the sequence-specific binding of the DnaA protein to binding through co-operative protein-protein interactions seems to be a common conserved strategy to generate oligomeric initiator complexes bound to multiple sites within the chromosomal, plasmid and virial origins.

Using a functional fusion of DnaN to enhanced green fluorescent protein, we examined the subcellular localization of the replisome machinery in the vegetative mycelium and aerial mycelium of the multinucleoid organism Streptomyces coelicolor. Chromosome replication took place in many compartments of both types of hypha, with the apical compartments of the aerial mycelium exhibiting the highest replication activity. Within a single compartment, the number of "current" ongoing DNA replications was lower than the expected chromosome number, and the appearance of fluorescent foci was often heterogeneous, indicating that this process is asynchronous within compartments and that only selected chromosomes undergo replication.Streptomycetes, which are gram-positive soil bacteria known for their ability to produce many valuable antibiotics and other secondary metabolites, are among the most striking examples of multicellular bacteria. Their hyphae grow by tip extension, forming a branched vegetative mycelium in which septation occurs at some distance from the growing hyphal tips (4, 23). Compartments of the vegetative hyphae contain several uncondensed copies of the large (8-to 9-Mbp), linear chromosome (2, 10). During further development, Streptomyces colonies form an aerial mycelium, in which long chains of exospores are formed. Rapidly growing aerial hyphae may contain up to 50 uncondensed chromosomes in one tip compartment. After an aerial hypha has stopped growing, a large number of FtsZ rings form a regular ladder in the long tip compartment, giving rise to sporulation septa that delimit prespore compartments (25). At this stage the chromosomes condense and are segregated into unigenomic prespore compartments. The prespore compartments eventually metamorphose into chains of physically separate spores.The model organism Streptomyces coelicolor A3 (2) is genetically the best-studied streptomycete (1). Although the S. coelicolor life cycle and its regulators have been extensively investigated, little is known about the dynamics of chromosome replication during this complex process. So far, Streptomyces chromosome replication has been studied only in the young vegetative mycelium using pulse-labeling with [ 3 H]thymidine (17), but the localization of replisome machinery in a single compartment has not been addressed. Visualization of a replisome(s) within single compartments of both vegetative and aerial hyphae should shed some light on important features of the multinucleoid prokaryotic cell.The use of replication proteins tagged with green fluorescent protein (GFP) has provided an opportunity for direct observation of chromosome dynamics in vivo in a single bacterial cell (11,19). Visualization of the replicating machinery has been achieved by fusing GFP to various DNA polymerase III holoenzyme subunits, including ␣ (PolC) (19) and (DnaX) (11) in Bacillus subtilis, (HolB) and ␦Ј (HolC) in Caulobacter crescentus (13), and ␤ (DnaN) (15) in Escherichia coli. Recently, systematic localization of over 100 proteins fused t...