Jolanta Zakrzewska-Czerwińska

Functional domains of DnaA proteins

Functional domains of the initiator protein DnaA of Escherichia coli have been defined. Domain 1, amino acids 1-86, is involved in oligomerization and in interaction with DnaB. Domain 2, aa 87-134, constitutes a flexible loop. Domain 3, aa 135-373, contains the binding site for ATP or ADP, the ATPase function, a second interaction site with DnaB, and is required for local DNA unwinding. Domain 4 is required and sufficient for specific binding to DNA. We show that there are three different types of cooperative interactions during the DNA binding of DnaA proteins from E. coli, Streptomyces lividans, and Thermus thermophilus: i) binding to distant binding sites; ii) binding to closely spaced binding sites; and iii) binding to non-canonical binding sites.

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.

Alignment of multiple chromosomes along helical ParA scaffolding in sporulating Streptomyces hyphae

The dynamic, mitosis‐like segregation of bacterial chromosomes and plasmids often involves proteins of the ParA (ATPase) and ParB (DNA‐binding protein) families. The conversion of multigenomic aerial hyphae of the mycelial organism Streptomyces coelicolor into chains of unigenomic spores requires the synchronous segregation of multiple chromosomes, providing an unusual context for chromosome segregation. Correct spatial organization of the oriC‐proximal region prior to septum formation is achieved by the assembly of ParB into segregation complexes (Jakimowicz et al., 2005; J Bacteriol 187: 3572–3580). Here, we focus on the contribution of ParA to sporulation‐associated chromosome segregation. Elimination of ParA strongly affects not only chromosome segregation but also septation. In wild type hyphae about to undergo sporulation, immunostained ParA was observed as a stretched double‐helical filament, which accompanies the formation of ParB foci. We show that ParA mediates efficient assembly of ParB complexes in vivo and in vitro, and that ATP binding is crucial for ParA dimerization and interaction with ParB but not for ParA localization in vivo. We suggest that S. coelicolor ParA provides scaffolding for proper distribution of ParB complexes and consequently controls synchronized segregation of several dozens of chromosomes, possibly mediating a segregation and septation checkpoint.

Classification of thermophilic streptomycetes, including the description of Streptomyces thermoalcalitolerans sp. nov.

A polyphasic taxonomic study was undertaken to clarify relationships within and between representative thermophilic alkalitolerant streptomycetes isolated from soil and appropriate marker strains. The resultant data, notably those from DNA-DNA relatedness studies, support the taxonomic integrity of the validly described species Streptomyces thermodiastaticus, Streptomyces thermoviolaceus and Streptomyces thermovulgaris. However, the genotypic and phenotypic data clearly show that Streptomyces thermonitrificans Desai and Dhala 1967 and S. thermovulgaris (Henssen 1957) Goodfellow et al. 1987 represent a single species. On the basis of priority, S. thermonitrificans is a later subjective synonym of S. thermovulgaris. Similarly, 10 out of the 11 representative thermophilic alkalitolerant isolates had a combination of properties consistent with their classification as S. thermovulgaris. The remaining thermophilic alkalitolerant isolate, Streptomyces strain TA56, merited species status. The name Streptomyces thermoalcalitolerans sp. nov. is proposed for this strain. A neutrophilic thermophilic isolate, Streptomyces strain NAR85, was identified as S. thermodiastaticus.

Architecture of bacterial replication initiation complexes : orisomes from four unrelated bacteria

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.

Rhodium(III) complexes with polypyridyls and pyrazole and their antitumor activity

Abstract Synthesis and properties of rhodium complexes with nitrogen ligands [RhCl 2 (Hpz) 4 ][RhCl 4 (Hpz) 2 ] ( 1 ), [RhCl 3 (tpy)] ( 2 ), [RhCl 3 (tpta)]·H 2 O ( 3 ) and [Rh(tpy) 2 (Him)]Cl 3 ·3H 2 O ( 4 ), have been described. X-ray structures of complexes 1 – 3 have been determined. IR, UV–Vis and 1 H NMR spectra of the complexes have been discussed. Cytostatic activity of the complexes against HCV29T tumor cells increases in the series: 1 3 2 4 . The cytostatic activity of complex 4 is greater than that of cisplatin. Interaction of the complexes with DNA has been investigated.

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.

oriC-encoded instructions for the initiation of bacterial chromosome replication

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 Escherichia 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.

HobA – a novel protein involved in initiation of chromosomal replication in Helicobacter pylori

Replication of the bacterial chromosome is initiated by the binding of the DnaA protein to a unique DNA region, called oriC. Many regulatory factors in numerous species act by controlling the ability of DnaA to bind and unwind DNA, but the Helicobacter pylori genome does not contain homologues to any of these factors. Here, we describe HobA, a novel protein essential for initiation of H. pylori chromosome replication, which is conserved among, and unique to, epsilon proteobacteria. We demonstrate that HobA interacts specifically via DnaA with the oriC–DnaA complex. We postulate that HobA is essential for correct formation and stabilization of the orisome by facilitating the spatial positioning of DnaA at oriC. Consistent with its function, overexpression of hobA had no effect on growth of H. pylori, whereas depletion of HobA led to growth arrest and failure to initiate replication. In conclusion, HobA may be the first identified of a new group of initiation factors common to epsilon proteobacteria.

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.