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Dendritic cells (DCs) play a key-role in the immune response against intracellular bacterial pathogens, including mycobacteria. Monocyte-derived dendritic cells (MoDCs) are considered to behave as inflammatory cell populations. Different immunomagnetic methods (positive and negative) can be used to purify monocytes before their in vitro differentiation and their culture behavior can be expected to be different. In this study we evaluated the reactivity of two dendritic cell populations towards the Bacillus Calmette-Guérin (BCG) antigen. Monocytes were obtained from the blood of healthy donors, using positive and negative immunomagnetic separation methods. The expression of DC-SIGN, CD86, CD80, HLA-DR and CD40 on MoDCs was estimated by flow cytometry. The level of IL-12p70, IL-10 and TNF-α was measured by ELISA. Neither of the tested methods affected the surface marker expression of DCs. No significant alteration in immunological response, measured by cytokine production, was noted either. After BCG stimulation, the absence of IL-12, but the IL-23 production was observed in both cell preparations. Positive and negative magnetic separation methods are effective techniques to optimize the preparation of monocytes as the source of MoDCs for potential clinical application.

FtsZ, a bacterial homolog of tubulin, forms a structural element called the FtsZ ring (Z ring) at the predivisional midcell site and sets up a scaffold for the assembly of other cell division proteins. The genetic aspects of FtsZ-catalyzed cell division and its assembly dynamics in Mycobacterium tuberculosis are unknown. Here, with an M. tuberculosis strain containing [FtsZ.sub.TB]; tagged with green fluorescent protein as the sole source of FtsZ, we examined FtsZ structures under various growth conditions. We found that midcell Z rings are present in approximately 11% of actively growing cells, suggesting that the low frequency of Z rings is reflective of their slow growth rate. Next, we showed that SRI-3072, a reported [FtsZ.sub.TB] inhibitor, disrupted Z-ring assembly and inhibited cell division and growth of M. tuberculosis. We also showed that M. tuberculosis cells grown in macrophages are filamentous and that only a small fraction had midcell Z rings. The majority of filamentous cells contained nonring, spiral-like FtsZ structures along their entire length. The levels of FtsZ in bacteria grown in macrophages or in broth were comparable, suggesting that Z-ring formation at midcell sites was compromised during intracellular growth. Our results suggest that the intraphagosomal milieu alters the expression of M. tuberculosis genes affecting Z-ring formation and thereby cell division.

The research study which was aimed at characterizing the chromosomal origin of replication (oriC) region of Mycobacterium smegmatis (M smegtatis) was conducted. The methods used were the recombinant DNA techniques, analysis of oriC activity, construction of oriC plasmids, site-directed mutagenesis, oriC copy number determination and stability experiments. Results suggested that M smegtatis oriC replication could involve interactions of DnaA protein with DnaA boxes and the DnaA boxes and AT nucleotide rich cluster are important to oriC activity.

The products of the recF, recO, and recR genes are thought to interact and assist RecA in the utilization of single-stranded DNA precomplexed with single-stranded DNA binding protein (Ssb) during synapsis. Using immunoprecipitation, size-exclusion chromatography, and Ssb protein affinity chromatography in the absence of any nucleotide cofactors, we have obtained the following results: (i) RecF interacts with RecO, (ii) RecF interacts with RecR in the presence of RecO to form a complex consisting of RecF, RecO, and RecR (RecF-RecO-RecR); (iii) RecF interacts with Ssb protein in the presence of RecO. These data suggested that RecO mediates the interactions of RecF protein with RecR and with Ssb proteins. Incubation of RecF, RecO, RecR, and Ssb proteins resulted in the formation of RecF-RecO-Ssb complexes; i.e., RecR was excluded. Preincubation of RecF, RecO, and RecR proteins prior to addition of Ssb protein resulted in the formation of complexes consisting of RecF, RecO, RecR, and Ssb proteins. These data suggest that one role of RecF is to stabilize the interaction of RecR with RecO in the presence of Ssb protein. Finally, we found that interactions of RecF with RecO are lost in the presence of ATP. We discuss these results to explain how the RecF-RecO-RecR complex functions as an anti-Ssb factor.

The Escherichia coli RecF protein preferentially binds gapped DNA (gDNA) when adenosine (gamma-thio) triphosphate is present. In the order of RecF binding preference, gDNA is followed by double-stranded DNA (dsDNA), while single-stranded DNA (ssDNA) is the least preferred. Considering that gDNA has components found in both ssDNA and dsDNA, it appears that ATP confers binding specificity to dsDNA-ssDNA junctions whenever RecF interacts with DNA.

A 4-kb chromosomal DNA fragment from Mycobacterium smegmatis that has a dnaA gene was studied to analyze mycobacteria's method of starting replication. Analysis of the nucleotide sequence showed homologies with the rpmH gene, which encodes the ribosomal protein L34, the dnaA gene, which encodes the replication initiator protein DnaA, and the 5' end of the dnaN gene, which encodes the beta subunit of DNA polymerase III.

A study was undertaken to identify the cofactors interacting with RecF protein involved in DNA recombination and repair. Using 8-azido-ATP, a photo-affinity analog of DNA, it was shown that RecF binds ATP. Photo-cross-linking in the presence of DNA is specific and both ssDNA and dsDNAstimulate photo-cross-linking. Moreover, stable complexes of RecF and dsDNA areobtained in the presence of ATP or its analog, ATP-gamma-S.

A study was conducted on the enzymatic properties of the RecA803 protein. This protein is a partial suppressor of the recombinational repair defect of recF mutations.RecA803 was compared with the wild-type protein and exhibited greater DNA-dependent adenosine triphosphatase (ATPase) activity. RecA803's ATPase activity was also found to be resistant to inhibition by the single-stranded dNA binding protein and it also had a quicker rate of association with ssDNA.

Abstract: We provide genetic evidence to show that the Mycobacterium tuberculosis FtsZ and FtsW proteins interact, and that these interactions are biologically relevant. Furthermore, we show by fluorescence microscopy that Mycobacterium smegmatis FtsW is part of its septasomal complex and colocalizes with FtsZ to the midcell sites. Colocalization experiments reveal that approximately 27% of the cells with septal Z-rings contain FtsW whereas 93% of the cells with FtsW bands are associated with FtsZ indicating that FtsW is late recruit to the septum, as in Escherichia coli. Our results suggest that mycobacterial FtsZ can localize to the septum independent of FtsW, and that interactions of FtsW with FtsZ are critical for the formation of productive FtsZ-rings and the cell division process in mycobacteria. [Copyright &y& Elsevier]

Examines the physiological consequences associated with overproduction of Mycobacterium tuberculosis FtsZ in mycobacterial hosts. Initiator of cell division process; Production of long filamentous cells; Absence of the signs for cellular constrictions.

Determines the DNA fragment containing dnaA gene region of Mycobacterium avium and the autonomous replication activities of its 5' and 3' flanking regions. Sequence analyses of the dnaA region; Autonomous replication activity of M. avium DNA fragments containing ribosomal protein L34; Replication initiation process of M. avium oriC.

To begin to understand the role of Mycobacterium smegmatis dnaA in DNA replication, the dnaA gene was characterized at the genetic level. Western analyses revealed that DnaA accounts for approximately 0·18% of the total cellular protein during both the active and stationary growth periods. Expression of antisense dnaA RNA reduced viability, indicating that dnaA is an essential gene in replication. To further understand the role(s) of dnaA in replication, a conditionally expressing strain was constructed in which expression of dnaA was controlled by acetamide. Growth in the presence of 0·2% acetamide elevated the intracellular levels of DnaA and increased cell length, but did not affect viability. Visualization of DNA by fluorescence microscopy revealed that DnaA-overproducing cells were multinucleoidal, indicating a loss of synchrony between the replication and cell-division cycles. Withdrawal of acetamide resulted in the depletion of the intracellular levels of DnaA, reduced viability and gradually blocked DNA synthesis. Acetamidestarved cells were very filamentous, several times the size of the parent cells and showed either abnormal or multi-nucleoid morphology, indicating a blockage in cell-division events. The addition of acetamide to the starved cells restored their viability and shortened the lengths of their filaments back to the size of the parent cells. Thus, both increasing and decreasing the levels of DnaA have an effect on the cells, indicating that the level of DnaA is critical to the maintenance of coordination between DNA replication and cell division. It is concluded that DNA replication and cell-division processes in M. smegmatis are linked, and it is proposed that DnaA has a role in both of these processes. [ABSTRACT FROM AUTHOR]

Examines the origin of replication region in clinical strains of Mycobacterium tuberculosis. Use of plasmid-based oriC-dependent replication assay; Interactions of the DnaA protein with the CCGTTCACA DnaA box; Replacement by homologous recombination of the CCGTTCACA DnaA box sequence of the laboratory strain M. tuberculosis.