Mycobacterium tuberculosis ClpX Interacts with FtsZ and Interferes with FtsZ Assembly

FtsZ assembly at the midcell division site in the form of a Z-ring is crucial for initiation of the cell division process in eubacteria. It is largely unknown how this process is regulated in the human pathogen Mycobacterium tuberculosis. Here we show that the expression of clpX was upregulated upon macrophage infection and exposure to cephalexin antibiotic, the conditions where FtsZ-ring assembly is delayed. Independently, we show using pull-down, solid-phase binding, bacterial two-hybrid and mycobacterial protein fragment complementation assays, that M. tuberculosis FtsZ interacts with ClpX, the substrate recognition domain of the ClpXP protease. Incubation of FtsZ with ClpX increased the critical concentration of GTP-dependent polymerization of FtsZ. Immunoblotting revealed that the intracellular ratio of ClpX to FtsZ in wild type M. tuberculosis is approximately 1∶2. Overproduction of ClpX increased cell length and modulated the localization of FtsZ at midcell sites; however, intracellular FtsZ levels were unaffected. A ClpX-CFP fusion protein localized to the cell poles and midcell sites and colocalized with the FtsZ-YFP protein. ClpX also interacted with FtsZ mutant proteins defective for binding to and hydrolyzing GTP and possibly for interactions with other proteins. Taken together, our results suggest that M. tuberculosis ClpX interacts stoichiometrically with FtsZ protomers, independent of its nucleotide-bound state and negatively regulates FtsZ activities, hence cell division.     

Stability of nitroblue tetrazolium-based alkaline phosphatase substrates.

This report demonstrates the stability of NBT substrate after multiple exposures to alkaline phosphatase. Perhaps more important than the ability to reuse substrates, the report provides some insight into the mechanisms by which tetrazoliums are reduced and evidence for the formation of an intermediary product, i.e., a half-formazan that is reduced more rapidly. (J Histochem Cytochem 49:1189-1190, 2001)     

Expression of the Chlamydia trachomatis major outer membrane protein-encoding gene in Escherichia coli: role of the 3' end in mRNA stability

The major outer membrane protein (MOMP)-encoding gene (omp1) of Chlamydia trachomatis has been cloned into Escherichia coli and partially sequenced. This recombinant gene expresses a full-length 40-kDa product, which is recognized by a monoclonal antibody directed against the species-specific epitope of MOMP. The recombinant omp1 is expressed in either insertion orientation, indicating that it utilizes its own promoter system. The endogenous omp1 promoter possesses a relatively low activity despite the high level of MOMP expression. Deletion of a 520-bp fragment at the 3' end encoding 39 amino acids (aa) at the C terminus and the remainder of the noncoding region leads to a significant decrease in mRNA stability and loss of protein synthesis. When the MOMP-encoding plasmid was introduced into E. coli minicells, it expressed 40- and 43-kDa proteins; however, inhibition of post-translational processing by ethanol revealed only a 43-kDa protein. These data indicate that the unprocessed omp1 gene product contains a 22-aa leader sequence which is cleaved during translocation to the outer membrane, to yield a processed 40-kDa protein. The recombinant MOMP was localized to the outer membrane E. coli fraction, comparable to the location of the native C. trachomatis protein.     

A minority of 46,XX true hermaphrodites are positive for the Y-DNA sequence including SRY

Summary A total of 30 cases of 46,XX true hermaphroditism was analysed for Y-DNA sequences including the recently cloned gene for male testis-determination SRY. In 3 cases, a portion of the Y chromosome including SRY was present and, in 2 cases, was localised, to Xp22 by in situ hybridisation. Since previous studies have shown that the majority of XX males are generated by an X-Y chromosomal interchange, the Xp22 position of the Yp material suggests that certain cases of hermaphroditism can arise by the same meiotic event. The phenotype in the 3 SRY-positive cases may be caused by X-inactivation resulting in somatic mosaicism of testis-determining factor expression giving rise to both testicular and ovarian tissues. Autosomal or X-linked mutation(s) elsewhere in the sex-determining pathway may explain the phenotype observed in the remaining 27 SRY-negative cases.     

Nonmetric cranial variation and biological distances between samples of six nontribal populations of India

Sixty-nine nonmetrical morphological variants of the cranium have been studied in six samples of non-tribal, state populations in India, and their incidence reported. Using C.A.B. Smith’s angular transformation of frequencies, the multivariate Thetasquare distances and their respective standard deviations have been presented. On the basis of nonmetrical cranial variation, it is clear that the samples from Uttar Pradesh, Andhra Pradesh and Bihar are closer to each other but distant from Madhya Pradesh, Karnataka and Maharashtra. On the other hand, Karnataka and Maharashtra samples are quite close to each other and both, in turn, are comparatively closer to Madhya Pradesh than to Uttar Pradesh, Andhra Pradesh and Bihar. Madhya Pradesh sample emerges as the most divergent group among the six population samples studied. This, in general, is in conformity with the picture that emerges from various analysis of morphometric and other biological data on various populations of India.     

Coimmobilization of substrate and biocatalyst: A method for bioconversion of poorly soluble substances in water milieu

Coimmobilization of biocatalyst and substrate was studied as a method to increase the conversion rate in systems with substrates of extremely low solubility in water. The system studied was the conversion of hydrocortisone to prednisolone by Arthrobacter simplex. As a matrix for coimmobilization, alginate turned out to be superior to agar and agarose. After the reaction was complete, the beads were solubilized, andthe cells recovered for reuse, by centrifugation, whereas the prednisolone was extracted from the supernatant.     

Activation of mitochondrial glycerol 3-phosphate dehydrogenase by cadmium ions

Mitochondrial glycerol 3-phosphate dehydrogenase (EC 1.1.2.1.) requires Ca2+ ions for its activity. Cadmium ions also have activatory effect on the enzyme. They activate the glycerol 3-phosphate dehydrogenase in a very narrow concentration range (1-2 mmol/l). As contrasted with calcium, strong inhibitory effect occurred at higher concentrations (3-4 mmol/l). The inhibition induced by cadmium ions was completely reversible by washing of the mitochondria.     

Hydrolysis of milk lactose by immobilized beta-galactosidase-hen egg white powder

Immobilized beta-galactosidase was obtained by crosslinking the enzyme with hen egg white using 2% glutaraldehyde. The gel obtained could be lyophilized to give a dry enzyme powder. The pH optimum of both the soluble and immobilized enzyme was found to be 6.8. The immobilized enzyme showed a higher K(m) for the substrates. The extent of enzyme inhibition by galactose was reduced upon immobilization. The stability towards inactivation by heat, urea, gamma irradiation, and protease treatment were enhanced. The bound enzyme as tested in a batch reactor could be used repeatedly for the hydrolysis of milk lactose. The possible application of this system for small-scale domestic use has been suggested.     

Extractive bioconversion in aqueous two-phase systems

Summary The transformation of hydrocortisone to prednisolone was studied in aqueous two-phase systems, as a model for the extractive bioconversion of fine chemicals. The bacterium, Arthrobacter simplex, was able to grow in the two-phase system and the cells could be revitalized after a period of use. Use of aqueous two-phase systems made it possible to operate the reaction at higher substrate concentrations than in pure buffer solution. An adsorptive method to remove the product from the top phase was tested and shown to be both efficient and compatible with the overall process. In order to reduce the costs of operation in aqueous two-phase systems, a cheaper starch-based polymer, Reppal-PES, was successfully used as a substitute for dextran.Dedicated to Professor Dr. Georg Manecke on the occasion of his 70th birthday