Antagonists of Hsp16.3, a low-molecular-weight mycobacterial chaperone and virulence factor, derived from phage-displayed peptide libraries

The persistence of Mycobacterium tuberculosis is a major cause of concern in tuberculosis (TB) therapy. In the persistent mode the pathogen can resist drug therapy, allowing the possibility of reactivation of the disease. Several protein factors have been identified that contribute to persistence, one of them being the 16-kDa low-molecular-weight mycobacterial heat shock protein Hsp16.3, a homologue of the mammalian eye lens protein alpha-crystallin. It is believed that Hsp16.3 plays a key role in the persistence phase by protecting essential proteins from being irreversibly denatured. Because of the close association of Hsp16.3 with persistence, an attempt has been made to develop inhibitors against it. Random peptide libraries displayed on bacteriophage M13 were screened for Hsp16.3 binding. Two phage clones were identified that bind to the Hsp16.3 protein. The corresponding synthetic peptides, an 11-mer and a 16-mer, were able to bind Hsp16.3 and inhibit its chaperone activity in vitro in a dose-dependent manner. Little or no effect of these peptides was observed on alphaB-crystallin, a homologous protein that is a key component of human eye lens, indicating that there is an element of specificity in the observed inhibition. Two histidine residues appear to be common to the selected peptides. Nuclear magnetic resonance studies performed with the 11-mer peptide indicate that in this case these two histidines may be the crucial binding determinants. The peptide inhibitors of Hsp16.3 thus obtained could serve as the basis for developing potent drugs against persistent TB.     

A simple protocol for isolation of fungal DNA

Genomic DNA was isolated from as little as 2 mg dry biomass of Magnaporthe grisea by microwave treatment within 30 s. The quantity of DNA was good enough for PCR analysis and Dot blot hybridization. This technique can be used for various studies, such as DNA fingerprinting to study the population structure of the phytopathogen in different regions, and for a quick screening of M. grisea transformants.     

Cloning of Rhizobium meliloti Locus sxf C Involved in Symbiosis and Phage Sensitivity

A symbiotic mutant of Rhizobium meliloti Rmd438 (sxf C:: Tn5) which was phage resistant against RMP64, failed to utilize galactose as carbon source as reported earlier (21). The Bg/ll gene bank of wild type R. meliloti was mobilized into Rmd438 and a clone pSP676 which complemented for phage sensitivity was isolated. In order to characterize this clone, a Bg/ll and EcoRI map was constructed. The insert of 13.2 kb had three Bg/ll fragments of 4.0, 3.6 and 5.6 kb in this order. All three fragments were subcloned on the vector pRK290 and mobilized into Sxf-mutants. The complementation for phage sensitivity, symbiosis and galactose utilization properties are discussed.     

PI5P4Ks drive metabolic homeostasis through peroxisome-mitochondria interplay

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Photosynthetic control of nitrate metabolism inPhormidium uncinatum,a cyanobaterium

The impact of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) was studied on growth, Hill reaction, nitrate uptake, enzymes of nitrate utilization, and of oxidative pentose pathway by phototrophically growingPhormidium uncinatum and its DCMU-resistant (DCMUR) mutant. The growth-inhibitory action of DCMU was apparently the consequence of an inactivation of photosystem II (PS II) reaction and of reduction of nitrate utilization owing to an inhibition of nitrite reductase (NiR) activity. Mutation to this herbicide rendered both the processes insensitive to DCMU. Nevertheless, nitrate transport, nitrate reduction to nitrite, and ammonia assimilation of both the strains remained rather unaffected by DCMU. Photosynthetically inactive cells of the two strains exhibited higher activity levels of glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) than their phototrophic cultures.These data suggest that photosynthesis regulates nitrate utilization in this cyanobacterium at nitrite reduction level and that nitrate uptake and reduction to nitrite are relaxed from this control and conditionally sustained by oxidative breakdown of reserve glycogen.