Mechanistic Insight into the Enzymatic Reduction of Truncated Hemoglobin N of Mycobacterium tuberculosis: ROLE OF THE CD LOOP AND PRE-A MOTIF IN ELECTRON CYCLING*

Many pathogenic microorganisms have evolved hemoglobin-mediated nitric oxide (NO) detoxification mechanisms, where a globin domain in conjunction with a partner reductase catalyzes the conversion of toxic NO to innocuous nitrate. The truncated hemoglobin HbN of Mycobacterium tuberculosis displays a potent NO dioxygenase activity despite lacking a reductase domain. The mechanism by which HbN recycles itself during NO dioxygenation and the reductase that participates in this process are currently unknown. This study demonstrates that the NADH-ferredoxin/flavodoxin system is a fairly efficient partner for electron transfer to HbN with an observed reduction rate of 6.2 μm/min⁻¹, which is nearly 3- and 5-fold faster than reported for Vitreoscilla hemoglobin and myoglobin, respectively. Structural docking of the HbN with Escherichia coli NADH-flavodoxin reductase (FdR) together with site-directed mutagenesis revealed that the CD loop of the HbN forms contacts with the reductase, and that Gly⁴⁸ may have a vital role. The donor to acceptor electron coupling parameters calculated using the semiempirical pathway method amounts to an average of about 6.4 10⁻⁵ eV, which is lower than the value obtained for E. coli flavoHb (8.0 10⁻⁴ eV), but still supports the feasibility of an efficient electron transfer. The deletion of Pre-A abrogated the heme iron reduction by FdR in the HbN, thus signifying its involvement during intermolecular interactions of the HbN and FdR. The present study, thus, unravels a novel role of the CD loop and Pre-A motif in assisting the interactions of the HbN with the reductase and the electron cycling, which may be vital for its NO-scavenging function.     

Expression of astrocytic genes coding for proteins implicated in neural excitation and brain edema is altered after acute liver failure

In vitro and in vivo studies have suggested that reduced astrocytic uptake of neuronally released glutamate, alterations in expression of glial fibrillary acidic protein (GFAP) and aquaporin‐4 (AQP‐4) contribute to brain edema in acute liver failure (ALF). However, there is no evidence to date to suggest that these alterations occur in patients with ALF. We analyzed the mRNA expression of excitatory amino acid transporters (EAAT‐1, EAAT‐2), GFAP, and AQP‐4 in the cerebral cortex obtained at autopsy from eight patients with ALF and from seven patients with no evidence of hepatic or neurological disorders by real‐time PCR, and protein expression was assessed using immunoblotting and immunohistochemistry. We demonstrated a significant decrease in GFAP mRNA and protein levels in ALF patients compared to controls. While the loss of EAAT‐2 protein in ALF samples was post‐translational in nature, EAAT‐1 protein remained within normal limits. Immunohistochemistry confirmed that, in all cases, the losses of EAAT‐2 and GFAP were uniquely astrocytic in their localization. AQP‐4 mRNA expression was significantly increased and its immunohistochemistry demonstrated increased AQP‐4 immunoreactivity in the glial end‐feet process surrounding the microvessels. These findings provide evidence of selective alterations in the expression of genes coding for key astrocytic proteins implicated in central nervous system (CNS) excitability and brain edema in human ALF.

     

Roles of Bacillus subtilis DprA and SsbA in RecA-mediated Genetic Recombination

Bacillus subtilis competence-induced RecA, SsbA, SsbB, and DprA are required to internalize and to recombine single-stranded (ss) DNA with homologous resident duplex. RecA, in the ATP·Mg²⁺-bound form (RecA·ATP), can nucleate and form filament onto ssDNA but is inactive to catalyze DNA recombination. We report that SsbA or SsbB bound to ssDNA blocks the RecA filament formation and fails to activate recombination. DprA facilitates RecA filamentation; however, the filaments cannot engage in DNA recombination. When ssDNA was preincubated with SsbA, but not SsbB, DprA was able to activate DNA strand exchange dependent on RecA·ATP. This work demonstrates that RecA·ATP, in concert with SsbA and DprA, catalyzes DNA strand exchange, and SsbB is an accessory factor in the reaction. In contrast, RecA·dATP efficiently catalyzes strand exchange even in the absence of single-stranded binding proteins or DprA, and addition of the accessory factors marginally improved it. We proposed that the RecA-bound nucleotide (ATP and to a lesser extent dATP) might dictate the requirement for accessory factors.     

Functional Genomic Characterization of Virulence Factors from Necrotizing Fasciitis-Causing Strains of Aeromonas hydrophila

The genomes of 10 Aeromonas isolates identified and designated Aeromonas hydrophila WI, Riv3, and NF1 to NF4; A. dhakensis SSU; A. jandaei Riv2; and A. caviae NM22 and NM33 were sequenced and annotated. Isolates NF1 to NF4 were from a patient with necrotizing fasciitis (NF). Two environmental isolates (Riv2 and -3) were from the river water from which the NF patient acquired the infection. While isolates NF2 to NF4 were clonal, NF1 was genetically distinct. Outside the conserved core genomes of these 10 isolates, several unique genomic features were identified. The most virulent strains possessed one of the following four virulence factors or a combination of them: cytotoxic enterotoxin, exotoxin A, and type 3 and 6 secretion system effectors AexU and Hcp. In a septicemic-mouse model, SSU, NF1, and Riv2 were the most virulent, while NF2 was moderately virulent. These data correlated with high motility and biofilm formation by the former three isolates. Conversely, in a mouse model of intramuscular infection, NF2 was much more virulent than NF1. Isolates NF2, SSU, and Riv2 disseminated in high numbers from the muscular tissue to the visceral organs of mice, while NF1 reached the liver and spleen in relatively lower numbers on the basis of colony counting and tracking of bioluminescent strains in real time by in vivo imaging. Histopathologically, degeneration of myofibers with significant infiltration of polymorphonuclear cells due to the highly virulent strains was noted. Functional genomic analysis provided data that allowed us to correlate the highly infectious nature of Aeromonas pathotypes belonging to several different species with virulence signatures and their potential ability to cause NF.     

Rotala sahyadrica sp. nov. (Lythraceae) from Western Ghats,India

A new aquatic species, Rotala sahyadrica S. P. Gaikwad, Sardesai et S. R. Yadav sp. nov. collected from a fresh water lake on a high altitude lateritic plateau ecosystem of the Western Ghats, India is described and illustrated here. It is similar to R. tenella (Guill. & Perr.) Hiern but differs in having cleistogamous and chasmogamous flowers, nectary glands on calyx tube and 8–12 multi‐cellular black trichomes in the axils of bracteoles.     

Plant growth promoting rhizobacteria alleviate salinity induced negative effects on growth, oil content and physiological status in Mentha arvensis

This study was aimed to investigate the effect of inoculation on three salt-tolerant, plant-growth-promoting rhizobacteria (PGPR) STR2 (Bacillus pumilus), STR8 (Halomonas desiderata) and STR36 (Exiguobacterium oxidotolerans), for their growth-promoting potential and efficacy in augmenting salt tolerance in Mentha arvensis, an essential oil-bearing crop and natural source of l-menthol, under varying levels of NaCl stress (0, 100, 300 and 500 mM) imposed through irrigating water. Increase in the levels of salt concentration led to a decrease in the growth, fresh weight, leaf–stem ratio, oil content and yield. However, the negative effects of salinity were observed to be convalesced in the PGPR inoculated plants. At salinity levels of 100 and 300 mM NaCl, H. desiderata inoculated plants recorded the highest herb yield, whereas at 500 mM NaCl, the plants inoculated with E. oxidotolerans yielded maximum herb. The oil content in non-inoculated, salt-stressed plants was observed to be 0.46, 0.42 and 0.35 % at 100, 300 and 500 mM NaCl, respectively, whereas the plants inoculated with H. desiderata recorded the oil content of 0.71 and 0.60 and 0.48 % at similar levels of NaCl stress, respectively. The halotolerant PGPR minimized the deleterious effects of salt toxicity producing at par or higher yields at lower and medium salinity levels (100, 300 mM NaCl) than the un-inoculated non-salt-stressed plants through improved foliar nutrient uptake and enhanced antioxidant machinery. Based on the results of the experiments reported herein, the use of salt-tolerant, plant-growth-promoting bacteria may provide an effective means of facilitating M. arvensis growth in salt-stressed environments.