The dual-function Rel(Mtb) protein from Mycobacterium tuberculosis catalyzes both the synthesis and hydrolysis of (p)ppGpp, the effector of the stringent response. In our previous work [Avarbock, D., Avarbock, A., and Rubin, H. (2000) Biochemistry 39, 11640], we presented evidence that the Rel(Mtb) protein might catalyze its two opposing reactions at distinct active sites. In the study presented here, we purified and characterized fragments of the 738-amino acid Rel(Mtb) protein and confirmed the hypothesis that amino acid fragment 1-394 contains both synthesis and hydrolysis activities, amino acid fragment 87-394 contains only (p)ppGpp synthesis activity, and amino acid fragment 1-181 contains only (p)ppGpp hydrolysis activity. Mutation of specific residues within fragment 1-394 results in the loss of synthetic activity and retention of hydrolysis (G241E and H344Y) or loss of hydrolytic activity with retention of synthesis (H80A and D81A). The C-terminally cleaved Rel(Mtb) fragment proteins have basal activities similar to that of full-length Rel(Mtb), but are no longer regulated by the previously described Rel(Mtb) activating complex (RAC). Residues within the C-terminus of Rel(Mtb) (D632A and C633A) are shown to have a role in interaction with the RAC. Additionally, size exclusion chromatography indicates Rel(Mtb) forms trimers and removal of the C-terminus results in monomers. The C-terminal deletion, 1-394, which exists as a mixture of monomers and trimers, will dissociate from the trimer state upon the addition of substrate. Furthermore, the trimer state of fragment 1-394 appears to be a catalytically less efficient state than the monomer state.

The mechanism of action of clofazimine (CFZ), an antimycobacterial drug with a long history, is not well understood. The present study describes a redox cycling pathway that involves the enzymatic reduction of CFZ by NDH-2, the primary respiratory chain NADH:quinone oxidoreductase of mycobacteria and nonenzymatic oxidation of reduced CFZ by O 2 yielding CFZ and reactive oxygen species (ROS). This pathway was demonstrated using isolated membranes and purified recombinant NDH-2. The reduction and oxidation of CFZ was measured spectrally, and the production of ROS was measured using a coupled assay system with Amplex Red. Supporting the ROS-based killing mechanism, bacteria grown in the presence of antioxidants are more resistant to CFZ. CFZ-mediated increase in NADH oxidation and ROS production were not observed in membranes from three different Gram-negative bacteria but was observed in Staphylococcus aureus and Saccharomyces cerevisiae, which is consistent with the known antimicrobial specificity of CFZ. A more soluble analog of CFZ, KS6, was synthesized and was shown to have the same activities as CFZ. These studies describe a pathway for a continuous and high rate of reactive oxygen species production in Mycobacterium smegmatis treated with CFZ and a CFZ analog as well as evidence that cell death produced by these agents are related to the production of these radical species.

Type-II NADH-menaquinone oxidoreductase (NDH-2) is an essential respiratory enzyme of the pathogenic bacterium Mycobacterium tuberculosis (Mtb) that plays a pivotal role in its growth. In the present study, we expressed and purified highly active Mtb NDH-2 using a Mycobacterium smegmatis expression system, and the steady-state kinetics and inhibitory actions of phenothiazines were characterized. Purified NDH-2 contains a non-covalently bound flavin adenine dinucleotide cofactor and oxidizes NADH with quinones but does not react with either NADPH or oxygen. Ubiquinone-2 (Q2) and decylubiquinone showed high electron-accepting activity, and the steady-state kinetics and the NADH-Q2 oxidoreductase reaction were found to operate by a ping-pong reaction mechanism. Phenothiazine analogues, trifluoperazine, Compound 1, and Compound 2 inhibit the NADH-Q2 reductase activity with IC 50 ‫؍‬ 12, 11, and 13 M, respectively. Trifluoperazine inhibition is non-competitive for NADH, whereas the inhibition kinetics is found to be uncompetitive in terms of Q2.The Gram-positive bacterium Mycobacterium tuberculosis (Mtb) 3 causes tuberculosis, one of the leading causes of morbidity and mortality in the world. Each year nine million active cases of the disease are diagnosed, accounting for three million deaths. Multidrug-resistant tuberculosis and the existence of "persistent" organisms that are tolerant to antibiotics exacerbate the problem, for which more effective and efficient treatments need to be urgently developed. Mtb is traditionally considered an obligate aerobe, yet during the normal course of events in the infectious cycle, the bacillus is able to survive in conditions of low oxygen and nutrient concentrations, such as those postulated to exist within granulomas. Mtb adapts its metabolic activity, cellular transcription, and protein expression accordingly (1). It is therefore of great importance to understand how Mtb generates ATP under a variety of environmental conditions. Type-II NADH-dehydrogenase (NDH-2) is a critical enzyme in the life cycle of Mtb. The enzyme has been purified from Saccharomyces cerevisiae (2), Escherichia coli (3, 4), Bacillus subtilis (5), Methyloccocus capsulatus (6), Corynebacterium glutamicum (7, 8), Acidianus ambicalens (9, 10), and Sulfolobus metallicus (11) and is, in general, composed of a single polypeptide chain, which contains a flavin as a sole cofactor. It is noteworthy that this enzyme is not found in mitochondria. The essential role of NDH-2 in Mtb is supported by extensive evidence from biochemical (12) and transcriptional studies (13), gene deletion analysis, investigation of bacterial growth in various media and under various culture conditions, and animal experiments (12). Mtb contains two copies of ndh genes (ndh and ndhA). The Mtb NDH-2 and NDH-2A share 67% sequence identity, and the genes are separated by 17 kb. Mtb NDH-2 is highly homologous to those of Mycobacterium leprae and Mycobacterium smegmatis with 91 and 81% amino acid sequence identity, respectively. A strain of Mtb...

Growth of three different soil isolates of Cladosporium resinae on a large number of carbon compounds was found to be inhibited by n-hexane and n-heptane whereas n-octane and all higher liquid n-alkanes had no effect on growth. In addition, n-hexane and, to a lesser extent, n-heptane caused rapid inhibition of glucose incorporation and considerable loss of potassium and protein from the cells. The phenomenon is unlikely to be due to removal of membrane lipids such as sterols and phospholipids by the n-alkanes, but rather to a limited disorganization of the cell membrane resulting in rapid loss of selective permeability.

Mycobacterium tuberculosis (Mtb) remains the deadliest bacterial pathogen worldwide, causing an estimated 1.7 million deaths in 2004 among an infected population of approximately 2 billion people, according to the World Health Organization (WHO). Therapeutic options are limited to a few drugs that are becoming increasingly ineffective. Multidrug-resistant (MDR) Mtb strains are prevalent globally, fueled by inadequate patient compliance of drug intake. Recently, a high incidence of extensively drug-resistant (XDR) strains resistant to all currently used drugs was reported among patients with the human immunodeficiency virus (HIV) in KwaZulu Natal, South Africa [1]. The high mortality rate and short survival time of patients with XDR Mtb was especially alarming. The emergence of XDR mycobacteria emphasizes the urgent need for the identification of novel targets and development of new drugs. New potential drug targets exist in the Mtb respiratory chain. Certain classes of drugs have long been shown to exert significant tuberculocidal activity, such as the phenothiazines [2, 3]. Phenothiazines inhibit one of the key enzymes of the respiratory chain; type II NADH:menaquinone oxidoreductase or NDH-2 [4]. The effectiveness of this class of drugs against Mtb justifies further research into the respiratory chain, with the aim of elucidating its physiologic roles in in vitro and in vivo survival, and discovering new (sub)classes of drugs that can safely serve as inhibitors for clinical use. In this chapter, we critically review the recent advances in this field, with particular emphasis on NDH-2, and underscore the kinds of research further needed for drug development.

Long-chain saturated fatty acids (C 13 to C 18 ) and fatty alcohols (C 12 to C 18 ) were well utilized by three different soil isolates of Cladosporium resinae as the sole carbon and energy sources in static liquid cultures. Shorter-chain compounds, down to C 5 , did not support growth and were in fact toxic towards the fungus growing on glucose. Rapid and considerable potassium efflux, protein leakage, and inhibition of endogenous respiration were observed in the presence of the shorter fatty acids and alcohols. Possible mechanisms and significance of the toxicity are discussed.

Four different isolates of Cladosporium resinae from Australian soils were tested for their ability to utilize liquid n -alkanes ranging from n -hexane to n -octadecane under standard conditions. The isolates were unable to make use of n -hexane, n -heptane, and n -octane for growth. In fact, these hydrocarbons, particularly n -hexane, exerted an inhibitory effect on spore germination and mycelial growth. All higher n -alkanes from n -nonane to n -octadecane were assimilated by the fungus, although only limited growth occurred on n -nonane and n -decane. The long chain n -alkanes (C 14 to C 18 ) supported good growth of all isolates, but there was no obvious correlation between cell yields and chain lengths of these n -alkanes. Variation in growth responses to individual n -alkane among the different isolates was also observed. The cause of this variation is unknown.

Glucose transport in Cladosporium resinae was studies with the aid of the non-metabolizable glucose analogue 3-0-methyl-D-glucose (3-0-MG). 3-0-MG, transported as a free sugar without phosphorylation, was found to inhibit glucose uptake competitively. Conversely, glucose was a competitive inhibitor of 3-0-MG uptake. Moreover, both glucose and 3-0-MG were able to bring about rapid counterflow of intracellular 3-0-MG. Thus, glucose and 3-0-MG share the same entry and exit systems. The transport of 3-0-MG is carrier mediated and energy dependent as shown by saturation kinetics, strong temperature dependence, accumulation of unaltered 3-0-MG against a concentration gradient, and inhibition of uptake by NaN,, NaCN, and 2,4-dinitrophenol. The glucose transport system appeared to be constitutive for glucose transport in cells grown on fructose, galactose, mannose, xylose, or glucose. There was no derepressible low-Km glucose transport system in C. resinae. n-Hexane and n-heptane were found to inhibit 3-0-MG uptake rapidly at temperatures above 20 C. Over 50% inhibition of the uptake rate occurred after only 10 min of incubation with n-hexane at 30 C. The percentage of inhibition in the presence of n-hexane, compared to controls in the absence of n-hexane, was found to increase with increasing temperature. Longer-chain n-alkanes (C8 to C1,) had no significant effect on uptake. The efflux of intracellular 3-0-MG, which appeared to occur by facilitated diffusion, was not affected by any of the n-alkanes tested including n-hexane. The filamentous fungus Cladosporium resinae is generally believed to be the major cause of corrosion in jet aircraft fuel systems today (13). Previous work from this laboratory has shown that C. resinae can utilize C, to C1, n-alkanes for growth, whereas the shorter-chain n-alkanes, particularly n-hexane and n-heptane, inhibit growth of the organism on glucose (20). The toxicity is apparently due to the direct action of the short-chain n-alkanes on the cell membrane (21). This paper presents the kinetic characteristics of glucose transport, a typical membrane-mediated process, and the effects of n-alkanes on the glucose transport system in C. resinae. MATERIALS AND METHODS Organism. Isolate 35A, which was isolated from Australian soil and identified as C. resinae f. avellaneum by D. G. Parbery, School of Agriculture, University of Melbourne, Australia, was used in all experiments. It was maintained on Bushnell-Hass glucose-agar slants (4). Media and cultivation method. Cells were grown at 30 C in static 1-liter Fernbach flasks containing 180