Structure of the Cellulose Synthase Complex of Gluconacetobacter hansenii at 23.4 Å Resolution.

Bacterial crystalline cellulose is used in biomedical and industrial applications, but the molecular mechanisms of synthesis are unclear. Unlike most bacteria, which make non-crystalline cellulose, Gluconacetobacter hansenii extrudes profuse amounts of crystalline cellulose. Its cellulose synthase (AcsA) exists as a complex with accessory protein AcsB, forming a 'terminal complex' (TC) that has been visualized by freeze-fracture TEM at the base of ribbons of crystalline cellulose. The catalytic AcsAB complex is embedded in the cytoplasmic membrane. The C-terminal portion of AcsC is predicted to form a translocation channel in the outer membrane, with the rest of AcsC possibly interacting with AcsD in the periplasm. It is thus believed that synthesis from an organized array of TCs coordinated with extrusion by AcsC and AcsD enable this bacterium to make crystalline cellulose. The only structural data that exist for this system are the above mentioned freeze-fracture TEM images, fluorescence microscopy images revealing that TCs align in a row, a crystal structure of AcsD bound to cellopentaose, and a crystal structure of PilZ domain of AcsA. Here we advance our understanding of the structural basis for crystalline cellulose production by bacterial cellulose synthase by determining a negative stain structure resolved to 23.4 Å for highly purified AcsAB complex that catalyzed incorporation of UDP-glucose into ß-1,4-glucan chains, and responded to the presence of allosteric activator cyclic diguanylate. Although the AcsAB complex was functional in vitro, the synthesized cellulose was not visible in TEM. The negative stain structure revealed that AcsAB is very similar to that of the BcsAB synthase of Rhodobacter sphaeroides, a non-crystalline cellulose producing bacterium. The results indicate that the crystalline cellulose producing and non-crystalline cellulose producing bacteria share conserved catalytic and membrane translocation components, and support the hypothesis that it is the extrusion mechanism and order in linearly arrayed TCs that enables production of crystalline cellulose.

AcsA-AcsB: The core of the cellulose synthase complex from Gluconacetobacter hansenii ATCC23769.

The gram-negative bacterium, Gluconacetobacter hansenii, produces cellulose of exceptionally high crystallinity in comparison to the cellulose of higher plants. This bacterial cellulose is synthesized and extruded into the extracellular medium by the cellulose synthase complex (CSC). The catalytic component of this complex is encoded by the gene AcsAB. However, several other genes are known to encode proteins critical to cellulose synthesis and are likely components of the bacterial CSC. We have purified an active heterodimer AcsA-AcsB from G. hansenii ATCC23769 to homogeneity by two different methods. With the purified protein, we have determined how it is post-translationally processed, forming the active heterodimer AcsA-AcsB. Additionally, we have performed steady-state kinetic studies on the AcsA-AcsB complex. Finally through mutagenesis studies, we have explored the roles of the postulated CSC proteins AcsC, AcsD, and CcpAx.

From Zn to Mn: the study of novel manganese-binding groups in the search for new drugs against tuberculosis.

In most eubacteria, apicomplexans, and most plants, including the causal agents for diseases such as malaria, leprosy, and tuberculosis, the methylerythritol phosphate pathway is the route for the biosynthesis of the C(5) precursors to the essential isoprenoid class of compounds. Owing to their absence in humans, the enzymes of the methylerythritol phosphate pathway have become attractive targets for drug discovery. This work investigates a new class of inhibitors against the second enzyme of the pathway, 1-deoxy-D-xylulose 5-phosphate reductoisomerase. Inhibition of this enzyme may involve the chelation of a crucial active site Mn ion, and the metal-chelating moieties studied here have previously been shown to be successful in application to the zinc-dependent metalloproteinases. Quantum mechanics and docking calculations presented in this work suggest the transferability of these metal-chelating compounds to Mn-containing 1-deoxy-D-xylulose 5-phosphate reductoisomerase enzyme, as a promising starting point to the development of potent inhibitors.

Metabolon formation in dhurrin biosynthesis.

Synthesis of the tyrosine derived cyanogenic glucoside dhurrin in Sorghum bicolor is catalyzed by two multifunctional, membrane bound cytochromes P450, CYP79A1 and CYP71E1, and a soluble UDPG-glucosyltransferase, UGT85B1 (Tattersall, D.B., Bak, S., Jones, P.R., Olsen, C.E., Nielsen, J.K., Hansen, M.L., Høj, P.B., Møller, B.L., 2001. Resistance to an herbivore through engineered cyanogenic glucoside synthesis. Science 293, 1826-1828). All three enzymes retained enzymatic activity when expressed as fluorescent fusion proteins in planta. Transgenic Arabidopsis thaliana plants that produced dhurrin were obtained by co-expression of CYP79A1/CYP71E1-CFP/UGT85B1-YFP and of CYP79A1/CYP71E1/UGT85B1-YFP but not by co-expression of CYP79A1-YFP/CYP71E-CFP/UGT85B1. The lack of dhurrin formation upon co-expression of the two cytochromes P450 as fusion proteins indicated that tight interaction was necessary for efficient substrate channelling. Transient expression in S. bicolor epidermal cells as monitored by confocal laser scanning microscopy showed that UGT85B1-YFP accumulated in the cytoplasm in the absence of CYP79A1 or CYP71E1. In the presence of CYP79A1 and CYP71E1, the localization of UGT85B1 shifted towards the surface of the ER membrane in the periphery of biosynthetic active cells, demonstrating in planta dhurrin metabolon formation.

A polyketide synthase catalyzes the last condensation step of mycolic acid biosynthesis in mycobacteria and related organisms.

Mycolic acids are major and specific constituents of the cell envelope of Corynebacterineae, a suborder of bacterial species including several important human pathogens such as Mycobacterium tuberculosis, Mycobacterium leprae, or Corynebacterium diphtheriae. These long-chain fatty acids are involved in the unusual architecture and impermeability of the cell envelope of these bacteria. The condensase, the enzyme responsible for the final condensation step in mycolic acid biosynthesis, has remained an enigma for decades. By in silico analysis of various mycobacterial genomes, we identified a candidate enzyme, Pks13, that contains the four catalytic domains required for the condensation reaction. Orthologs of this enzyme were found in other Corynebacterineae species. A Corynebacterium glutamicum strain with a deletion in the pks13 gene was shown to be deficient in mycolic acid production whereas it was able to produce the fatty acids precursors. This mutant strain displayed an altered cell envelope structure. We showed that the pks13 gene was essential for the survival of Mycobacterium smegmatis. A conditional M. smegmatis mutant carrying its only copy of pks13 on a thermosensitive plasmid exhibited mycolic acid biosynthesis defect if grown at nonpermissive temperature. These results indicate that Pks13 is the condensase, a promising target for the development of new antimicrobial drugs against Corynebacterineae.

Oxidative-stress-induced T lymphocyte hyporesponsiveness is caused by structural modification rather than proteasomal degradation of crucial TCR signaling molecules.

In several human pathologies (e.g. cancer, rheumatoid arthritis, AIDS and leprosy) oxidative stress induces T cell hyporesponsiveness. Hyporesponsive T cells often appear to display impaired expression of some (e.g. TCR-zeta, p56(lck) and LAT) but not all (e.g. TCR-alphabeta and CD3-epsilon) crucial TCR-proximal signaling molecules but the underlying mechanisms have as yet not been identified. Using an in vitro system for oxidative-stress-induced T cell hyporesponsiveness we here report two sequential effects of oxidative stress on TCR signaling molecules: protein alterations and proteasomal degradation. We have identified the C-terminal part of TCR-zeta and the membrane-proximal domain of p56(lck) as potential targets for modifications induced by reactive oxygen species. Oxidative-stress-exposed proteins were differentially susceptible to proteasomal degradation: whereas modified TCR-zeta was relatively resistant, reactive oxygen species (ROS)-altered LAT and p56(lck) were much more susceptible. Importantly, we found that T cell hyporesponsiveness best correlated with ROS-dependent protein alteration since inhibition of proteasomal degradation did not restore function. Finally, our data provide an explanation for the paradox of reduced TCR-zeta signals combined with unaltered TCR-alphabeta and CD3-epsilon expression levels: the TCR-zeta chain in hyporesponsive T cells is still expressed but no longer detectable by certain mAb recognizing ROS-sensitive epitopes.

Characterization of a Mycobacterium smegmatis mutant lacking penicillin binding protein 1.

The ponA gene of Mycobacterium smegmatis encodes a 95-kDa penicillin binding protein, PBP1, that is similar to PBP1s of Mycobacterium tuberculosis and Mycobacterium leprae. Transposon disruption of ponA in M. smegmatis resulted in a PBP1-deficient mutant that was sensitive to beta-lactam antibiotics, was more permeable to glycine, and grew slowly in liquid culture.

Dual multimodular class A penicillin-binding proteins in Mycobacterium leprae.

The ponA gene of cosmid L222 of the Mycobacterium leprae genome library encodes a multimodular class A penicillin-binding protein (PBP), PBP1. The PBP, labelled with a polyhistidine sequence, has been produced in Escherichia coli, extracted from the membranes with 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane-sulfonate (CHAPS) and purified by Ni2(+)-nitrilotriacetic acid-agarose chromatography. In contrast to the pon1-encoded class A PBP1, PBP1 undergoes denaturation at temperatures higher than 25 degrees C, it catalyzes acyl transfer reactions on properly structured thiolesters, and it binds penicillin with high affinity.

Gene knockout reveals a novel gene cluster for the synthesis of a class of cell wall lipids unique to pathogenic mycobacteria.

Surface-exposed unusual lipids containing phthiocerol and phenolphthiocerol are found only in the cell wall of slow-growing pathogenic mycobacteria and are thought to play important roles in host-pathogen interaction. The enzymology and molecular genetics of biosynthesis of phthiocerol and phenolphthiocerol are unknown. We postulate the domain organization of a set of multifunctional enzymes and a cluster of genes (pps) that would encode these enzymes for the biosynthesis of phthiocerol and phenolphthiocerol. A cosmid containing the postulated pps gene cluster was identified by screening a genomic library of Mycobacterium bovis BCG with the postulated homologous domains from mycocerosic acid synthase and fatty acid synthase genes as probes. Homologous cosmids were also identified in the genomic libraries of Mycobacterium tuberculosis and Mycobacterium leprae. M. bovis BCG was transformed with a pps disruption construct, made from the BCG cosmid by introducing the hygromycin resistance gene as the positive-selectable marker and the sacB gene as the counter-selectable marker. Gene disruption by homologous recombination with double crossover was confirmed by polymerase chain reaction and Southern hybridization. Chromatographic analysis showed that the phenolphthiocerol derivative, mycoside B, and phthiocerol dimycocerosates were not produced by the gene knockout mutants. This result confirms the identity of the pps genes. With the identification of the pps gene clusters in both M. tuberculosis and M. leprae, it should be possible to test the postulated roles of these unique lipids in tuberculosis and leprosy.

Identification and overexpression in Escherichia coli of a Mycobacterium leprae gene, pon1, encoding a high-molecular-mass class A penicillin-binding protein, PBP1.

Cosmid B577, a member of the collection of ordered clones corresponding to the genome of Mycobacterium leprae, contains a gene, provisionally called pon1, that encodes an 821-amino-acid-residue high-molecular-mass class A penicillin-binding protein, provisionally called PBP1. With similar amino acid sequences and modular designs, M. leprae PBP1 is related to Escherichia coli PBP1a and PBP1b, bienzymatic proteins with transglycosylase and transpeptidase activities. When produced in E. coli, His tag-labelled derivatives of M. leprae PBP1 adopt the correct membrane topology, with the bulk of the polypeptide chain on the surface of the plasma membrane. They defy attempts at solubilization with all the detergents tested except cetyltrimethylammonium bromide. The solubilized PBP1 derivatives can be purified by affinity chromatography on Ni2+-nitrilotriacetic acid agarose. They have low affinities for the usual penicillins and cephalosporins.