11th International Congress on Molecular Plant-Microbe Interactions held at St. Petersburg--a report

The report is a short summary of the most interesting presentations at the 11th International Congress on Molecular Plant-Microbe Interactions held during July 18-27, 2003 at St. Petersburg, Russia. The key elements from several sessions on the legume-Rhizobium interactions have been discussed.     

The mechanism of Mycobacterium tuberculosis alkylhydroperoxidase AhpD as defined by mutagenesis,crystallography, and kinetics

AhpD, a protein with two cysteine residues, is required for physiological reduction of the Mycobacterium tuberculosis alkylhydroperoxidase AhpC. AhpD also has an alkylhydroperoxidase activity of its own. The AhpC/AhpD system provides critical antioxidant protection, particularly in the absence of the catalase-peroxidase KatG, which is suppressed in most isoniazid-resistant strains. Based on the crystal structure, we proposed recently a catalytic mechanism for AhpD involving a proton relay in which the Glu118 carboxylate group, via His137 and a water molecule, deprotonates the catalytic residue Cys133 (Nunn, C. M., Djordjevic, S., Hillas, P. J., Nishida, C., and Ortiz de Montellano, P. R. (2002) J. Biol. Chem. 277, 20033-20040). A possible role for His132 in subsequent formation of the Cys133-Cys130 disulfide bond was also noted. To test this proposed mechanism, we have expressed the H137F, H137Q, H132F, H132Q, E118F, E118Q, C133S, and C130S mutants of AhpD, determined the crystal structures of the H137F and H132Q mutants, estimated the pKa values of the cysteine residues, and defined the kinetic properties of the mutant proteins. The collective results strongly support the proposed catalytic mechanism for AhpD.     

Saccharomyces cerevisiae nucleolar protein Nop7p is necessary for biogenesis of 60S ribosomal subunits

To identify new gene products that participate in ribosome biogenesis, we carried out a screen for mutations that result in lethality in combination with mutations in DRS1, a Saccharomyces cerevisiae nucleolar DEAD-box protein required for synthesis of 60S ribosomal subunits. We identified the gene NOP7that encodes an essential protein. The temperature-sensitive nop7-1 mutation or metabolic depletion of Nop7p results in a deficiency of 60S ribosomal subunits and accumulation of halfmer polyribosomes. Analysis of pre-rRNA processing indicates that nop7 mutants exhibit a delay in processing of 27S pre-rRNA to mature 25S rRNA and decreased accumulation of 25S rRNA. Thus Nop7p, like Drs1p, is required for essential steps leading to synthesis of 60S ribosomal subunits. In addition, inactivation or depletion of Nop7p also affects processing at the A0, A1, and A2 sites, which may result from the association of Nop7p with 35S pre-rRNA in 90S pre-rRNPs. Nop7p is localized primarily in the nucleolus, where most steps in ribosome assembly occur. Nop7p is homologous to the zebrafish pescadillo protein necessary for embryonic development. The Nop7 protein contains the BRCT motif, a protein-protein interaction domain through which, for example, the human BRCA1 protein interacts with RNA helicase A.     

Preliminary investigation: the impact of the NCAA Wrestling Weight Certification Program on weight cutting

Historically, wrestling is a sport dependent on weight. Three tragic deaths in late 1997 prompted the National Collegiate Athletic Association (NCAA) to make a Wrestling Weight Certification Program (WWCP) mandatory to foster a safe competitive environment. One institution examined the impact of this program on weight cutting. Thirty-two NCAA Division I wrestlers completed the WWCP in the 1998-1999 season and 29 in 1999-2000. Eighteen (56%) of 32 wrestlers in 1998-1999 weighed in 10 or more pounds above the previous year's competition weight. Whereas, 28% weighed in 20 or more pounds above the previous year's competition weight. Weekly weight loss for the wrestlers in 1998-1999 revealed a substantial loss during the first week, possibly demonstrating the use of time-tested techniques for weight loss. However, in 1999-2000, the first week weight loss was less pronounced, with 65.8% of the weight being lost during the second half of the WWCP. Therefore, these wrestlers may be breaking the sport historic cycle of weight fluctuations through the WWCP.     

The crystal structure of Mycobacterium tuberculosis alkylhydroperoxidase AhpD, a potential target for antitubercular drug design

The resistance of Mycobacterium tuberculosis to isoniazid is commonly linked to inactivation of a catalase-peroxidase, KatG, that converts isoniazid to its biologically active form. Loss of KatG is associated with elevated expression of the alkylhydroperoxidases AhpC and AhpD. AhpD has no sequence identity with AhpC or other proteins but has alkylhydroperoxidase activity and possibly additional physiological activities. The alkylhydroperoxidase activity, in the absence of KatG, provides an important antioxidant defense. We have determined the M. tuberculosis AhpD structure to a resolution of 1.9 A. The protein is a trimer in a symmetrical cloverleaf arrangement. Each subunit exhibits a new all-helical protein fold in which the two catalytic sulfhydryl groups, Cys-130 and Cys-133, are located near a central cavity in the trimer. The structure supports a mechanism for the alkylhydroperoxidase activity in which Cys-133 is deprotonated by a distant glutamic acid via the relay action of His-137 and a water molecule. The cysteine then reacts with the peroxide to give a sulfenic acid that subsequently forms a disulfide bond with Cys-130. The crystal structure of AhpD identifies a new protein fold relevant to members of this protein family in other organisms. The structural details constitute a potential platform for the design of inhibitors of potential utility as antitubercular agents and suggest that AhpD may have disulfide exchange properties of importance in other areas of M. tuberculosis biology.     

Semi automated production of a set of different recombinant GST-Streptag fusion proteins

We describe a high-throughput procedure for the large-scale production of recombinant GST-Streptag fusion proteins. This three-step process, comprising cloning, expression and purification, simultaneously produces up to 96 different proteins in a multi-well format with high yield and purity. Two complementary oligonucleotides, together encoding a specific peptide sequence are annealed and directly ligated into a pre-digested pGEX-2T plasmid carrying an N-terminal GST-tag and a C-terminal Streptag. Following expression, a multichannel pipetting robot purifies the resulting fusion proteins within 2 h by affinity chromatography on Streptactin Macroprep mini-columns.     

Bystander effects: a concept in need of clarification

An increasing body of evidence indicates that the response to genotoxic agents such as radiation or drugs is a group phenomenon, rather than the summed response of individual independent cells to injury. Thus, a complex contagion-like response may spread beyond the initial impact of an agent to enlarge its effect. This indirect effect, termed "Bystander Effect," is multifaceted and may play a significant role in the therapy of tumors and in carcinogenesis. A better understanding of this phenomenon is needed in order to modulate treatment protocols to therapeutic advantage and to provide more rational guidelines for the evaluation of environmental hazards.     

Proteome analysis of differentially displayed proteins as a tool for the investigation of symbiosis

Two-dimensional gel electrophoresis was used to identify differentially displayed proteins expressed during the symbiotic interaction between the bacterium Sinorhizobium meliloti strain 1021 and the legume Melilotus alba (white sweetclover). Our aim was to characterize novel symbiosis proteins and to determine how the two symbiotic partners alter their respective metabolisms as part of the interaction, by identifying gene products that are differentially present between the symbiotic and non-symbiotic states. Proteome maps from control M. alba roots, wild-type nodules, cultured S. meliloti, and S. meliloti bacteroids were generated and compared. Over 250 proteins were induced or up-regulated in the nodule, compared with the root, and over 350 proteins were down-regulated in the bacteroid form of the rhizobia, compared with cultured cells. N-terminal amino acid sequencing and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry peptide mass fingerprint analysis, in conjunction with data base searching, were used to assign putative identity to nearly 100 nodule, bacterial, and bacteroid proteins. These included the previously identified nodule proteins leghemoglobin and NifH as well as proteins involved in carbon and nitrogen metabolism in S. meliloti. Bacteroid cells showed down-regulation of several proteins involved in nitrogen acquisition, including glutamine synthetase, urease, a urea-amide binding protein, and a PII isoform, indicating that the bacteroids were nitrogen proficient. The down-regulation of several enzymes involved in polyhydroxybutyrate synthesis and a cell division protein was also observed. This work shows that proteome analysis will be a useful strategy to link sequence information and functional genomics.     

Effect of steroid hormone deprivation on the expression of ecto-ATPase in distinct brain regions of female rats

Abundant evidence indicates that ATP and adenosine act as neurotransmitters or co-transmitters, influencing nerve cell physiology in various ways. Therefore, regulation of ATP-metabolizing enzymes is essential for the normal development and function of neuronal tissue. In the present study we have examined the effect of gonadal (OVX) or adrenal (ADX) steroid hormone deprivation on the activity and expression of synaptic membrane ecto-ATPase in three extrahypothalamic brain areas of female rats, primarily not associated with reproductive function. It was shown that OVX significantly increased ecto-ATPase activity and the relative abundance of this enzyme in the hippocampal (Hip) and caudate nucleus (CN), but not in brain stem (BS) membrane preparations. ADX was followed by an upregulation of the enzyme activity and its relative abundance in all the brain areas investigated. The highest enzyme activity and the most profound effects of OVX and ADX were detected in the CN. The results obtained indicate that ADX and OVX upregulate the expression of ecto-ATPase, potentiating the production of adenosine in synaptic cleft thus modulating the activity of numerous neurotransmitter systems in distinct areas of the CNS.