E. De Rossi

The TB structural genomics consortium: a resource for Mycobacterium tuberculosis biology

The TB Structural Genomics Consortium is an organization devoted to encouraging, coordinating, and facilitating the determination and analysis of structures of proteins from Mycobacterium tuberculosis. The Consortium members hope to work together with other M. tuberculosis researchers to identify M. tuberculosis proteins for which structural information could provide important biological information, to analyze and interpret structures of M. tuberculosis proteins, and to work collaboratively to test ideas about M. tuberculosis protein function that are suggested by structure or related to structural information. This review describes the TB Structural Genomics Consortium and some of the proteins for which the Consortium is in the progress of determining three-dimensional structures.

Genetic transformation of intact cells of Bacillus subtilis by electroporation

Plasmid DNAs were introduced by electroporation into Bacillus subtilis PB1424 as an alternative to competent-cell or protoplast transformation. The maximum electroporation efficiency was 10(4) transformants/microgram DNA. Parameters including growth phase of cells, ionic strength of the suspending medium, concentration and size of plasmid DNAs, amplitude and duration of the pulse, were evaluated in order to determine conditions that improved transformation efficiency.

Gene arrangement and organization in a ∼ 76 kb fragment encompassing the oriC region of the chromosome of Mycobacterium leprae

A continuous 75627 bp segment of the Mycobacterium leprae chromosome spanning the oriC region was sequenced. The gene order at this locus was similar to that found in the replication origin region of many other prokaryotes, particularly Mycobacterium tuberculosis and Streptomyces coelicolor. As in the case of several Gram-positive bacteria, essential genes involved in basic cellular functions, such as DNA or RNA metabolism (dnaA, dnaB, dnaN, gyrB, gyrA, pcnB, recF, rnpA, ssb), cell wall synthesis (ponA, pbpA) and probably cell division (gidB, rodA) were found. Strikingly, the gidA gene was absent from this part of the genome and there was no rRNA operon near oriC. The gyrA gene harbours an intein coding sequence indicating that protein splicing is required to produce the mature A subunit of DNA gyrase. Among the many other noteworthy features were ORFs encoding putative serine/threonine protein kinases and a protein phosphatase, three tRNA genes, one M. leprae-specific repetitive element and a glnQ pseudogene.

The katE gene, which encodes the catalase HPII of Mycobacterium avium

Disseminated Mycobacterium avium‐Mycobacterium intracellular disease is a prevalent opportunistic infection in patients with acquired immune deficiency syndrome (AIDS). These pathogens are generally resistant to isoniazid (INH), a powerful antituberculosis drug. It is now generally accepted that the INH susceptibility of Mycobacterium tuberculosis results from the transformation of the drug into a toxic derivative, as a result of the action of the enzyme catalase‐peroxidase (HPI), encoded by the katG gene. It has been speculated that the presence of a second catalase (HPII) in some mycobacterial species, but lacking in M. tuberculosis, may impair the action of INH. In this report, the nucleotide sequence of the M. avium katE gene, encoding catalase HPII, is described. This enzyme shows strong similarity to Escherichia coli catalase HPII and eukaryotic catalases. All amino acids previously postulated as participating directly in catalysis by liver catalase and most of the amino acids binding the prosthetic group are conserved in M. avium catalase HPII. The enzyme is expressed in E. coli and is inhibited by 3‐amino ‐l,2,4 triazole (AT). Furthermore, Southern blot hybridizations and polymerase chain reaction experiments demonstrate the distribution of katE gene in several mycobacterial species.

New shuttle vector for cloning in Bacillus stearothermophilus

Cloning vector plasmid pRP9 was constructed on the basis of the broad host-range plasmid pLM6. pRP9 was a small plasmid (2.9 kb), possessed a convenient polyrestriction site sequence and efficiently transformed Bacillus subtilis, Bacillus stearothermophilus and Escherichia coli. Furthermore, pRP9 presented a very high segregational stability in Bacillus hosts. Also, the structural stability in Bacillus strains, grown under selective pressure, of pRP9 carrying a 3-kb fragment, was high. No single-stranded and high-molecular weight pRP9 DNA was found in B. stearothermophilus. The host/vector systems described possessed all the properties required for efficient gene cloning.

Characterization of Gram-positive broad host-range plasmids carrying a thermophilic replicon

The cryptic plasmid pBC1 (1.6 kb) isolated from Bacillus coagulans Zu1961 was genetically marked with the genes for chloramphenicol and ampicillin resistance (CmR and ApR) from the Escherichia coli plasmid pJH101. The recombinant vector obtained (pCP49, 7.0 kb) replicated and expressed CmR in B. subtilis and CmR and ApR in E. coli. Different shuttle vectors for Gram+ bacteria were also constructed by inserting pBC1 into the Staphylococcus aureus plasmid pC194. The smallest of these, pLM6 (2.8 kb), containing essentially pBC1 and the chloramphenicol acetyl transferase gene from pC194, replicated in B. subtilis at a copy number of 60. By electroporation, these plasmids were introduced and stably maintained in B. subtilis, B. amyloliquefaciens, S. aureus, S. carnosus and Lactobacillus reuteri.

Molecular characterization of the genes encoding acetohydroxy acid synthase in the cyanobacterium Spirulina platensis

The enzyme acetohydroxy acid synthase (AHS), which catalyses the first common step in the biosynthesis of isoleucine, leucine and valine, has been demonstrated to be present in Spirulina platensis in two isoenzymic forms. The complete nucleotide sequences of the genes ilvX and ilvW encoding these two enzymes have been determined. Sequence analysis revealed the presence of two open reading frames, of 1836 and 1737 nucleotides for ilvX and ilvW, respectively. The predicted amino acid sequences of the two isoenzymes, compared with the Synechococcus PCC 7942 AHS enzyme and the large subunits of the Escherichia coli AHSI, II, III isoenzymes, revealed a notable degree of similarity. A small subunit has not been identified for either of the S. platensis AHS isoenzymes. Analysis by Northern blot hybridization demonstrated that the ilvX and ilvW genes are transcribed to give mRNA species of approximately 2.15 kb and 1.95 kb, respectively.