Michael W. Mather

Cytochromecaa3 from the thermophilic bacteriumThermus thermophilus: A member of the heme-copper oxidase superfamily

The subject of this short review is the cytochromec oxidase (caa3) from the thermophilic bacteriumThermus thermophilus. First, some of the extensive physical and enzymological results obtained with this enzyme are reviewed, and two experiments are described, involving isotope substitutions in combination with Mössbauer and ENDOR spectroscopies, which have provided novel insight into the active sites of the enzyme. Second, we summarize recent molecular genetic work showing thatThermus cytochromecaa3 is abona fide member of the superfamily of heme-copper oxidases. Finally, we present a rough three-dimensional model and speculate about certain features of the metal-binding sites.

Plasmid-associated aggregation inThermus thermophilus HB8

Thermus thermophilus HB8, a moderate thermophile, exhibits visible aggregation when growing on a rich broth. Strain HB8 also contains two cryptic plasmids. We isolated cured strains from HB8 and observed that loss of the 47-MDa plasmid was correlated with loss of aggregation. An enrichment procedure was developed for aggregating cells and used to demonstrate that aggregation was restored upon transformation of a cured strain with plasmid DNA. The aggregation phenotype of transformed cells was variably stable; most did not retain either the plasmid or the phenotype for prolonged periods of growth. Hybridization experiments using a partial sequence from the 47-MDa plasmid suggested the presence of a repeated DNA sequence on this plasmid and on the chromosome. This is the first report of a phenotype associated with a plasmid from a Thermus strain.

Isolation and Partial Sequence of the A‐Protein Gene of Thermus thermophilus Cytochrome c1aa3

Thermus thermophilus is a strictly aerobic eubacterium that grows optimally near 70°C. Its respiratory system is very similar to that of eukaryotic mitochondria, and the organism has proven to be a particularly good source of stable, comparatively simple respiratory enzymes [cf. Refs. 1 and 21. There are a t least two terminal oxidases: The recently discovered cytochrome ba: and cytochrome c,aa3? Cytochrome ba, is an analog of aa, in which the heme A of cytochrome a is replaced with protoporphyrin IX (heme B) while its other redox components appear to be largely identical to those of the now classical mammalian cytochrome aa, [cf. Zimmerman et ~ 1 . ~ 1 ; it has only a single 35-kDa protein subunit. Cytochrome qua3 consists of two polypeptides. The -33-kDa C-protein covalently binds one heme C, while the -55-kDa protein is thought to bind the four canonical redox centers of aa,, two heme A, and two Cu [cf. Refs. 1 and 21. Toward our goal of unequivocally establishing the distribution of the metal centers in cytochrome c,aa,, we have isolated the structural gene of the A-protein.

Recovery and cloning of genomic DNA fragments from dried agarose gels

Publisher Summary This chapter describes a method for the cloning of bacterial genes that circumvents the need to prepare and maintain genomic DNA libraries. Genomic DNA samples, highly enriched in specific restriction fragments, are isolated from dried agarose gels and used directly for cloning. The method is rapid and technically simple and reduces the possibility that a DNA sequence of interest might be missed because of underrepresentation in a library. The use of the autoradiogram with labeled marker DNA fragments as a template for cutting out the region of the preparative gel containing the target DNA allows precise excision of the gel band containing the DNA of interest. This minimizes the amount of screening subsequently required by reducing the carryover of nontarget genomic DNA. This compares favorably to the many thousands of colonies one must screen in the case of a bacterial genomic library, but can undoubtedly be improved on by using narrower excision slots.

Molecular modeling studies on the proposed NaCl-induced dimerization of Chromatium vinosum high-potential iron protein.

Previous work (Dunham, W.R., Hagen, W.R., Fee, J.A., Sands, R.H., Dunbar, J.B., Humblet, C. (1991) An investigation of Chromatium vinosum high-potential iron-sulfur protein by EPR and Mössbauer spectroscopy; evidence for a freezing-induced dimerization in NaCl solutions, Biochimica Biophysica Acta 1079, 253-262) suggested that under specific solution conditions and slow freezing times, samples of oxidized Chromatium vinosum (Cv) high-potential, iron-sulfur protein (HiPIP) form dimeric structures that exhibit characteristic spin-spin interaction in the EPR spectrum. In that study, it was also shown that two HiPIP molecules could approach each other along their Fe1-S4 axes to a distance of approximately 13-14 A, as required by an analysis of the spin-spin physics. This is made possible because of a flattened surface on one side of the molecule within which S4 may, depending on side-chain motions, interact with solvent (Carter, C.W., Jr., Kraut, J., Freer, S.T., Alden, R.A., Sieker, L.C., Adman, E.T., Jensen, L.H. (1972) A comparison of Fe4S4 clusters in high potential iron protein and in ferredoxin, Proc. Natl. Acad. Sci. USA 69, 3527-3529). Here we describe a computer generated, hypothetical model of this proposed dimeric structure which suggests an energetically favorable interaction between two Cv HiPIP molecules and could account for the experimental observations. Two Cv HiPIP molecules brought together along their Fe1-S4 axes and maintained at a center-to-center distance of 14 A can be rotated with respect to each other so as to create complementary interactions between two glutamine residues, two phenylalanine residues, and two leucine residues, and an energetically unfavorable interaction between two arginine residues. Energy minimization calculations using the program XPLOR indicate that this arrangement may provide an overall energetically favorable interaction between the two HiPIP molecules that is strengthened by site-specific binding of Na and Cl ions.