James A. Fuchs

Crystal structure of a Y35G mutant of bovine pancreatic trypsin inhibitor.

The structure of a Y35G mutant of bovine pancreatic trypsin inhibitor (BPTI) was solved by molecular replacement and was refined by both simulated annealing and restrained least-squares at 1.8 A resolution. The crystals belong to the space group P42212, with unit cell dimensions a = b = 46.75 A, c = 50.61 A. The final R-factor is 0.159 and the deviation from ideality for bond distances is 0.02 A. The structure of the mutant differs from that of the native protein, showing an overall root-mean-square (r.m.s.) difference of 1.86 A for main-chain atoms. However, the change is mostly localized in the two loops (respective r.m.s. values of 2.04 A and 3.93 A) and the C terminus (r.m.s. 6.79 A), while the core of the protein is well conserved (r.m.s. 0.45 A). The change in the loop regions can be clearly attributed to the mutation while the difference in the C terminus might be only due to a different crystal packing. Seventy water molecules were included in the model but only seven of them are shared with the native structure. Thermal parameters are showing a good correlation with those for the wild-type of BPTI.

Multiple cis‐acting sites positively regulate Escherichia coli nrd expression

Regulation of nrd expression in Escherichia coli by cis‐acting elements was found to be more complex than previously reported. At least five upstream sites appear to positively regulate nrd expression including a Fis binding site, a DnaA binding site, an AT‐rich region, an inverted repeat and a 10 bp site between the AT‐rich region and the inverted repeat. Double mutants defective in these sites indicate that all sites tested act independently when regulating nrd expression. As the decrease in nrd expression in exponentially growing cultures paralleled the decrease observed in DNA synthesis‐inhibited cultures for all single and double mutants, we concluded that nrd is regulated by the same mechanism in these physiological states. As mutants unable to induce nrd expression during inhibition of DNA synthesis also fail to exhibit cell cycle‐regulated nrd expression, we conclude that cell cycle nrd regulation is controlled by these same sites. Site‐directed mutagenesis was used to show that the absence of an increase in nrd expression during DNA inhibition previously observed for deletion of the AT‐rich region results from deletion of both the Fis binding site and the AT‐rich region.

Null mutation in the stringent starvation protein of Escherichia coli disrupts lytic development of bacteriophage P1

As initial steps toward understanding the regulation and function of the stringent starvation protein (SSP) of Escherichia coli, we have isolated the ssp gene (encoding SSP), defined the operon in which ssp is found, and created insertion-deletion mutations of the ssp gene in recBC, sbc and recD strains by linear DNA transformation. During attempts to move the insertion-deletion structure to other strains by P1 transduction, we found that P1 was unable to form plaques on hosts lacking an intact ssp gene. The delta ssp mutation, however, did not affect transduction of the delta ssp strains and mutant strains were able to support lysogenic P1. When P1 lytic growth was induced, an increase in P1 DNA was detected without lysis or plaque formation. Examination of proteins synthesized in the delta ssp host during induction revealed the absence of P1 late gene products. Also, the apparent continued synthesis of early gene products during late time points was observed in the delta ssp host. The results reported here suggest that the defect in P1 lytic growth brought about by the absence of SSP occurs at the point at which bacteriophage P1 shifts from early to late gene expression. We also report the results of experiments on stable RNA synthesis following amino acid (aa) starvation induced by serine hydroxamate, and experiments on stable RNA synthesis following resupplementation of a limiting aa. These experiments show that SSP is not involved in stable RNA synthesis. Additionally, complementation studies have shown that ssp is identical to the previously described pog gene of E. coli.

Involvement of thioredoxin in sulfoxide reduction by mammalian tissues

Sulindac, a sulfoxide with antiinflammatory activity, is reduced to the corresponding sulfide by rat hepatic cytosolic enzymes requiring NADPH for maximal activity. This reaction is inhibited by insulin, L-cystine, glutathione disulfide and 5,5′-dithiobis(2-nitrobenzoic acid), all of which are known to interact with the thioredoxin system comprised of NADPH, thioredoxin reductase and thioredoxin. Sodium arsenite, a known inhibitor of thioredoxin reductase, also inhibited sulindac reduction. Rat hepatic cytosolic fractions from which thioredoxin had been removed by chromatography on Sephadex G-50 showed minimal sulfoxide reductase activity; activity could be restored by addition of purified Escherichiacoli thioredoxin or dithiothreitol. These findings are the first demonstration of thioredoxin-dependent sulfoxide reduction by mammalian tissues.

Characterization and regulation of glutathione S-transferase gene from Schizosaccharomyces pombe.

A glutathione S-transferase (GST) gene has been cloned from Schizosaccharomyces pombe for the first time. The nucleotide sequence determined was found to contain 2030 base pairs including an open reading frame of 229 amino acids that would encode a protein of a molecular mass of 27017 Da. The cloned GST gene was expressed and was found to function in S. pombe, Saccharomyces cerevisiae, and Escherichia coli. The plasmid pGT207 encoding the S. pombe GST gene appeared to be able to accelerate the growth of a wild type S. pombe culture. In a culture of S. pombe containing plasmid pGT207, the growth was inhibited less by mercuric chloride than in a culture with vector alone. The 1088 bp region upstream from the GST gene as well as the region encoding the N-terminal 14 amino acids was transferred into the promoterless beta-galactosidase gene of plasmid YEp357R to yield the fusion plasmid pYSH2000. beta-Galactosidase synthesis was induced by cadmium chloride, mercuric chloride, hydrogen peroxide, and menadione. It was also induced by high temperature. These results suggest that the cloned S. pombe GST gene is involved in the oxidative stress response.

A 45 BP INVERTED REPEAT IS REQUIRED FOR CELL CYCLE REGULATION OF THE ESCHERICHIA COLI NRD OPERON

Expression of β‐galactosidase from a nrd–lacZ fusion was used to determine the role in nrd regulation of an inverted sequence upstream of the promoter. Removal or replacement of a 45 bp inverted repeat with an altered sequence including a 48 bp perfect inverted repeat resulted in a mutant phenotype that was low in nrd expression in an exponentially growing culture and that did not increase during DNA synthesis inhibition. Changing the 22 bp in the upstream half of the inverted repeat resulted in the same phenotype, whereas changing the 22 bp in the downstream half of the inverted repeat decreased nrd expression to a lesser extent in an exponentially growing culture and had only a smaller effect on nrd expression during DNA synthesis inhibition. As other mutants with the phenotype of the upstream inverted repeat mutant were found to lack cell cycle regulation, expression of nrd–lac mRNA produced from a plasmid with this mutation in the nrd–lacZ fusion gene was compared with nrd mRNA produced from the chromosomal nrd gene in a synchronized culture. The results indicated that the upstream half of the nrd inverted repeat contains a cis‐acting element essential for nrd cell cycle regulation.

Production of nitrite and N2O by the ammonia-oxidizing nitrifiers

Nitrosomonas is an obligately autotrophic and aerobic bacterium which produces energy for growth from the following reactions: NH3 + O2 + 2e- → NH2OH (ammonia monoxygenase, “AMO”) (8); NH2OH + H2O → 4e- + 4H+ + HNO2 (hydroxylamine oxidoreductase, “HAO”) (3) and 1/2 O2 + 2e- + 2H+ → H2O (terminal oxidase).

Bacterial carbonic anhydrases.

In contrast to animal and plant carbonic anhydrases, relatively little is known about carbonic anhydrases in bacteria. Carbonic anhydrase activity has been well documented in a few bacterial species and its presence has been inferred on the basis of gene sequence homologies in several others, but their functions are generally not known. Two exceptions are the carbonic anhydrase (CA) that is a part of the cyn operon in Escherichia coli and the CA in the cyanobacterium Synechococcus PCC7942, both of which serve known specialized roles, i.e. prevention of CO2 depletion during cyanate degradation and concentration of CO2 for photosynthesis, respectively. The focus of this chapter will be on the properties and function of these two bacterial carbonic anhydrases. Since the function of the carbonic anhydrase in cyanobacteria is presumably closely related to the role of carbonic anhydrase in photosynthesis in higher plants, which is reviewed elsewhere in this book, more emphasis is placed on the carbonic anhydrase in E. coli. Although not eubacterial, a short section is included on the carbonic anhydrase from the Archaebacterium Methanosarcina thermophila. The distribution, properties, and possible functions and wider occurrence of carbonic anhydrases in other bacterial species are reviewed in the last section.

Characterization, expression and regulation of a third gene encoding glutathione S-transferase from the fission yeast.

A third gene encoding glutathione S-transferase (GSTIII) was cloned from the fission yeast Schizosaccharomyces pombe. The nucleotide sequence determined was found to contain 2110 base pairs including an open reading frame of 242 amino acids that would encode a protein of a molecular mass of 26,620 Da. The cloned GSTIII gene could be expressed in S. pombe, S. cerevisiae and Escherichia coli cells which gave 1.4-, 2.1-, and 3.0-fold higher GST activity in an assay using 1-chloro-2,4-dinitrobenzene as a substrate, respectively. The cloned GSTIII gene caused higher survivals of S. pombe cells on solid media with cadmium chloride or mercuric chloride. The GSTIII protein has 16% and 18% homologies with the GSTI and GSTII proteins, respectively. To independently monitor the regulation of the GSTIII gene, its 1168 bp upstream region and N-terminal 33 amino acid-coding region was fused into the promoterless beta-galactosidase gene of the shuttle vector YEp357. The synthesis of beta-galactosidase from the fusion plasmid pGY357 was greatly enhanced by cadmium chloride (50 microM), cupric chloride (10 microM), aluminum chloride (5 mM, 10 mM), mercuric chloride (1 microM), and zinc chloride (10 mM). However, the synthesis of beta-galactosidase from the fusion plasmid pGY357 was not affected by superoxide-generating menadione, and o-dinitrobenzene, whereas they could significantly induce the expression of the GSTI and GSTII genes of S. pombe. The overproduced Pap1 inhibited the induction of beta-galactosidase synthesis from the fusion plasmid pGY357 by cadmium chloride, which is opposite to the previously known role of Pap1 in the response to oxidative stress. Our results collectively indicate that the three GST genes of S. pombe are subjected to different regulatory mechanisms. The major role of the GSTIII protein in S. pombe may be the detoxification of various metals.

A second stress-inducible glutathione S-transferase gene from Schizosaccharomyces pombe

A second glutathione S-transferase gene (GST II) was isolated from the chromosomal DNA of the fission yeast Schizosaccharomyces pombe. The nucleotide sequence determined contains 1908 bp including an open reading frame of 230 amino acids that would encode a protein of a molecular mass of 26843.4 Da. The amino acid sequence of the putative GST II is very homologous with that of the previously isolated GST gene (GST I) located in the same chromosome III of S. pombe. The cloned GST II gene produces the functional GST in S. pombe, and it gives much higher GST in the stationary phase than in the exponential phase. Regulation of the GST II gene was studied using the GST II-lacZ fusion. The synthesis of beta-galactosidase from the fusion plasmid is greatly enhanced by the treatments with oxidative stresses such as menadione and mercuric chloride. It is also induced by o-dinitrobenzene, one of the GST substrates. NO-generating S-nitroso-N-acetylpenicillamine has a weak induction effect on the expression of GST II gene. These results indicate that the S. pombe GST II gene is involved in the oxidative stress response and detoxification. However, physiological meaning on the existence of the two similar GST genes in S. pombe remains unknown yet.