Increased cellular levels of reactive oxygen species are known to arise during exposure of organisms to elevated metal concentrations, but the consequences for cells in the context of metal toxicity are poorly characterized. Using two-dimensional gel electrophoresis, combined with immunodetection of protein carbonyls, we report here that exposure of the yeast Saccharomyces cerevisiae to copper causes a marked increase in cellular protein carbonyl levels, indicative of oxidative protein damage. The response was time dependent, with total-protein oxidation peaking approximately 15 min after the onset of copper treatment. Moreover, this oxidative damage was not evenly distributed among the expressed proteins of the cell. Rather, in a similar manner to peroxide-induced oxidative stress, copperdependent protein carbonylation appeared to target glycolytic pathway and related enzymes, as well as heat shock proteins. Oxidative targeting of these and other enzymes was isoformspeci¢c and, in most cases, was also associated with a decline in the proteins' relative abundance. Our results are consistent with a model in which copper-induced oxidative stress disables the £ow of carbon through the preferred glycolytic pathway, and promotes the production of glucose-equivalents within the pentose phosphate pathway. Such re-routing of the metabolic £ux may serve as a rapid-response mechanism to help cells counter the damaging e¡ects of copper-induced oxidative stress. ß

Pseudomonas putida utilizes the catBC operon for growth on benzoate as a sole carbon source. This operon is positively regulated by the CatR protein, which is encoded from a gene divergently oriented from the catBC operon. The catR gene encodes a 32.2-kilodalton polypeptide that binds to the catBC promoter region in the presence or absence of the inducer cis-cis-muconate, as shown by gel retardation studies. However, the inducer is required for transcriptional activation of the catBC operon. The catR promoter has been localized to a 385-base-pair fragment by using the broad-host-range promoter-probe vector pKT240. This fragment also contains the catBC promoter whose -35 site is separated by only 36 nucleotides from the predicted CatR translational start. Dot blot analysis suggests that CatR binding to this dual promoter-control region, in addition to inducing the catBC operon, may also regulate its own expression. Data from a computer homology search using the predicted amino acid sequence of CatR, deduced from the DNA sequence, showed CatR to be a member of a large class of procaryotic regulatory proteins designated the LysR family. Striking homology was seen between CatR and a putative regulatory protein, TfdS.

The pca branch of the beta-ketoadipate pathway in Pseudomonas putida is responsible for the complete degradation of p-hydroxybenzoate through ortho cleavage of the initial pathway metabolite, protocatechuate. The pcaR regulatory locus has been found to be required for both induction of all of the genes within the pca regulon (pcaBDC, pcaIJ, and pcaF) and the chemotactic response of the bacteria to aromatic compounds. Insertional inactivation mutagenesis, using Tn5 and mini-Tn5 transposons, was used to locate, clone, and sequence this pcaR regulatory gene. The pcaR gene product, when overexpressed in Escherichia coli, possessed a specific affinity for the pcaIJ promoter region and demonstrated that the entire PcaR protein was required for this function. The deduced amino acid sequence of the PcaR regulatory peptide bears little resemblance to its counterpart in the other branch of the pathway, CatR, but exhibits significant homology to its regulatory antecedent, PobR, which regulates the initial breakdown of p-hydroxybenzoate into protocatechuate. Comparisons of the pcaIJ and pcaR promoter regions revealed conservation of a 15-bp sequence centered around the -10 region in both sequences. This, together with previously defined deletional studies with the pcaIJ promoter region, suggests that PcaR exerts its regulatory effect through protein-DNA interactions within this region, which would be unusually close to the transcriptional start site of pcaIJ for a positive regulator.

No abstract

No abstract

Oxidative damage in microbial cells occurs during exposure to the toxic metal chromium, but it is not certain whether such oxidation accounts for the toxicity of Cr. Here, a Saccharomyces cerevisiae sod1D mutant (defective for the Cu,Zn-superoxide dismutase) was found to be hypersensitive to Cr(VI) toxicity under aerobic conditions, but this phenotype was suppressed under anaerobic conditions. Studies with cells expressing a Sod1p variant (Sod1 H46C ) showed that the superoxide dismutase activity rather than the metal-binding function of Sod1p was required for Cr resistance. To help identify the macromolecular target(s) of Cr-dependent oxidative damage, cells deficient for the reduction of phospholipid hydroperoxides (gpx3D and gpx1D/gpx2D/gpx3D) and for the repair of DNA oxidation (ogg1D and rad30D/ogg1D) were tested, but were found not to be Cr-sensitive. In contrast, S. cerevisiae msraD (mxr1D) and msrbD (ycl033cD) mutants defective for peptide methionine sulfoxide reductase (MSR) activity exhibited a Cr sensitivity phenotype, and cells overexpressing these enzymes were Cr-resistant. Overexpression of MSRs also suppressed the Cr sensitivity of sod1D cells. The inference that protein oxidation is a primary mechanism of Cr toxicity was corroborated by an observed~20-fold increase in the cellular levels of protein carbonyls within 30 min of Cr exposure. Carbonylation was not distributed evenly among the expressed proteins of the cells; certain glycolytic enzymes and heat-shock proteins were specifically targeted by Cr-dependent oxidative damage. This study establishes an oxidative mode of Cr toxicity in S. cerevisiae, which primarily involves oxidative damage to cellular proteins.

SecA, an essential component of the Sec machinery, exists in a soluble and a membrane form in Escherichia coli. Previous studies have shown that the soluble SecA transforms into pore structures when it interacts with liposomes, and integrates into membranes containing SecYEG in two forms: SecAS and SecAM; the latter exemplified by two tryptic membrane-specific domains, an N-terminal domain (N39) and a middle M48 domain (M48). The formation of these lipid-specific domains was further investigated. The N39 and M48 domains are induced only when SecA interacts with anionic liposomes. Additionally, the N-terminus, not the C-terminus of SecA is required for inducing such conformational changes. Proteolytic treatment and sequence analyses showed that liposome-embedded SecA yields the same M48 and N39 domains as does the membrane-embedded SecA. Studies with chemical extraction and resistance to trypsin have also shown that these proteoliposome-embedded SecA fragments exhibit the same stability and characteristics as their membrane-embedded SecA equivalents. Furthermore, the cloned lipid-specific domains N39 and M48, but not N68 or C34, are able to form partial, but imperfect ring-like structures when they interact with phospholipids. These ring-like structures are characteristic of a SecA pore-structure, suggesting that these domains contribute part of the SecA-dependent protein-conducting channel. We, therefore, propose a model in which SecA alone is capable of forming a lipid-specific, asymmetric dimer that is able to function as a viable protein-conducting channel in the membrane, without any requirement for SecYEG.

The organization and transcriptional control of chromosomal cat genes (required for dissimilation of catechol by the ␤-ketoadipate pathway) in the Pseudomonas putida biotype strain (ATCC 12633) are reported. Nucleotide sequence reveals that catR is separated by 135 bp from the divergently transcribed catBC,A; catC begins 21 nucleotides downstream from catB, and catA begins 41 nucleotides downstream from catC. This contrasts with the gene arrangement in other bacteria, in which catA lies several kilobases upstream from catB. Properties of Tn5 mutants confirmed earlier suggestions that catR is a transcriptional activator and indicated that catA is activated by CatR independently of its activation of catBC. CatR binds to both a DNA fragment containing the catR-catB intergenic region and another DNA fragment containing catC. Pseudomonas strain RB1 resembles P. putida in some respects. Divergence of the two Pseudomonas chromosomes was revealed as nucleotide substitution of about 10% after alignment of known portions of catR,BC,A. Divergent transcriptional controls are suggested by a cluster of nucleotide sequence modifications in Pseudomonas strain RB1 which disrupt a stem-loop structure directly upstream of catB in the P. putida chromosome. Abrupt divergence of the catR,BC,A nucleotide sequences was achieved during evolution by insertion of an 85-bp palindromic genetic element uniquely positioned downstream from P. putida catR and counterpoised by insertion of a similar palindromic sequence in the Pseudomonas strain RB1 catB-catC intergenic region. Properties of the palindromic genetic element suggest that it may serve functions analogous to those of repetitive extragenic palindromic sequences and enteric repetitive intergenic consensus sequences in enteric bacteria.