Mutations in yeast ribosomal proteins S28 and S4 affect the accuracy of translation and alter the sensitivity of the ribosomes to paromomycin.

Ribosomal proteins S12, S5 and S4 of Escherichia coli are essential for the control of translational accuracy. Their yeast equivalents, i.e., S28, S4 and S13, have also been implicated in this process. Using a poly(U)-dependent cell-free translation system, we determined the accuracy of translation and the sensitivity to antibiotic paromomycin of yeast ribosomes carrying mutant ribosomal proteins S28 and/or S4. Our results confirm by quantitative biochemical methods previous genetic data showing that proteins S28 and S4 are involved in the decoding activity of the ribosome and interact to control translational accuracy. We find that the suppressor mutation SUP44 in yeast S4, decreased the accuracy of translation. To examine the effect of mutant S28, we disrupted RPS28B and introduced in RPS28A the same substitutions that cause hyperaccurate translation or antibiotic resistance in bacteria. Three of these substitutions (Lys-62-->Asn, Thr or Gln) similarly increased translational accuracy in vitro or antibiotic resistance. In the presence of the SUP44 mutation, these substitutions partially reversed the decrease of translational accuracy caused by SUP44. However, the Lys-62-->Arg substitution decreased translational accuracy and caused antibiotic sensitivity both in nonsuppressor and in SUP44 haploids. These results establish the role of Lys-62 of S28 in optimizing translational accuracy and provide a more precise view of the functional role of two important ribosomal proteins.

Bacterial virulence as a target for antimicrobial chemotherapy.

As bacterial resistance to currently used antibiotics increases, so too must efforts to identify novel agents and strategies for the prevention and treatment of bacterial infection. In the past, antimicrobial drug discovery efforts have focused on eradicating infection by either cidal or static agents, resulting in clearance of the bacterium from the infected host. To this end, drug discovery targets have been those proteins or processes essential for bacterial cell viability. However, inhibition of the interaction between the bacterium and its host may also be a target. During establishment of an infection, pathogenic bacteria use carefully regulated pathways of conditional gene expression to transition from a free-living form to one that must adapt to the host milieu. This transition requires the regulated production of both extracellular and cell-surface molecules, often termed virulence factors. As the biological imperatives of the invading organism change during the course of an infection, the expression of these factors is altered in response to environmental cues. These may be changes in the host environment, for example, pH, metabolites, metal ions, osmolarity, and temperature. Alternatively, effector molecules produced by the bacterium to sense changing cell density can also lead to changes in virulence gene expression. Although the mechanisms of pathogenesis among different bacteria vary, the principles of virulence are generally conserved. Bacterial virulence may therefore offer unique opportunities to inhibit the establishment of infection or alter its course as a method of antimicrobial chemotherapy.

Expression of the AsbA1, OXA-12, and AsbM1 beta-lactamases in Aeromonas jandaei AER 14 is coordinated by a two-component regulon.

Aeromonas jandaei AER 14 (formerly Aeromonas sobria AER 14) expresses three inducible beta-lactamases, AsbA1, OXA-12 (AsbB1), and AsbM1. Mutant strains that constitutively overexpress all three enzyme simultaneously, suggesting that they share a common regulatory pathway, have been isolated. Detectable expression of the cloned genes of AsbA1 and OXA-12 in some Escherichia coli K-12 laboratory strains is achieved only in the presence of a blp mutation. These mutations map to the cre operon at 0 min, which encodes a classical two-component regulatory system of unknown function. Two regulatory elements from A. jandaei which permit high-level constitutive expression of OXA-12 in E. coli were cloned. Both loci encode proteins with characteristics of response regulator proteins of two-component regulatory systems. One of these loci, designated blrA, bestowed constitutive expression of all three beta-lactamases in A. jandaei AER 14 when present on a multicopy plasmid, confirming its role in the regulatory pathway of beta-lactamase production in this organism.

A novel cloning strategy reveals the gene for the yeast homologue to Escherichia coli ribosomal protein S12.

Using a novel technique designed to identify genes of Saccharomyces cerevisiae which carry introns, we have cloned two genes encoding ribosomal protein S28. Although the genes differ by 15 nucleotides within their coding regions, they are predicted to encode identical proteins of 145 amino acids. The predicted amino acid sequence of S28 contains significant homology to ribosomal protein S25 of Tetrahymena thermophila and to ribosomal protein S12 of several archaebacteria, suggesting a relationship to S12 of Escherichia coli. Dot matrix analysis confirmed that regions of S12, especially those implicated in the accuracy of translation, have been conserved in S28 of S. cerevisiae. Either RPS28A or RPS28B alone can support growth, but heterozygous disruption of both genes abolishes the ability to sporulate. Haploids harboring a disruption of both genes cannot survive without an intact gene on a plasmid. RPS28A maps to the right arm of chromosome VII and RPS28B to the right arm of chromosome XVI.

rna12+, a gene of Saccharomyces cerevisiae involved in pre-rRNA maturation. Characterization of a temperature-sensitive mutant, cloning and sequencing of the gene.

RNA12-1 is a dominant temperature-sensitive (Ts) yeast mutant which has previously been reported to exhibit a defect in RNA accumulation at 37 degrees C. We further characterized this mutant through analyses of rRNA transcription rates and maturation. The results show that pre-rRNA is normally synthesized but that subsequent maturation is severely affected by a temperature upshift: the nascent rRNAs are under-methylated and little mature rRNA can be observed at 37 degrees C. Likewise, the accumulation of some mRNAs for ribosomal proteins is also prevented at 37 degrees C. The RNA12-1 mutation is recessive at 32 degrees C, which made it possible to clone the wild-type rna12+ gene by complementation of the Ts phenotype with plasmids from a multicopy yeast genomic library. The predicted gene product is a protein of 96,630 Da with no significant sequence similarity to any known proteins. Gene disruption is not lethal at either the permissive or the restrictive temperature. The gene is located on chromosome XIII, downstream of the ADH2 gene and 10 cM from the ADE4 gene. Furthermore, the mutant allele RNA12-1 was cloned and sequenced. A point mutation found in this allele leads to dominant thermosensitivity at 37 degrees C when the mutant gene is introduced into a wild-type strain. Taken together, these data suggest that the rna12+ gene product plays a dispensable role in early maturation of pre-rRNA but that its mutant gene product can interfere with the normal function of other proteins required for pre-rRNA maturation.

Rehabilitation of the Puerto Rican addict: a cultural perspective.

Drug addiction has assumed major proportions in the Hispanic population. This paper questions whether treatment programs in Puerto Rican communities adequately relate their rehabilitative services to the realities of Hispanic culture. The experience of a clinic in the South Bronx is reviewed, and it is suggested that programs need to be aware of cultural differences, provide Hispanic staff to treat Hispanic patients, and build up acknowledged strengths, values, and folkways.

A mutation in either dsbA or dsbB, a gene encoding a component of a periplasmic disulfide bond-catalyzing system, is required for high-level expression of the Bacteroides fragilis metallo-beta-lactamase, CcrA, in Escherichia coli.

The metallo-beta-lactamase gene, ccrA, from Bacteroides fragilis is functionally expressed in Escherichia coli only in the presence of a genomic mutation in iarA or iarB (increased ampicillin resistance), identified in this study as dsbA or dsbB, respectively. DsbA and DsbB are components of a periplasmic protein disulfide bond-catalyzing system. Data indicated that DsbA interacted with CcrA, creating aberrant disulfide bond linkages that render CcrA proteolytically unstable. Mutations in dsbA or dsbB permissive for CcrA expression eliminated or greatly reduced DsbA activity, allowing CcrA to assume a disulfide bond-free and proteolytically stable conformation.

Locus of control and initiation of detoxification among male methadone maintenance patients.

Two hypotheses were derived linking locus of control to voluntary, "completion of treatment" detoxification from methadone maintenance: (1) methadone maintenance patients with an internal locus of control will be more likely to indicate a willingness to begin detoxification, and (2) among patients indicating a willingness to begin, those with an internal locus of control would be more likely to actually begin. Subjects were 115 male methadone patients. A nonsignificant trend was found in support of the first hypothesis, while the second was reversed at a statistically significant level (r = -.30, p less than .012).

An accuracy center in the ribosome conserved over 2 billion years.

The accuracy of translation in Escherichia coli is profoundly influenced by three interacting ribosomal proteins, S12, S4, and S5. Mutations at lysine-42 of S12, originally isolated as causing resistance to streptomycin, increase accuracy. Countervailing "ribosomal ambiguity mutations" (ram) in S4 or S5 decrease accuracy. In the eukaryotic ribosome of Saccharomyces cerevisiae, mutations in SUP46 and SUP44, encoding the proteins equivalent to S4 and S5, lead to omnipotent suppression--i.e., to less accurate translation. The evolution of ribosomal protein S12 can be traced, by comparison with archaebacteria and Tetrahymena, to S28 of S. cerevisiae, even though the two proteins share only very limited regions of homology. However, one region that has been conserved contains a lysine residue whose mutation leads to increased accuracy in E. coli. We have introduced into S28 of yeast the same amino acid substitutions that led to the original streptomycin-resistant mutations in E. coli. We find that they have a profound effect on the accuracy of translation and interact with SUP44 and SUP46, just as predicted from the E. coli model. Thus, the interplay of these three proteins to provide the optimal level of accuracy of translation has been conserved during the 2 billion years of evolution that separate E. coli from S. cerevisiae.

Identification and analysis of bacterial protein secretion inhibitors utilizing a SecA-LacZ reporter fusion system.

Protein secretion is an essential process for bacterial growth, yet there are few if any antimicrobial agents which inhibit secretion. An in vivo, high-throughput screen to detect secretion inhibitors was developed based on the translational autoregulation of one of the central protein components, SecA. The assay makes use of a SecA-LacZ fusion reporter construct in Escherichia coli which is induced when secretion is perturbed. Several compounds, including two natural product extracts, which had the ability to induce the reporter fusion were identified and the MICs of these compounds for Staphylococcus aureus strain MN8 were found to be < or =128 microg/ml. Enzyme-linked immunosorbent assay, Western blotting, and immunoprecipitation techniques were used to analyze the affects of these compounds on protein secretion. Six representative compounds presented here appear to be bona fide secretion inhibitors but were found to have deleterious effects on membranes. It was concluded that, while the method described here for identifying inhibitors of secretion is valid, screens such as this, which are directed against the membrane-bound portion of a pathway, may preferentially identify compounds which affect membrane integrity.