Cardiolipin biosynthesis and remodeling enzymes are altered during development of heart failure.

Cardiolipin (CL) is responsible for modulation of activities of various enzymes involved in oxidative phosphorylation. Although energy production decreases in heart failure (HF), regulation of cardiolipin during HF development is unknown. Enzymes involved in cardiac cardiolipin synthesis and remodeling were studied in spontaneously hypertensive HF (SHHF) rats, explanted hearts from human HF patients, and nonfailing Sprague Dawley (SD) rats. The biosynthetic enzymes cytidinediphosphatediacylglycerol synthetase (CDS), phosphatidylglycerolphosphate synthase (PGPS) and cardiolipin synthase (CLS) were investigated. Mitochondrial CDS activity and CDS-1 mRNA increased in HF whereas CDS-2 mRNA in SHHF and humans, not in SD rats, decreased. PGPS activity, but not mRNA, increased in SHHF. CLS activity and mRNA decreased in SHHF, but mRNA was not significantly altered in humans. Cardiolipin remodeling enzymes, monolysocardiolipin acyltransferase (MLCL AT) and tafazzin, showed variable changes during HF. MLCL AT activity increased in SHHF. Tafazzin mRNA decreased in SHHF and human HF, but not in SD rats. The gene expression of acyl-CoA: lysocardiolipin acyltransferase-1, an endoplasmic reticulum MLCL AT, remained unaltered in SHHF rats. The results provide mechanisms whereby both cardiolipin biosynthesis and remodeling are altered during HF. Increases in CDS-1, PGPS, and MLCL AT suggest compensatory mechanisms during the development of HF. Human and SD data imply that similar trends may occur in human HF, but not during nonpathological aging, consistent with previous cardiolipin studies.

Clinical and microbiological aspects of linezolid resistance mediated by the cfr gene encoding a 23S rRNA methyltransferase.

The cfr (chloramphenicol-florfenicol resistance) gene encodes a 23S rRNA methyltransferase that confers resistance to linezolid. Detection of linezolid resistance was evaluated in the first cfr-carrying human hospital isolate of linezolid and methicillin-resistant Staphylococcus aureus (designated MRSA CM-05) by dilution and diffusion methods (including Etest). The presence of cfr was investigated in isolates of staphylococci colonizing the patient's household contacts and clinical isolates recovered from patients in the same unit where MRSA CM-05 was isolated. Additionally, 68 chloramphenicol-resistant Colombian MRSA isolates recovered from hospitals between 2001 and 2004 were screened for the presence of the cfr gene. In addition to erm(B), the erm(A) gene was also detected in CM-05. The isolate belonged to sequence type 5 and carried staphylococcal chromosomal cassette mec type I. We were unable to detect the cfr gene in any of the human staphylococci screened (either clinical or colonizing isolates). Agar and broth dilution methods detected linezolid resistance in CM-05. However, the Etest and disk diffusion methods failed to detect resistance after 24 h of incubation. Oxazolidinone resistance mediated by the cfr gene is rare, and acquisition by a human isolate appears to be a recent event in Colombia. The detection of cfr-mediated linezolid resistance might be compromised by the use of the disk diffusion or Etest method.

Regulation of phospholipid biosynthesis in {\it Saccharomyces cerevisiae\/} by CDP-diacylglycerol synthase

Amine-containing phospholipid synthesis in Saccharomyces cerevisiae starts with the conversion of CDP-diacylglycerol (CDP-DAG) and serine to phosphatidylserine (PS) while phosphatidylinositol (PI) is formed from CDP-DAG and inositol (derived from inositol-1-phosphate). In this study a gene (CDS1) encoding CDP-DAG synthase in S. cerevisiae was isolated and identified. The CDS1 gene encodes the majority, if not all, of the synthase activity, and is essential for cell growth. Overexpression of the CDS1 gene resulted in an elevation in the apparent initial rate of synthesis and also steady-state level of PI relative to PS in both wild type yeast and the cds1 mutant. Down-regulation of CDS1 expression resulted in an inositol excretion phenotype and an opposite effect on the above phospholipid synthesis in the cds1 mutant. This regulation of phospholipid biosynthesis is mediated by changes of the phospholipid biosynthetic enzymes via a mechanism independent of the expression of the INO2-OPI1 regulatory genes. Reduction in the level of CDP-DAG synthase activity resulted in an increase in PS synthase activity which followed a similar change in the CHO1/PSS (encodes PS synthase) mRNA level. INO1 (encodes inositol-1-phosphate synthase) mRNA also increased but only after CDP-DAG synthase activity fell below the wild type level. PI synthase activity followed the decrease of the CDP-DAG synthase activity, but there was no parallel change in the level of PIS1 mRNA. A G$\sp{305}$/A$\sp{305}$ point mutation within the CDS1 gene which causes the cdg1 phenotype was identified. A human cDNA clone encoding CDP-DAG synthase activity was characterized by complementation of the yeast cds1 null mutant. ^

Regulation of pyridoxal $5\prime$-phosphate biosynthesis in {\it Escherichia coli\/} K-12

Vitamin B$\sb6$ (or pyridoxal 5$\sp\prime$-phosphate, PLP) is an essential, ubiquitous coenzyme that affects many aspects of amino acid and cellular metabolism in all organisms. The goal of this thesis is to examine the regulation of PLP biosynthesis in Escherichia coli K-12. First, PdxH oxidase is a PLP biosynthetic enzyme, which uses molecular oxygen as an electron acceptor under aerobic assay conditions. To test if facultative anaerobic E. coli uses another enzyme to replace the function of PdxH oxidase anaerobically, suppressors of a pdxH null mutant were isolated anaerobically after 2-aminopurine or spontaneous mutagenesis. Only one specific bypass mutation in another PLP biosynthetic gene pdxJ was found, suggesting that PdxH oxidase is able to function anaerobically and PdxT utilizes D-1-deoxyxyulose as a substrate. Second, regulation of the serC (pdxF)-aroA operon, which is involved the biosynthesis of L-serine, PLP and aromatic compounds was examined. A serC (pdxF) single gene transcript and a serC (pdXf)-aroA cotranscript initiated at P$\sb{serC\ (pdxF)}$ upstream of serC (pdxF) were detected. The expression of the operon is activated by leucine responsive regulatory protein (LRP) and repressed by cAMP receptor protein-cAMP complex (CRP$\cdot$cAMP) at the transcriptional level. LRP activates the operon by directly binding to the upstream consensus box. Binding of CRP$\cdot$cAMP to the upstream CRP box diminishes the activation effect of LRP. However, deletion of the CRP box did not affect the repression of CRP$\cdot$cAMP, suggesting that CRP$\cdot$cAMP may repress the operon indirectly by stimulating the activity or level of an unidentified repressor. The overall effect of this regulation is to maximize the expression of the operon when the cells are growing in minimal-glucose medium. In addition, the binding and the transcription of P$\sb{serC\ (pdxF)}$ by RNA polymerase require a supercoiled circular DNA, indicating that DNA supercoiling affects the transcription of the operon. Third, regulation of another PLP biosynthetic gene gapB was also examined. gapB is activated by CRP$\cdot$cAMP and repressed by catabolic repressor activator protein (CRA). However, the activation of CRP$\cdot$cAMP is epistatic to the repression of CRA. Due to the CRA repression, gapB was expressed at a low level in all the media tested, suggesting that it may be the rate-limiting step of PLP biosynthesis. In summary, unlike genes in many biosynthetic pathways, PLP biosynthetic genes are regulated by global regulators that are important for carbon and amino acid metabolism, instead of the end product(s) of the pathway. ^

Identification of antigen-encoding genes of Enterococcus faecalis during human infections and characterization of a gene cluster for the biosynthesis of an antigenic polysaccharide

Genomic libraries of two Enterococcus faecalis strains, OG1RF and TX52 (an isolate from an endocarditis patient), were constructed in Escherichia coli and were screened with serum from a rabbit immunized with surface proteins of an E. faecalis endocarditis isolate and sera from four patients with enterococcal endocarditis. Thirty-eight immunopositive cosmid clones reacted with at least two of the patient sera and contained distinct inserts based on their DNA restriction patterns. These were chosen for further subcloning in a pBluescript SK ($-$) vector. Each sublibrary was screened with one of the five sera. Analysis of sequences from the immunopositive subclones revealed similarities to a range of proteins, including bacterial virulence factors, transporters, two-component regulators, metabolic enzymes, and membrane or cell surface proteins. Fourteen subclones did not show significant similarity to any sequence in the databases and may contain novel genes. Thirteen of the immunopositive cosmid clones did not yield immunopositive subclones and one such cosmid clone, TX5159, produced an antigenic polysaccharide in Escherichia coli. The insert of TX5159 was found to contain a multicistronic gene cluster containing genes similar to those involved in the biosynthesis and export of polysaccharides from both Gram-positive and Gram-negative organisms. Insertions in several genes within the cluster abolished the immunoreactivity of TX5159. RT-PCR of genes within the cluster with total RNA from OG1RF showed that these genes are transcribed. The polysaccharide was detected in two recently reported E. faecalis mucoid strains using specific antibody, but not in the other strains tested. This is the first report on a gene cluster of E. faecalis involved in the biosynthesis of an antigenic polysaccharide. ^

The role of specific regions of ribosomal RNAs in translation termination

The ribosome is a molecular machine that produces proteins in a cell. It consists of RNAs (rRNAs) and proteins. The rRNAs have been implicated in various aspects of protein biosynthesis supporting the idea that they function directly in translation. In this study the direct involvement of rRNA in translation termination was hypothesized and both genetic and biochemical strategies were designed to test this hypothesis. As a result, several regions of rRNAs from both ribosomal subunits were implicated in termination. More specifically, the mutation G1093A in an RNA of the large subunit (23S rRNA) and the mutation C1054A in the small subunit RNA (16S rRNA) of the Escherichia coli ribosome, were shown to affect the binding of the proteins that drive termination, RF1 and RF2. These mutations also caused defects in catalysis of peptidyl-tRNA hydrolysis, the last step of termination. Furthermore, the mutations affected the function of RF2 to a greater extent than that of RF1, a striking result considering the similarity of the RFs. The major defect in RF2 function was consistent with in vivo characteristics of the mutants and can be explained by the inability of the mutant rRNA sites to activate the hydrolytic center, that is the catalytic site for peptidyl-tRNA hydrolysis. Consistent with this explanation is the possibility of a direct interaction between the G1093-region (domain II of 23S rRNA) and the hydrolytic center (most likely domains IV–VI of 23S rRNA). To test that interaction hypothesis selections were performed for mutations in domains IV–VI that compensated for the growth defects caused by G1093A. Several compensatory mutations were isolated which not only restored growth in the presence of G1093A but also appeared to compensate for the termination defects caused by the G1093A. Therefore these results provided genetic evidence for an intramolecular interaction that might lead to peptidyl-tRNA hydrolysis. Finally, a new approach to the study of rRNA involvement in termination was designed. By screening a library of rRNA fragments, a fragment of the 23S rRNA (nt 74-136) was identified that caused readthrough of UGA. The antisense RNA fragment produced a similar effect. The data implicated the corresponding segment of intact 23S rRNA in termination. ^

THE BIOSYNTHESIS AND CHARACTERIZATION OF MURINE LEUKEMIA VIRAL PROTEINS

Two murine leukemia viruses (MuLVs), Rauscher (R-MuLV) and Moloney (Mo-MuLV) MuLVs, were studied to identify the biosynthetic pathways leading to the generation of mature virion proteins. Emphasis was placed on the examination of the clone 1 Mo-MuLV infected cell system.^ At least three genetic loci vital to virion replication exist on the MuLV genome. The 'gag' gene encodes information for the virion core proteins. The 'pol' gene specifies information for the RNA-dependent-DNA-polymerase (pol), or reverse transcriptase (RT). The 'env' gene contains information for the virion envelope proteins.^ MuLV specified proteins were synthesized by way of precursor polyproteins, which were processed to yield mature virion proteins. Pulse-chase kinetic studies, radioimmunoprecipitation, and peptide mapping were the techniques used to identify and characterize the MuLV viral precursor polyproteins and mature virion proteins.^ The 'gag' gene of Mo-MuLV coded for two primary gene products. One 'gag' gene product was found to be a polyprotein of 65,000 daltons M(,r) (Pr65('gag)). Pr65('gag) contained the antigenic and structural determinants of all four viral core proteins--p30, p15, pp12 and p10. Pr65('gag) was the major intracellular precursor polyprotein in the generation of mature viral core proteins. The second 'gag' gene product was a glycosylated gene product (gPr('gag)). An 85,000 dalton M(,r) polyprotein (gPr85('gag)) and an 80,000 dalton M(,r) (gPr80('gag)) polyprotein were the products of the 'gag' genes of Mo-MuLV and R-MuLV, respectively. gPr('gag) contained the antigenic and structural determinants of the four virion core proteins. In addition, gPr('gag) contained peptide information over and above that of Pr65('gag). Pulse-chase kinetic studies in the presence of tunicamycin revealed a separate processing pathway of gPr('gag) that did not seem to involve the generation of mature virion core proteins. Subglycosylated gPr('gag) was found to have a molecular weight of 75,000 daltons (Pr75('gag)) for both Mo-MuLV and R-MuLV.^ The Mo-MuLV 'pol' gene product was initially synthesized as a read-through 'gag-pol' intracellular polyprotein containing both antigenic and structural determinants of both the 'gag' and 'pol' genes. This read-through polyprotein was found to be a closely spaced doublet of two similarly sized proteins at 220-200,000 daltons M(,r) (Pr220/200('gag-pol)). Pulse-chase kinetic studies revealed processing of Pr220/200('gag-pol) to unstable intermediate intracellular proteins of 145,000 (Pr145('pol)), 135,000 (Pr135('pol)), and 125,000 (Pr125('pol)) daltons M(,r). Further chase incubations demonstrated the appearance of an 80,000 dalton M(,r) protein, which represented the mature polymerase (p80('pol)).^ The primary intracellular Mo-MuLV 'env' gene product was found to be a glycosylated polyprotein of 83,000 daltons M(,r) (gPr83('env)). gPr83('env) contained the antigenic and structural determinants of both mature virion envelope proteins, gp70 and p15E. In addition, gPr83('env) contained unique peptide sequences not present in either gp70 or p15E. The subglycosylated form of gPr83('env) had a molecular weight of 62,000 daltons (Pr62('env)).^ Virion core proteins of R-MuLV and Mo-MuLV were examined. Structural homology was observed betwen p30s and p10s. Significant structural non-homology was demonstrated between p15s and pp12s. ^

Characterization of fecal contamination targeting 16S rRNA bacteroides molecular markers in the Santa Cruz River, Arizona

Understanding the origins, transport and fate of contamination is essential to effective management of water resources and public health. Individuals and organizations with management responsibilities need to understand the risks to ecosystems and to humans from contact with contamination. Managers also need to understand how key contaminants vary over time and space in order to design and prioritize mitigation strategies. Tumacacori National Historic Park (NHP) is responsible for management of its water resources for the benefit of the park and for the health of its visitors. The existence of microbial contaminants in the park poses risks that must be considered in park planning and operations. The water quality laboratory at the Maricopa Agricultural Center (in collaboration with stakeholder groups and individuals located in the ADEQ-targeted watersheds) identified biological changes in surface water quality in impaired reaches rivers to determine the sources of Escherichia coli (E. coli); bacteria utilizing innovative water quality microbial/bacterial source tracking methods. The end goal was to support targeted watershed groups and ADEQ towards E. coli reductions. In the field monitoring was conducted by the selected targeted watershed groups in conjunction with The University of Arizona Maricopa Agricultural Center Water Quality Laboratory. This consisted of collecting samples for Bacteroides testing from multiple locations on select impaired reaches, to determine contamination resulting from cattle, human recreation, and other contributions. Such testing was performed in conjunction with high flow and base flow conditions in order to accurately portray water quality conditions and variations. Microbial monitoring was conducted by The University of Arizona Water Quality Laboratory at the Maricopa Agricultural Center using genetic typing to differentiate among two categories of Bacteroides: human and all (total). Testing used microbial detection methodologies and molecular source tracking techniques.^

Structure and biosynthesis of a pyruvate -dependent enzyme---phosphatidylserine decarboxylase from {\it Escherichia coli\/}

Phosphatidylserine decarboxylase of E. coli, a cytoplasmic membrane protein, catalyzes the formation of phosphatidylethanolamine, the principal phospholipid of the organism. The activity of the enzyme is dependent on a covalently bound pyruvate (Satre and Kennedy (1978) J. Biol. Chem. 253, 479-483). This study shows that the enzyme consists of two nonidentical subunits, $\alpha$ (Mr = 7,332) and $\beta$ (Mr = 28,579), with the pyruvate prosthetic group in amide linkage to the amino-terminus of the $\alpha$ subunit. Partial protein sequence and DNA sequence analysis reveal that the two subunits are derived from a proenzyme ($\pi$ subunit, Mr = 35,893) through a post-translational event. During the conversion of the proenzyme to the $\alpha$ and $\beta$ subunits, the peptide bond between Gly253-Ser254 is cleaved, and Ser254 is converted to the pyruvate prosthetic group at the amino-terminus of the $\alpha$ subunit (Li and Dowhan (1988) J. Biol. Chem. 263, 11516-11522).^ The proenzyme cannot be detected in cells carrying either single or multiple copies of the gene (psd), but can be observed in a T7 RNA polymerase/promoter and transcription-translation system. The cleavage of the wild-type proenzyme occurs rapidly with a half-time on the order of 2 min. Changing of the Ser254 to cysteine (S254C) or threonine (S254T) slows the cleavage rate dramatically and results in mutants with a half-time for processing of around 2-4 h. Change of the Ser254 to alanine (S254A) blocks the cleavage of the proenzyme. The reduced processing rate with the mutations of the proenzyme is consistent with less of the functional enzyme being made. Mutants S254C and S254T produce $\sim$15% and $\sim$1%, respectively, of the activity of the wild-type allele, but can still complement a temperature-sensitive mutant of the psd locus. Neither detectable activity nor complementation is observed by mutant S254A. These results are consistent with the hydroxyl-group of the Ser254 playing a critical role in the cleavage of the peptide bond Gly253-Ser254 of the pro-phosphatidylserine decarboxylase, and support the mechanism proposed by Snell and co-workers (Recsei and Snell (1984) Annu. Rev. Biochem. 53, 357-387) for the formation of the prosthetic group of pyruvate-dependent decarboxylases. ^