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The fabM Gene Product of Streptococcus mutans Is Responsible for the Synthesis of Monounsaturated Fatty Acids and Is Necessary for Survival at Low pH. Elizabeth M. Fozo, 2004.Previously, it has been demonstrated that the membrane fatty acid composition of Streptococcus mutans is affected by growth pH (E . M . Fozo and R . G . Quivey, Jr., Appl . Environ . Microbiol . 70:929-936, 2004; R . G . Quivey, Jr., R . Faustoferri, K . Monahan, and R . Marquis, FEMS Microbiol . Lett . 189:89-92, 2000) . Specifically, the proportion of monounsaturated fatty acids increases when the organism is grown in acidic environments; if the shift to increased monounsaturated fatty acids is blocked by the addition of a fatty acid biosynthesis inhibitor, the organism is rendered more acid sensitive (E . M . Fozo and R . G . Quivey, Jr., Appl . Environ . Microbiol . 70:929-936, 2004) . Recently, work with Streptococcus pneumoniae has identified a novel enzyme, FabM, responsible for the production of monounsaturated fatty acids (H . Marrakchi, K . H . Choi, and C . O . Rock, J . Biol . Chem . 277:44809-44816, 2002) . Using the published S . pneumoniae sequence, a putative FabM was identified in the S . mutans strain UA159 . We generated a fabM strain that does not produce unsaturated fatty acids as determined by gas chromatography of fatty acid methyl esters . The mutant strain was extremely sensitive to low pH in comparison to the wild type; however, the acid-sensitive phenotype was relieved by growth in the presence of long-chain monounsaturated fatty acids or through genetic complementation . The strain exhibited reduced glycolytic capability and altered glucose-PTS activity . In addition, the altered membrane composition was more impermeable to protons and did not maintain a normal  pH .
The results suggest that altered membrane composition can
significantly affect the acid survival capabilities, as well as
several enzymatic activities, of S . mutans .
In Vitro Activities of Voriconazole in Combination with Three Other Antifungal Agents against Candida glabrata. Francesco Barchiesi, 2004.Candida glabrata has recently emerged as a significant pathogen involved in both superficial and deep-seated infections . In the present study, a checkerboard broth microdilution method was performed to investigate the in vitro activities of voriconazole (VOR) in combination with terbinafine (TRB), amphotericin B (AMB), and flucytosine (5FC) against 20 clinical isolates of C . glabrata . Synergy, defined as a fractional inhibitory concentration (FIC) index of  0.50, was observed in 75% of VOR-TRB, 10% of VOR-AMB, and 5% of VOR-5FC interactions . None of these combinations yielded antagonistic interactions (FIC index > 4) . When synergy was not achieved, there was still a decrease in the MIC of one or both drugs used in the combination . In particular, the MICs were reduced to
1.0 µg/ml as a result of the combination for all isolates for which the AMB MIC at the baseline was
 2.0 µg/ml . By a disk diffusion assay, the halo diameters produced by antifungal agents in combination were greater that those produced by each drug alone . Finally, killing curves showed that VOR-AMB exhibited synergistic interactions, while VOR-5FC sustained fungicidal activities against C . glabrata . These studies demonstrate that the in vitro activity of VOR against this important yeast pathogen can be enhanced upon combination with other drugs that have different modes of action or that target a different step in the ergosterol pathway . Further studies are warranted to elucidate the potential beneficial effects of such combination regimens in vivo .
A Salmonella enterica Serovar Typhimurium hemA Mutant Is Highly Susceptible to Oxidative DNA Damage. Maya Elgrably-Weiss, 2002.The first committed step in the biosynthesis of heme, an important cofactor of two catalases and a number of cytochromes, is catalyzed by the hemA gene product . Salmonella enterica serovar Typhimurium hemA26::Tn10d (hemA26) was identified in a genetic screen of insertion mutants that were sensitive to hydrogen peroxide . Here we show that the hemA26 mutant respires at half the rate of wild-type cells and is highly susceptible to the effects of oxygen species . Exposure of the hemA26 strain to hydrogen peroxide results in extensive DNA damage and cell death . The chelation of intracellular free iron fully abrogates the sensitivity of this mutant, indicating that the DNA damage results from the iron-catalyzed formation of hydroxyl radicals . The inactivation of heme synthesis does not change the amount of intracellular iron, but by diminishing the rate of respiration, it apparently increases the amount of reducing equivalents available to drive the Fenton reaction . We also report that hydrogen peroxide has opposite effects on the expression of hemA and hemH, the first and last genes of heme biosynthesis pathway, respectively . hemA mRNA levels decrease, while the transcription of hemH is induced by hydrogen peroxide, in an oxyR-dependent manner . The oxyR-dependent induction is suppressed under conditions that accelerate the Fenton reaction by a mechanism that is not yet understood . Dimer-Tetramer Transition between Solution and Crystalline States of Streptavidin and Avidin Mutants. Yael Pazy, 2003.The biotin-binding tetrameric proteins, streptavidin from Streptomyces avidinii and chicken egg white avidin, are excellent models for the study of subunit-subunit interactions of a multimeric protein . Efforts are thus being made to prepare mutated forms of streptavidin and avidin, which would form monomers or dimers, in order to examine their effect on quaternary structure and assembly . In the present communication, we compared the crystal structures of binding site W  K mutations in streptavidin and avidin . In solution, both mutant proteins are known to form dimers, but upon crystallization, both formed tetramers with the same parameters as the native proteins . All of the intersubunit bonds were conserved, except for the hydrophobic interaction between biotin and the tryptophan that was replaced by lysine . In the crystal structure, the binding site of the mutated apo-avidin contains 3 molecules of structured water instead of the 5 contained in the native protein . The lysine side chain extends in a direction opposite that of the native tryptophan, the void being partially filled by an adjacent lysine residue . Nevertheless, the binding-site conformation observed for the mutant tetramer is an artificial consequence of crystal packing that would not be maintained in the solution-phase dimer . It appears that the dimer-tetramer transition may be concentration dependent, and the interaction among subunits obeys the law of mass action .
Functional Analysis of the Gene Cluster Involved in Production of the Bacteriocin Circularin A by Clostridium beijerinckii ATCC 25752. Robèr Kemperman, 2003.A region of 12 kb flanking the structural gene of the cyclic antibacterial peptide circularin A of Clostridium beijerinckii ATCC 25752 was sequenced, and the putative proteins involved in the production and secretion of circularin A were identified . The genes are tightly organized in overlapping open reading frames . Heterologous expression of circularin A in Enterococcus faecalis was achieved, and five genes were identified as minimally required for bacteriocin production and secretion . Two of the putative proteins, CirB and CirC, are predicted to contain membrane-spanning domains, while CirD contains a highly conserved ATP-binding domain . Together with CirB and CirC, this ATP-binding protein is involved in the production of circularin A . The fifth gene, cirE, confers immunity towards circularin A when expressed in either Lactococcus lactis or E . faecalis and is needed in order to allow the bacteria to produce bacteriocin . Additional resistance against circularin A is conferred by the activity of the putative transporter consisting of CirB and CirD .
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