We isolated and characterized mutants of Bartonella bacilliformis that are resistant to the fluoroquinolone antibiotic ciprofloxacin, which targets the A subunit of DNA gyrase. Mutants had single point mutations in the gyrA gene that changed either Asp-90 to Gly or Asp-95 to Asn and had 3-or 16-fold higher resistance, respectively, to ciprofloxacin than did wild-type B. bacilliformis. Asp-95 is homologous to Asp-87 of Escherichia coli GyrA and is a common residue mutated in fluoroquinolone-resistant strains of other bacteria. This is the first report of a mutation at an Asp-90 homologue, which corresponds to Asp-82 in E. coli GyrA.
The secretion of PlcH and its homolog PlcN of Pseudomonas aeruginosa through the inner membrane depends upon a functional twin arginine translocase (Tat) system and a Tat signal sequence. Conserved twin arginine (Arg) residues within the Tat signal sequence consensus motif (S/TRRxFLK) are considered essential for the secretion of Tat substrates, but some exceptions (e.g., Lys and Arg) to the twin Arg residues in this motif have been noted. The roles of all three Arg residues within the PlcH RRRTFLK consensus motif were examined. Data are presented which indicate that Arg-9 and Arg-10 are essential for PlcH secretion across the inner membrane, but the mutation of Arg-8 (e.g., to Ala or Ser) had no observable effect on the localization of PlcH. In the signal sequence of PlcH and in all of its homologs in other bacteria, there are basic amino acid residues (Arg, Lys, and Gln) immediately adjacent to the signal peptidase cleavage site (Ala-X-Ala) that are not seen in Sec-dependent signal sequences. The mutation of these basic residues to Ala caused slightly decreased levels of extracellular PlcH, but normal localization was still observed. Deletion of the entire Tat signal sequence of PlcH not only resulted in the absence of detectable extracellular PlcH activity and protein but also caused a substantial decrease in the detectable level of plcH mRNA. Finally, data are presented which indicate that P. aeruginosa PlcH exhibits cross-species compatibility with the Escherichia coli Tat secretion machinery, but only when the E. coli Tat machinery is expressed in a P. aeruginosa host.
The
world depends on petroleum for everything from the plastics that contain
our food to the natural gas that heats our homes to the gasoline that
feed our cars’ engines. With rising prices of petroleum reflecting
demand for this finite resource, attention has been turned to alternative
sources of energy. Biodiesel, which exhibits many of the same properties
as conventional diesel but is derived from biological sources, is
an attractive alternative. Fats and oils are converted to biodiesel,
fatty acid methyl esters (FAMEs), by transesterification. FAMEs are
subsequently thermally cracked to form more lightweight transportation
fuels such as natural gas, kerosene, and possibly gasoline. We aim
to further understand the thermal cracking procedure, at an atomic
level, in hopes that this may aid in future engineering of viable
fuels. We will present our study on the effective computational modeling
of bond dissociations in the FAMEs methyl linoleate and methyl oleate,
which are the most common biodiesel products of soybeans and rapeseeds
(also known as canola seeds). We have employed quantum chemical methods,
including the density functionals B3LYP, M06-2X, and B97D; the wave
function-based MP2; and the composite CBS-QB3 method. Bond dissociation
in a 44-reaction database set for which experimental energies are
known is used to evaluate methods. We find that the M06-2X/6-31+G(d,p)
model chemistry provides results comparable to the composite CBS-QB3
method at a much reduced cost. Last, data are compiled for possible
bond dissociations in FAMEs methyl oleate and methyl linoleate.
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