Figure 1.
Growth of wild type Haloferax volcanii and the Δpcc1 mutant cells.
Growth curves comparing the growth rate of H133 (wild-type), Δpcc1 (HAN16) and Δpcc1 containing the complementing plasmid (HAN19). Results are a mean of 10 replicates, error bars indicates standard error of the mean.
Figure 2.
Advanced glycation products in wild type Haloferax volcanii and the Δpcc1 mutant cells.
A. Measurement of AGEs-specific fluorescence in the H133 (wild-type) and in Δpcc1 (HAN16). The emission spectrum from 400 nm to 480 nm upon excitation at 370 nm is presented. A representative sample is shown. B. Accumulation and secretion of AGEs. AGE-specific fluorescence is shown relative to the wild-type. The results represent three independent experiments, error bars indicates standard error of mean.
Figure 3.
Flow cytometry analysis of Haloferax volcanii H195 and H133 Δpcc1 mutant cells.
H195 (based on H133, with a bgaHa-Bb deletion, and a leuB- Ag1 allele, which has flow cytometry profiles highly similar to H133– data not shown) A. Cell size as determined by forward light scatter of H195, and of Δpcc1 (HAN16). B. Nucleic acid content as determined by acridine orange fluorescence of H195 (wild-type) and in Δpcc1 (HAN16).
Figure 4.
Nucleoside analysis analysis in wild type Haloferax volcanii and the Δpcc1 mutant cells.
tRNAs were extracted from each strain and hydrolyzed to nucleosides. The nucleoside content was determined by HPLC with detection by UV/Vis at 254 nm. Analyses were performed in triplicate from independent cultures. A. HPLC chromatographs of nucleosides from wild-type, wild-type with synthesized t6A added, or Δpcc1. t6A elutes at 24 minutes. B. Comparison of the t6A peak area of wild-type and Δpcc1. The ratios of Ψ-modified base/m22G were used to normalize tRNA concentrations across samples with t6A peak area of wild-type set at 100%. Δpcc1 contains approximately 19% less t6A than wild-type (P = 0.02).
Table 1.
Strains used in this study.