Article Figures & Data
Figures
- FIG. 1.
Bdellovibrio bacteriovorus life cycle depicted in eight stages. In the extracellular predatory phase (stage 1, free-living attack Bdellovibrio [Bd]), the predators search for (stage 2, prey location) and attach to (stage 3, attachment) prey cells. They enter the prey cell wall initiating the intraperiplasmic or replicative phase (stage 4, invasion) (24-26, 28). In the periplasm, they feed upon the cytoplasmic contents of the prey (stage 5, establishment or early bdelloplast) (10) and form what is called the bdelloplast. They elongate (stage 6, elongation or bdelloplast) and segmentate into approximately 6 to 10 smaller components (stage 7, division or late bdelloplast) (1, 15, 16), finally releasing the progeny by lysis of the prey (stage 8, release) (14, 20). Interestingly, some strains of Bdellovibrio can generate host-independent mutants that grow axenically (18). The inserts show fluorescence staining of bdelloplasts that distinguishes live cells from dead cells; BacLight-stained live bacteria with intact membranes are green (Bdellovibrio), and dead cells with disrupted membranes are red (prey, E. coli) in the indicated examples.
- FIG. 2.
Nudix sequences of the Bdellovibrio bacteriovorus genome. Bd0714 (NP_967680), Bd3179 (NP_969942), Bd2220 (NP_969058), Bd0236 (NP_967241), Bd2755 (NP_969548), and one modified signature sequence in Bd0654 (NP_967621). The black background indicates identity in a proline residue and in the Nudix signature sequence. Bd0654, the open reading frame with the modified signature sequence, has a lysine residue instead of the typical first glutamate in GX5EX7REUXEEXGU. The boxes show homologous residues. The alignment was done with ClustalW and the figure with ESPript (8).
- FIG. 3.
(A) Relative activity of the Bd0714 for possible nucleotide substrates; dGTPase activity was normalized to 100%. Black bars correspond to the colorimetric assay with added enzyme, gray bars correspond to all components of the assay except for inorganic pyrophosphatase (IPP) or calf intestinal phosphatase enzyme (CIP), and white bars correspond to the reaction carried out without the Bd0714 enzyme. (B) Relative activity of Bd0714 versus Bd0714-E70Q for deoxynucleoside triphosphates, with dGTP normalized to 100% activity.
- FIG. 4.
Kinetic data for Bd0714 and Bd0714-E70Q. (A) Initial velocities as a function of dGTP concentration. The continuous curves correspond to nonlinear least-square fits of the data to the Michaelis-Menten equation. (B) Plot of the k cat values of pH in the range of 7.5 to 9 for Bd0714 and Bd0714-E70Q.
- FIG. 5.
RT-PCR analysis of the Bd0714 gene in B. bacteriovorus HD100, the ΔBd0714 mutant, and host-independent B. bacteriovorus. Total RNA was extracted from E. coli ML35 prey (lane 1), host-independent B. bacteriovorus (lane 2), the attack-phase ΔBd0714 mutant (lane 3), and wild-type B. bacteriovorus HD100 (lane 4, free-living attack phase; lane 5, early bdelloplast; lane 6, bdelloplast; and lane 7, late bdelloplast) throughout the life cycle and used in RT-PCRs with primers specific for the Bd0714 gene. The rpsL gene was used as a load control for B. bacteriovorus RNA. The absence of an RT-PCR product in lane 1 indicates that the product seen for both the Bd0714 and rpsL genes corresponds to B. bacteriovorus RNA rather than to any possible contaminating E. coli RNA. MW, molecular weight marker.
Tables
- TABLE 1.
Mutation frequencies of the E.coli SB3 mutT mutants transformed with different plasmids
Plasmid No. of indicated mutants per 109 cells Streptomycin resistant Nalidixic acid resistant pTrc99A 491 ± 206 1,123 ± 257 pTrc99mutT 50 ± 26 79 ± 75 pTrc99Bd0714 7 ± 2 23 ± 2
Supplemental material
Files in this Data Supplement:
- Supplemental file 1
-
Supplemental methods.
Zipped PDF file, 124K. - Supplemental file 1
-
Table S1, E. coli strains and plasmids used; Table S2, primers used.
MS Word document, 31K.
- Supplemental file 1
-
Supplemental methods.
