Differential response of Bacillus subtilis ribosomal RNA operons to nutritional stress.
Item
-
Title
-
Differential response of Bacillus subtilis ribosomal RNA operons to nutritional stress.
-
Identifier
-
AAI9630502
-
identifier
-
9630502
-
Creator
-
Samarrai, Walied.
-
Contributor
-
Adviser: Rivka Rudner
-
Date
-
1996
-
Language
-
English
-
Publisher
-
City University of New York.
-
Subject
-
Biology, Molecular | Biology, Cell | Biology, Microbiology
-
Abstract
-
The expression of ribosomal RNA operons (rrn) of B. subtilis during fast and slow growth and the intrinsic promoter strength of P1 and P2 were evaluated by placing promoter bearing fragments from rrnO, rrnJ, rrnB, rrnD and a veg control at the amyE locus using pDH32/pDG26B containing a spoVG-lacZ fusion-gene with two large regions of the amyE gene. The levels of rrn operons expression as measured by {dollar}\beta{dollar}-galactosidase, dot blot and primer extension assays revealed definite heterogeneity; namely, strong (rrnO), intermediate (rrnJ), and weak (rrnB, rrnD). The intrinsic promoter strengths were determined from the relative abundance of RNA transcripts using primer extension analysis. The P2/P1 ratios were 9.10 and 12.5 for rrnO and rrnJ, respectively. Only P2 transcripts were detected for the weak promoters of rrnD and rrnB while veg produced only P1 transcripts. Similarly, the response to stringent control during amino acid starvation induced by serine hydroxmate (SH) or carbon limitation induced by {dollar}\alpha{dollar}-methyl glucoside ({dollar}\alpha{dollar}MG) was variable: rrnO and rrnJ showed a strong effect (8-15 fold decrease), rrnD and rrnB barely responded (1.3-fold decrease) and veg remained unchanged. Promoter elements P1-P2, P1 or P2 of rrnO and rrnJ lacking Upstream Activating Sequences (UAS) were synthesized using PCR, cloned in pDG268 and integrated into three genetic backgrounds ({dollar}relA\sp+{dollar}, {dollar}relA\sp-{dollar} and {dollar}relA\sp{lcub}(S){rcub}{dollar}; the latter is a suppressor that responds only to carbon limitation) revealed the importance of the upstream region in the maximal expression levels. Only the strong P2 elements responded to carbon-source limitation in the wild-type and the suppressor cells, while the weak P1 elements continued to function in all strains. When a {dollar}relA\sp+{dollar} strain was treated with SH, both P1 and P2 responded by showing a dramatic decrease in the synthesis of lacZ-mRNA and a concomitant accumulation of (p)ppGpp. In the {dollar}relA\sp-{dollar} and {dollar}relA\sp{lcub}\prime(S){rcub}{dollar} backgrounds RNA synthesis is relaxed. During rifampin challenge, (p)ppGpp accumulated with a concomitant decrease of GTP in all cell line. Mutants in the rpoB gene did not accumulate guanosine polyphosphates. Finally, growth rate regulation of rrnO was abolished in a double mutant ({dollar}relA\sp-{dollar}, rpoB) and not in the single mutant strains (either {dollar}relA\sp-{dollar} or rpoB). We conclude that in B. subtilis, (p)ppGpp is the inhibitor of rRNA synthesis even in the absences of amino acid starvation and is involved in growth-rate regulation.
-
Type
-
dissertation
-
Source
-
PQT Legacy CUNY.xlsx
-
degree
-
Ph.D.
