Download Investigating the role of an uncharacterized carboxy

Investigating the role of an uncharacterized carboxy-terminal
protease, CtpA, in Rhizobium leguminosarum bacteroid development
C5
Kerrigan B. Gilbert, Michael C. Bayda, and Christopher K. Yost
University of Regina, 3737 Wascana Parkway, Regina, Sask S4S 0A2
Creation of ctpA mutant
Mutated ctpA
gene cloned
into suicide
vector
pJQ200SK5
ctpA
Ab Resistance
37Mutant
copy
ctpA on
pJQ200SK5
yibQ
1. ctpA Promoter: a 296nt region upstream of ctpA has
promoter activity. A slight overlap between yibP and
ctpA may mean ctpA is also expressed by the yibP
promoter.
oriT
Plate counts for repeated attempts to mutate ctpA
2. Predicted secretion signal sequence is present in CtpA,
from amino acids 1 to 32.
3. Catalytic Sites:
Bolded amino acids: conserved in both R.
leguminosarum and Synechocystis sp. PCC6803
Amino acids highlighted in yellow: essential for CtpA
activity in Synechocystis sp. PCC68034
Number of Colonies
Displaying Mutant Phenotype
Tetracycline Cassette 6
- Double crossover event
0
Vector DNA
-Single crossover event using
internal ctpA fragment
0
Vector DNA
- Single crossover event instead
of double crossover
*On average 4 plates / mating
9.00
7.08
4,000
6.00
*
2,202
*
2.18
2,000
3.00
vector
Mate construct into R.
leguminosarum
3
Bacteroid
0
0
sacB
ctpA interrupted by:
2
37
suicide
Mutagenesis Results
ctpA
1
51
of
4,253
Miller Assay
Enzymatic Activity
* p=0.0001: significant decrease in gusA activity in bacteroids vs
free living cells as measured by Miller Assay
Summary
Ab Resistance
Methods and Results
yibP
Wildtype ctpA on
chromosome
Sma I
Free Living
6,000
Calculations for the rate of ß-glucuronidase activity using the
Miller Assay and as a function of total protein reveal that the
ctpA promoter is downregulated two- to three-fold in the
bacteroid.
To date, repeated attempts to mutate ctpA using two different
strategies have been unsuccessful suggesting that ctpA may
be essential in the free-living state.
Bioinformatic analysis reveals that ctpA may be part of a twogene operon with yibP. As well, a putative secretion signal
sequence is present, suggesting ctpA may be present in the
periplasm. Finally, the amino acids required for catalytic activity
of CtpA in Synechocystis are conserved in R. leguminosarum.
References
1Oke,
V., and S. Long (1999) Curr. Opin. Micro. 2: 641-646.
2Hengge,
R., and B. Bukau (2003) Mol. Microbiol. 46: 1451-1462.
Trust Sanger Centre Rhizobium leguminosarum biovar
viciae Sequencing Project:
http://www.sanger.ac.uk/Projects/R_leguminosarum/
4Inagaki, N. et al. (2001) J. Biol. Chem. 276: 30099-30105.
5Quandt, J.Q., and M.F. Hynes (1993) Gene 127: 15-21.
6Fellay, R., J. Frey, and H. Krisch. 1987. Gene 52: 147-154.
3Wellcome
522 / plate*
Rate of Reaction
(nmol/min/mg of protein)
Antibiotic
cassette
inserted into
ctpA at Sma I
site in the
centre of the
gene
Select for: Ab-Resistant and
Sucrose-Resistant colonies
Bioinformatic analysis3
gusA Assay Using Two Different Methods
Double Recombinant Event - Protease gene is interrupted by a
selectable marker ie. an antibiotic resistance gene
GmR
The bacterium Rhizobium leguminosarum exhibits two
distinct life cycles:
- as a free-living organism present in soil, and
- as a bacteroid found in nodules present on the roots
of legumes1.
The Rhizobial-legume relationship is an important
symbiosis in agriculture as it is a major source of global
nitrogen input.
R. leguminosarum is only able to fix nitrogen when in the
bacteroid form; therefore a better understanding of this
life cycle is necessary.
Since it has been demonstrated that proteases play a
key part in the cell cycle and development of other
bacteria2, CtpA, a protein uncharacterized in rhizobial
species, is being investigated to understand its role in
bacteroid development.
ctpA Gene Expression: gusA fusion to ctpA promoter
Miller Units
Introduction
Acknowledgements
This work was funded by an NSERC grant to CKY.