This document presents research on characterizing single amino acid variations in a non-specific nuclease from the ice-nucleating bacterium Pseudomonas syringae. The researchers cloned DNA fragments to create identical copies and expressed the nuclease recombinantly in Escherichia coli. They purified the nuclease using cation exchange and affinity chromatography and analyzed it using electrophoresis and kinetic assays. Results showed the nuclease was tolerant of EDTA and varying temperatures and pH. Evolutionary genetic variants of the nuclease were also created and analyzed. The discussion evaluates previous studies on nucleases and their cellular functions as well as the fine-tuning of enzyme functions over time. The conclusions state that Pseudomonas nucleases are very tolerant enzymes to adverse

1. Presented by:
JORDY GIAM KARLO DÍAZ DÍAZ
3° Semester l School of medicine l Bolivarian Pontifical University

2. INTRODUCTION
Pseudomonas is a genus
of straight or slightly
curved bacilli, Gram
negative, strict aerobics
although in some cases
they can use nitrate as an
electron acceptor
Pseudomona auriginosa

3. Pseudomona syringae
Pseudomonas syringae is
pathogenic of plants, it is
classified in pathovaries
according to its host specificity.
P. syringae is linked to the water
cycle and can reach the surface
of the leaves through
precipitation. It can live
epiphytically or cause disease.
This infection determines
significant economic losses and
can seriously affect both net
production and quality.

4. OVERALL OBJECTIVE
• Characterize the Single Amino AcidVariations
in a Non-specific Nuclease
from the Ice-Nucleating Bacterium
Pseudomonas syringae

5. METHODS
CLONING: create several identical
forms of a DNA fragment
Plasmid use
Presence of restriction endonucleases

6. METHODS
RECOMBINANT EXPRESSION:
Harvest  Balance buffer solution  cation
exchange chromatography  final affinity
chromatography  Electrophoresis
EscherichiaColi

7. METHODS
ELECTROPHORESIS:
• DNA, RNA, proteins and
other macromolecules
• Gel (polysaccharide 
agarose)
• Add electricity to form a
flow

8. METHODS
KINETICS:
• First the molar concentration is
calculated
• One unit of nuclease activity is
defined as ΔA260 min - 1 per mg of
protein
• "Solver" in Microsoft Excel
• Kcat (catalytic constant) =Vmax /
[E0]

9. RESULTS
FIGURE 2.
Production and
purification of the
recombinant
nuclease PsNuc

10. RESULTS
FIGURE 3. Substrate specificity, EDTA and salt stress, and influence of
temperature and pH on the activity of PsNuc

11. RESULTS
FIGURE 4.
Evolutionary-
based genetic
variants of
PsNuc.

12. DISCUSSION
Author What did he say? Is it fulfilled?
Wilkinson, S. G. (1967). Another reason to screen P.
syringae and related species for
EDTA-tolerant members of
nucleases is the known resistance
of several species within the
Pseudomonads to EDTA
Yes
Rangarajan, S., & Shankar,
V. (1999).
...Their cellular functions are often
incompletely understood, but it
has also been discussed which
nuclease tales are involved in
recombination, repair or
replication processes.
Yes
Conant, G. C., & Wolfe, K.
H. (2008)
Moreover, a fine-tuning process of
an existing (but maybe rather
weak) function might result in the
development of “not-so-new”
specificities of enzymes
Yes

13. CONCLUSIONS
1. The NSNs of pseudomonas are very
tolerant enzymes to adverse
environments
2. Not all NSN variants of the pseudomonas
have the same tolerance capabilities
against EDTA
EDTA