Bariloche protein symposium argentine society for biochemistry and molecular biology

44
BIOCELL, 27 (Suppl. I), 2003
MI-C1.
CHEMICAL MODIFICATION OF AN 
α-MANNOSYL-
TRANSFERASE FROM Acetobacter xylinum
Abdian, Patricia L.
1
; Barreras, Máximo
1
; Geremia, Roberto A.
2
and Ielpi, Luis
1
.
1
Fundación Instituto Leloir, Av. Patricias Argentinas 435, (1405)
Buenos Aires, Argentina and 
2
CERMO-CNRS, Université Joseph
Fourier, BP 53X, F-38041 Grenoble Cedex 9, France. E-mail:
[e-mail]
AceA is a non-processive 
α-1,3 mannosyltransferase from
Acetobacter xylinum, which transfers mannose from GDP-mannose
to polyprenyl-pyrophosphate linked cellobiose (Glc
2
-PP-Lip)
during the assembly of the repeat unit of the exopolysaccharide
acetan. AceA belongs to CaZY family 4 of retaining
glycosyltransferases. We have previously identified a series of
conserved residues, performed site-directed mutagenesis and found
that acidic aminoacids may be involved in sugar donor binding
and catalysis. We have now confirmed our findings by chemical
modification using the carboxyl specific reagent 1-ethyl-3-(3-
dimethyl-aminopropyl) carbodiimide (EDAC), resulting in a time-
and concentration-dependent inactivation of AceA. The semi-
logarithmic plots of residual enzyme activity vs time at different
EDAC concentrations resulted in a linear relationship, indicating
pseudo-first order rate kinetics. A log plot of inactivation rate
constants vs EDAC concentration indicated that at least one acidic
aminoacid is important for AceA activity. The effect of substrates
was investigated showing that a synthetic analog of the acceptor
Glc
2
-PP-Lip partially protected the enzyme from inactivation by
EDAC.
MI-C2.
RHODOBACTER CAPSULATUS SOD MUTANT DISPLAYS
INCREASED SPONTANEOUS DNA MUTAGENESIS
Bortolotti, Ana; Bittel, Cristian; Tabares, Leandro C. and Cortez, Néstor
IBR (CONICET & UNR), FCByF, UNR. Suipacha 531, S2002LRK-
Rosario. E-mail: [e-mail]
The purple bacterium Rhodobacter capsulatus can live under a
broad range of enviromental conditions. When illuminated in
anaerobiosis this microorganism synthesizes ATP through a cyclic
electron transport around a single photosystem, whereas in the
presence of air it shifts to a respiratory metabolism. In this
condition, reactive oxygen derivatives such as superoxide,
peroxides and the hydroxyl radical are produced by reduced
intermediates of the electron transport chain.
Superoxide dismutases (SOD) are the first line of antioxidant
defense in most aerobic organisms.
We have cloned the promoter region of the single SOD present in
Rhodobacter capsulatus and determined the transcription start site
by primer extension. Besides canonical –35 and –10 elements,
one putative binding site for the redox regulator RegA was
identified. A sod insertional mutant was studied to evaluate the
fisiological role of SOD. Lucigenin luminesence was determined
as measurement of intracellular superoxide level, indicating a two-
fold raise in the mutant strain.
The oxidative damage on Fe/S centers was evaluated by measuring
aconitase activity. An increased rate of spontaneous mutagenesis
suggests that superoxide itself or Fe released from Fe/S clusters
promoted DNA oxidative injury.
MI-C3.
THE RPON GENE OF BRUCELLA ABORTUS IS
IMPORTANT FOR THE BACTERIAL PERSISTENCE IN
MICE
Iannino, F.; Ciocchini A.; Vidal Russell, R.; Ugalde, R. A. and
Iñón de Iannino, N.
IIB -INTECH, UNSAM-CONICET, Buenos Aires, Argentina.
E-mail: [e-mail]
rpoN encodes the alternative sigma factor 
σ54, which is required
for transcription of a wide range of genes involved in diverse
physiological functions such as nitrogen metabolism, dicarboxylic
acid transport and xylene degradation. 
σ54 is also involved in the
regulation of virulence-related factors in both plant and animal
pathogens, including synthesis of flagellar and pilus components.
A gene with high homology to Agrobacterium tumefaciens 
σ54
was identified in Brucella abortus. Given the facts that RpoN
activates the expression of a wild variety of environmentally
regulated genes and is required for virulence in a variety of
pathogens we hypothesized that RpoN would play a central role
in B. abortus virulence.  In this study we describe the results of
experiments that involved the construction of a B. abortus rpoN
mutant in order to study the role of 
σ54 in B. abortus pathogenesis
in HeLa cells, murine macrophage J774 cell line and in Balb/c
mice. We concluded that B. abortus rpoN is not essential for
infection and multiplication in nonphagocytic or phagocitic cells,
however  rpoN may be important for the bacterial persistence in
Balb/c mice.
PL-C12.
PURIFICATION AND ANTIFUNGAL ACTIVITY  OF A
SUNFLOWER LIPID TRANSFER PROTEIN EXPRESSED
IN Escherichia coli AS A GST-FUSION
Espinosa Vidal, Esteban;  Martín, Mariana; de la Canal, Laura.
IIB, UNMdP, Buenos Aires, Argentina. E-mail: [e-mail]
Lipid transfer proteins (LTPs) are low-molecular-mass, basic,
antifungal proteins extensively studied in higher plants. A wide
array of limitations have been found to express antifungal protein
in vitro, but in order to produce enough protein to develop structural
and functional studies we have tempted the expression of an
antifungal protein in Escherichia coli. We have previously isolated,
characterized and cloned Ha-AP10, a potent sunflower antifungal
protein homologous to members of the LTP family. This work
describes the cloning of the PCR-amplified ADN encoding Ha-
AP10 into an Escherichia coli expression vector, and its expression
as a fusion with gluthathion-S-transferase (GST). The optimal
condition of the expression recombinant protein was determined
and a protein with the expected molecular mass was detected in
the soluble fraction. In addition, the specific antibody against
sunflower-extracted Ha-AP10 recognized the fusion protein
purified by affinity chromatografy. A preliminary characterization
of the antifungal activity of GST-Ha-AP10 showed it was unable
to inhibit the germination of Fusarium solani spores, which can
be a consequence of structural changes in the protein bound to
GST. When the fusion protein was cleaved with thrombin, small
amounts of Ha-AP10 were produced to enable a detailed
characterization. Although, low yields are obtained, the system
proved to be feasible. Scale-up process will allow the production
of the recombinant protein to perform structural and functional
studies.

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