Bariloche protein symposium argentine society for biochemistry and molecular biology

73
BIOCELL, 27 (Suppl. I), 2003
MI-P54.
RADIATION SURVIVAL STRATEGIES OF A GRAM
NEGATIVE YELLOW PIGMENTED BACTERIUM
Oppezzo O, Fernández Degiorgi C, Fernández R, Costa C, Dantur
K, Ibáñez J, Pizarro R.
CNEA. Departamento de Radiobiología. E-mail:
[e-mail]
We isolated a yellow pigmented nonfermentative Gram negative
bacterium which exhibited high resistance to the lethal effects of
sunlight. In order to study the mechanisms responsible for radiation
resistance in this organism, we assayed its ability to survive under
UV exposure. Survival curves obtained with the pigmented
bacterium irradiated at 254 and 313 nm showed increased
resistance when compared with those obtained for E.coli K12,
and a similar response was found at 365 nm. HPLC analysis of
pigments extracted from the isolated bacterium suggested the
presence of carotenoids. Protective effects of pigments were
evaluated studying survival curves obtained with bacteria grown
in the presence of an inhibitor of carotenoids synthesis and with a
color-less mutant derived from the isolated organism by chemical
mutagenesis. Pigments depletion had no influence at 254 and 313
nm, and slightly reduced resistance at 365 nm. Similar photo-
reactivation capacity was found in E.coli and the isolated organism,
which exhibited higher levels of catalase activity. The lethal effects
produced at the assayed wavelengths depend on different targets
and action mechanisms. The resistance at 254 and 313 nm suggests
that the ability to overcome DNA damage plays a key role in
radioresistance of the isolated bacterium.
MI-P55.
yhdO GENE OF Bacillus subtilis ENCODES AN ACYL-ACP
ACYLTRANSFERASE
Paoletti, Luciana E.; Schujman, Gustavo E. and de Mendoza, Diego.
IBR-CONICET. Facultad de Ciencias Bioquímicas y Farmacéuticas,
Rosario, Argentina. E-mail: [e-mail]
The first step in phospholipids biosynthesis is the acylation of
glycerol-3-phosphate to give phosphatidic acid, which is the
precursor of all membrane phospholipids. That step has been
characterized in Gram negative bacteria but the knowledge in Gram
positive microorganisms is scarce. That is why we begun the study
of  Bacillus subtilis acyltransferases. In E. coli, the genes plsB
and plsC encode for glycerolphosphate and acylglycerol phosphate
acyltransferases, respectively. The analysis of B. subtilis genome
revealed only one gene homologue to plsC, called yhdO, and no
homologues to plsB. To study the role of yhdO, we constructed the
strain LP15, which contains the sole copy of yhdO under the control
of an IPTG inducible promoter. In the absence of IPTG, growth of
strain LP15 stops and also does lysophospholipids and
phospholipids synthesis, as determined by one-dimensional TLC.
Expression of yhdO in E. coli cells  allowed growth of plsC but
not of plsB mutant cells. By gel shift assays we demonstrated that
the promoter region of yhdO is able to bind FapR, the global
regulator of fatty acid biosynthesis of Gram positive bacteria, and
by Western blot assays we found that YhdO is accumulated in
fapR
-
 cells. With these results we conclude that yhdO is the only
gene of B. subtilis that encodes for an acylglycerolphosphate
acyltransferase, that YhdO probably also has glycerolphosphate
acyltransferase activity, and that expression of yhdO gene is tightly
coordinated with expression of genes involved in fatty acid
biosynthesis.
MI-P56.
IN SILICO NORTHERN OF THE PROTEOLYTIC
MACHINERY OF PHYTOPHTHORA INFESTANS
París, Ramiro
1
; Govers, Francine
2
 and Lamattina, Lorenzo
1
.
1
IIB, FECyN, UNMdP, Argentina. 
2
PPD, WAU, Netherlands.
E-mail: [e-mail]
Members of the oomycete genus are the major pathogens of
innumerable crops, among them P. infestans that causes the most
important economic looses on potato fields around the world. Thus,
the basic knowledge of the P. infestans biology remains a priority
for many plant pathologists. Proteolysis is an essential metabolic
process required for protein processing and turnover. In particular,
proteases of pathogenic microorganisms are key component of
developmental stages and pathogenesis. Previously, we have
analyzed the intra and extracellular proteolytic activities in P.
infestans. We have found an extracellular serine activity and an
aspartic intracellular one. We have also cloned and expressed a
cDNA with acidic in vitro activity. Nowadays, the important
amount of information generated by genomic projects is demanding
to be deeply studied. In this work we analyzed a databank of 18
different P. infestans EST libraries (SYNGENTA-NCGR). A search
for high protease-homology rendered 110 entries, of 72822, with
homology to proteases [numbers of entries]: aspartic [5], serin [12],
cystein [28], subtillisin [7], metallo [5], ubiquitin specific [18]
and not identified [35]. The “in silico northern” analysis predicts
the differential expression of each clone during developmental
stages, potato-P. infestans interaction and stress conditions. Results
showed a broad view of the putative proteolityc machinery of P.
infestans and could be of interest for planning further research
directions. Supported by UNMdP, Conicet and ANPCyT, Argentina.
MI-P57.
PHENOTYPIC CHARACTERIZATION OF IRON
TRANSPORT MUTANTS (FUR
-
) OF BORDETELLA
BRONCHISEPTICA  SUGGESTS THAT VIR90 IS FUR
REGULATED
Passerini De Rossi, B.*; Friedman, L.; Franco, M.
Cátedra de Microbiología, Facultad de Farmacia y Bioquímica,
U.B.A., Buenos Aires, Argentina. *E-mail: [e-mail]
Bordetella pertussis and B. bronchiseptica undergo phenotypic
changes modulated by the bvgAS locus, which regulates the
expression of many virulence genes. We previously reported the
n-terminal sequence of vir90, a 90kda bvg-regulated omp of B.
Pertussis.  vir90 encodes for a protein with similarities to
ferrisiderophore receptors. We scanned the vir90 promoter region
and found a potential fur box. Vir90 was detected under high-iron
growth conditions in immunoblotting of OMPs, and its expression
increased 4-fold under low-iron conditions. In order to test if the
expression of vir90 is repressed by Fur, we selected manganese-
resistant deregulated iron transport mutants (Fur
-
). 40 mutants
were checked for positive Csaky assay for hydroxamate class
siderophore and hemolysis (Hly+ indicates Bvg+) in media with
iron. Two Hly+/Csaky+ mutants were cultured under both high-
iron and low-iron conditions and tested for constitutive production
of siderophore. Iron-regulated protein expression in OMPs from
these cultures was monitored by SDS-PAGE as well as
immunoblotting. The amount of Vir90 is incresed in these mutants
independently of the iron condition used, in agreement with
mutation in fur. These results suggest that expression of Vir90 is
fur regulated.

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