Bariloche protein symposium argentine society for biochemistry and molecular biology

63
BIOCELL, 27 (Suppl. I), 2003
MI-P14.
IDENTIFICATION OF NOVEL PhoP-REGULATED GENES
IN  Salmonella enterica
Cabeza, M. Laura; Aguirre, Andrés; Spinelli, Silvana; Soncini,
Fernando C. and García Véscovi, Eleonora.
Instituto de Biología Molecular y Celular de Rosario (IBR-
CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas
(UNR).  Rosario, Argentina. E-mail: [e-mail]
Detection of magnesium plays a fundamental role in Salmonella
pathogenesis because it triggers the activation of the PhoP/PhoQ
major virulence signal-transduction pathway.  Mg
2+
 is the signal
sensed by the membrane-protein PhoQ that results in changes of
the phosphorylation level of the response regulator PhoP.  This in
turn determines the expression of the PhoP-activated genes (pags).
We took advantage of the microarray technology to analyze the
PhoP-PhoQ regulon in Salmonella enterica serovar Typhimurium.
We analyzed the transcriptional profile of the wild-type strain
compared to 
∆phoPQ or to ∆phoPQ/pPhoP grown in inducing or
repressing concentrations of Mg
2+
.  In this way we identified new
putative PhoP regulated genes.  In order to contrast this approach,
we made cromosomal lacZ transcriptional fusions to these genes
and confirmed that the genes under study were under PhoP control.
Interestingly, we determined that four independent loci displayed
homology with genes coding for transcription factors.  Among them
we unravel that the activity of a novel two-component system RstA/
RstB depends upon PhoP/PhoQ. However, the tested genes
appeared to be neither co-regulators nor intermediates in the PhoP
signal pathway to activate the already known indirectly regulated
pags, indicating the presence of yet unidentified genes belonging
to the Mg
2+
 stimulon.
MI-P15.
LicA, A PROTEIN INVOLVED IN THE VIRULENCE OF
Brucella  spp
Carrica M., Cravero S.L. and Rossetti O.L.
Instituto de Biotecnología, INTA, Hurlingham, Argentina. E-mail:
[e-mail]
Brucella spp. are facultatively intracellular bacteria that persist
and multiply in the macrophages of their mammalian hosts,
producing brucellosis, a zoonosis distributed worldwide. The
genome of three species of Brucella has been sequenced, and their
analysis has shown the absence of functional sequences for most
of the classical virulence factor. We are interested in identify
virulence factors in Brucella  through the inactivation of
uncharacterised genes and evaluating their virulence in animal
models.
We report the identification of the ORF BMEI0489 as a gene that
contributes to the bacterial virulence. A null mutant of this ORF
in the virulent B. abortus S2308 showed a severely attenuate
phenotype in the BALB/c mice model, and it recovered the full
virulence when was complemented in trans whit the wild type
ORF. This ORF was named iivA (involved in virulence gene), and
encodes for a protein of 11 kDa which presents a high content of
alfa-helix, as deduced by prediction of the secondary structure
using different algorithms and confirmed by circular dichroism of
the recombinant protein. Northern blot analysis suggested that
iivA is monocystronic and BLAST homology searches indicated
that IivA have uncharacterised homologues in others 
α-
proteobacteria.
Using chemical crosslinking and a bacterial two-hybrid system
we showed that Iiva can assembly in homomultimers. The
molecular bases of Iiva function could contribute to understand
the intimate mechanisms of the Brucella virulence.
MI-P16.
ASCOMYCETOUS YEASTS ASSOCIATED WITH PHOEBE
POPHYRIA AT THE PARK “SIERRA DE SAN JAVIER”,
TUCUMÁN, ARGENTINA.  PHYSIOLOGICAL AND
MOLECULAR CHARACTERIZATION
H.F. Pajot
1
, L.I.C. de Figueroa
1,2
.
1
PROIMI, Av. Belgrano y Pje. Caseros, 4000. Tucumán, Argentina.
2
Universidad Nacional de Tucumán. Tucumán, Argentina. E-mail:
[e-mail]
“Yungas Andinas” is the name of the mountain forests in northwest
Argentina. “Sierra de San Javier” Park belongs to the Uiversidad
Nacional de Tucumán and is located in this area.
We studied specific yeast communities associated with “Laurel
del Monte” (Phoebe pophyria). The objetive was to isolate yeast
strains and analyze their metabolic and genetic diversity. Yeasts
was isolated from  alive and dead parts of plants. A preliminary
identification of isolated yeasts was made according to conventional
techniques. These studies showed some limitations for
identification of new isolates, since there was not a 100% matching
with reference strains profiles. This work was followed by using
molecular techniques based on amplification, restriction fragment
length polymorphism analysis (RFLP) and sequence analysis of
rDNA. Phylogenetic studies based on 26S  sequences showed that
the isolated yeast belongs to the ascomycetous yeasts.
Hanseniaspora opuntiae,  Kodamaea ohmerii, Candida haemulonii
and Pichia rabaulensis  strains isolated show some physiological
or molecular differences with type strains.
MI-P17.
UPTAKE OF COPPER BY YEASTS ISOLATED FROM
COPPER POLLUTED AREA OF ARGENTINA
L.B.Villegas
1
, M.J.Amoroso
1
, O.I.Villegas
3
 and L.I.C.de Figueroa
2
.
1
PROIMI, Tucumán, Argentina. E-mail: [e-mail]
2
Microbiología Superior, Fac. Bioqca., Qca. y Fcia., U.N.T,
Argentina. 
3
Química Analítica, Fac. Qca., Bioqca. y Fcia.,
U.N.S.L, Argentina.
Copper, is utilized as an essential cofactor in critical biological
processes. However, excess copper is highly toxic to most
organisms. Microorganisms may be used to remediate wastewaters
or soils contaminated with cooper. This capacity can be used to
concentrate, remove and recover copper from different
contaminated sites. The aim of this study was to analyze the growth
and uptake copper capacity of two isolated yeasts from copper
polluted area in Northwest Argentina.  Isolates designated as RCL-
3 and RCL-11 were obtained from a sediment sample of a Argentina
mine containing high copper level. Yeasts were cultured in minimal
liquid medium, yeast nitrogen-base containing Cu
2+
 as CuSO
4
, at
0.1, 0.2, 0.5 and 1 mM. As positive control, cultures without this
heavy metal were performed. Cultures were incubated at 30
o
C,
250 rpm and pH 5. The growth of yeast was followed by turbidity
at 620 nm, and measured by counting cells number. Total copper
concentration in the supernatants of the culture media and
intracellular were determined by atomic absorption
spectrophotometer. The growth rates decreased with the increase
copper concentration, but the uptake of copper percentage incresed
when the copper concentrations were smaller. The uptake of copper
by yeast is important for understanding how these microorganisms
develop mechanisms for surviving in presence of toxic copper
concentration.

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