Bariloche protein symposium argentine society for biochemistry and molecular biology

78
BIOCELL, 27 (Suppl. I), 2003
MI-P74.
BIOFILM GROWTH OF Pseudomonas aeruginosa
MISMATCH REPAIR DEFICIENT CELLS CORRELATES
WITH EMERGENCE OF MUCOID AND SMALL-COLONY
ADHERENT VARIANTS
Moyano AJ, Argaraña CE, and Smania AM.
CIQUIBIC-CONICET, Facultad de Ciencias Químicas, UNC.
Córdoba, ARGENTINA. E-mail: [e-mail]
Pseudomonas aeruginosa is an opportunistic human pathogen
capable of forming specialized communities known as biofilm.
Particularly in Cystic Fibrosis (CF), a biofilm mode of growth is
thought to be responsible for persistent lung infections conferring
protective advantage against antibiotic therapy, oxidative stress,
and immune system attack. Moreover, emergence of mucoid and
highly adherent small-colony variants within CF lung have been
demonstrated to play a key role in biofilm formation and
consequently in P. aeruginosa pathogenesis. Recently, the
observation of a high proportion of mutator P. aeruginosa isolates
from CF patients leads to the association between hypermutability
and P. aeruginosa adaptability. In this work we show new evidences
supporting this postulation. By performing an analysis of P.
aeruginosa growth in continuous-flow culture chamber, a higher
frequency of small adherent variants emerged from biofilms formed
by a hypermutator P. aeruginosa mutant compared with isogenic
wild-type strain biofilms. In addition, when biofilms were exposed
to sublethal levels of H
2
O
2
, treatment known to induce the
formation of mucoid variants in vitro, we observed the emergence
of mutS mucoid variants at a frequency significantly high respect
to the wild-type strain. Our results suggest that hypermutability
favor the acquisition of the more adaptable characteristic P.
aeruginosa CF phenotypes and could explain the high frequency
of hypermutability observed in P. aeruginosa CF isolates.
MI-P75.
NEW INSIGHTS INTO THE CONTROL OF mAGNESIUM
HOMEOSTASIS IN Salmonella enterica
Spinelli, Silvana; Pontel, Lucas; García Véscovi, Eleonora; and
Soncini, Fernando C.
Instituto de Biología Molecular y Celular de Rosario (CONICET),
and Facultad de Ciencias Bioquímicas y Farmacéuticas (UNR),
Rosario, Argentina. E-mail: [e-mail].
Magnesium plays a fundamental role in Salmonella pathogenesis.
It controls the activation of the PhoP/PhoQ regulatory system,
signaling the bacteria  its specific location inside the host.  In
response to Mg
2+
 limitation, the PhoP/PhoQ regulatory system
triggers the expression of two high-affinity Mg
2+
 transporter genes,
mgtA and mgtCB.  Mutations in mgtA or in mgtCB greatly affect
the ability of Salmonella to grow in low Mg
2+
 environments.
We uncovered a novel checkpoint in the Mg
2+
 deprivation response.
This mechanism can detect this cellular stress bypassing PhoQ
and posing an additional control over the expression of the Mg
2+
transporters.  We have demonstrated that in mgtA the responsive
element for the PhoQ-independent regulatory mechanism maps to
the 5’UTR.  We further characterized this region using site directed
mutagenesis, and examined the level (transcriptional/
posttranscriptional) at which this system exerts its control.  The
study of this homeostatic network will provide new insights in the
mechanisms that allow Salmonella to proliferate within its host.
MI-P76.
ISOLATION AND CHARACTERIZATION OF A SUCROSE
- PHOSPHATE PHOSPHATASE FROM AGROBACTERIUM
TUMEFACIENS
Torres, Leticia L.; Cumino, Andrea C.; Curatti, Leonardo and
Salerno, Graciela L.
Centro de Investigaciones Biológicas (FIBA), Mar del Plata,
Argentina. E-mail:  [e-mail]
 Agrobacterium tumefaciens is an 
α-protebacterium able to induce
the crown gall disease in plants. It has a chemoorganotroph
metabolism, exploiting a variety of carbohydrates, organic acids
and amino acids as carbon sources. Sequence analysis of A.
tumefaciens C-58 genome revealed the existence of a Glucosyl-
Transferase-Domain (GTD) and a Phospho-Hydrolase-Domain
(PHD),  characteristic of sucrose biosynthesis related proteins
present in oxygenic photosynthetic organisms. The Agrobacterium
PHD is encoded by a 747 bp orf  whose deduced amino acid
sequence shares 22-28 % identity with cyanobacterial and plant
sucrose- phosphate phosphatase (SPP), enzyme that catalyzes the
last step in sucrose synthesis. The present study describes the first
isolation, structural analysis and functional characterization of a
prokaryotic SPP gene present in a non-photosynthetic organism.
The 747-orf expressed in E. coli produced a fully active SPP (248-
amino-acid). Kinetic, biochemical and molecular properties of the
recombinant protein were similar to those of SPP purified from
Agrobacterium cells.  It is a monomeric enzyme as its predicted
Mr (27,262 Da) was similar to the native Mr. Agrobacterium SPP,
similarly to orthologous enzymes, is highly specific for sucrose-6-
P (Km 1.0 
± 0.1 mM) and it is inhibited by common phosphatase
inhibitors (fluoride, molibdate and vanadate). Moreover, the
conserved sequence motifs identified in all described SPP, are
present in Agrobacterium SPP. Supported by CONICET, Fundación
Antorchas, Univ. Nac. de Mar del Plata, and FIBA.
MI-P77.
THROMBOCYTOPENIA DURING THE ACUTE PHASE OF
CHAGAS’ DISEASE IS INDUCED BY THE TRANS-
SIALIDASE
Tribulatti Ma. Virginia
1
, Mucci Juan
1
, Leguizamón, Ma. Susana
2
,
Campetella Oscar
1
.
1
Instituto de InvestigacionesBiotecnológicas, Univ, Nacional de
San Martín. 
2
Facultad de Medicina, Univ. de Buenos Aires.
E-mail: [e-mail]
Thrombocytopenia and bone marrow hypoplasia have been
described during the acute phase of Chagas’ disease. At present
there is no molecular explanation for these observations. The
parasite sheds an enzime, the trans-sialidase (TS), which transfers
sialic acid residues between glycoproteins. By another hand, it is
known that the sialic acid content of the platelet surface is related
to their rapid removal from circulation. Also, platelets were
involved in parasite clearance. Here we investigated whether the
TS shed by the parasite is associated with the thrombocytopenia.
The iv. administration of 10 µg of TS induces transient
thrombocytopenia and the passive transfusion of pre-treated
platelets results in their rapid removal from blood. Both
experiments were reproduced in splenectomized mice, suggesting
that Kuppfer cells are the major responsible of platelet clearance.
To test this hypothesis, mice were depleted from phagocytes with
clodronate-containing liposomes. The absence of thrombocytopenia
in these mice, after TS injection, demonstrates the active
participation of Kuppfer cells in the removal of altered platelets
by the enzime action. Finally, a correlation between the percentage
of desialilation of platelet surface and their disappearance from
blood was observed. All this results contribute for the
comprehension about the molecular mechanisms involved in
Chagas’ disease pathogenicity.

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