Bariloche protein symposium argentine society for biochemistry and molecular biology

71
BIOCELL, 27 (Suppl. I), 2003
MI-P46.
CHARACTERIZATION OF THE POLYHYDROXY-
ALKANOATES (PHAs) DEGRADATION ACTIVITY BY AN
ISOLATED PSEUDOMONA SPP. FROM EARTH-WORM
COMPOST
Miyazaki SS, Sarti GC, Galelli MI, Bondesio ME, Casas C,
Trinchero MF, and Monzón MA.
Laboratorio de Agroalimentos, Facultad de Agronomía, UBA,
Ciudad Autónoma de Buenos Aires. E-mail:
[e-mail]
Polyhydroxyalkanoates (PHAs) are accumulated as an intracellular
organic carbon storage by different bacteria, under unbalance
growth conditions. The thermoplastic characteristics of these
polyesters and the biodegradability has attracted much attention
as environmentally degradable plastic materials.
An extracelular PHAs depolymerase of Pseudomona spp. isolated
from earth worm compost was characterized. The enzyme was
secreted by the bacterium to the culture medium only when was
cultivated on poly 3-hydroxybutyrate-co-3-hydroxyvalerate (p3HB-
co-3HV) as the sole carbon source.  When yeast extract was added
to the culture broth, Pseudomona spp. grew well but the
depolymerase activity was not detected in the culture supernatant.
The secreted enzyme was purified by chromatographic method
fast performance liquid chromatography (FPLC). The molecular
mass was determined as 45.000 by polyacrylamide gel
electrophoresis in the presence of sodium dodecylsulfate. The
purified enzyme was stable at temperature below 35ºC and at pH
value of 7,0 and can depolymerized PHAs.  Kinetics of enzymatic
activities were measured on PHAs films and commercial PHAs
powders (they were used after purification and aged). The purified
enzyme has higher affinity for PHAs films.
MI-P47.
ACTIVITY OF ENTEROCIN CRL35 DISPLAYED AT   SUB-
INHIBITORY CONCENTRATIONS. POSSIBLE
IMPLICATIONS
Carlos J. Minahk, Fernando Dupuy  and Roberto D. Morero.
Departamento de Bioquímica de la Nutrición, Instituto Superior
de Investigaciones Biológicas (CONICET/UNT), Argentina. E-
mail: [e-mail]
The  in vitro interaction between eight conventional antibiotics
namely ciprofloxacin, nalidixic acid, cephalexin, ampicillin,
vancomycin, tetracycline, erythromycin, and chloramphenicol with
sub-lethal concentrations of Enterocin CRL35 was evaluated. The
combination studies showed positive interactions between
tetracycline, erythromycin, and chloramphenicol with the cationic
peptide. The three antibiotics are excluded by efflux pumps
dependent on the membrane proton gradient as could be
demonstrated in everted vesicles derived from Listeria cells.
Enterocin CRL35 even at sub-lethal concentrations induced the
dissipation of both components of the proton motive force (
∆p)
i.e. transmembrane electrical potential and pH gradient, as could
be demostrated by the inhibitory activity of this bacteriocin on
two 
∆p-driven transport processes: the leucine transport and the
active efflux of ethidium bromide. Moreover, the growth rate of
Listeria innocua 7 was noticeably altered when this fluorescent
was present alongside sub-lethal concentration of Enterocin
CRL35, meanwhile, both compounds did not affect the bacterial
growth when they were added separately, indicating a positive
interaction between them, presumably as a consequence of the 
∆p
depolarization. Taking into account all these results, we propose
that the cationic peptide increase the effectiveness of some clinical
antibiotics through the depolarization of 
∆p, indispensable factor
for the extrusion of these compounds.
MI-P48.
CHARACTERIZATION OF AN  ANAPLASMA MARGINALE
EXPORTED ANKYRIN-LIKE PROTEIN
Rosalía Moretta, Silvina Wilcowsky, Susana Toroni de Echaide,
Marisa D. Farber.
INTA. E-mail: [e-mail]
Anaplasmosis is the tick - borne disease caused by Anaplasma
marginale (order Rickettsiales, family Anaplasmataceae), an
obligate intraerythrocytic parasite. The disease is clinically
characterized by fever, marked haemolytic anaemia,
haemoglobinaemia, icterus, weight loss, abortion and often death.
Using the PhoA fussion system we identified several exported
proteins. Among them we selected an ORF coding for a protein
with a conserved domain of ankyrin. Ankyrins were first identified
as protein mediating interactions between cytoeskeletal and
transmembrane proteins, mainly in red blood cells. However a
conserved domain of ankyrin was found to occur in a large number
of functionally diverse proteins. The selected ORF was expressed
in E coli and the purified recombinant protein was used to obtain
specific mouse serum. Polimorfism studies were performed in
different isolations by PCR amplification and restriction enzyme
analysis and each of them were confirmed by sequence.  Southern
blot analysis was developed to verify the presence in the weakly
virulent related organism Anaplasma centrale. Futher funtional
studies will be developed in order to figure out the role of the
ankyrin like protein in A marginale.
MI-P49.
CORDOBA NATIVE PGPR STRAINS: BIOCHEMICAL
AND GENETIC CHARACTERIZATION
Fischer S, Zacchi L, Principe A, Alvarez F, Marty C, Jofré E, and
Mori G.
Universidad Nacional de Río Cuarto. Córdoba. Argentina. E-mail:
[e-mail]
Over 120.000 has. of cultivable  lands on Rio Cuarto region are
under salt effects due mainly  to the deficient drainage of superficial
and subterranean  water. The objective of the present study was to
isolate salt tolerant strains, native of Cordoba soils, able to promote
the growth of important regional cultures. Wheat, maize and
agropyro were used as tramp hosts, from whose rizosphere and
endorhizosphere bacteria were isolated. These strains  were tested
in order to establish their PGPR role in vitro (N
2
 fixation,
siderophore and AIA production, phosphate solubilization). Those
strains showing two or three PGPR characteristics were selected
and studied on their ability to promote wheat, agropyro and maize
growth in greenhouse conditions. Some isolates produced a
significant increment in root and shoot fresh and dry weight
parameters. From the 16s DNA sequencing analysis, one of the
strains isolated from agropyro was assigned to the genus
Microbacterium (99% homology) whereas two isolated from wheat
showed high homology with Pseudomonas genus. Microbacterium
demonstrated to be salt, pH and temperature tolerant. Other strains
inhibited  Sclerotinia sclerotiorum and S. minor.

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