Bariloche protein symposium argentine society for biochemistry and molecular biology

79
BIOCELL, 27 (Suppl. I), 2003
MI-P78.
PROTECTIVE ACTION OF ppGpp ON MICROCIN J25
SENSITIVE STRAINS
Vincent, Paula; Pomares, Fernanda;, Farías, Ricardo and
Salomón, Raúl.
INSIBIO e Instituto de Qca Biológica (UNT-CONICET). Tucumán.
Argentina. E-mail: [e-mail]
Stringent response is mediated by the alarmone ppGpp. Many
reports have shown that ppGpp has a significant role in growth
rate control and gene expression during stationary phase. Sensitive
strains exhibit an increased resistance to the peptide antibiotic
microcin J25 in stationary phase. This result led us to suppose
that accumulation of ppGpp during stationary phase could have a
protective action against the antibiotic. To test this hypothesis we
transformed E. coli AB1133, which is hypersusceptible to microcin
J25, with a plasmid that overproduces ppGpp (pALS13). We
observed that for AB1133 colony counts dropped five orders of
magnitude, while AB1133 (pALS13) remained unaffected. The
experiment was repeated using another ppGpp-overproducing
plasmid, pCNB0209R, with similar results. A direct interaction
of ppGpp with the microcin molecule can be ruled out, since
extracts of ppGpp-overproducing strains did not exert any
protective effect against microcin in vitro. It is known that MccJ25
targets RNA polymerase and reduces transcription. Therefore, we
did  in vivo transcription experiments, using AB1133 y AB1133
(pALS13) in the presence and in the absence of microcin. In
contrast to the control, microcin had no effect on transcription
activity in the strain harboring the ppGpp-overproducing plasmid.
We conclude that ppGpp, either directly or indirectly, protects RNA
polymerase from inhibition by microcin J25.
MI-P79.
DEVELOPMENT OF A NEW SYSTEM FOR Babesia bovis
STRAIN IDENTIFICATION USING MOLECULAR
MARKERS
Wilkowsky S
1
, Dominguez M
1
, Farber M
1
, Echaide I
2
, Valentíni I
2
,
Alcaraz E
3
, Cetra B
3
, Suarez C
4
, and Florin-Christensen M.
1
1
CICVyA-INTA Castelar, Argentina; 
2
INTA Rafaela, Argentina;
3
INTA Mercedes, Argentina and 
4
ADRU-USDA, Pullman, USA.
E-mail: [e-mail]
Prevention against the bovine hemoparasite Babesia bovis is
achieved by vaccination with attenuated strains. Vaccine failures
could be attributed to vaccine mishandling or to infection with
field strains against which the vaccinial strains are not protective.
Strain-specific molecular markers could be useful tools to
discriminate this and, also, they could serve for epidemiological
studies. The B. bovis Variable Merozoite Surface Antigen-2
(VMSA) family is encoded by five genes in the Mexican Mo7
strain, namely msa-1, -2a
1
, -2a
2
, -2b and -2c. Homologues of some
of these genes are present in the Argentine strains R1A, S2P and
M1A and the Texan strain T2B. We hypothesized that
polymorphism among members of the VMSA family could be
reflected in different restriction patterns. We tested our hypothesis
by PCR-amplifying msa-2a
1
, a
2
 and b from DNA of the Mo7, R1A,
S2P, M1A and T2B strains. PCR-products were digested with
BsPMI and analyzed by polyacrylamide gel electrophoresis
followed by silver staining. The results showed that all strains
produced differential banding patterns. These data strongly suggest
that these genes may be adequate molecular markers for strain
comparison. Analysis of a larger number of samples from cattle
from different regions of Argentina and its neighboring countries
is now under way.
Supported by ANPCyT and Fundacion Antorchas, Argentina.
MI-P80.
NATIVE FLUORESCENT Pseudomonas AS BIOCONTROL
AGENTS OF ALFALFA SEEDLING DISEASES
Yanes M
1
, De La Fuente L
2
, Altier N
3
, and Arias A.
1
1
Lab. Biología Microbiana IIBCE. Montevideo, Uruguay. 
2
WSU.
USA. 
3
INIA-Las Brujas. Uruguay. E-mail: [e-mail]
Alfalfa plays an important rol in the Uruguayan agriculture.
Seedling diseases caused by soilborne fungal pathogens affect
alfalfa establishment. Biological control of these pathogens using
native fluorescent Pseudomonas can be an excellent alternative.
A collection of fluorescent Pseudomonas isolates was obtained
from the rhizosphere of alfalfa and its antagonistic activity in vitro
against Pythium debaryanum was evaluated. We found that 5.1%
of isolates from Colonia, 5% from Paysandú and 25.5% from
Tacuarembó were antagonist. The presence of biosynthetic loci
for antibiotics with biocontrol activity found in fluorescent
Pseudomonas was screened by PCR. Only 5% of isolates exhibit
these biosynthetic loci. Production of biosurfactant compounds,
screened by drop collapse test, was observed in 52% of isolates
from Colonia, 60% from Paysandú and 40% from Tacuarembó.
Growth chamber assays conducted to evaluate the ability of
selected isolates to suppress damping-off in vivo revealed that
33% are able to protect alfalfa against P. debaryanum. Selected
isolates were analyzed by fingerprinting using rep-PCR with
primers BOX and ERIC. Distinct genomic fingerprints were
observed, whit little correlation regarding phenotypic characters.
The development of a bacterial inoculant, based on antagonistic
strains, will have a relevant impact on alfalfa establishment.
Financed by Fondo Clemente Estable.
MI-P81.
GENETIC AND PHENOTYPIC STABILITY OF NDV
ISOLATES
Zanetti, Flavia; Junco, Mariano; Berinstein, Analía and Carrillo,
Elisa.
Instituto de Biotecnología, CICVyA, INTA Castelar and CONICET,
Buenos Aires, Argentina. E-mail: [e-mail].
In 1997, Argentina was declared virulent Newcastle Disease Virus
(NDV)-free for commercial poultry. However, the disease has a
global distribution with a wide host range, and wild birds are
considered natural reservoirs for the virus. The difference in
virulence between strains is determined primarily by the fusion
(F) protein cleavage site aa sequence (less virulent has fewer basic
aa) and, by the size of the viral hemagglutinin-neuraminidase (HN)
protein. The objective of the present study was to evaluate the
stability of NDV isolated from wild birds, during sequential
passages in chicken embryo. The isolates were inoculated in the
allantoic cavity of embryonated SPF chicken eggs. After incubation
for 5 days, the eggs were chilled and the allantoic fluids collected
and inoculated as described up to 20 passages. La Sota, which is
an NDV low virulent strain, was included in the experiment as a
control. The F protein cleavage site amino acid sequence changed
from 
112
GKQGRL
117
 to 
112
GRQKRF
117
 after 6/7 passages. The
HN C-terminal nucleotide sequence changed from a 616 aa HN to
a 571 aa HN after 20 passages. Mean Death Time (MDT) of 83 hs
was obtained from viruses after 20 passages whereas original
isolates couldn´t kill all the embryos inoculated in the same test.
F protein cleavage site, HN C-terminal and MDT of La Sota strain
were not  modified after twenty passages. This results showed an
increment in virulence of NDV isolated from wild birds after these
viruses were passaged in a different host.

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