Bariloche protein symposium argentine society for biochemistry and molecular biology

61
BIOCELL, 27 (Suppl. I), 2003
MI-P6.
NEUTRAL LIPIDS ACCUMULATION IN STREPTOMYCES
COELICOLOR A3(2) AND ITS RELATIONSHIP WITH
ACTINORHODIN BIOSYNTHESIS
Ana Arabolaza, Hector Alvarez, Silvia Altabe and Hugo Gramajo.
IBR. UNR-CONICET, Suipacha 531, Rosario, 2000. Argentina.
E-mail: [e-mail]
The capability for biosynthesis and storage of neutral lipids is
widely distributed among  eukariotic organisms suchs as yeast,
fungi, plants and animals, but the occurrence of TAG  in bacteria
has only rarely been described. A few examples of substantial
TAG accumulation have been reported for species mainly belonging
to the actinomycetes. The main function of bacterial TAG seems
to be as a reserve compoud, but for Streptomyces it has been
suggested that neutral lipids might act as the carbon source for
those polyketide antibiotics sinthesized from acetyl- and malonyl-
CoA precursors. S coelicolor produces a wide variety of secondary
metabolites, including the blue-pigmented polyketide actinorhodin
antibiotic. In our laboratory we are investigating the relationship
between the neutral lipids accumulation and the actinorhodin
production as a funtion of the external carbon source. We used
different culture conditions to analyse and compare the pattern of
TAG formation. Cuantification of TAG and actinorhodin production
along the growth curve showed that the TAG levels continue
increasing in conditions where no significant amounts of pigment
was detected. We could perform for the first time a preparative
isolation of lipid inclusion from the S coelicolor M145 strain under
different growth condition. We could analyze their composition
by TLC and by GC-mass spectrometry.
In other experiment, we cloned the SCO0958 gene, wich showed
a high homology with a recently described bifunctional enzyme
from  Acinetobacter calcoaceticus ADP1 exhibiting acyl-
CoA:diacylglycerol acyltransferase activity. Further studies will
allow to determine the potencial rol of this gene product in the
biosynthesis of TAG.
MI-P7.
EARLY IMMUNE RESPONSES IN BOVINES
EXPERIMENTALLY INFECTED WITH THE
HEMOPARASITE  BABESIA BOVIS
Asenzo, Gustavo
1
; Dominguez, Mariana
1
; Rodríguez, Anabel
1
;
Echaide, Ignacio
2
; Wilkowsky, Silvina
1
; Suarez, Carlos
3
;
Zamorano, Patricia
1 
and Florin-Christensen, Mónica
1
.
1
CICVyA-INTA-Castelar, Argentina; 
2
INTA-Rafaela, Argentina and
3
ADRU-USDA, Pullman, USA. E-mail: [e-mail]
We have studied the early stages of the immunological responses
developed in Babesia bovis-experimentally infected bovines
against several antigens that have been postulated as vaccine and
diagnostic candidates. To this end, bovines were inoculated with
a vaccinial (R1A) or a pathogenic (S2P) strain of B. bovis and
serum samples were collected at different time points up to 66
days post infection. Recombinant forms of the following antigens
of B. bovis R1A strain were produced in a prokaryotic expression
system and purified by affinity chromatography: the merozoite
surface antigens (MSA) 1, 2a
1
, 2b and 2c and the rhoptry associated
protein-1. An indirect ELISA test was developed for each of these
antigens. The titers obtained so far show that these tests are able
to detect the appearance of specific antibodies against each of the
antigens between days 15 and 42 post infection. In addition,
increased and decreases of antibody titers were observed along
the time period investigated. Further studies are needed to reveal
if these could be due to antigenic variations in the parasites during
infection. Importantly, our ELISA tests were able to detect
antibodies both in infections with the homologous R1A and the
heterologous S2P strains, demonstrating the presence of conserved
B-cell epitopes in all studied antigens between these two strains.
Supported by ANPCyT PICT 98-083838 and Fundacion Antorchas.
MI-P8.
IN VITRO SYNTHESIS OF XANTHAN REPETITIVE SUBUNIT
USING RECOMBINANT GLYCOSYLTRANSFERASES
Barreras, Máximo; Abdian, Patricia L. and Ielpi, Luis.
Fundación Instituto Leloir. Patricias Argentinas 435 (1405).
Buenos Aires. E-mail: [e-mail]
Glycosyltransferases (GTs) are enzymes involved in the synthesis
of polysaccharides. The bacterial mannosyltransferase GumH,
glucuronosyltransferase GumK and mannosyltransferase GumI are
involved in sequential steps in the synthesis of the pentasaccharidic
subunit of xanthan, an exopolysaccharide produced by
Xanthomonas campestris. Recombinant His-Tagged GumH, GumK
and GumI were cloned, overexpressed and purified to homogeneity.
By incubating these recombinant enzymes with its substrates, we
achieved xanthan repetitive subunit production in a cell-free assay
for the first time. Incubations were carried out in the presence of
affinity-resin immobilized or soluble GTs, in the presence of all
or a combination of them, and using different glycolipidic acceptors
involved in different xanthan biosynthetic steps.
Immunoprecipitations using polyclonal or commercial antibodies
were carried out in order to ascertain whether these enzymes
physically interact or rather act as isolated biosynthetic units. This
is a first approach towards understanding the functioning of these
enzymes as a multienzymatic complex, and how interaction
between these GTs is affecting its function.
MI-P9.
MICROBIAL COMMUNITY STRUCTURE IN POLLUTED
MARINE SEDIMENTS DETERMINED BY CULTURE-
INDEPENDENT METHODS
Basile, Laura
1
; Lozada, Mariana
1
; Luzzatto, Diego
2
; Figuerola,
Eva
1
; Penchaszadeh, Pablo
2
; Erijman, Leonardo
1
.
1
INGEBI-CONICET, 
2
Laboratorio  de Biología Reproductiva de
Invertebrados. FCEyN-UBA. Buenos Aires, Argentina. E-mail:
[e-mail]
Knowledge about the microbial community structure in polluted
marine sediments is required to develop tools for predicting and
monitoring natural attenuation. We have conducted culture-
independent profiling of microbial communities present in heavily
polluted sediments in the Mar del Plata harbor area. Surface
sediment samples at depths of 10 to 20 m were collected with a
dredge in five sampling stations, representative of different kind
of pollution. A procedure based on dipersion, separation and
filtration, combined with DAPI staining, was used for enumeration
of total cells in all sampled sediments. The number of DAPI-
detected bacterium-like particles ranged from 4,7x10
8
 to 6.9x10
10
per g of sediment. Bead mill homogenization in the presence of a
phosphate-buffered containing SDS, followed by agarose gel
electrophoresis in the presence of polyvinylpyrrolydone (PVP) was
used for DNA recovery and removal of PCR inhibitors from crude
extracts. Numerical comparison of 16S ribosomal DNA (rDNA)-
based denaturing gradient gel electrophoresis (DGGE) profiles of
Bacteria and Archaea was used to analyse the microbial community
structures. Sequencing of cloned bands complemented DGGE data.
Comparison of 16S rDNA profiles from polluted and nonpolluted
sediments points to the relationship between microbial diversity
and the degree and kind of pollution.

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