Bariloche protein symposium argentine society for biochemistry and molecular biology

77
BIOCELL, 27 (Suppl. I), 2003
MI-P70.
AN ALTERNATIVE MECHANISM OF ACTION OF
MICROCIN J25 IN Escherichia coli STRAINS
Vincent, Paula; Bellomio, Augusto; Arcuri, Beatriz F. de; Farías,
Ricardo; Morero, Roberto and Salomón, Raul.
INSIBIO e Instituto de Qca. Biológica (CONICET-UNT). Tucumán.
Argentina. E-mail: [e-mail]
By using serial dilution assays we have observed striking
differences in sensitivity to microcin J25 (MccJ25) between E.
coli strains. We have previously established that RNA polymerase
(RNAP) is the target of MccJ25 and that the antibiotic inhibits
transcription in vivo and in vitro. E. coli strain SBG231 harbors a
mutation in rpoC (the gene encoding largest subunit of RNAP)
which makes this strain completely resistant to the antibiotic. The
mutation was transduced to the MccJ25-hypersusceptible strain
AB1133. One of the transductants, named PA232, was studied
further. Transfer of the mutation was confirmed by genetic
complementation tests and in vivo transcription experiments.
Notably, strain PA232 did not become completely resistant to
MccJ25. This could be explained by assuming the existence in
strain AB1133 of an alternative mode of action of the antibiotic,
which would not be operative in strain SBG231. This result could
also explain the differential sensitivity of E. coli strains. Previous
work from our laboratory showed that in Salmonella strains MccJ25
acts on the cytoplasmic membrane by dissipating the electric
potential and affecting the cell respiration. In the present study
we have not detected an effect on cell respiration in PA232.
However, the possibility that the membrane be the putative second
target in hypersusceptible E. coli strains cannot be discarded.
MI-P71.
PHYSIOLOGICAL AND  MOLECULAR
CHARACTERIZATION OF YEASTS PRESENT DURING
SPONTANEOUS CIDER FERMENTATIONS
Barbagelata R, Lopes C, Giraudo de Van Broock M, Caballero A
and Sangorrín M.
Univ. Nac Comahue. Neuquén. E-mail:  [e-mail]
The transformation of must apple into cider by spontaneous
alcoholic fermentations is the result of the sequential development
and metabolic activity of various species of yeasts originated from
apple and cidery surfaces. The aim of this work was to isolate, to
characterize and to compare yeast microbiota from fermenting
apple musts in two cideries. Initial musts from both cideries, were
also fermented in laboratory. The samples were took at three stages
of spontaneous alcoholic fermentation: initial, middle and end.
Yeast characterization at species and strain level was performed
according to conventional physiological, morphological and
molecular methods (RFLP of the ITS1-5.8S-ITS2 and mtDNA-
RFLP). Differential killer sensitivity was also used as a tool for
fingerprinting at strain level. Pichia membranaefasciens, Pichia
kluyveri, Dekkera anomala, Torulaspa delbrueckii and Kloeckera
apiculata were identified in the initial stages of fermentation from
both cideries. Saccharomyces cerevisiae turned out the most among
those isolated at middle and final stages. The killer phenotype
(K
+
) was observed in 80% of cider yeasts. The dominant strains in
both industrial and laboratory vinification processes were just a
few and the same. The quality of the final products was very
similar, with the exception of the volatile acidity. We can conclude
that there exist a great diversity of wild yeast in the cideries with
particular roles in the fermentation processes.
MI-P72.
BIOCHEMICAL CHARACTERIZATION OF CITM
RESPONSIBLE FOR THE OXALACETATE
DECARBOXILASE ACTIVITY IN Lactococcus lactis CRL264
Sender, Pablo D.; Martín, Mauricio; Peirú, Salvador and Magni,
Christian.
Departamento de Microbiología, FCByF, U.N.R., IBR-CONICET,
Suipacha 531, Rosario, Argentina. E-mail: [e-mail]
Only a few numbers of malic enzymes have been studied in
bacteria. These enzymes are in some cases associated with operons
involved in several cellular processes. In Lactococcus lactis, citM
encodes for a malic enzyme and it is co-transcribed with other
genes of the citrate metabolic pathway (citMCDEFXG operon). In
this work the citM enzyme from L. lactis was expressed in a
heterologic host and was purified. In vitro studies let us
determinate its oxalacetate decarboxylase activity and the kinetics
parameters (Km 1.53 mM, Sp Act 12,6 
µmol  . min
-1 
. mg of
protein
-1
).  E. coli EJ1321
 
(dme, tme, pck) unable to grow in
minimal medium with succinate as the sole carbon source was
transformed with the plasmid pQE264 (citM is under the control
of IPTG). E. coli cells grow with succinate as
 
sole carbon source
only in the presence of IPTG. Summing up, the obtained results
provide evidence to conclude that citM gene which is a part of  cit
operon, encodes the malic enzyme with oxalacetate decarboxylase
activity necessary to piruvate production in the citrate metabolic
pathway in L. lactis.
MI-P73.
CAROTENOID PIGMENTS IN RHODOCOCCUS OPACUS
PD630
Silva, Roxana A.; Barría, Mabel E.; Mazzuca, Marcia; Alvarez,
Héctor M.
Depto. Bioquímica. Universidad Nacional de la Patagonia San
Juan Bosco, km 4- Ciudad Universitaria, (9000) Comodoro
Rivadavia, Chubut, Argentina. E-mail: [e-mail]
Rhodococcus opacus PD630 is a pigmented actinomycete
bacterium able to produce and accumulate triacylglycerols into
intracellular inclusions. The aim of this study was to determine
the identity and location of pigments in strain PD630.The
production of salmon-pink pigmentation by strain PD630 was
constitutive and non-light induced. Different chemical techniques
were performed for the identification of pigments. A mixture of
different compounds like carotenes (
γ- carotene) and
oxocarotenoids (xanthophylls) has been detected on the basis of
their UV/visible spectra in hexane in comparison with references
and thin layer chromatography. Several colourless mutants was
produced by chemical mutagenesis suggesting a common
biosynthetic pathway for the different kind of pigments in R. opacus
PD630. Lipid inclusions containing triacylglycerols and cellular
envelope were the main site of localization of pigments in strain
PD630 as revealed by centrifugation of free-cell extracts in
discontinuous glycerol density gradients. Preliminary results
suggested that strain PD630 has a sophisticated antioxidant defence
system protecting different lipophilic compartments since mixtures
of carotenoids are more effective than single compound.

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