Bariloche protein symposium argentine society for biochemistry and molecular biology

46
BIOCELL, 27 (Suppl. I), 2003
MI-C8.
TRANSCRIPTIONAL REGULATION OF SUCROSE
BIOSYNTHESIS  IN Anabaena sp., A NITROGEN- FIXING
CYANOBACTERIUM
Cumino, Andrea C.; Giarrocco, Laura E.; Salerno, Graciela L.
Centro de Investigaciones Biológica (FIBA), Mar del Plata,
Argentina. E-mail: [e-mail]
In Anabaena sp. we have previously identified the biosynthesis of
sucrose through the sequential action of sucrose-phosphate
synthase (SPS) and sucrose-phosphate phosphatase (SPP), and
have reported the presence of two SPS genes (spsA,  spsB) and
one SPP gene (sppA). In this work we show that the expression of
those genes are subjected to transcriptional regulation in response
to changes in the nitrogen source, to salt stress and to the
physiological cell stage. RT-PCR and primer extension experiments
demonstrated that spsA is highly transcribed in osmotic stress,
stationary phase and nitrogen-starved cells. In contrast, spsB
expression is only increased under nitrogen deficiency. Anabaena
sp. PCC 7120 sucrose biosynthesis genes showed multiple
transcripts with different 5' ends analyzed by primer extension,
suggesting multiple transcription start sites. Primer extension
experiments also revealed an upstream sequence in the spsB
putative promoter related to a nitrogen transcriptional regulator
binding site. Thus, sucrose synthesis is regulated through a high
transcriptional control at the first step of the pathway.
Supported by Fundación Antorchas, CONICET, Univ. Nac. de Mar
del Plata and FIBA.
MI-C9.
IDENTIFICATION BY MICROARRAYS OF A NOVEL
DELETION IN A MEMBER OF Mycobacterium tuberculosis
COMPLEX:  M.microti
K. Caimi, C. García-Pelayo, F. Bigi, M.I.Romano, S.Gordon  and
A.Cataldi.
E-mail: [e-mail]
The  Mycobacterium tuberculosis complex consists of slow-
growing, pathogenic mycobacteria. The group includes M.
tuberculosis, the agent of human tuberculosis; M. bovis, which
causes both bovine and human tuberculosis; M. africanum, which
causes human tuberculosis on the African continent; and M.
microti, which is pathogenic for the vole, a wild rodent. M. microti
has been used as a live vaccine against tuberculosis in man and
cattle. However, recent reports suggest that some strains can cause
disease in humans. Deletions were clearly a driving force in the
genome evolution of the tubercle bacilli. Using comparative
genomic approaches, 16 regions of difference (RD1–16), ranging
in size from ~2 to 12.7 kb, have been described in M. bovis and
BCG strains relative to M. tuberculosis H37Rv, with 5 regions
deleted from M. tuberculosis H37Rv. To explore the M. microti
genome we used a M. tuberculosis H37Rv genomic DNA
microarray to detect gene deletions among M. microti isolates. A
number of deletions were identified that correlated with those
described previously by Brodin et al. A novel M. microti deletion
was also found (MiD4) which removes 5 genes that code for ESAT-
6 family antigens and PE-PPE proteins. Southern blot experiments
showed that this region was also deleted from M. pinnipedii, a
mycobacterium isolated from seals that is closely related to M.
microti. Genes encoding ESAT-6 antigens and PE-PPE proteins
appear to be frequently deleted from M. microti, and the
implications of this are discussed.
MI-C10.
VIBRIO CHOLERAE-INDUCED APOPTOSIS  OF
MAMMALIAN CELLS MEDIATED BY EL TOR
HAEMOLYSIN
Saka Héctor A., Bidinost Carla, Echenique José, Chinen Isabel*,
Bonacci Gustavo and Bocco José L.
Dpto de Bioquímica Clínica. Fac. Cs. Químicas. Universidad
Nacional de Córdoba. *INEI-ANLIS “Dr. C.G. Malbrán”. E-mail:
[e-mail]
El Tor Haemolysin (ETH) is a pore-forming toxin encoded by the
hlyA gene of V. cholerae O1 biotype El Tor and most of V. cholerae
non-O1/ non-O139 (VCN) isolates. Previous studies demonstrated
that ETH is able to induce cytolytic, enterotoxic and vacuolating
activity, suggesting that this toxin may contribute to the
pathogenesis of gastroenteritis caused by V. cholerae strains lacking
the cholera toxin (CT). In order to explore further mechanisms of
cell damage, the potential involvement of apoptosis triggered by
ETH was investigated. To this end, COS-7 cell monolayers were
exposed to sterile culture supernatant from a clinical VCN strain,
lacking CT, or from its isogenic hlyA  null mutant. At different
times post-incubation the apoptosis phenomenon was analyzed by
DNA fragmentation, flow cytometry and TUNEL. The parental
strain but not its isogenic hlyA  null mutant induced
internucleosomal DNA fragmentation, hypodiploidia and TdT-
mediated incorporation of fluorescent d-UTP, demonstrating the
apoptotic state of treated cells. Apoptosis was also observed after
exposure to 75 ng/mL of purified ETH. Furthermore, apoptotic
activity of both parental strain and purified ETH was completely
abolished after pre-incubation with an anti-ETH antibody. These
results demonstrate that ETH is capable to induce apoptosis of
mammalian COS-7 cells and points to previously unknown
interactions between V. cholerae and its host.
MI-C11.
CHARACTERIZATION OF AN ADAPTIVE ACID-
TOLERANCE MECHANISM IN Streptococcus pneumoniae:
ANALYSIS OF QUORUM-SENSING MUTANTS AND
IDENTIFICATION OF ACID-INDUCED PROTEINS
Cortes, Paulo; Piñas, Germán E. and Echenique, José R.
Departamento Bioquímica Clínica, Facultad Cs. Químicas, UNC.
E-mail: [e-mail]
During the invasion process, S. pneumoniae has to overcome
different stress conditions due to host defence mechanisms, like
pH decrease produced by phagosomes. The aim of this work was
to study a putative acid tolerance response (ATR) in pneumococcus.
We found that log- phase cells, grown previously at neutral pH,
survived 2 hours at pH 4.4. In contrast, stationary-phase cells
required a pre-incubation in an acetate-bufferized medium at pH
5.6 during 1 h at 37
o
C to tolerate this pH condition. With the
purpose to study the cellular density effect on ATR induction, we
analysed  comE mutants. The comE gene encodes a response
regulator that belongs to the quorum-sensing system ComCDE,
involved in competence regulation. The null mutant, comE::km,
showed no significant effect on ATR, however, the hyperactive
mutant  comE38KE repressed either acidic or log-phase ATR
induction. These results demonstrate that ATR induction is
controlled by the ComE pathway. In addition, we searched acid-
induced proteins in different proteic fractions obtained by acetone
precipitation and urea solubilization. The proteic band patterns
were compared by PAGE-SDS, and we found four proteins clearly
induced by pH 5.6 (30, 70, 80 and 120 kDa). At present, we
identified by peptide sequencing and pneumococcal genome
analysis two proteins: an ATP-dependent protease and a fructose
aldolase. In the future, we will determine the impact of these genes
on ATR induction by mutagenesis assays.

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