Bariloche protein symposium argentine society for biochemistry and molecular biology

47
BIOCELL, 27 (Suppl. I), 2003
MI-C12.
A SALMONELLA SPECIFIC TRANSCRIPTIONAL
REGULATOR THAT REPONDS TO GOLD
Checa, Susana K.; Botta, Pablo E.; Spinelli, Silvana V. and
Soncini, Fernando C.
Instituto de Biología Molecular y Celular de Rosario (IBR-
CONICET), Fac. Cs. Bioquímicas y Farmaceúticas (U.N.R.),
Rosario, Argentina. E-mail: [e-mail]
The MerR family is a group of transcriptional regulators with
similar N-terminal helix-turn-helix DNA binding regions and C-
terminal effectors binding regions that respond specifically to
certain environmental stimuli, such as heavy metals, oxidative
stress or antibiotics. We searched for new transcriptional regulators
in Salmonella enterica serovar Typhimurium and identified a gene
with high homology in sequence to members of the merR family
of metalloregulatory proteins. This gene, hmrR, was located within
a  Salmonella specific region of the genome. hmrR forms a two-
gene operon with a gene coding for a putative metal-transporting
P-type ATPase (hmrT), and is located upstream to a gene showing
high similarity to a family of metal binding proteins (hmrB).
We showed here that HmrR regulates the expression of the two
neighbouring genes, and probably its own synthesis. By measuring
β-galactosidase activity, we found that expression of these genes
is strongly regulated by gold, but responds poorly to copper or
silver ions, as well as other heavy metals. We mapped hmrT and
hmrB promoters by primer extension analysis and noticed that
their promoter/operator regions have signature elements that
distinguish promoters controlled by transcriptional regulators of
the MerR family: a suboptimal spacing between -10 and -35
elements and sequences of dyad symmetry. To our knowledge,
this is the first example of a metalloregulatory protein that responds
specifically to gold.
MI-C13.
REGULATION OF SPORULATION AND ENTEROTOXIN
PRODUCTION OF THE GAS GANGRENE PRODUCER
Clostridium perfringens TYPE A FOOD-POISONING
Philippe Valeria, Orsaria Lelia, and Grau Roberto.
Facultad de Ciencias Bioquímicas y Farmacéuticas / IBR-
CONICET- Rosario. E-mail: [e-mail]
The spore forming, anaerobic bacterium Clostridium perfringens
is responsible for the human gangrene apart from being an
important cause of gastrointestinal and histotoxic infections. The
enterotoxigenic pathology is due to the production of a potent toxin
(CPE) that induces cell death and the symptoms of enterotoxemia,
intestinal cramping, and diarrhea. Expression of cpe is temporally
and genetically linked to sporulation. Here we examined the
molecular mechanism of spore formation and its role in CPE
production. An isogenic spo0A knockout mutant of C. perfringens
was constructed by allelic exchange. The spo0A knockout mutant
failed to produce Spo0A and it was completely deficient in
sporulation and CPE production. By complementation of the spo0A
knock out mutant with a recombinant plasmid carrying the intact
spo0A gene it was possible to restore full efficiency in sporulation
and CPE formation. By the expression of a clostridial DNA library
in a Bacillus subtilis strain deficient in the expression of the sensor
sporulation kinases KinA and KinB it was possible to identify a
clostridial clone that restored Bacillus sporulation. DNA analysis
indicated the presence of a putative sensor histidine kinase coding
gene (cp-kinA) involved in inorganic metabolism. A cp-kinA knock
out mutant strain of C. perfringens cpe
+
 was severely affected in
sporulation and CPE production. The overall results are relevant
for the development of antidotes to prevent gastrointestinal diseases
and the gas gangrene.
MI-C14.
EFFECT OF THE MUTATIONS ON THE Cys RESIDUES
IN THE Escherichia coli NADH-DEHYDROGENASE-2
ACTIVITIES
Volentini, Sabrina; Solbiati, José; Rapisarda, Viviana; Rodríguez
Montelongo, Luisa and Farías, Ricardo.
Dpto. Bioquímica de la Nutrición del INSIBIO. Instituto de
Química Biológica “Dr. B. Bloj” CONICET-UNT. Chacabuco 461
(4000) San Miguel de Tucumán. E-mail: [e-mail]
NADH dehydrogenase-2 (NDH-2) of Escherichia coli is a
membrane-bound flavoprotein linked to the respiratory chain. Our
previous studies showed that purified NDH-2 has a Cu(I)-bound
and exhibits Cu(II)-reductase activity supported by NADH.
Moreover, bioinformatic analysis indicated that this protein has a
cysteine-rich domain (domain III) conserved in primary and
secondary structure with the HMA (Heavy  Metal Associated)
domains that could be the site of copper binding. Domain III
presents the Cys
315
XXCys
318
 and the MetXXCys
339
 motifs. In order
to study the participation of all the Cys present in domain III on
the NDH-2 activities, we prepared a set of plasmids bearing ndh
gene mutagenized in the three Cys and then transformed into strain
IY12 that lacks the NADH- dehydrogenase activities (nuo and
ndh). We found that the mutations affected the NADH-
dehydrogenase and the NADH-oxidase activities of the
membranes, compared with one preparation containing the wild-
type protein. We saw that both activities decreased about 80% in
the C318S mutant, and almost disappeared in the double mutant
(C315S-C318S). On other hand, the activities increased about 50%
in C339S mutants. These results indicating that this mutations
affected some of the NDH-2 activities, suggest that Cys
315
XXCys
318
and MetXXCys
339
 motifs are involved on the functionality of the
enzyme directly or due to the Cu (I) putatively bound there.
MI-C15.
THE SPECIFICITY AND ARCHITECTURE OF ACYL-COA
CARBOXYLASE 
βββββ SUBUNIT IN STREPTOMYCES
COELICOLOR A3(2)
L. Diacovich
1
, G. Gago
1
, S-C. (S) Tsai
2,3
, C. Khosla
2
, and H.
Gramajo
1
.
1
Facultad de Ciencias Bioquimicas y Farmaceuticas- IBR. UNR-
CONICET, Suipacha 531, Rosario, 2000. Argentina. 
2
Departments
of Chemistry, Biochemistry and Chemical Engineering, Stanford
University. 
3
Department of Molecular Biology and Biochemistry,
and Department of Chemistry, University of California, Irvine.
E-mail: [e-mail]
Two acyl-CoA carboxylase complexes, acetyl-CoA carboxylase
(ACC) and propionyl-CoA carboxylase (PCC) have been
characterized in Streptomyces coelicolor whose main physiological
role is to provide malonyl- and methylmalonyl-CoA for both fatty
acid and polyketide biosynthesis. Both complexes share the same
biotinylated 
α
.
 subunit, AccA2 (biotin carboxyl carrier protein,
BCCP), that also contains a biotin carboxylase domain. The 
β
.
and the 
ε subunits are specific for each of the complexes (AccB-
AccE and PccB-PccE to ACC y PCC respectively). The 
β subunit
has transcaboxylase activity and the 
ε subunit forms a subcomplex
with the 
β subunit that increases drastically the activity of the
enzymatic complex. ACC and PCC in S. coelicolor are 1 MDa
multienzyme complexes containing at least 18 polypeptide chains.
The 
β subunit, PccB and AccB are 360 kDa homo-hexamers. Apo
and substrate-bound crystal structures of PccB hexamer were
resolved to 2.0 – 2.4 Å. Overall, the hexamer assembly of the core
360 kD 
β
.
 subunit forms a large ring-shaped complex as two stacks
of trimers related by two-fold symmetry. The structural studies
shed light on the molecular basis of substrate recognition and the
nature of the assembly.

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