Bariloche protein symposium argentine society for biochemistry and molecular biology

66
BIOCELL, 27 (Suppl. I), 2003
MI-P26.
MUTATIONS OF MICROCIN J25 THAT AFFECT ENTRY
OF THE ANTIBIOTIC INTO Escherichia coli CELLS
de Cristóbal, R.E.; Zenoff, A.M.; Farías, R.N.; Solbiati, J.O., and
Salomón R.A.
INSIBIO (CONICET-UNT). Chacabuco 461, (4000) Tucumán,
Argentina. E-mail: [e-mail]
Microcin J25 (MccJ25) is a 2,107-Da cyclopeptide antibiotic of
21  amino acids produced by Escherichia coli. MccJ25 can enter
sensitive cells by binding to specific membrane receptors: FhuA,
in the outer membrane, and TonB and SbmA, in the inner
membrane. E. coli RNA polymerase is the intracellular target of
MccJ25. We are interested in identifying which residues within
MccJ25 are critical for interaction with the target site and with
the membrane receptors. MccJ25 shows a predominance of
uncharged and hydrophobic residues over the charged residues
Glu and His. We have mutagenized the unique His residue in
MccJ25 in order to obtain 3 different analogs in which His has
been replaced by Ala, Lys or Arg, respectively. This mutant peptides
were purified by HPLC and their effect on RNA polymerase was
assessed  in vitro. No significant difference on RNA polymerase
activity was noted as compared with the control, wild-type
microcin. We also performed in vivo assays to calculate the minimal
inhibitory concentration (MIC) of each one of the mutant microcins
against  E. coli strains where the different membrane receptors
were overexpressed or not. From these experiments we found that
E. coli cells are more resistant to the mutant microcins HisxAla
and HisxArg than to the wild-type microcin and the HisxLys
mutant. Additional experiments showed that the interaction with
the inner membrane receptor SbmA is affected in these mutants.
MI-P27.
ABUNDANCE OF nagAc NAPHTHALENE DIOXYGENASE
GENE MEASURED USING REAL-TIME PCR IS
CORRELATED WITH NAPHTHALENE
CONCENTRATIONS IN FRESHWATER SEDIMENTS
Dionisi, Hebe M.*; Chewning, Christopher S.; Morgan, Katherine H.;
Menn, Fu-M.; Easter, James P.; Vulava, Vijay M. and Sayler, Gary S.
Center for Environmental Biotechnology. University of Tennessee,
Knoxville, TN, U.S.A. *Present Address: Centro Nacional
Patagónico. E-mail:[e-mail]
Chattanooga Creek is one of the most polluted waterways in the
southeastern United States. Chemical analysis of creek sediments
indicated moderate to high levels of polycyclic aromatic
hydrocarbons, with naphthalene concentrations between 0.1 and
105.64 mg/kg of dry sediment. The objective of this study was to
quantify genes encoding for the naphthalene dioxygenase enzyme
using TaqMan real-time PCR. An assay was designed and
optimized for the quantification of nagAc-like genes using
conserved regions in this gene cloned from Ralstonia sp. U2 and
41 related sequences from pure cultures and uncultured bacteria.
The assay indicated the presence of 4.10 ± 0.66 x 10
3
 to 2.87 ±
0.34 x 10
5
 copies of nagAc-like gene per ?g of DNA extracted
from sediments of Chattanooga Creek. These values corresponded
to 1.15 ± 0.56 x 10
5
 to 5.35 ± 0.41 x 10
7
 copies of this target per g
of dry sediment. A positive correlation was found between
naphthalene concentrations and copies of nagAc target per 
µg of
DNA (r
2
 = 0.747) and per g of dry sediment (r
2
 = 0.586). A real-
time PCR system targeting the 16S rRNA gene of most bacteria
indicated the presence of approximately equal concentrations of
this target (1.31 ± 0.42 x 10
9
) per 
µg of DNA in all analyzed
samples. These results indicate significant differences in the
population of Bacteria carrying nagAc-like genes in sediments of
Chattanooga Creek with different naphthalene concentrations.
MI-P28.
DEVELOPMENT OF A COMPETITIVE ELISA TEST FOR
BABESIA BOVIS INFECTIONS BASED ON MEROZOITE
SURFACE ANTIGEN-2C
Dominguez M.
1
; Zabal O.
1
; Wilkowsky S.
1
; Rodriguez A.
1
; Asenzo
G.
1
; Farber M.
1
; Echaide I.
2
; Echaide S.T. de
2
; Suarez C.
3
 and
Florin-Christensen M.
1
.
1
CICVyA-INTA Castelar, Argentina; 
2
INTA Rafaela, Argentina;
3
ADRU-USDA, Pullman, USA. E-mail: [e-mail]
Previous studies have shown that Babesia bovis merozoite surface
antigen-2c (MSA-2c) has a high degree of genetic and antigenic
conservation among geographically distant strains. This antigen
thus appears as an adequate diagnostic candidate for bovine
babesiosis. We have produced a set of monoclonal antibodies
against recombinant MSA-2c (rMSA-2c) from the Argentine R1A
strain, expressed in a prokaryotic system. One of them, MAb
H9P2C2, was shown to react in Western blots with a parasite
protein of the expected MSA-2c size. MAb H9P2C2 and rMSA-
2c were employed to develop a competitive ELISA test. This test
essentially consisted of binding of rMSA-2c to Immulon 2HB
plates, blocking, incubations with a) serial dilutions of different
sera from control or B. bovis-naturally or experimentally infected
bovines, b) MAb H9P2C2, c) peroxidase-conjugated anti-mouse
IgG and d) OPD-H
2
O
2
 colorimetric substrate, and optical density
readings at 490nm. All tested B. bovis positive sera from
experimental and natural infections significantly reduced binding
of MAb H9P2C2 to rMSA-2c, while no reduction was observed
with negative sera. These results indicate that this cELISA could
be an adequate diagnostic tool for bovine babesiosis.
Standardization of the test conditions and validation with a larger
number of samples are currently under progress. (Supported by
ANPCyT PICT 98-083838 and Fundacion Antorchas, Argentina).
MI-P29.
NOVEL GENES INVOLVED IN THE RESISTANCE OF
ANTIMICROBIAL PEPTIDES IN Salmonella enterica
Espariz, Martín; Barchiesi, Julieta; and Soncini, Fernando C.
Instituto de Biología Molecular y Celular de Rosario (CONICET),
and Facultad de Ciencias Bioquímicas y Farmacéuticas (UNR),
Rosario. E-mail: [e-mail]
Studies that provide new insight into the relationship between the
host and their pathogens are necessary to solve the growing problem
of resistance to conventional antibiotics. The bacterial genes
involved in resistance to antimicrobial peptides are ideal targets
for the action of novel antibacterial agents. The detailed knowledge
of their mechanism of action will entail the development of
alternative therapies. In Salmonella enterica serovar Typhimurium
it has been demonstrated that resistance to antimicrobial peptides
is essential to colonize the tissues of the infected host. We
performed a genetic analysis of an operon that increased the
bacterial resistance to the antimicrobial peptides protamine and
cecropin P1, and that is essential for virulence. Interestingly, an
insertional mutant of this operon showed increased resistance to
polymyxin B, resembling a PmrA/PmrB-dependent effect. As
expected, the LPS extracted from this strain showed an
electrophoresis pattern similar to the one that characterizes PmrA
constitutive strains. Surprisingly, its expression was independent
of either PmrA-PmrB, or PhoP-PhoQ. In addition, the mutant was
able to replicate in macrophages, indicating that resistance to
antimicrobial peptides is not associated to macrophage survival.
The analysis of this novel locus will broaden the knowledge of
the actions taken by Salmonella to resist the initial antimicrobial
assault in order to prosper in the infection process.

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