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Papers by Paula Vincent

A) Liquid aerated minimal M9 medium cultures of wild-type strain (blue squares), <i&g... more A) Liquid aerated minimal M9 medium cultures of wild-type strain (blue squares), entE strain (green circles) and entE strain in the same media but supplemented with 100 µM FeCl3 (red triangles). Growth (OD600) was determined at the indicated times. B) Lawn growth of wt and entE E. coli strains on M9A. A stationary phase culture of entE E. coli strain was serially diluted (10−1 to 10−4) and an aliquot of these dilutions was applied on M9A or M9A supplemented with 100 µM FeCl3. As control, the same dilutions of a wt strain overnight culture were applied on M9A medium. Lawn growth was compared at 8 hours of incubation. C) Colony growth of wt and entE E. coli on LBA. A stationary phase culture of entE E. coli strain was serially diluted and an aliquot of dilutions 10−6 to 10−8 were applied on LBA or LBA supplemented with 100 µM FeCl3. As control, the same dilutions of an overnight culture of the wt strain were applied on LBA medium. After overnight incubation, colonies sizes were compared. D) Activity of the rhyB promoter estimated by β-galactosidase activity as an indirect measure of the intracellular iron content (The higher the promoter expression, the lower the iron content <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084734#pone.0084734-Ma2" target="_blank">[48]</a>). Both wild-type strain and entE mutant respond to iron addition. The plasmid pALM23 carries the ryhB- lacZ fusion.</p

Agronomy, 2021

Salinity is a major detrimental factor for plant growth and crop productivity that could be allev... more Salinity is a major detrimental factor for plant growth and crop productivity that could be alleviated by the use of plant growth promoting bacteria (PGPB) with a protective role in such stressful conditions. In this study, four native strains of the genus Pseudomonas were isolated from both a strongly saline soil and the rhizosphere of soybean plants grown in a slightly saline soil. These isolates were able to tolerate high NaCl concentration, showed efficient adhesion to biotic and abiotic surfaces and efficiently colonized the rhizosphere of soybean grown in slightly saline soil. In these conditions, the four strains outperformed Pseudomonas putida KT2440, a strain known as a good root colonizer of different plants. Inoculation with all the isolates improved seed germination and vigor index, particularly in saline conditions, and one of them also had a positive effect on shoot length and phenological state of soybean plants grown in slightly saline soil. Our results suggest that ...

International Journal of Food Microbiology, 2021

The use of bacteriocins is a promising alternative to improve food security through the biocontro... more The use of bacteriocins is a promising alternative to improve food security through the biocontrol of food pathogens and spoilage microorganisms. Gram-negative produced microcin J25(G12Y), known as (MccJ25(G12Y)) is a variant of the well-studied and characterized antimicrobial peptide, microcin J25 (MccJ25). In the present work, we explored the activity of this microcin against Gram-negative bacteria linked to foodborne diseases. We evaluated the in vitro antimicrobial activity of MccJ25(G12Y) in solid medium against a collection of pathogenic and food-altering strains and studied its activity and stability in meat and dairy food systems. We show that MccJ25(G12Y) exhibited the same in vitro antimicrobial spectrum as its parental microcin (MccJ25) against different Gram-negative foodborne pathogens and spoilage strains. We highlight that low concentrations of MccJ25(G12Y) between 0.45 and 29.4 μM were able to inhibit a substantial number of pathogens, including Salmonella, Escherichia, Shigella and Enterobacter genus. We also demonstrate the antimicrobial effectiveness of the peptide against Escherichia coli O157:H7 NCTC 12900, Enterobacter cloacae CECT 194, and Salmonella enterica CECT 4396 in fish and beef burgers and yogurt. MccJ25(G12Y) was added or not to food matrices inoculated with the foodborne pathogens at 105 CFU/g or mL. Afterward, food products were stored at 4 °C and selective media for the specific enumeration were used to determine the antimicrobial susceptibility of each pathogen to MccJ25(G12Y). The viability of the three pathogens was significantly reduced in the different food biological environments. In yogurt, the peptide decreased E. coli numbers on day 5 by about 4 log 10 CFU/mL as compared to non-treated samples. For S. enterica and E. cloacae no viable cells were detected at the end of the treatment. Adding MccJ25(G12Y) to fish burgers decreased E. cloacae numbers during storage 2 log10 CFU/g on the first day, reaching a difference of about 5 log 10 CFU/g after 10 days compared to non-treated control. Finally, the peptide decreased E. coli O157:H7 numbers on the beef burgers samples during storage on day 10 by about 3 log 10 CFU/g as compared to non-treated samples. The stability analysis demonstrated that MccJ25(G12Y) is capable of remaining active in these food matrices for a considerable time during the storage at refrigeration temperatures. These results reinforce the studies on the potential applicability of this microcin as a biopreservative in the food industry.

Applied Microbiology and Biotechnology, 2020

New strategies to improve crop yield include the incorporation of plant growth-promoting bacteria... more New strategies to improve crop yield include the incorporation of plant growth-promoting bacteria in agricultural practices. The non-pathogenic bacterium Pseudomonas putida KT2440 is an excellent root colonizer of crops of agronomical importance and has been shown to activate the induced systemic resistance of plants in response to certain foliar pathogens. In this work, we have analyzed additional plant growth promotion features of this strain. We show it can tolerate high NaCl concentrations and determine how salinity influences traits such as the production of indole compounds, siderophore synthesis, and phosphate solubilization. Inoculation with P. putida KT2440 significantly improved seed germination and root and stem length of soybean and corn plants under saline conditions compared to uninoculated plants, whereas the effects were minor under non-saline conditions. Also, random transposon mutagenesis was used for preliminary identification of KT2440 genes involved in bacterial tolerance to saline stress. One of the obtained mutants was analyzed in detail. The disrupted gene encodes a predicted phosphoethanolamine-lipid A transferase (EptA), an enzyme described to be involved in the modification of lipid A during lipopolysaccharide (LPS) biosynthesis. This mutant showed changes in exopolysaccharide (EPS) production, low salinity tolerance, and reduced competitive fitness in the rhizosphere.

PloS one, 2017

Seed inoculation with plant growth promoting rhizobacteria (PGPR) is an ideal tool to supply the ... more Seed inoculation with plant growth promoting rhizobacteria (PGPR) is an ideal tool to supply the soil with a high density of beneficial microorganisms. However, maintaining viable microorganisms is a major problem during seed treatment and storage. In this work, an evaluation was made of the effect of bacterial immobilization in nanofibers on the stability (viability and maintenance of beneficial properties) of two potential PGPR, Pantoea agglomerans ISIB55 and Burkholderia caribensis ISIB40. Moreover, the impact of soybean seed coating with nanofiber-immobilized rhizobacteria on bacterial survival during seed storage and on germination and plant growth parameters was determined. Bacterial nanoimmobilization and subsequent seed coating with nanofiber-immobilized rhizobacteria were carried out by electrospinning. The results demonstrate that this technique successfully immobilized P. agglomerans ISIB55 and B. caribensis ISIB40 because it did not affect the viability or beneficial pro...

Journal of Bacteriology, 2006

Escherichia coli microcin J25 (MccJ25) is a plasmid-encoded antibiotic peptide consisting of 21 l... more Escherichia coli microcin J25 (MccJ25) is a plasmid-encoded antibiotic peptide consisting of 21 l -amino acid residues (G 1 -G-A-G-H 5 -V-P-E-Y-F 10 -V-G-I-G-T 15 -P-I-S-F-Y 20 -G). E. coli RNA polymerase (RNAP) is the intracellular target of MccJ25. MccJ25 enters cells after binding to specific membrane transporters: FhuA in the outer membrane and SbmA in the inner membrane. Here, we studied MccJ25 mutants carrying a substitution of His 5 by Lys, Arg, or Ala. The inhibitory effects on cellular growth and in vitro RNAP activity were determined for each mutant microcin. The results show that all mutants inhibited RNAP in vitro. However, the mutants were defective in their ability to inhibit cellular growth. Experiments in which the FhuA protein was bypassed showed that substitutions of MccJ25 His 5 affected the SbmA-dependent transport. Our results thus suggest that MccJ25 His 5 located in the lariat ring is involved, directly or indirectly, in specific interaction with SbmA and is n...

Journal of Bacteriology, 2007

Microcin J25 (MccJ25) uptake by Escherichia coli requires the outer membrane receptor FhuA and th... more Microcin J25 (MccJ25) uptake by Escherichia coli requires the outer membrane receptor FhuA and the inner membrane proteins TonB, ExbD, ExbB, and SbmA. MccJ25 appears to have two intracellular targets: (i) RNA polymerase (RNAP), which has been described in E. coli and Salmonella enterica serovars, and (ii) the respiratory chain, reported only in S. enterica serovars. In the current study, it is shown that the observed difference between the actions of microcin on the respiratory chain in E. coli and S. enterica is due to the relatively low microcin uptake via the chromosomally encoded FhuA. Higher expression by a plasmid-encoded FhuA allowed greater uptake of MccJ25 by E. coli strains and the consequent inhibition of oxygen consumption. The two mechanisms, inhibition of RNAP and oxygen consumption, are independent of each other. Further analysis revealed for the first time that MccJ25 stimulates the production of reactive oxygen species (O 2 · − ) in bacterial cells, which could be t...

Journal of Bacteriology, 2013

SbmA protein has been proposed as a dimeric secondary transporter. The protein is involved in the... more SbmA protein has been proposed as a dimeric secondary transporter. The protein is involved in the transport of microcins B17 and J25, bleomycin, proline-rich antimicrobial peptides, antisense peptide phosphorodiamidate morpholino oligomers, and peptide nucleic acids into the Escherichia coli cytoplasm. The sbmA homologue is found in a variety of bacteria, though the physiological role of the protein is hitherto unknown. In this work, we carried out a functional and structural analysis to determine which amino acids are critical for the transport properties of SbmA. We created a set of 15 site-directed sbmA mutants in which single conserved amino acids were replaced by glycine residues. Our work demonstrated that strains carrying the site-directed mutants V102G, F219G, and E276G had a null phenotype for SbmA transport functions. In contrast, strains carrying the single point mutants W19G,

Journal of Bacteriology, 2005

In the present study, we showed that yojI , an Escherichia coli open reading frame with an unknow... more In the present study, we showed that yojI , an Escherichia coli open reading frame with an unknown function, mediates resistance to the peptide antibiotic microcin J25 when it is expressed from a multicopy vector. Disruption of the single chromosomal copy of yojI increased sensitivity of cells to microcin J25. The YojI protein was previously assumed to be an ATP-binding-cassette-type exporter on the basis of sequence similarities. We demonstrate that YojI is capable of pumping out microcin molecules. Thus, one obvious explanation for the protective effect against microcin J25 is that YojI action keeps the intracellular concentration of the peptide below a toxic level. The outer membrane protein TolC in addition to YojI is required for export of microcin J25 out of the cell. Microcin J25 is thus the first known substrate for YojI.

Journal of Antimicrobial Chemotherapy, 2007

To study the possible therapeutic utility of microcin J25 (MccJ25), a peptide RNA polymerase inhi... more To study the possible therapeutic utility of microcin J25 (MccJ25), a peptide RNA polymerase inhibitor. Methods: We subjected the antibiotic to two types of assays. First, with an ex vivo assay, we evaluated the stability and efficacy of MccJ25 in complex fluid biomatrices such as human whole blood, plasma and serum, compared with that in conventional laboratory media. Antimicrobial efficacy of MccJ25 was assessed by quantitative culture 2 h after inoculation of the biomatrices with a Salmonella Newport target organism and compared with that of MccJ25-free controls. Second, the antibiotic was tested in a mouse model of Salmonella infection. The latter was induced by intraperitoneal inoculation of 10 6 cfu of Salmonella Newport and the treatment with MccJ25 was initiated at 2 h post-infection. Results: MccJ25 retained full activity after 24 h of incubation in whole blood, plasma or serum. In addition, it did not show any haemolytic activity. In whole blood, homologous plasma and serum, introduction of MccJ25 was associated with a significant reduction in cfu versus the respective peptide-free controls. The counts of viable bacteria in the spleen and liver of mice treated with MccJ25 at a total dosage of 3 mg/mouse during either 24 h (0.5 mg/mouse every 4 h) or 6 days (0.5 mg/mouse every 24 h) significantly decreased by two or three orders of magnitude (P 0.05) compared with those in control mice. Conclusions: Collectively, these findings indicate that the biological activity of MccJ25 is not affected in complex biological matrices. The potent in vitro activity of MccJ25 against Salmonella translates into good in vivo efficacy in a mouse infection model.

FEMS Microbiology Letters, 2004

Escherichia coli microcin J25 (MccJ25) is a 2107-Da peptide antibiotic whose uptake into E. coli ... more Escherichia coli microcin J25 (MccJ25) is a 2107-Da peptide antibiotic whose uptake into E. coli is mediated by the outermembrane receptor FhuA and the inner membrane proteins TonB, ExbB, ExbD, and SbmA. A survey of the sensitivity of several Salmonella enterica serovars showed that the antibiotic was highly active against some serovars, while S. Typhimurium, S. Derby, and some S. Enteritidis strains were completely resistant. Resistant strains became hypersensitive to MccJ25 when given the fhuA gene of E. coli, indicating that insensitivity is due to the inability of the FhuA protein to mediate penetration of MccJ25. Whereas in E. coli MccJ25 targets RNA polymerase, in S. Typhimurium it inhibits not only RNA synthesis but also cell respiration. Fluorescence viability staining showed that S. Typhimurium cells exposed to MccJ25 remain viable but are unable to form colonies.

FEMS Microbiology Letters, 2009

Escherichia coli microcin J25 (MccJ25) is a lasso-peptide antibiotic comprising 21 L-amino acid r... more Escherichia coli microcin J25 (MccJ25) is a lasso-peptide antibiotic comprising 21 L-amino acid residues (G 1-G-A-G-H 5-V-P-E-Y-F 10-V-G-I-G-T 15-P-IS -F-Y 20-G). MccJ25 has two independent substrates: RNA-polymerase (RNAP) and the membrane respiratory chain. The latter is mediated by oxygen consumption inhibition together with an increase of superoxide production. In the present paper, the antibiotic MccJ25 was engineered by substituting Tyr 9 or Tyr 20 with phenylalanine. Both mutants were well transported into the cells and remained active on RNAP. Only the Y9F mutant lost the ability to overproduce superoxide and inhibit oxygen consumption. The last results confirm that the Tyr 9 , and not Tyr 20 , is involved in the MccJ25 action on the respiratory chain target.

Current Medicinal Chemistry, 2009

I-G-T15-P-IS -F-Y20-G), excreted to the medium by an Escherichia coli strain. MccJ25 is active on... more I-G-T15-P-IS -F-Y20-G), excreted to the medium by an Escherichia coli strain. MccJ25 is active on Gram-negative bacteria related to the producer strain, including some pathogenic strains. The four-plasmid genes mcjABCD, are involved in MccJ25 production: mcjA encodes a 58-residue precursor, mcjB and mcjC codify two processing enzymes required for the in vivo synthesis of the mature peptide and mcjD encodes the immunity protein (McjD), a member of the super family of ABC transporters. Immunity is mediated by active efflux of the peptide, keeping its intracellular concentration below a critical level. YojI, a chromosomal protein with ATP-binding-cassette-type exporter homology, is also able to export MccJ25. The E. coli outer membrane protein, TolC, is necessary for MccJ25 secretion mediated by either McjD or YojI. The uptake of MccJ25 is dependent on the outer-membrane receptor FhuA and the four inner-membrane proteins TonB, ExbD, ExbB and SbmA. At least two mechanisms described the action of MccJ25 on the target cells: (1) inhibition of the RNA-polymerase (RNAP) activity by obstructing the secondary channel, and consequently, preventing the access of the substrates to its active sites; and (2) operating on the cell membrane, MccJ25 disrupts the electric potential inhibiting the oxygen consumption in Salmonella enterica. MccJ25 also inhibits oxygen consumption and the respiratory chain enzymes in E. coli throughout the increasing of ROS concentration. Nevertheless the exact mechanism of this phenomenon must be elucidated. The MccJ25 exhibits a prolonged antimicrobial activity in a mouse infection model, suggesting a noteworthy potential for therapeutic uses.

Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 2002

The aim of the present research was to explore the capacity of PreR-Co to process prorenin purifi... more The aim of the present research was to explore the capacity of PreR-Co to process prorenin purified from kidney and Ž. corpora lutea CL and to study its action on extrarenal tissues. The PreR-Co was obtained from plasma as a single Ž. electrophoretic band by NH SO precipitation, gel filtration, anti-rat albumin immunoaffinity, and ion-exchange 4 2 4 Ž. chromatography. Prorenin free of renin was obtained after NH SO precipitation, gel filtration, and ion-exchange 4 2 4 chromatography by a passage through an affinity gel of H-77 Sepharose. SDS-PAGE of supernatant and of acidic elution from gel, exhibited a single band of 43 kDa and 35 kDa, respectively; both recognized by the specific anti rat renin antibody. The isolated renin was not attacked by PreR-Co; on the contrary prorenin was completely activated. The product of PreR-Co-activated prorenin showed an analogous MW to that of renin and was recognized by the specific antibody. In addition to processing kidney prorenin, PreR-Co was able to cleave inactive renin from ovary, CL, uterus and adrenal gland homogenates. However, the amount of active renin generated from these tissues was lower than those produced by trypsin activation. PreR-Co is a good candidate for the role of the enzyme involved in tissues prorenin activation.

Clinical and Experimental Hypertension, 2000

The amount of renal prorenin in models of hypertension in rats was studied by using a novel enzym... more The amount of renal prorenin in models of hypertension in rats was studied by using a novel enzyme (PreR-Co). Ten microgrames of PreR-Co promoted a complete conversion of inactive renin, and during the first 15-min incubation the reaction was under initial velocity conditions. The enzyme-substrate reaction obeyed Michaelis-Menten kinetics, with a Vmax of 0.97 x 10(-5) pmol Ang I/min and a Km of 5.03 x 10(-5) pmol prorenin. The difference between the total renin concentration (TRC) and active renin concentration (ARC) in the normal rat kidney (356.4 +/- 20.6 and 105.3 +/- 7.6 ng Ang I/mg tissue/h respectively), indicated that inactive renin comprised 70% of TRC. In the aortic coarctation model, inactive renin comprised 68 % of TRC in the right kidney and no or very little prorenin was found in the left kidney. In the Goldblatt 2-kidney, 1-clip rats, the right kidney prorenin comprised 61% of the TRC and 54% in the clamped left kidney. After DOCA-Salt treatment prorenin was almost absent in the rat kidneys. In conclusion, we have developed an easy and sensitive method to measure inactive renin in the kidney that may be useful to study the biochemical events of renin maturation in physiological and pathological states.