Study of the cwaRS-ldcA Operon Coding a Two-Component System and a Putative L,D-Carboxypeptidase in Lactobacillus paracasei.

The cell surface is the primary recognition site between the bacterium and the host. An operon of three genes, LSEI_0219 (cwaR), LSEI_0220 (cwaS), and LSEI_0221 (ldcA), has been previously identified as required for the establishment of Lactobacillus paracasei in the gut. The genes cwaR and cwaS encode a predicted two-component system (TCS) and ldcA a predicted D-alanyl-D-alanine carboxypeptidase which is a peptidoglycan (PG) biosynthesis enzyme. We explored the functionality and the physiological role of these three genes, particularly their impact on the bacterial cell wall architecture and on the bacterial adaptation to environmental perturbations in the gut. The functionality of CwaS/R proteins as a TCS has been demonstrated by biochemical analysis. It is involved in the transcriptional regulation of several genes of the PG biosynthesis. Analysis of the muropeptides of PG in mutants allowed us to re-annotate LSEI_0221 as a putative L,D-carboxypeptidase (LdcA). The absence of this protein coincided with a decrease of two surface antigens: LSEI_0020, corresponding to p40 or msp2 whose implication in the host epithelial homeostasis has been recently studied, and LSEI_2029 which has never been functionally characterized. The inactivation of each of these three genes induces susceptibility to antimicrobial peptides (hBD1, hBD2, and CCL20), which could be the main cause of the gut establishment deficiency. Thus, this operon is necessary for the presence of two surface antigens and for a suitable cell wall architecture.

Identification and transcriptional profile of Lactobacillus paracasei genes involved in the response to desiccation and rehydration.

Lactobacillus paracasei is able to persist in a variety of natural and technological environments despite physico-chemical perturbations, in particular alternations between desiccation and rehydration. However, the way in which it adapts to hydric fluctuations and the genetic determinants involved are not clearly understood. To identify the genes involved in adaptation to desiccation, an annotated library of L. paracasei random transposon mutants was screened for viability after desiccation (25% relative humidity, 25 °C). We found 16 genes that have not been described as being involved in this response. Most of them are linked to either the transport of molecules or to cell wall structure and function. Our screening also identified genes encoding DNA related enzymes and an alarmone necessary for L. paracasei survival. Subsequently, the expression of the identified genes was measured at five stages of the dehydration-rehydration process to decipher the chronology of genetic mechanisms. They were classified into four different transcriptional profiles: genes upregulated during both desiccation and rehydration phases, genes upregulated during the desiccation phase only, genes downregulated during both desiccation and rehydration and genes downregulated only during the rehydration stage. Thus, genetic response to hydric fluctuations seems to occur during desiccation and can continue or not during rehydration. The genes identified should contribute to improve the stabilization of Lactobacillus starters in dry state.

New Genes Involved in Mild Stress Response Identified by Transposon Mutagenesis in Lactobacillus paracasei.

Lactic acid bacteria (LAB) are associated with various plant, animal, and human niches and are also present in many fermented foods and beverages. Thus, they are subjected to several stress conditions and have developed advanced response mechanisms to resist, adapt, and grow. This work aimed to identify the genes involved in some stress adaptation mechanisms in LAB. For this purpose, global reverse genetics was applied by screening a library of 1287 Lactobacillus paracasei transposon mutants for mild monofactorial stresses. This library was submitted independently to heat (52°C, 30 min), ethanol (170 g.L-1, 30 min), salt (NaCl 0.8 M, 24 h), acid (pH 4.5, 24 h), and oxidative (2 mM H2O2, 24 h) perturbations which trigger mild monofactorial stresses compatible with bacterial adaptation. Stress sensitivity of mutants was determined either by evaluating viability using propidium iodide (PI) staining, or by following growth inhibition through turbidity measurement. The screening for heat and ethanol stresses lead respectively to the identification of 63 and 27 genes/putative promoters whose disruption lead to an increased sensitivity. Among them, 14 genes or putative promoters were common for both stresses. For salt, acid and oxidative stresses, respectively 8, 6, and 9 genes or putative promoters were identified as essential for adaptation to these unfavorable conditions, with only three genes common to at least two stresses. Then, RT-qPCR was performed on selected stress response genes identified by mutant screenings in order to evaluate if their expression was modified in response to stresses in the parental strain. Eleven genes (membrane, transposase, chaperone, nucleotide and carbohydrate metabolism, and hypothetical protein genes) were upregulated during stress adaptation for at least two stresses. Seven genes, encoding membrane functions, were upregulated in response to a specific stress and thus could represent potential transcriptomic biomarkers. The results highlights that most of the genes identified by global reverse genetics are specifically required in response to one stress and that they are not differentially transcribed during stress in the parental strain. Most of these genes have not been characterized as stress response genes and provide new insights into the adaptation of lactic acid bacteria to their environment.

Conidiation of Penicillium camemberti in submerged liquid cultures is dependent on the nitrogen source.

OBJECTIVE: To study the ability of a commercial Penicillium camemberti strain, used for Camembert type cheese ripening, to produce conidia during growth in liquid culture (LC), in media containing different sources of nitrogen as, industrially, conidia are produced by growth at the surface of a solid state culture because conidiation in stirred submerged aerobic LC is not known. RESULTS: In complex media containing peptic digest of meat, hyphae ends did not differentiate into phialides and conidia. Contrarily, in a synthetic media containing KNO3 as sole nitrogen source, hyphae ends differentiated into phialides producing 0.5 × 10(7) conidia/ml. Conidia produced in LC were 25 % less hydrophobic than conidia produced in solid culture, and this correlates with a seven-times-lower expression of the gene rodA encoding hydrophobin RodA in the mycelium grown in LC. CONCLUSION: Conidiation of P. camembertii is stimulated in iquid medium containing KNO3 as sole source of nitrogen and therefore opens up opportunities for using liquid medium in commercial productions.

Rapid 96-well plates DNA extraction and sequencing procedures to identify genome-wide transposon insertion sites in a difficult to lyse bacterium: Lactobacillus casei.

Random transposon mutagenesis followed by adequate screening methods is an unavoidable procedure to characterize genetics of bacterial adaptation to environmental changes. We have recently constructed a mutant library of Lactobacillus casei and we aimed to fully annotate it. However, we have observed that, for L. casei which is a difficult to lyse bacterium, methods used to identify the transposon insertion site in a few mutants (transposon rescue by restriction and recircularization or PCR-based methods) were not transposable for a larger number because they are too time-consuming and sometimes not reliable. Here, we describe a method for large-scale and reliable identification of transposon insertion sites in a L. casei mutant library of 9250 mutants. DNA extraction procedure based on silica membranes in 96-column format was optimized to obtain genomic DNA from a large number of mutants. Then reliable direct genomic sequencing was improved to fit the obtained genomic DNA extracts. Using this procedure, readable and identifiable sequences were obtained for 87% of the L. casei mutants. This method extends the applications of a library of this type, reduces the number of insertions needed to be screened, and allows selection of specific mutants from an arrayed and stored mutant library. This method is applicable to any already existing mutant library (obtained by transposon or insertional mutagenesis) and could be useful for other bacterial species, especially for highly lysis-resistant bacteria species such as lactic acid bacteria.

Functional genomics of Lactobacillus casei establishment in the gut.

Although the composition of the gut microbiota and its symbiotic contribution to key host physiological functions are well established, little is known as yet about the bacterial factors that account for this symbiosis. We selected Lactobacillus casei as a model microorganism to proceed to genomewide identification of the functions required for a symbiont to establish colonization in the gut. As a result of our recent development of a transposon-mutagenesis tool that overcomes the barrier that had prevented L. casei random mutagenesis, we developed a signature-tagged mutagenesis approach combining whole-genome reverse genetics using a set of tagged transposons and in vivo screening using the rabbit ligated ileal loop model. After sequencing transposon insertion sites in 9,250 random mutants, we assembled a library of 1,110 independent mutants, all disrupted in a different gene, that provides a representative view of the L. casei genome. By determining the relative quantity of each of the 1,110 mutants before and after the in vivo challenge, we identified a core of 47 L. casei genes necessary for its establishment in the gut. They are involved in housekeeping functions, metabolism (sugar, amino acids), cell wall biogenesis, and adaptation to environment. Hence we provide what is, to our knowledge, the first global functional genomics analysis of L. casei symbiosis.

Production of conidia of Penicillium camemberti in liquid medium through microcycles of conidiation.

Microcycle conidiation is a survival mechanism of fungi encountering unfavorable conditions. In this phenomenon, asexual spores germinate secondary spores directly without formation of mycelium. As Penicillium camemberti conidia have the ability to produce conidiophores after germination in liquid culture induced by a thermal stress (18 and 30 °C), our work has aimed at producing conidia through this mean. Incubation at 18 and 30 °C increased the swelling of conidia and their proportion thereby producing conidiophores. Our results showed that the microcycle of conidiation can produce 5 × 10(8) conidia ml(-1) after 7 days at 18 °C of culture. The activity of these conidia was checked through culture on a solid medium. Conidia produced by microcycle conidiation formed a normal mycelium on the surface of solid media and 25 % could still germinate after 5 months of storage.

Sensitivity to vinyl phenol derivatives produced by phenolic acid decarboxylase activity in Escherichia coli and several food-borne Gram-negative species.

Ferulic, p-coumaric, and caffeic acids are phenolic acids present in soil, food, and gut, which have antimicrobial effects. Some Gram (+) bacteria metabolize these phenolic acids into vinyl derivatives due to phenolic acid decarboxylase activity (PAD) involved in the phenolic acid stress response (PASR). In this study, the antimicrobial activity of phenolic acids and their vinyl derivatives was tested on a panel of desirable and undesirable food-borne bacteria, especially Gram (-) species of Salmonella, Enterobacter, Klebsiella, and Pseudomonas, most of them without PAD activity. Native and engineered Escherichia coli strains either expressing or not PAD activity were included. Gram (-) bacteria of the panel were not significantly inhibited by phenolic acids at 3 mM, but were dramatically inhibited by the corresponding vinyl derivatives. On the contrary, Gram (+) bacteria displaying the PASR face the toxicity of phenolic acids by PAD activity and are not inhibited by vinyl phenols. In E. coli, the genes aaeB and marA, encoding efflux pumps for antimicrobial compounds, are upregulated by the addition of p-coumaric acid, but not by its derivative 4-vinyl phenol (p-hydroxystyrene). These results suggest that phenolic acids and their vinyl phenol derivatives produced by PAD (+) species could have a significant impact on undesirable or pathogenic food-borne Gram (-) bacteria in complex microbial ecosystems.

Development of an efficient in vivo system (Pjunc-TpaseIS1223) for random transposon mutagenesis of Lactobacillus casei.

The random transposon mutagenesis system P(junc)-TpaseIS(1223) is composed of plasmids pVI129, expressing IS1223 transposase, and pVI110, a suicide transposon plasmid carrying the P(junc) sequence, the substrate of the IS1223 transposase. This system is particularly efficient in Lactobacillus casei, as more than 10,000 stable, random mutants were routinely obtained via electroporation.

Genetic and biochemical analysis of PadR-padC promoter interactions during the phenolic acid stress response in Bacillus subtilis 168.

Bacillus subtilis 168 is resistant to phenolic acids by expression of an inducible enzyme, the phenolic acid decarboxylase (PadC), that decarboxylates these acids into less toxic vinyl derivatives. In the phenolic acid stress response (PASR), the repressor of padC, PadR, is inactivated by these acids. Inactivation of PadR is followed by a strong expression of padC. To elucidate the functional interaction between PadR and the padC promoter, we performed (i) footprinting assays to identify the region protected by PadR, (ii) electrophoretic mobility shift assays (EMSAs) with a modified padC promoter protected region to determine the interacting sequences, and (iii) random mutagenesis of padR to identify amino acid residues essential for the function of PadR. We identified an important consensus dyad sequence called IR1-2 (ATGT-8N-ACAT) overlapping a second dyad element (GTGT-8N-ACAT) that we named dIR1-2bis. The entire dIR1-2bis/IR1-2 sequence permits binding of two PadR dimers in EMSAs, which may be observed for bacteria grown under noninduced conditions where the padC promoter is completely repressed. Three groups of modified PadRs giving a PASR phenotype were characterized in vivo. The DNA sequences of certain mutant padR alleles indicate that important residues are all located in the region containing the coiled-coil leucine zipper domain that is involved in dimerization. These substitutions reduce the affinity of PadR binding to the padC promoter. Of particular interest are residue L128, located at the center of the putative coiled-coil leucine zipper domain, and residue E97, which is conserved among all PadRs.

New insights into the effect of medium-chain-length lactones on yeast membranes. Importance of the culture medium.

In hydrophobic compounds biotechnology, medium-chain-length metabolites often perturb cell activity. Their effect is usually studied in model conditions of growth in glucose media. Here, we study whether culture on lipids has an impact on the resistance of Yarrowia lipolytica to such compounds: Cells were cultured on glucose or oleate and submitted to gamma-dodecalactone. After a 60-min exposure to 3 g L(-1), about 80% of the glucose-grown cells (yeast extract peptone dextrose (YPD) cells) had lost their cultivability, 38% their membrane integrity, and 31% their reducing capacity as shown with propidium iodide and methylene blue, respectively. For oleate-grown cells, treatment at 6 g L(-1) did not alter cultivability despite some transient loss of membrane integrity from 3 g L(-1). It was shown with diphenylhexatriene and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene that oleate-grown cells had membranes more fluid and less sensitive to the lactone-induced fluidization. Analyses revealed also higher contents of ergosterol but, for YPD- and minimum-oleate-grown cells (YNBO cells), the addition of lactone provoked a decrease in the concentration of ergosterol in a way similar to the depletion by methyl-beta-cyclodextrin and an important membrane fluidization. Ergosterol depletion or incorporation increased or decreased, respectively, cell sensitivity to lactone. This study shows that the embedment of oleate moieties into membranes as well as higher concentrations of sterol play a role in the higher resistance to lactone of oleate-grown cells (YPO cells). Similar oleate-induced increase in resistance was also observed for Rhodotorula and Candida strains able to grow on oleate as the sole carbon source whereas Saccharomyces and Sporidiobolus cells were more sensitive after induction.

Inactivation of PadR, the repressor of the phenolic acid stress response, by molecular interaction with Usp1, a universal stress protein from Lactobacillus plantarum, in Escherichia coli.

The phenolic acid decarboxylase gene padA is involved in the phenolic acid stress response (PASR) in gram-positive bacteria. In Lactobacillus plantarum, the padR gene encodes the negative transcriptional regulator of padA and is cotranscribed with a downstream gene, usp1, which encodes a putative universal stress protein (USP), Usp1, of unknown function. The usp1 gene is overexpressed during the PASR. However, the role and the mechanism of action of the USPs are unknown in gram-positive bacteria. Therefore, to gain insights into the role of USPs in the PASR; (i) a usp1 deletion mutant was constructed; (ii) the two genes padR and usp1 were coexpressed with padA under its own promoter as a reporter gene in Escherichia coli; and (iii) molecular in vitro interactions between the PadR, Usp1, and the padA promoter were studied. Although the usp1 mutant strain retained phenolic acid-dependent PAD activity, it displayed a greater sensitivity to strong acidic conditions compared to that of the wild-type strain. PadR cannot be inactivated directly by phenolic acid in E. coli recombinant cultures but is inactivated by Usp1 when the two proteins are coexpressed in E. coli. The PadR inactivation observed in recombinant E. coli cells was supported by electrophoretic mobility shift assays. Although Usp1 seems not to be absolutely required for the PASR, its capacity to inactivate PadR indicates that it could serve as an important mediator in acid stress response mechanisms through its capacity to interact with transcriptional regulators.

Phenolic acid-mediated regulation of the padC gene, encoding the phenolic acid decarboxylase of Bacillus subtilis.

In Bacillus subtilis, several phenolic acids specifically induce expression of padC, encoding a phenolic acid decarboxylase that converts these antimicrobial compounds into vinyl derivatives. padC forms an operon with a putative coding sequence of unknown function, yveFG, and this coding sequence does not appear to be involved in the phenolic acid stress response (PASR). To identify putative regulators involved in the PASR, random transposon mutagenesis, combined with two different screens, was performed. PadR, a negative transcriptional regulator of padC expression, was identified. padR is not located in the vicinity of padC, and the expression of padR is low and appears constitutive. This is in contrast with what occurs in other gram-positive bacteria, in which padR is autoregulated and induced by phenolic acids. Further screening of the transposon library failed to identify genes other than padR involved in the PASR. Modest inactivation of padR by phenolic acids was obtained in recombinant Escherichia coli expressing padC and padR, and this translates into induction of decarboxylase activity. Gel shift promoter binding assays performed with and without MgCl(2), and with and without phenolic acids, demonstrated that phenolic acids were able to abolish the binding of PadR to the yveFG-padC promoter in the absence of MgCl(2). Altogether, our results indicate that (i) PadR is inactivated directly by phenolic acids in vitro, (ii) inhibition of PadR in response to phenolic acids may occur without the need for a sensor-like effector in B. subtilis, and (iii) phenolic acids are able to modulate PadR activity in E. coli in the absence of any additional effector.

Epidemiological analysis of Salmonella enterica from beef sampled in the slaughterhouse and retailers in Dakar (Senegal) using pulsed-field gel electrophoresis and antibiotic susceptibility testing.

Seventy-eight isolates of Salmonella spp. isolated from beef sampled from the official city slaughterhouse and from retailers in Dakar, Senegal were analyzed using serotyping, antimicrobial testing and macrorestriction profiling by Pulsed-Field Gel Electrophoresis (PFGE). These analyses were done to identify clonal relationships and potential transmission routes in beef channel. XbaI macrorestriction allowed defining 17 genotypes among the six main analyzed serotypes: Salmonella bredeney (3 genotypes), S. muenster (6), S. waycross (1), S. corvallis (3), S. kentucky (1) and S. brandenburg (3). The cross analysis of PFGE profiles and origin of the beef samples reveals a wide range of contamination sources in the beef channel in Dakar. Comparison of PFGE and antimicrobial resistance types shows that the Salmonella contamination sources are equally shared by the slaughterhouse (56% of the isolates) and by the distribution channel (44% of the isolates) by handlings and houseflies.

Cloning and expression of genes involved in conidiation and surface properties of Penicillium camemberti grown in liquid and solid cultures.

Based on bioinformatic data on model fungi, the rodA and wetA genes encoding, respectively, a RodA hydrophobin protein and the WetA protein involved in conidiation mechanisms, were PCR-cloned and characterized for the first time in Penicillium camemberti. These results, completed by a sequence of the brlA gene (available in GenBank), which encodes a major transcriptional regulator also involved in the conidiation mechanism, were used to compare, by qRT-PCR, the expression of the three genes in liquid and solid cultures in a synthetic medium. While expression of the brlA and wetA genes increased dramatically in both culture conditions after 4 days of growth, expression of the rodA gene increased only with conidiation and in the solid culture, and this expression was correlated with production and secretion of a RodA protein outside the hyphae, which became very hydrophobic. In liquid cultures, no production of RodA occurred in mycelia, which remained hydrophilic, and no conidiation was detected despite formation of swellings at the tips of hyphae. The absence of conidiation in liquid culture correlated with the lack of rodA gene expression, which could be regulated by the medium composition independently of brlA and wetA genes expression.

Kinetics and intensity of the expression of genes involved in the stress response tightly induced by phenolic acids in Lactobacillus plantarum.

In Lactobacillus plantarum, PadR, the negative transcriptional regulator of padA encoding the phenolic acid decarboxylase, is divergently oriented from padA. Moreover, it forms an operonic structure with usp1, a genewhose products display homology with proteins belonging to the UspA family of universal stress proteins. PadR is inactivated by the addition of p-coumaric, ferulic or caffeic acid to the culture medium. In order to better characterize the stress response of this bacterium to phenolic acids, we report here the kinetics and quantitative expression by qRT-PCR of the 3 genes from the padA locus. The expression of the 3 genes is very low in the non-induced condition, while the addition of 1.2 mMp-coumaric acid induces an increase in the expression of padA, padR and usp1 by factors of 8,000, 37 and 13, respectively. These maximum relative transcript levels are obtained after 5 min of induction at the end of the exponential growth phase, while phenolic acid decarboxylase activity, not detectable before induction, is increased by a factor of 8,000 in 10 min. The apparent half-life of padA mRNA is about 1.4 min. The padA-padR system displays dynamic characteristics that are valuable to the development of tools for gene expression in this bacterium.

The proteome and transcriptome analysis of Bacillus subtilis in response to salicylic acid.

Phenolic acids that are present in plant-soil ecosystems can be considered as toxins which induce specific stress responses in microorganisms. In this paper, we have analyzed the global response of the soil bacterium Bacillus subtilis to salicylic acid using proteomics and transcriptomics. The results demonstrate that salicylic acid caused predominantly the induction of the SigmaB-dependent general stress response in B. subtilis which is not related to the acidic conditions. Treatment of B. subtilis with growth-inhibitory concentrations of 4 mM salicylic acid caused protein damage in B. subtilis as reflected by the induction of the CtsR and Spx regulons. Both phenolic acid decarboxylases (pads) of B. subtilis padC and bsdBCD (yclBCD) were induced by 4 mM salicylic acid that were previously shown to be involved in decarboxylation and detoxification of different phenolic acids. Deletion of the putative LysR-type regulator encoded by the divergently transcribed bsdA (yclA) gene upstream of the bsdBCD operon revealed that BsdA is the transcriptional activator of bsdBCD expression in response to salicylic acid. Phenotype analysis of bsdA and padC single and double mutants demonstrated that both pads confer resistance to salicylic acid in B. subtilis.

Relationship between the presence of the citrate permease plasmid and high electron-donor surface properties of Lactococcus lactis ssp. lactis biovar. diacetylactis.

Some strains of Lactococcus lactis subspecies possess a citrate permease that enables them to utilize citrate and to produce diacetyl. Such strains are classified as diacetylactis biovariants (L. lactis ssp. lactis biovar. diacetylactis). We investigated the electron-donor surface properties of L. lactis strains and observed that the diacetylactis biovariants presented increased adhesion to electron-acceptor solvents (microbial adhesion to solvents electron-donor characteristics of cells of <27% for L. lactis and about 50% for L. lactis ssp. lactis biovar diacetylactis). We investigated the properties of a pCitP- derivative and observed for a diacetylactis biovariant strain a loss of the electron-donor characteristics falling from 47% for a pCitP+ strain to 8% for its pCitP- derivative. This suggests that the presence of high electron-donor characteristics on the surface of L. lactis results to a large extent from the presence of the citrate permease plasmid.

Prevalence and antibiotic-resistance of Salmonella isolated from beef sampled from the slaughterhouse and from retailers in Dakar (Senegal).

A study was made of Salmonella contamination in beef sampled from a slaughterhouse and from retailers in Dakar, Senegal. The serotypes as well as antibiotic-resistance patterns of the Salmonella isolates were determined. A total of 435 meat samples (236 from the slaughterhouse, 199 from retailers) were tested. Among them, 275 (63%) were positive for Salmonella, 43% (101/236) from the slaughterhouse and 87% (174/199) from the retailers. Furthermore, 97% of the investigated retailers had at least one beef sample contaminated by Salmonella. The 286 Salmonella isolates were divided into 51 serotypes. The most prevalent serotypes were Salmonella bredeney (25%), S. muenster (8%), S. waycross (7%), S. corvallis (4%) and S. kentucky (4%). About 62% of the isolates were resistant to nitrofurans. Resistance rates were lower to streptomycin (22%), sulfamethoxazole (15%), spectinomycin (1%), chloramphenicol (1%), and tetracycline (0,4%) while low-level resistance to quinolones was detected. About 16% of the Salmonella strains were multiresistant to two or more antibiotic families. Finally, ten resistance profiles have been identified. This study shows the huge spread of Salmonella in the beef production chain in Dakar, Senegal. Finally, this study provides the very first data about Salmonella prevalence in sub-saharian Africa.

Random transposon mutagenesis of Lactobacillus plantarum by using the pGh9:IS S1 vector to clone genes involved in the regulation of phenolic acid metabolism.

The lactic acid bacterium Lactobacillus plantarum possesses a promising inducible padA promoter that controls the expression of the padA gene encoding a phenolic acid decarboxylase, and which is transcriptionally regulated by phenolic acids. A strategy was followed in order to clone genes involved in the transcriptional regulation of the padA gene. The pGh9:IS S1 plasmid was used to perfect the mutagenesis of L. plantarum by transposition. This plasmid transposed randomly in the L. plantarum NC8 chromosome, with a frequency of 0.03% at a non-permissive replicating temperature of 42 degrees C. A L. plantarum mutant strain harbouring the transcriptional fusion padA:cat that displayed a chloramphenicol acetyl transferase (CAT) activity induced by p-coumaric acid was constructed. This strain was mutated by transposition with pGh9:IS S1 in order to select mutants with a constitutive CAT activity therefore able to grow on medium containing chloramphenicol without the phenolic acid inducer. Four mutants were identified and three of them harboured an inserted single copy of the pGh9:IS S1 vector. Analysis of IS S1 target sites allowed the identification of two genes: one encoding a putative protein that displays similarity with histidine-binding protein, the other belongs to the ATP-dependent RNA helicase family, protein which are usually involved in gene expression.