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Unité de Recherche en Biologie Moléculaire, University of Namur, Namur, Belgium
Received 17 December 2004/ Returned for modification 7 March 2005/ Accepted 10 May 2005
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ABSTRACT |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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INTRODUCTION |
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The availability of complete bacterial genome sequences has opened up doors to the development of new strategies to study the biology of microorganisms (59). For instance, the complete Brucella melitensis sequence (15) allowed us to conduct a systematic study of several families of regulators in this organism. Bacteria of the genus Brucella are facultative intracellular gram-negative coccobacilli that are pathogenic for domestic animals and occasionally for humans (57). Some transcriptional regulators of Brucella that have a role in the control of virulence have already been identified. Indeed, the two-component system BvrR/BvrS controls cell invasion, intracellular survival, and expression of two outer membrane proteins (25, 58). Some transcriptional regulators required for Brucella's virulence were also identified in several screens in cellular models and in BALB/c mice (19, 31, 35, 36). Random screening for attenuated mutants may not be viewed as a comprehensive analysis that would allow the identification of all regulators involved in the virulence of the model tested. We therefore conducted a systematic disruption strategy for genes putatively coding for transcriptional regulators in B. melitensis 16M.
To identify regulators of virulence genes in Brucella, we selected transcriptional regulator families whose members are known to be involved in host-bacteria interactions (AraC, ArsR, Crp/Fnr, DeoR, GntR, IclR, LysR, MerR, RpiR, and TetR families). Sigma factors and the two-component systems are under investigation elsewhere.
Three families, AraC, ArsR, and IclR families, were chosen for their involvement in virulence (9, 44, 64). Since previous data (37, 61) suggested that Brucella and Rhizobiaceae, which are capable of establishing symbiosis with plants, could share common molecular mechanisms for host-bacterium interactions, we also selected members of the GntR and MerR families. Indeed, Sinorhizobium and its relatives are phylogenetically close to Brucella (42). Rhizobia have the ability to infect the roots of leguminous plants, leading to a symbiotic interaction and eliciting the formation of new organs, the nodules. In these organs, bacteria reduce atmospheric nitrogen into ammonia, which is used by the plants as a nitrogen source (62). The RpiR family was selected because a homolog is found in a cluster of genes putatively involved in the rhizopine metabolism. Rhizopine is thought to be a signal molecule produced by Rhizobium when it is residing in the nodules induced in the roots of leguminous plants (62). Two mutants of Brucella with a disrupted gene involved in rhizopine metabolism are attenuated (18, 36), supporting the hypothesis that Brucella and Rhizobium could share common strategies to interact with their host (61). Members of the Crp, LysR, and TetR families of transcriptional regulators are involved in virulence and in symbiosis (26, 33, 51, 53, 65). Some regulators of the DeoR family are involved in sugar metabolism, but many proteins of that family have no experimentally defined function (43, 66). Nevertheless, the regulator AccR represses opine catabolism and conjugal transfer of the Ti (pTIC58) plasmid in Agrobacterium tumefaciens (4).
In this report, we selected 94 transcriptional regulators grouped in 10 families (Table 1). We successfully mutated 88 genes, and we evaluated the virulence of the mutants in mice by a new method based on signature-tagged mutagenesis (STM) screening. We identified 10 regulators required for Brucella during the first week of infection in mice. The analysis of these regulators allowed us to better understand the environment encountered by the bacteria in a mouse model of infection. We also studied the expression of the virB operon in the library of the 88 mutants, leading to the identification of five new regulators of the virB operon.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The remaining predictions are based on homology to transcriptional regulators from other bacteria described in the literature. In this way, we identified 174 potential transcriptional regulators.
The library
of mutants for regulators (LiMuR) and B. melitensis database
website addresses are as follows:
http://urbm59.urbm.fundp.ac.be/
dharbi/aPAGe/PHP/basic/pCDS/limur.php
and
http://serine.urbm.fundp.ac.be/seqbruce/GENOMES/Brucella_melitensis/,
respectively.
Bacterial strains and plasmids. B. melitensis 16M was obtained from A. Macmillan, Central Veterinary Laboratory, Weybridge, United Kingdom. We selected a spontaneous nalidixic acid (Nal)-resistant (Nalr) mutant of this strain that was used as the parental strain for all experiments. B. melitensis strains were grown on solid or liquid 2YT medium with appropriate antibiotics. The Escherichia coli strains used in this study were S17-1 (56) and DH10B (Gibco BRL). E. coli strains were grown on Luria-Bertani (LB) medium with appropriate antibiotics. Antibiotics were used at the following concentrations for E. coli and B. melitensis: kanamycin (Kan), 50 µg/ml; chloramphenicol (Cm), 20 µg/ml; Nal, 25 µg/ml. The plasmids used in this study were pSKOriTKan (12), pBBRMCS1lacZ (S. Léonard, unpublished data), and pMR10Cat Gateway (R. Hallez, unpublished data).
Matings. Matings were performed by mixing equal volumes (100 µl) of liquid cultures of E. coli S17-1 donor cells (optical density at 600 nm of 0.6) and the B. melitensis 16M Nalr recipient strain (overnight culture) on a 0.22-µm-pore-size filter. The filter was left for 1 h on a 2YT (log/liter yeast extract, 16 g/liter tryptone, 5 g/liter NaCl) plate without antibiotics and then transferred onto a 2YT plate containing Kan and Nal (or Cm and Nal for replicative plasmids). After 1 week of incubation at 37°C, the exconjugates were replicated on a 2YT plate containing Nal and Kan (or a 2YT plate containing Nal and Cm).
Molecular techniques. DNA manipulations were performed according to standard techniques (2). Restriction enzymes were purchased from Roche, and primers were purchased from Sigma-Aldrich. Internal fragments were initially amplified by PCR from B. melitensis 16M genomic DNA with primers different for each gene, with common 5'-end sequences, that are as follows: Fint (5'-ATCTCTAGA-3') and Rint (5'-ATCGTCGACCTA-3') for forward and reverse primers, respectively. These sequences contain an in-frame stop codon, a restriction site (XbaI or SalI), and an EcoRV half-site. High-fidelity PCR was performed using Pwo polymerase (Boehringer Mannheim) or Pfx polymerase (Gibco BRL). The cycling conditions were 2 min at 94°C followed by 30 cycles of 94°C for 30 s, 60°C for 30 s, 72°C for 30 s, and 72°C for 6 min. The PCR products were purified using a Concert Rapid PCR purification system (Gibco BRL). The amplified product was then inserted in the EcoRV-digested pSKOriTKan vector in the opposite orientation compared to the Plac promoter to avoid expression of a 3' fragment of the disrupted coding sequences (CDS) in Brucella. The inserts were sequenced using the dye terminator method (BigDye Terminator kit; Perkin-Elmer) with an ABI 377 sequencer. The construction was introduced into B. melitensis 16M (Nalr) from E. coli S17-1 by mating. A single crossover then leads to the disruption of the wild-type locus on the chromosome (Fig. 1). Integrative mutants were selected on a medium with kanamycin and nalidixic acid. One mutant was constructed by allelic replacement, with the arsR6 CDS being replaced by a kanamycin resistance cassette.
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Southern blot analysis. The gene disruption was confirmed by Southern blot analysis using a Kan probe. After chromosomal DNA was extracted, it was digested overnight with HindIII and SspI (or HindIII and EcoRI) restriction enzymes, separated by electrophoresis in 1% agarose, and transferred to positively charged nylon membranes. The Kan cassette was excised from pUC4K (Pharmacia) using EcoRI digestion and was labeled by using a RadPrime DNA labeling system (Invitrogen), and it was used as the DNA probe for Southern hybridization under stringent conditions.
The PTM method. (i) Screening of the mutants. Mutants were grown at 37°C in 200 µl of 2YT medium in 96-well microtiter plates for 24 h. The bacteria were then pooled (9 to 12 mutants/pool), centrifuged at 4,000 x g for 10 min, and resuspended in 2 ml of phosphate-buffered saline (PBS). The bacterial suspension was then diluted to a final concentration of 5 x 105 CFU in 100 µl of PBS. The number of bacteria was counted by plating dilutions on 2YT plates. The bacterial suspension (100 µl) was injected intraperitoneally into 7-week-old male BALB/c mice (n = 2 mice). The remaining part of the suspension was used for genomic DNA preparation. One week after the infection, animals were sacrificed, and the spleens were removed aseptically. For recovery of bacteria, the spleens were homogenized in PBS-0.1% Triton X-100 (Roche), and dilutions were plated on 2YT medium. Plates containing approximately 105 clones were used for DNA extraction. The plasmid-tagged mutagenesis (PTM) method was tested on 85 mutants. The three remaining mutants were used to infect two mice to complete the testing of virulence for all LiMuR mutants.
(ii) Amplification and labeling of DNA tags. For both the input and the output pools, bacteria from plates containing approximately 105 clones were resuspended in 3 ml PBS and then centrifuged, and genomic DNA was extracted from the pelleted bacteria. Tags were initially amplified by PCR from genomic DNA with the primers Fint (specific to each gene coding for a regulator) and RpSKKan(5'-CCGCTCTAGAACTAGTGGATC-3'). The amplicons were purified with a Concert Rapid PCR purification system (Gibco BRL), and a fraction (approximately 150 ng) was used as a template for a second PCR including [32P]dCTP to radiolabel the tags. The cycling conditions for both PCRs were as follows: 2 min at 94°C followed by 30 cycles of 94°C for 30 s, 52°C for 30 s, 72°C for 30 s, and 72°C for 6 min. The reactions were performed with Taq polymerase from Biotools.
For dot blots, 100 µl of a 1:10 dilution of the PCR products (approximately 4 ng) was transferred to a positively charged nylon membrane (Hybond N+; Amersham) with a dot blot apparatus (Bio-Rad). After a short prehybridization reaction (in 0.25 M Na2HPO4, 1 mM EDTA, sodium dodecyl sulfate [10%], and formamide [40%]) of 5 min, the hybridization reaction was performed overnight.
No labelings were detected in the input pool for 17 mutants (of the 85 mutants tested), which may be due to the impossibility to amplify the signature tags in the multiple PCR. We therefore infected two mice with each of these 17 mutants, and 1 week postinfection (p.i.), we evaluated the number of CFU in spleen and compared it to that of a wild-type control, as described below.
Infection in BALB/c mice. Seven-week-old male BALB/c mice (n = 4 mice) were inoculated intraperitoneally with 0.1 ml of a suspension containing 5 x 105 CFU of each bacterial strain harvested with PBS from a 24-h 2YT liquid culture. One week after infection, mice were sacrificed for spleen collection. The spleens were homogenized in 2 ml of PBS-0.1% Triton X-100 (Roche), and serial dilutions of the homogenates were plated on 2YT agar to determine bacterial counts. For the wild-type train, 5 to 6 log CFU were recovered using this procedure. The arbitrary attenuation cutoff was at 0.8 log.
Infection of macrophages and HeLa cells. Subconfluent monolayers (105) of murine J774 macrophages or human HeLa cells were inoculated with bacteria diluted to 6 x 106 CFU ml–1 in cell culture medium. Plates were centrifuged for 10 min at 1,000 x g at room temperature and placed in a 5% CO2 atmosphere at 37°C. After 1 h, wells were washed three times with PBS and incubated for 48 h with cell culture medium supplemented with 50 µg/ml of gentamicin. At the end of infection time, the monolayers were washed three times with PBS and treated for 10 min with 200 µl of 0.1% Triton X-100 (Roche) in PBS. Serial dilutions of the lysates were plated onto 2YT plates for determination of CFU. Each infection was performed in triplicate. We used the wild-type strain as a positive control (we obtained about 5 log CFU at 48 h p.i. in HeLa cells and macrophages) and the vjbR mutant as a control of attenuation in cellular models of infection (14a).
Western blot analysis. Bacteria were pregrown in rich medium (2YT) with chloramphenicol, diluted to an optical density at 600 nm of 0.05 in 10 ml of 2YT medium, and grown during 24 h (the final optical density at 600 nm was about 1). After inactivation at 80°C for 1 h, bacteria were pelleted and resuspended in PBS at a concentration of 1010 CFU/ml. Forty microliters of each total cell lysate was subjected to electrophoresis on 12% sodium dodecyl sulfate-polyacrylamide gels and transferred to Hybond-C nitrocellulose membranes (Amersham Pharmacia Biotech) by the semidry transfer technique (2). Immunodetection of proteins in total cell lysates was first performed with polyclonal VirB9 antiserum (diluted 1/2,500), and the mutants showing a difference in VirB9 abundance compared to that of the wild type were retested using a polyclonal VirB8 antiserum (diluted 1/2,500) (49). The monoclonal anti-Omp89 antibody A5310B2 (8) (diluted 1/1,000) was used as a loading control. Bound antibodies were detected by chemiluminescence with peroxidase-conjugated secondary antibodies and the ECL Western blotting reagent RPN2209 as recommended by the manufacturer (Amersham Pharmacia Biotech).
ß-Galactosidase assay. The sequence located upstream of the virB1 coding sequence (including the ATG itself) was amplified by PCR from genomic DNA of B. melitensis 16M (Nalr) with the primers FpvirB (5'-TCTAGAGCTAGCTGAAATCCAGGCGTT-3') and RpvirB (5'-GGATCCACCATAGGATCGTCTCCTTCTCA-3') containing the XbaI and BamHI restriction sites, respectively. The PCR product was cloned into the pGEM-T easy vector for sequencing. This construct was digested by XbaI and BamHI, and the insert was cloned into the corresponding sites of the broad-host-range plasmid pBBRMCS1lacZ.
Bacteria were pregrown in 2YT medium supplemented with chloramphenicol, diluted to an optical density at 600 nm of 0.05 in 5 ml of 2YT medium (containing chloramphenicol), and grown during 24 h. ß-Galactosidase activity (Miller units) was determined on permeabilized bacteria (41).
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Systematic disruption of transcriptional regulators. We constructed the mutants by integration of a plasmid in the target CDS (integrative disruption) using an internal fragment of the CDS as the site of homologous recombination (Fig. 1A). Each mutant was validated by Southern blot using a probe hybridizing in the kanamycin resistance gene and two restrictions of the genomic DNA. We have obtained 87 integrative mutants of transcriptional regulators. One additional mutant (arsR6) was obtained by replacement of the target CDS with a Kanr cassette.
The complete collection of mutants, called LiMuR, is available to the scientific community in a 96-well format. Two versions of LiMuR are available, one with the E. coli clones carrying plasmids used for disruption, allowing the targeted mutations in other Brucella strains, and the other being the B. melitensis mutants. A website linked to the B. melitensis database was established to simplify the search for information.
Identification of regulators involved in Brucella's virulence. In an effort to identify regulators required for Brucella virulence, the ability of LiMuR mutants to survive and replicate in a mouse model of infection was investigated. For obvious bioethic reasons, it was desirable to minimize the number of mice sacrificed for each mutant to test. Thus, we developed a method to test several mutants simultaneously in one animal and then to identify mutants unable to survive. Our strategy is based on STM screening (28), and we called it the PTM method (Fig. 1). We divided the different integrative mutants into nine pools (9 to 12 mutants per pool), and each pool was injected into two mice. This pool size is a compromise between the advantage of mixing many clones and the limitation of multiplex PCR (V. Haine, unpublished data). Considering that each mutant has a plasmid integrated at a specific locus, we used the junction between the disrupted CDS and the integrated plasmid as a tag to differentiate the mutants. In order to check if we are able to identify underrepresented clones, we made preliminary tests by mixing a known amount of mutants in pools, and we determined that the PTM method was able to identify mutants 10- to 100-fold diluted in a pool (data not shown).
First, 9 to 12 mutants were grouped in an input pool (Fig. 1B). Genomic DNA of this pool was extracted, and the tags were amplified and radiolabeled in a PCR. These labeled tags were hybridized on membranes where internal fragments of the target genes without plasmidic sequence are spotted (Fig. 1A). The pools were then injected into mice by the intraperitoneal route. In order to identify regulators involved in the acute phase of infection, mice were killed 1 week p.i., the spleens were recovered and homogenized, and the residual brucellae were allowed to grow on a rich medium, forming the output pool. The tags were also amplified on the output pool, radiolabeled in a PCR, and hybridized on the membranes. The comparison of the input and output membranes allowed the identification of mutants that are unable to survive in mice (Fig. 1B). Like for the STM method, those attenuated mutants present a marked decrease of labeling for the two output pools. The LiMuR mutants were tested by the PTM method, which allowed us to select eight mutants displaying a growth defect in the two mice tested. Twenty signature tags were not amplified from the input pool (see Materials and Methods). We therefore infected two mice for each of these 20 mutants, and 1 week p.i., we evaluated the number of CFU in spleen and compared it to that of a wild-type control. Among these 20 newly tested mutants, we identified 4 attenuated mutants. After this first step of screening, a set of 12 attenuated mutants was therefore identified.
The 12 individual
mutants were used to infect groups of four mice intraperitoneally, and
the CFU were counted in the spleen after 1 week of infection. We
confirmed attenuation for 10 of the 12 selected mutants (Table
2). These 10 mutants display between 0.8 and 2 log of attenuation when
expressed as average CFU per spleen. Surprisingly, among these 10
attenuated mutants, 6 mutants belong to the GntR family, which is much
more than would be expected by chance (P < 0.01 in a
2 test).
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Implication of the mutants in VirB expression. In principle, the availability of the LiMuR allows simple screenings for the identification of factors involved in the control of a particular gene or operon. For example, an abundance of a given protein or activity of a selected promoter could be individually evaluated for each mutant of the library.
Numerous data have highlighted a crucial role of the virB operon in the intracellular survival and multiplication of several Brucella species (14, 45, 55). Moreover, several studies showed the importance of this operon in B. abortus pathogenicity in mice (29, 55). In an effort to evaluate the use of LiMuR to identify regulators of a gene of interest, we analyzed virB expression in the 88 mutants available in LiMuR.
We first carried out a Western blotting of whole-cell extracts to investigate whether the mutants produced VirB proteins by using VirB8 and VirB9 polyclonal antisera generated in rabbits (49). We performed a two-step screening in which the abundance of VirB9 and VirB8 was tested (see Materials and Methods). Five mutants showed a difference in VirB8 production compared to the parental strain (Fig. 2). Indeed, VirB8 was either absent or weakly produced in four mutants (arsR6, araC8, deoR1, and gntR4) and overexpressed in one mutant (araC2).
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Among the 10 attenuated mutants identified here, six disrupted genes belong to the GntR family and three belong to the LysR family. The GntR family of regulators currently comprises about 300 members that are involved in the regulation of many different biological processes. This family is overrepresented in Brucella compared to Sinorhizobium meliloti, in which only the LysR family predominates (20). A transcriptional regulator of the GntR family is described to be involved in Rhizobium meliloti symbiosis (MocR) (48). A mutant in a gene coding for a GntR-like regulator (GntR18, which was not mutated in this work) was found to be attenuated in an STM screen of B. melitensis in BALB/c mice (36). LysR-type transcriptional regulators (LTTRs) are widely distributed in diverse genera of prokaryotes (52). Most LTTRs are transcriptional activators, but they usually negatively regulate their own expression (52). A member of this family (LysR21) was shown to be required for Brucella pathogenesis in human macrophages (19), but according to our results, a lysR21 mutant is not attenuated after 1 week of infection in mice. Many virulence factors are regulated by LTTRs, and it is not surprising that some regulators required for the virulence of Brucella belong to the LysR family (16, 22, 24, 26).
It was surprising that among the 10 mutants attenuated in BALB/c mice, only two were attenuated in their virulence in cellular models since it was reported that most mutants identified during an STM screen in this mouse model were also attenuated in cellular models (35, 36). We propose that this discrepancy is linked to the fact that only regulator-coding genes are mutated here. Indeed, if the function of a regulator is to transduce a particular signal present in the mouse but absent in cultured cells, e.g., linked to innate immunity, to modify the expression of virulence-associated genes, then a mutant of this regulator will be attenuated in mice but not in cellular models of infection.
Our PTM screen allowed us to identify a mutant which is strongly attenuated in mice as well as in cellular models (HeLa cells and J774 macrophages). This mutant is disrupted in a gene coding for the transcriptional regulator ArsR6. ArsR6 displays 63% amino acid identity with the Sinorhizobium meliloti NolR protein. NolR is known to repress expression of the nodulation genes. Moreover, a proteomic study showed that nolR encodes a global regulatory protein (7, 32). In response to environmental stresses, NolR regulates diverse factors such as heat shock proteins, protein synthesis, and cell growth. The putative global role of ArsR6 in Brucella could explain that an arsR6 mutant is attenuated in all models tested, since other Brucella mutants affected for responses to various stresses are also attenuated in such models (46, 58, 60).
For the other genes identified here as attenuated in mice, we examined their homologs and their genomic context. GntR1 is weakly homologous to UxuR of Geobacillus stearothermophilus (28% of identity over 65 residues), which represses the uxuAB operon involved in the catabolism of glucuronate (54). Furthermore, in the B. melitensis genome, homologs of these uxuAB genes are close to gntR1 as well as the uxaC gene. The UxuAB and UxaC proteins are involved in the Ashwell pathway, which converts glucuronate into 2-keto-3-deoxygluconate (KDG). KDG is phosphorylated by the KDG kinase to give KDGP (2-keto-3-deoxy-6-phosphogluconate). The pathway then converges with the Entner-Doudoroff pathway, and KDGP is finally cleaved by KDGP aldolase to yield glyceraldehyde-3-phosphate and pyruvate (38). These two latter compounds can enter the pentose phosphate pathway and the citrate cycle, respectively. The requirement of a functional gntR1 gene for survival and/or proliferation in BALB/c mice spleen suggests that the absence of an appropriate control of the Ashwell pathway unsettled the sugar metabolism of the bacteria in mice.
The lysR18 gene is surrounded with genes involved in the catabolism of purines (guanine deaminase and xanthine dehydrogenase) (34). Furthermore, homologs of lysR18 in Mesorhizobium loti and Sinorhizobium meliloti are also next to the orthologous genes required for purines catabolism. It may be suggested that LysR18 is involved in the control of purine catabolism genes. Until now, only the synthesis of purines was shown to be required for the virulence of Brucella in both cellular and murine models (1, 11, 30, 31).
Since the LiMuR mutants constructed are useful to study the regulation of a particular gene, we studied the regulation of the virB operon as an example. The virB operon is known to be crucial for the pathogenicity of Brucella (14, 29, 45, 55).
We first carried out Western blottings to analyze the production of VirB proteins using polyclonal antisera against B. suis VirB9 and VirB8. Expression of the VirB8 and VirB9 proteins is reduced in four mutants (araC8, arsR6, deoR1, and gntR4) and overproduced in one mutant (araC2) in comparison with the parental strain during growth in bacteriological medium. The arsR6 and araC2 mutations have only a slight effect on PvirB activity, and moreover, the araC2 mutation is shown to induce higher amounts of VirB8 and VirB9. Additional data are required to test the role of ArsR6 and AraC2 regulators of the virB promoter.
We propose that GntR4, DeoR1, and AraC8 are direct or indirect activators of virB transcription. However, it may not be excluded that these regulators, as AraC2 and ArsR6, may also act at another, i.e., posttranscriptional, regulation level. The GntR4 transcriptional factor was also selected in our screen as required for the pathogenesis of Brucella in mice after 1 week of infection. A B. suis deoR1 mutant is strongly attenuated in human macrophages (31), which is consistent with a possible role of deoR1 in virB activation in this cellular model. Until now, the AraC8 regulator was never described as important for the virulence of Brucella. Additional data are required to investigate the role of AraC8 in virulence control.
It will be interesting to investigate possible links between AraC8, DeoR1, and GntR4 and other regulators of the virB operon, such as the quorum-sensing regulator VjbR, which is an important activator of VirB (14a), or the integration factor (IHF), which was recently shown to interact directly with the virB promoter in B. abortus (55a). Furthermore, the increasing number of regulators involved in virB control suggests the presence of a network of regulators. The PTM method developed in this work allows study of the pathogenesis of several mutants simultaneously in one animal. This method could be systematically applied to other bacterial pathogens. The PTM method was successful on 80% of the integrative mutants, but this percentage is similar to the one observed by Hensel et al. in their first STM screen with Salmonella enterica serovar Typhimurium (28). The LiMuR is a resource available to the scientific community that will be an interesting tool for the study of a given process or a given promoter. Indeed, here we only tested the virulence at 1 week of infection in a mouse model, but other virulence models may be tested using a similar strategy, i.e., using the PTM method. In addition, other processes may be studied, such as resistance to H2O2 and polymyxin B. The regulation of particular genes or operons may also be studied by using promoter-reporter fusions or antibodies directed against proteins of interest. The LiMuR may also be extended to other regulators. First, there are six mutants that were not obtained in the course of this study, one of them being reluctant to disruption. That demonstrated the very low proportion of essential genes in the regulator families tested. Finally, there are 80 remaining genes putatively coding for transcriptional regulators, and for the sake of completeness of LiMuR, it would be interesting to mutate them. The availability of the B. melitensis ORFeome (i.e., a collection of all predicted CDS of a genome, each inserted in a plasmid) could provide new gene inactivation strategies (17).
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ACKNOWLEDGMENTS |
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We thank Leila Lahlimi for the LiMuR database. We thank Régis Hallez and Sandrine Léonard for the construction of the pMR10GW (pMR10Cat Gateway) and pBBRMCS1lacZ plasmids. The sequencing at URPhyM (University of Namur) is also acknowledged.
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FOOTNOTES |
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