Michel Débarbouillé

Characterization of the levanase gene of Bacillus subtilis which shows homology to yeast invertase

SummaryThe structural gene for the enzyme levanase of Bacillus subtilis (SacC) was cloned in Escherichia coli. The cloned gene was mapped by PBS1 transduction near the sacL locus on the B. subtilis chromosome, between leu4 and aroD. Expression of the enzyme was demonstrated both in B. subtilis and in E. coli. The presence of sacC allowed E. coli to grow on sucrose as the sole carbon source. The complete nucleotide sequence of sacC was determined. It includes an open reading frame of 2,031 bp, coding for a protein with calculated molecular weight of 75,866 Da, including a putative signal peptide similar to precursors of secreted proteins found in Bacilli. The apparent molecular weight of purified levanase is 73 kDa. The sacC gene product was characterized in an in vitro system and in a minicellproducing strain of E. coli, confirming the existence of a precursor form of levanase of about 75 kDa. Comparison of the predicted aminoacid sequence of levanase with those of the two other known β-D-fructofuranosidases of B. subtilis indicated a homology with sucrase, but not with levansucrase. A stronger homology was detected with the N-terminal region of yeast invertase, suggesting the existence of a common ancestor.

Bacitracin and nisin resistance in Staphylococcus aureus: A novel pathway involving the BraS/BraR two-component system (SA2417/SA2418) and both the BraD/BraE and VraD/VraE ABC transporters

Two‐component systems (TCSs) are key regulatory pathways allowing bacteria to adapt their genetic expression to environmental changes. Bacitracin, a cyclic dodecylpeptide antibiotic, binds to undecaprenyl pyrophosphate, the lipid carrier for cell wall precursors, effectively inhibiting peptidoglycan biosynthesis. We have identified a novel and previously uncharacterized TCS in the major human pathogen Staphylococcus aureus that we show to be essential for bacitracin and nisin resistance: the BraS/BraR system (Bacitracin resistance associated; SA2417/SA2418). The braRS genes are located immediately upstream from genes encoding an ABC transporter, accordingly designated BraDE. We have shown that the BraSR/BraDE module is a key bacitracin and nisin resistance determinant in S. aureus. In the presence of low antibiotic concentrations, BraSR activate transcription of two operons encoding ABC transporters: braDE and vraDE. We identified a highly conserved imperfect palindromic sequence upstream from the braDE and vraDE promoter sequences, essential for their transcriptional activation by BraSR, suggesting it is the likely BraR binding site. We demonstrated that the two ABC transporters play distinct and original roles in antibiotic resistance: BraDE is involved in bacitracin sensing and signalling through BraSR, whereas VraDE acts specifically as a detoxification module and is sufficient to confer bacitracin and nisin resistance when produced on its own. We show that these processes require functional BraD and VraD nucleotide‐binding domain proteins, and that the large extracellular loop of VraE confers its specificity in bacitracin resistance. This is the first example of a TCS associated with two ABC transporters playing separate roles in signal transduction and antibiotic resistance.

Investigation of the Staphylococcus aureus GraSR Regulon Reveals Novel Links to Virulence, Stress Response and Cell Wall Signal Transduction Pathways

The GraS/GraR two-component system has been shown to control cationic antimicrobial peptide (CAMP) resistance in the major human pathogen Staphylococcus aureus. We demonstrated that graX, also involved in CAMP resistance and cotranscribed with graRS, encodes a regulatory cofactor of the GraSR signaling pathway, effectively constituting a three-component system. We identified a highly conserved ten base pair palindromic sequence (5′ ACAAA TTTGT 3′) located upstream from GraR-regulated genes (mprF and the dlt and vraFG operons), which we show to be essential for transcriptional regulation by GraR and induction in response to CAMPs, suggesting it is the likely GraR binding site. Genome-based predictions and transcriptome analysis revealed several novel GraR target genes. We also found that the GraSR TCS is required for growth of S. aureus at high temperatures and resistance to oxidative stress. The GraSR system has previously been shown to play a role in S. aureus pathogenesis and we have uncovered previously unsuspected links with the AgrCA peptide quorum-sensing system controlling virulence gene expression. We also show that the GraSR TCS controls stress reponse and cell wall metabolism signal transduction pathways, sharing an extensive overlap with the WalKR regulon. This is the first report showing a role for the GraSR TCS in high temperature and oxidative stress survival and linking this system to stress response, cell wall and pathogenesis control pathways.

Staphylococcus aureus Transcriptome Architecture: From Laboratory to Infection-Mimicking Conditions

Staphylococcus aureus is a major pathogen that colonizes about 20% of the human population. Intriguingly, this Gram-positive bacterium can survive and thrive under a wide range of different conditions, both inside and outside the human body. Here, we investigated the transcriptional adaptation of S. aureus HG001, a derivative of strain NCTC 8325, across experimental conditions ranging from optimal growth in vitro to intracellular growth in host cells. These data establish an extensive repertoire of transcription units and non-coding RNAs, a classification of 1412 promoters according to their dependence on the RNA polymerase sigma factors SigA or SigB, and allow identification of new potential targets for several known transcription factors. In particular, this study revealed a relatively low abundance of antisense RNAs in S. aureus, where they overlap only 6% of the coding genes, and only 19 antisense RNAs not co-transcribed with other genes were found. Promoter analysis and comparison with Bacillus subtilis links the small number of antisense RNAs to a less profound impact of alternative sigma factors in S. aureus. Furthermore, we revealed that Rho-dependent transcription termination suppresses pervasive antisense transcription, presumably originating from abundant spurious transcription initiation in this A+T-rich genome, which would otherwise affect expression of the overlapped genes. In summary, our study provides genome-wide information on transcriptional regulation and non-coding RNAs in S. aureus as well as new insights into the biological function of Rho and the implications of spurious transcription in bacteria.

The use of gene fusions to study the expression of malT the positive regulator gene of the maltose regulon.

Abstract The technique of Casadaban (1976a) has been adapted to the selection of Escherichia coli strains in which lacZ is fused to malT, the positive regulator gene of the maltose regulon. Genetic evidence is presented that each of these strains codes for a malT-lacZ hybrid protein, which is endowed with β-galactosidase activity. The procedure used to characterize the fusion strains also allows us to establish the direction of malT transcription, which is clockwise on the circular genetic map of E. coli. From the results regarding the expression of the malT-lacZ hybrid genes in the fusion strains it is concluded that the expression of malT, like that of araC in the l -arabinose system is sensitive to catabolite repression but that, unlike that of araC, it is not autoregulated.

A DNA sequence containing the control sites for gene malT and for the malPQ operon

SummaryThe order of 802 base pairs was established in a DNA segment containing the promoter for malPQ which is one of the three maltose operons, and the promoter for malT, the positive regulator gene of the maltose regulon. The determination of the amino-terminal sequence of the MalT protein allowed us to identify the beginning of the malT gene on the sequence. The position of the malP gene was deduced from the published amino-terminal sequence of maltodextrin phosphorylase. A total of 611 base pairs separate the initiation codons for these two genes, which are transcribed in opposite directions. This large intergenic region does not code for any polypeptide of significant size. The main features of this sequence are discussed in terms of the regulation known to operate on malT and malPQ expression.

Staphylococcus aureus FepA and FepB Proteins Drive Heme Iron Utilization in Escherichia coli

EfeUOB-like tripartite systems are widespread in bacteria and in many cases they are encoded by genes organized into iron-regulated operons. They consist of: EfeU, a protein similar to the yeast iron permease Ftrp1; EfeO, an extracytoplasmic protein of unknown function and EfeB, also an extracytoplasmic protein with heme peroxidase activity, belonging to the DyP family. Many bacterial EfeUOB systems have been implicated in iron uptake, but a prefential iron source remains undetermined. Nevertheless, in the case of Escherichia coli, the EfeUOB system has been shown to recognize heme and to allow extracytoplasmic heme iron extraction via a deferrochelation reaction. Given the high level of sequence conservations between EfeUOB orthologs, we hypothesized that heme might be the physiological iron substrate for the other orthologous systems. To test this hypothesis, we undertook characterization of the Staphylococcus aureus FepABC system. Results presented here indicate: i) that the S. aureus FepB protein binds both heme and PPIX with high affinity, like EfeB, the E. coli ortholog; ii) that it has low peroxidase activity, comparable to that of EfeB; iii) that both FepA and FepB drive heme iron utilization, and both are required for this activity and iv) that the E. coli FepA ortholog (EfeO) cannot replace FepA in FepB-driven iron release from heme indicating protein specificity in these activities. Our results show that the function in heme iron extraction is conserved in the two orthologous systems.

Use of deletions created in vitro to map transcriptional regulatory signals in the malA region of Escherichia coli

The malA region of Escherichia coli contains one of the three maltose operons, namely malPQ, and the positive regulatory gene, malT. Gene malT and the malPQ operon are transcribed in opposite directions, in a divergent manner. The distance separating the transcription start-points in the two directions was previously shown to be 513 base-pairs. We are now presenting a deletion analysis of this unexpectedly long intergenic region. Two sets of deletions were created in vitro, by using exonuclease BAL31. One set comprised deletions centered on a HincII restriction site located in the malPQ promoter, and extending towards gene malT. The other set was centered on an EcoRI site, which had been introduced close to the beginning of the malT cistron, and extended towards gene malP. These deletions, initially created on plasmids, were transferred onto the bacterial chromosome. By studying the phenotype resulting from the presence of these deletions, we concluded that: (1) all of the DNA sequences required for expression of malT and malPQ are within 100 base-pairs of the respective transcription start-points for these genes; (2) a sequence located more than 120 base-pairs upstream from the malT transcription start-point plays a role in limiting malT expression; and (3) a remaining DNA segment, 150 to 300 base-pairs in length, and centrally located in the inter-promoter region, seems to play no role in the expression of malT or malPQ.

Structural Basis for Feed-Forward Transcriptional Regulation of Membrane Lipid Homeostasis in Staphylococcus aureus

The biosynthesis of membrane lipids is an essential pathway for virtually all bacteria. Despite its potential importance for the development of novel antibiotics, little is known about the underlying signaling mechanisms that allow bacteria to control their membrane lipid composition within narrow limits. Recent studies disclosed an elaborate feed-forward system that senses the levels of malonyl-CoA and modulates the transcription of genes that mediate fatty acid and phospholipid synthesis in many Gram-positive bacteria including several human pathogens. A key component of this network is FapR, a transcriptional regulator that binds malonyl-CoA, but whose mode of action remains enigmatic. We report here the crystal structures of FapR from Staphylococcus aureus (SaFapR) in three relevant states of its regulation cycle. The repressor-DNA complex reveals that the operator binds two SaFapR homodimers with different affinities, involving sequence-specific contacts from the helix-turn-helix motifs to the major and minor grooves of DNA. In contrast with the elongated conformation observed for the DNA-bound FapR homodimer, binding of malonyl-CoA stabilizes a different, more compact, quaternary arrangement of the repressor, in which the two DNA-binding domains are attached to either side of the central thioesterase-like domain, resulting in a non-productive overall conformation that precludes DNA binding. The structural transition between the DNA-bound and malonyl-CoA-bound states of SaFapR involves substantial changes and large (>30 Å) inter-domain movements; however, both conformational states can be populated by the ligand-free repressor species, as confirmed by the structure of SaFapR in two distinct crystal forms. Disruption of the ability of SaFapR to monitor malonyl-CoA compromises cell growth, revealing the essentiality of membrane lipid homeostasis for S. aureus survival and uncovering novel opportunities for the development of antibiotics against this major human pathogen.

Distinct control sites located upstream from the levansucrase gene of Bacillus subtilis

The sacR regulatory region, which modulates the expression of sacB, the structural gene for ievansucrase, was separated into two parts: an upstream region which carries a constitutive promoter and a downstream region which carries a palindromic structure. Three types of fusions were constructed in which the aphA3 gene coding for kanamycin resistance of Streptococcus faecalis was placed downstream from different deleted sacR regions. Other fusions were constructed by inserting a promoter from phage SPO1 upstream from the sacB gene and part of the sacA region. A third kind of fusion was constructed in which the palindromic structure was flanked by a heterologous promoter and a heterologous structural gene. After introduction of these fusions into the chromosomal DNA of mutants affected in sacB regulation, it was possible to reveal different targets for the regulatory genes sacU, sacQ and sacS: the sacU and sacQ genes act on a region located near or just upstream from the promoter, and the sacS gene, which is involved in the Induction process, acts on the palindromic structure.