The TetR family of transcriptional repressors.

We have developed a general profile for the proteins of the TetR family of repressors. The stretch that best defines the profile of this family is made up of 47 amino acid residues that correspond to the helix-turn-helix DNA binding motif and adjacent regions in the three-dimensional structures of TetR, QacR, CprB, and EthR, four family members for which the function and three-dimensional structure are known. We have detected a set of 2,353 nonredundant proteins belonging to this family by screening genome and protein databases with the TetR profile. Proteins of the TetR family have been found in 115 genera of gram-positive, alpha-, beta-, and gamma-proteobacteria, cyanobacteria, and archaea. The set of genes they regulate is known for 85 out of the 2,353 members of the family. These proteins are involved in the transcriptional control of multidrug efflux pumps, pathways for the biosynthesis of antibiotics, response to osmotic stress and toxic chemicals, control of catabolic pathways, differentiation processes, and pathogenicity. The regulatory network in which the family member is involved can be simple, as in TetR (i.e., TetR bound to the target operator represses tetA transcription and is released in the presence of tetracycline), or more complex, involving a series of regulatory cascades in which either the expression of the TetR family member is modulated by another regulator or the TetR family member triggers a cell response to react to environmental insults. Based on what has been learned from the cocrystals of TetR and QacR with their target operators and from their three-dimensional structures in the absence and in the presence of ligands, and based on multialignment analyses of the conserved stretch of 47 amino acids in the 2,353 TetR family members, two groups of residues have been identified. One group includes highly conserved positions involved in the proper orientation of the helix-turn-helix motif and hence seems to play a structural role. The other set of less conserved residues are involved in establishing contacts with the phosphate backbone and target bases in the operator. Information related to the TetR family of regulators has been updated in a database that can be accessed at www.bactregulators.org.

Members of the IclR family of bacterial transcriptional regulators function as activators and/or repressors.

Members of the IclR family of regulators are proteins with around 250 residues. The IclR family is best defined by a profile covering the effector binding domain. This is supported by structural data and by a number of mutants showing that effector specificity lies within a pocket in the C-terminal domain. These regulators have a helix-turn-helix DNA binding motif in the N-terminal domain and bind target promoters as dimers or as a dimer of dimers. This family comprises regulators acting as repressors, activators and proteins with a dual role. Members of the IclR family control genes whose products are involved in the glyoxylate shunt in Enterobacteriaceae, multidrug resistance, degradation of aromatics, inactivation of quorum-sensing signals, determinants of plant pathogenicity and sporulation. No clear consensus exists on the architecture of DNA binding sites for IclR activators: the MhpR binding site is formed by a 15-bp palindrome, but the binding sites of PcaU and PobR are three perfect 10-bp sequence repetitions forming an inverted and a direct repeat. IclR-type positive regulators bind their promoter DNA in the absence of effector. The mechanism of repression differs among IclR-type regulators. In most of them the binding sites of RNA polymerase and the repressor overlap, so that the repressor occludes RNA polymerase binding. In other cases the repressor binding site is distal to the RNA polymerase, so that the repressor destabilizes the open complex.

Characterization of the RND family of multidrug efflux pumps: in silico to in vivo confirmation of four functionally distinct subgroups.

We have developed a generalized profile that identifies members of the root-nodulation-cell-division (RND) family of efflux pumps and classifies them into four functional subfamilies. According to Z-score values, efflux pumps can be grouped by their metabolic function, thus making it possible to distinguish pumps involved in antibiotic resistance (group 1) from those involved in metal resistance (group 3). In silico data regarding efflux pumps in group 1 were validated after identification of RND efflux pumps in a number of environmental microbes that were isolated as resistant to ethidium bromide. Analysis of the Pseudomonas putida KT2440 genome identified efflux pumps in all groups. A collection of mutants in efflux pumps and a screening platform consisting of 50 drugs were created to assign a function to the efflux pumps. We validated in silico data regarding efflux pumps in groups 1 and 3 using 9 different mutants. Four mutants belonging to group 2 were found to be more sensitive than the wild-type to oxidative stress-inducing agents such as bipyridyl and methyl viologen. The two remaining mutants belonging to group 4 were found to be more sensitive than the parental to tetracycline and one of them was particularly sensitive to rubidium and chromate. By effectively combining in vivo data with generalized profiles and gene annotation data, this approach allowed the assignment, according to metabolic function, of both known and uncharacterized RND efflux pumps into subgroups, thereby providing important new insight into the functions of proteins within this family.

Physiological and transcriptomic characterization of a fliA mutant of Pseudomonas putida KT2440.

Pseudomonas putida KT2440 encodes 23 alternative sigma factors. The fliA gene, which encodes σ(28) , is in a cluster with other genes involved in flagella biosynthesis and chemotaxis. Reverse transcriptase-PCR revealed that this cluster is comprised of four independent transcriptional units: flhAF, fleNfliA, cheYZA and cheBmotAB. We generated a nonpolar fliA mutant by homologous recombination and tested its motility, adhesion to biotic and abiotic surfaces, and responses to various stress conditions. The mutant strain was nonmotile and exhibited decreased capacity to bind to corn seeds, although its ability to colonize the rhizosphere of plants was unaffected. The mutant was also affected in binding to abiotic surfaces and its ability to form biofilms decreased by almost threefold. In the fliA mutant background expression of 25 genes was affected: two genes were upregulated and 23 genes were downregulated. In addition to a number of motility and chemotaxis genes, the fliA gene product is also necessary for the expression of some genes potentially involved in amino acid utilization or stress responses; however, we were unable to assign specific phenotypes linked to these genes since the fliA mutant used the same range of amino acids as the parental strain, and was as tolerant as the wild type to stress imposed by heat, antibiotics, NaCl, sodium dodecyl sulfate, H2 O2 and benzoate. Based on the sequence alignment of promoters recognized by FliA and genome in silico analysis, we propose that P. putidaσ(28) recognizes a TCAAG-t-N12 -GCCGATA consensus sequence located between -34 and -8 and that this sequence is preferentially associated with an AT-rich upstream region.

A general profile for the MerR family of transcriptional regulators constructed using the semi-automated Provalidator tool.

Provalidator is a web-based tool that facilitates the design and validation of generalized profiles of protein families in prokaryotes. This tool combines the nearly full automation of profile building with a search for family members in all available databases. The tool is useful for assigning a given protein to a specific family, and is also useful for genome mining in annotated prokaryotic genomes. The tool is freely available at http://www.bactregulators.org. As proof of concept we constructed a profile that best defines the MerR family of transcriptional regulators. The profile created includes functional residues that are part of the helix-turn-helix DNA binding domain and accessory elements defined as wings 1 and 2, suggesting that members of the MerR family of regulators may exhibit conserved 3D structure in the region that defines the family profile. The profile defined for MerR was used to search for members of this family in the Swiss-Prot and TrEMBL databases, and also to identify members of the family in the genome of Pseudomonas putida. One of these identified regulators was found to be involved in zinc tolerance, showing the usefulness of identifying family members and assigning phenotypes.

BacTregulators: a database of transcriptional regulators in bacteria and archaea.

MOTIVATION: The BacTregulators database is intended to collect and to integrate information on proteins belonging to defined families of transcriptional regulators in prokaryotes. RESULTS: The BacTregulators database currently contains data on two families of transcriptional regulators: AraC-XylS and TetR. The proteins included in the BacTregulators database have been identified by screening 123 genomes from archaea and bacteria and the SWISS-PROT and TrEMBL databases with profiles defining each family. As the result of an integration process, we have included 1326 different protein sequences from the AraC-XylS family and 1487 different protein sequences from the TetR family. The definition of an entry in BacTregulators is based on protein sequence, source organism, genome element and position in this genome element. The BacTregulators site allows the user to retrieve protein sequences, functional features and experimental evidence supporting the functions, references and the three-dimensional structure of the regulator when available. BacTregulators supplies an innovative tool that allows the researcher to obtain an integrated report that shows the data corresponding to other entries which are related by sequence similarity to the query entry. BacTregulators detects and classifies the regulators belonging to AraC-XylS and TetR families present in prokaryotic genomes, and thus contributes to a more accurate annotation of regulators in genomes. The information collected on each protein in the family can be useful to characterize a new regulator or compile information on the biological properties of a known regulator. AVAILABILITY: The BacTregulators is available at www.bactregulators.org