Regulation of the Mycobacterium tuberculosis PE/PPE genes.

The genome of Mycobacterium tuberculosis encodes approximately 170 members of the unique mycobacterial PE and PPE gene families. Evidence suggests members of these families are surface-associated cell wall proteins that may provide a diverse antigenic profile and affect immunity. To determine if the expression patterns of PE/PPE genes are consistent with a role in antigenic variability, we analyzed microarray data from 132 experimental conditions for expression of PE/PPE genes. Whole genome expression patterns show that the PE/PPE genes are regulated in a variable and largely independent manner. Gene expression profiling of 15 unique conditions identified differential regulation of 128 of the 169 PE/PPE genes. Expression of the PE/PPE genes appears to be controlled by a variety of independent mechanisms. These data indicate that differential expression of the PE/PPE genes has the potential to provide a dynamic antigenic profile during the course of changing microenvironments within the host.

Energy transfer between fluorescent proteins using a co-expression system in Mycobacterium smegmatis.

The goal of this study was to establish a two-plasmid co-expression system for Mycobacterium smegmatis. Two vectors with compatible origins of replication and a polylinker, which allows modular cloning of promoters and genes, were constructed and used to clone genes encoding a blue fluorescent protein (BFP) and a green fluorescent protein (GFP). A 160-fold variation of GFP expression levels in M. smegmatis was achieved by combining three promoters with different copy numbers of the vectors. An efficient energy transfer between BFP and GFP in M. smegmatis was observed by fluorescence measurements and demonstrated that these genes were simultaneously expressed from both vectors. Thus, these vectors will be valuable for all strategies where co-expression of proteins in M. smegmatis is needed, e.g. for constructing a two-hybrid system or for deleting essential genes.

Reprogramming of the macrophage transcriptome in response to interferon-gamma and Mycobacterium tuberculosis: signaling roles of nitric oxide synthase-2 and phagocyte oxidase.

Macrophage activation determines the outcome of infection by Mycobacterium tuberculosis (Mtb). Interferon-gamma (IFN-gamma) activates macrophages by driving Janus tyrosine kinase (JAK)/signal transducer and activator of transcription-dependent induction of transcription and PKR-dependent suppression of translation. Microarray-based experiments reported here enlarge this picture. Exposure to IFN-gamma and/or Mtb led to altered expression of 25% of the monitored genome in macrophages. The number of genes suppressed by IFN-gamma exceeded the number of genes induced, and much of the suppression was transcriptional. Five times as many genes related to immunity and inflammation were induced than suppressed. Mtb mimicked or synergized with IFN-gamma more than antagonized its actions. Phagocytosis of nonviable Mtb or polystyrene beads affected many genes, but the transcriptional signature of macrophages infected with viable Mtb was distinct. Studies involving macrophages deficient in inducible nitric oxide synthase and/or phagocyte oxidase revealed that these two antimicrobial enzymes help orchestrate the profound transcriptional remodeling that underlies macrophage activation.

Regulation of the Mycobacterium tuberculosis hypoxic response gene encoding alpha -crystallin.

Unlike many pathogens that are overtly toxic to their hosts, the primary virulence determinant of Mycobacterium tuberculosis appears to be its ability to persist for years or decades within humans in a clinically latent state. Since early in the 20th century latency has been linked to hypoxic conditions within the host, but the response of M. tuberculosis to a hypoxic signal remains poorly characterized. The M. tuberculosis alpha-crystallin (acr) gene is powerfully and rapidly induced at reduced oxygen tensions, providing us with a means to identify regulators of the hypoxic response. Using a whole genome microarray, we identified >100 genes whose expression is rapidly altered by defined hypoxic conditions. Numerous genes involved in biosynthesis and aerobic metabolism are repressed, whereas a high proportion of the induced genes have no known function. Among the induced genes is an apparent operon that includes the putative two-component response regulator pair Rv3133c/Rv3132c. When we interrupted expression of this operon by targeted disruption of the upstream gene Rv3134c, the hypoxic regulation of acr was eliminated. These results suggest a possible role for Rv3132c/3133c/3134c in mycobacterial latency.

Identification of a stability determinant on the edge of the Tet repressor four-helix bundle dimerization motif.

Isofunctional tetracycline repressor (TetR) proteins isolated from different bacteria show a sequence identity between 38 and 88% of the residues. Their active state is a homodimer formed by a four-alpha-helix bundle as the main interaction motif. We utilize this sequence variation of isofunctional proteins to determine residues contributing to the stability of the four-helix bundle. The thermodynamic stabilities of two TetR proteins with 63% sequence identity were determined by urea-induced reversible denaturation followed by fluorescence and circular dichroism. Both methods yield identical results. The deltaG(o)U (H2O) values are 60 and 75 kJ x mol(-1). We have constructed TetR hybrid proteins derived from these wild types to identify the determinant leading to the 15 kJ x mol(-1) stability difference. Successive size reduction of the exchanged portion yielded two single residues affecting the overall protein stability. The P184Q exchange leads to a more stable protein, whereas the G181D exchange located at the solvent's exposed edge of the four-helix bundle is solely responsible for the reduced stability. Additional mutants based on crystal structures of TetR do not reveal any hint for steric interference of the Asp181 side chain with neighboring residues. Thus, this is an example for the role played by surface-exposed turn residues for the stability of four-helix bundles. We assume that the larger conformational flexibility of Gly and the reduction of the negative surface charge could favor formation of the turn on the edge of the four-helix bundle.

Functional Genomics of Mycobacterium tuberculosis Using DNA Microarrays.

Completion of the sequence of the entire genome of strain H37Rv was a benchmark for Mycobacterium tuberculosis research (1). This achievement ushers in the era of genome-wide functional and comparative genomics for this organism. At present, the most powerful enabling technology of the postgenomic era is microarray-based hybridization. Microarrays, by whatever means they are fabricated, contain surface-bound representations of each open reading frame (ORF) of a sequenced genome. Thus, they provide a method for parallel sampling of thousands of different genes within a complex pool of nucleic acids. Microarray gene capacity readily accommodates the number of ORFs in the relatively small genomes of bacteria and yeast and, in principle, can accommodate the entire genetic repertoire of complex multicellular animals. Below, we discuss our fabrication and use of an M. tuberculosis microarray, containing representations of each of the identified 3924 ORFs of this organism. We will describe two applications of this method. In the first-microarray-based gene response, i.e., transcript profiling - we ask the question: which genes are selectively expressed under a particular condition of growth, in a particular host compartment or as a result of inhibition of a metabolic or biosynthetic pathway? In the second, comparative genomics, we use a microarray containing the ORFs of one strain or species to identify ORFs deleted or absent from a second strain or species whose genome sequence may not have been determined. In this manner, microarray-based comparative genomics seeks to learn the ORF-by-ORF relatedness of two similar, but nonidentical organisms whose biological differences are under investigation. Examples of each application have been applied to M. tuberculosis (2,3).

Structural basis of gene regulation by the tetracycline inducible Tet repressor-operator system.

The tetracycline repressor (TetR) regulates the most abundant resistance mechanism against the antibiotic tetracycline in grain-negative bacteria. The TetR protein and its mutants are commonly used as control elements to regulate gene expression in higher eukaryotes. We present the crystal structure of the TetR homodimer in complex with its palindromic DNA operator at 2.5 A resolution. Comparison to the structure of TetR in complex with the inducer tetracycline-Mg2+ allows the mechanism of induction to be deduced. Inducer binding in the repressor core initiates conformational changes starting with C-terminal unwinding and shifting of the short helix a6 in each monomer. This forces a pendulum-like motion of helix a4, which increases the separation of the attached DNA binding domains by 3 A, abolishing the affinity of TetR for its operator DNA.

Generation of conditional mutants in higher eukaryotes by switching between the expression of two genes.

A regulatory system for the in-depth study of gene functions in higher eukaryotic cells has been developed. It is based on the tetracycline-controlled transactivators and reverse tTA, which were remodeled to discriminate efficiently between two different promoters. The system permits one to control reversibly the activity of two genes, or two alleles of a gene, in a mutually exclusive way, and also allows one to abrogate the activities of both. This dual regulatory circuit, which can be operated by a single effector substance such as doxycycline, overcomes limitations of conventional genetic approaches. The conditional mutants that can now be generated will be useful for the study of gene function in vitro and in vivo. In addition, the system may be of value for a variety of practical applications, including gene therapy.

Solvent-exposed residues in the Tet repressor (TetR) four-helix bundle contribute to subunit recognition and dimer stability.

Dimerization specificity of Tet repressor (TetR) can be altered by changes in the core of the four-helix bundle that mediates protein-protein recognition. We demonstrate here that the affinity of subunit interaction depends also on the solvent-exposed residues at positions 128 and 179'-184', which interact across the dimerization surface. TetR(B) and (D), two naturally occurring sequence variants, differ at position 128 with respect to the monomer-monomer distances in the crystal structures and the charge of the amino acids, being glutamate in TetR(B) and arginine in TetR(D). In vivo analysis of chimeric TetR(B/D) variants revealed that the single E128R exchange does not alter the dimerization specificity of TetR(B) to the one of TetR(D). When combined with specificity mutations in alpha10, it is, however, able to increase dimerization efficiency of the TetR(B/D) chimera with TetR(D). A loss of contact analysis revealed a positive interaction between Arg-128 and residues located at positions 179'-184' of the second monomer. We constructed a hyperstable TetR(B) variant by replacing residues 128 and 179-184 by the respective TetR(D) sequence. These results establish that in addition to a region in the hydrophobic core residues at the solvent-exposed periphery of the dimerization surface participate in protein-protein recognition in the TetR four-helix bundle.

Crystal structure of the tet repressor in complex with a novel tetracycline, 9-(N,N-dimethylglycylamido)- 6-demethyl-6-deoxy-tetracycline.

The tetracycline analog 9-(N, N-dimethylglycylamido)-6-demethyl-6-deoxy-tetracycline (9glyTc) belongs to a new group of tetracyclines called glycylcyclines. They are strong antibiotics showing reduced sensitivity against the major tetracycline resistance mechanisms. We have determined the crystal structure of 9glyTc in complex with Tet repressor class D, TetR(D), at 2.4 A resolution. Sterical hindrance at the entrance of the tetracycline binding tunnel of TetR by the bulky and charged glycyl amido substituent interferes with conformational changes required for the mechanism of induction, and leads to decreased induction efficiency as observed for point mutations of amino acid residues located in the neighbourhood to the glycylamido moiety of bound 9glyTc.

Crystallization and preliminary X-ray analysis of the Tet-repressor/operator complex.

Three crystal forms of the repressor protein TetR class D in complex with the palindromic 17 bp operator sequence containing T overhangs on both sides were obtained by hanging-drop vapor-diffusion methods using PEG 4000 and PEG monomethylether 5000 as precipitants. Although the crystallization conditions were very similar, up to three different crystal forms were observed in the same drop. The space groups are monoclinic C2, P21 and hexagonal P6122. The asymmetric units of the latter two crystal forms contain one repressor-operator complex. The crystal structures of these forms were solved by molecular replacement using the Tet-repressor molecule of the complex with tetracycline as a search model.

Conformational changes necessary for gene regulation by Tet repressor assayed by reversible disulfide bond formation.

We constructed and characterized four Tet repressor (TetR) variants with engineered cysteine residues which can form disulfide bonds and are located in regions where conformational changes during induction by tetracycline (tc) might occur. All TetR mutants show nearly wild-type activities in vivo, and the reduced proteins also show wild-type activities in vitro. Complete and reversible disulfide bond formation was achieved in vitro for all four mutants. The disulfide bond in NC18RC94 immobilizes the DNA reading head with respect to the protein core and prevents operator binding. Formation of this disulfide bond is possible only in the tc-bound, but not in the operator-bound conformation. Thus, these residues must have different conformations when bound to these ligands. The disulfide bonds in DC106PC159' and EC107NC165' immobilize the variable loop between alpha-helices 8 and 9 located near the tc-binding pocket. A faster rate of disulfide formation in the operator-bound conformation and a lack of induction after disulfide formation show that the variable loop is located closer to the protein core in the operator-bound conformation and that a movement is necessary for induction. The disulfide bond in RC195VC199' connects alpha-helices 10 and 10' of the two subunits in the dimer and is only formed in the tc-bound conformation. The oxidized protein shows reduced operator binding. Thus, this bond prevents formation of the operator-bound conformation. The detection of conformational changes in three different regions is the first biochemical evidence for induction-associated global internal movements in TetR.

Conformational changes of the Tet repressor induced by tetracycline trapping.

The X-ray crystal structure analysis of inducer-free Tet repressor, TetR, at 2.4 A resolution identifies one of two openings of the tunnel-like binding site as the entrance for the inducer tetracycline-Mg2+, [Mg Tc]+. Recognition and binding of the inducer unleashes conformational changes leading to the induced state of TetR. In the first step, the C-terminal turn of alpha-helix 6 unwinds, thereby altering the orientation of alpha-helix 4. This different orientation of alpha-helix 4 is stabilized by a series of hydrogen bonds mediated through a chain of eight water molecules. The alpha-helix 4 connects the DNA-binding domain (alpha-helices 1 to 3) to the rigid TetR core, and thus regulates gene expression through its respective orientations.

Determinants of protein-protein recognition by four helix bundles: changing the dimerization specificity of Tet repressor.

Homo- and heterodimerization is essential for the activity of many proteins, particularly transcription factors. One widely distributed structural motif for protein recognition is the four helix bundle. To understand the molecular details determining specificity of subunit recognition in a dimer formed by a four helix bundle, we investigated Tet repressor (TetR) sequence variants TetR(B) and TetR(D), which do not form heterodimers. We used molecular modeling to identify residues with the potential to determine recognition of subunits. Directed mutagenesis of these residues in TetR(B) by the TetR(D) sequence resulted in chimeric TetR(B/D) repressors with new subunit recognition specificities. The single LS192 exchange in TetR(B/D)192 in the center of the helix bundle leads to a relaxed specificity since this variant dimerizes with TetR(B) and (D). To construct a variant with a new specificity it was not sufficient to mutate the contacting residue, F197, in the other subunit. Instead, it was necessary to exchange two more residues in the vicinity of F197 and S192. The resulting TetR(B/D)188, 192,193,197 forms dimers with TetR(D) but not with TetR(B), indicating that four amino acid exchanges are sufficient to change subunit recognition. These results establish that targeted alterations in the structural complementarity of protein-protein interaction surfaces can be used to construct new recognition specificities. However, it is not sufficient to adjust the complementary residues since the surrounding amino acids contribute essentially to protein-protein recognition.

The role of the variable region in Tet repressor for inducibility by tetracycline.

A set of deletions and substitutions to alanine was introduced into the loop separating helices alpha8 and alpha9 of Tn10 Tet repressor (TetR). This region appears as an unstructured loop in the crystal structure of the TetR(D).([Mg-tc]+)2 complex and is the only internal segment of variable length in an alignment of Tet repressors from seven different resistance determinants. In vivo analysis of 10 mutants shows that this loop is important for inducibility by tetracycline (tc), whereas DNA binding is not or only marginally affected. All deletions have an induction-deficient TetRS phenotype, but the corresponding substitutions do not or only slightly affect inducibility. The purified mutant TetR proteins have a reduced affinity for tc in vitro that correlates with their lack of inducibility. The association rate of [Mg-tc]+ to the TetR mutants is enhanced. Since none of the mutated residues contacts tc directly in the crystal structure, we propose that the length of the loop is important for the structural transition between a closed, tc binding and an open, operator binding conformation of TetR. We propose that the deletions in the loop shift the equilibrium between both forms toward the open, operator binding conformation.

Fast large-scale purification of tetracycline repressor variants from overproducing Escherichia coli strains.

We constructed a plasmid for overexpression of Tn10 Tet repressor (TetR) by placing a synthetic tetR gene under control of the Pc promoter. Active TetR is expressed up to 30% of the total soluble cell protein. A protocol containing anion-exchange, cation-exchange, and size-exclusion chromatography steps is described for the large-scale purification of milligram amounts of TetR in three days. Cation-exchange chromatography already yields almost homogenous TetR. Purification of about fifty TetR mutants demonstrates that this protocol is generally applicable. No correlation between net charge of TetR variants and elution behaviour was detected for the anion-exchange column. On the other hand, TetR mutants with increased negative charge in their DNA binding domain eluted at lower NaCl concentration from the cation-exchange column. The applicability of this purification protocol to the wide variety of TetR variants suggests that it can be used for the rapid purification of other DNA binding proteins as well.

Tetracyclines: antibiotic action, uptake, and resistance mechanisms.

Tetracyclines probably penetrate bacterial cells by passive diffusion and inhibit bacterial growth by interfering with protein synthesis or by destroying the membrane. A growing number of various bacterial species acquire resistance to the bacteriostatic activity of tetracycline. The two widespread mechanisms of bacterial resistance do not destroy tetracycline: one is mediated by efflux pumps, the other involves an EF-G-like protein that confers ribosome protection. Oxidative destruction of tetracycline has been found in a few species. Several efflux transporters, including multidrug-resistance pumps and tetracycline-specific exporters, confer bacterial resistance against tetracycline. Single amino acids of these carrier proteins important for tetracycline transport and substrate specificity have been identified, allowing the mechanism of tetracycline transport to begin to emerge.

Extracellular expression of native human anti-lysozyme fragments in Staphylococcus carnosus.

Xylose-inducible vectors have been constructed for extracellular production of antibody fragments in Staphylococcus carnosus. The pre-pro sequence of S. hyicus lipase was taken as secretional signal sequence, and the S. xylosus Xyl repressor was used to confer xylose inducibility of transcription. Cleavage sites for the IgA protease were engineered between the pre-pro sequence and the antibody fragments to permit removal of the pro sequence. Extracellular expression of the light chain and the Fd fragment of a chimeric Fab fragment containing the variable regions of the anti-lysozyme antibody D1.3 was achieved with these vectors. The pro sequence could be removed from the expression product by IgA protease treatment. When the light chain and the Fd fragment were co-secreted as a protein fusion they accumulated in a structure capable of heterodimerization after IgA cleavage. This fusion contains the pre-pro sequence followed by the light chain, a second IgA site and the Fd fragment.