Wolfgang Hillen

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 gram-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 Å 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 α6 in each monomer. This forces a pendulum-like motion of helix α4, which increases the separation of the attached DNA binding domains by 3 Å, abolishing the affinity of TetR for its operator DNA.

Carbon catabolite repression in bacteria.

Carbon catabolite repression (CCR) is a regulatory mechanism by which the expression of genes required for the utilization of secondary sources of carbon is prevented by the presence of a preferred substrate. This enables bacteria to increase their fitness by optimizing growth rates in natural environments providing complex mixtures of nutrients. In most bacteria, the enzymes involved in sugar transport and phosphorylation play an essential role in signal generation leading through different transduction mechanisms to catabolite repression. The actual mechanisms of regulation are substantially different in various bacteria. The mechanism of lactose-glucose diauxie in Escherichia coli has been reinvestigated and was found to be caused mainly by inducer exclusion. In addition, the gene encoding HPr kinase, a key component of CCR in many bacteria, was discovered recently.

Protein kinase‐dependent HPr/CcpA interaction links glycolytic activity to carbon catabolite repression in Gram‐positive bacteria

CcpA, the repressor/activator mediating carbon catabolite repression and glucose activation in many Gram‐positive bacteria, has been purified from Bacillus megaterium after fusing it to a His tag. CcpA‐his immobilized on a Ni‐NTA resin specifically interacted with HPr phosphorylated at seryl residue 46. HPr, a phosphocarrier protein of the phosphoenolpyruvate: glycose phosphotransferase system (PTS), can be phosphorylated at two different sites: (i) at His‐15 in a PEP‐dependent reaction catalysed by enzyme I of the PTS; and (ii) at Ser‐46 in an ATP‐dependent reaction catalysed by a metabolite‐activated protein kinase. Neither unphosphorylated HPr nor HPr phosphorylated at His‐15 nor the doubly phosphorylated HPr bound to CcpA. The interaction with seryl‐phosphorylated HPr required the presence of fructose 1,6‐bisphosphate. These findings suggest that carbon catabolite repression in Gram‐positive bacteria is a protein kinase‐triggered mechanism. Glycolytic intermediates, stimulating the corresponding protein kinase and the P‐ser‐HPr/CcpA complex formation, provide a link between glycolytic activity and carbon catabolite repression. The sensitivity of this complex formation to phosphorylation of HPr at His‐15 also suggests a link between carbon catabolite repression and PTS transport activity.

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.

Catabolite repression in Bacillus subtilis: a global regulatory mechanism for the gram-positive bacteria?

Three components involved in catabolite repression (CR) of gene expression in Bacillus have been identified. The cis‐acting catabolite responsive element (CRE), which is present in many genes encoding carbon catabolic enzymes in various species of the Gram‐positive bacteria, mediates CR of several genes in Bacillus subtilis, Bacillus megaterium, and Staphylococcus xylosus. CR of most genes regulated via CRE is also affected by the trans‐acting factors CcpA and HPr. Similarities between CcpA and Lac and Gal repressors suggest binding of CcpA to CRE. HPr, a component of the phosphoenol pyruvate:sugar phosphotransferase system, undergoes regulatory phosphorylation at a serine residue by a fcuctose‐1,6‐diphosphate‐activated kinase. A mutant of HPr, which is not phosphorylatable at this position because of an exchange of serine to alanine, lacks CR of several catabolic activities. This mutant phenotype is similar to the one exhibited by a ccpA mutant. Direct protein‐protein interaction between CcpA and HPr(Ser‐P) was recently demonstrated and constitutes a link between metabolic activity and CR.

Global control of sugar metabolism: a Gram-positive solution

Bacteria utilise carbon sources in a strictly controlled hierarchical manner for which they have developed global control mechanisms that govern and coordinate carbon source-specific regulation. This is achieved via carbon catabolite repression (CCR), which is the result of global transcriptional control and inducer exclusion. A common mechanism for transcriptional control has evolved within the group of low-GC Gram-positive bacteria, including lactic acid bacteria. The seryl-phosphorylated form of the phosphotransferase HPr (HPr-ser-P) mediates CCR in concert with the pleiotropic regulator CcpA (catabolite control protein) by repressing or activating catabolite-controlled genes. HPr-ser-P can concomitantly trigger inducer exclusion by inhibition of carbohydrate-specific permeases. Histidyl-phosphorylated HPr (HPr-his P) is required for the transport of many carbon sources by the phosphotransferase system (PTS). In addition, HPr-his P controls carbohydrate-specific regulators and catabolic enzymes by phosphorylation. Thus, the ratio of HPr-his P/HPr-ser-P determines utilisation of a particular carbon source. This ratio is mainly adjusted by the bifunctional HPr kinase/phosphatase (HPrK/P), which itself is controlled by the metabolic state of the cell. As a result, the information about the metabolic state of the cell is combined with signals scoring the availability of carbon sources to fine-tune the expression of catabolic genes with the goal to optimise growth rate in any given mixture of nutrients. This review summarises the current understanding of carbon catabolite regulation in low-GC Gram-positive bacteria with special emphasis on lactic acid bacteria.

A novel protein kinase that controls carbon catabolite repression in bacteria

HPr(Ser) kinase is the sensor in a multicomponent phosphorelay system that controls catabolite repression, sugar transport and carbon metabolism in Gram‐positive bacteria. Unlike most other protein kinases, it recognizes the tertiary structure in its target protein, HPr, a phosphocarrier protein of the bacterial phosphotransferase system and a transcriptional cofactor controlling the phenomenon of catabolite repression. We have identified the gene (ptsK) encoding this serine/threonine protein kinase and characterized the purified protein product. Orthologues of PtsK have been identified only in bacteria. These proteins constitute a novel family unrelated to other previously characterized protein phosphorylating enzymes. The Bacillus subtilis kinase is shown to be allosterically activated by metabolites such as fructose 1,6‐bisphosphate and inhibited by inorganic phosphate. In contrast to wild‐type B. subtilis, the ptsK mutant is insensitive to transcriptional regulation by catabolite repression. The reported results advance our understanding of phosphorylation‐dependent carbon control mechanisms in Gram‐positive bacteria.

Analysis of a cis-active sequence mediating catabolite repression in gram-positive bacteria.

One form of catabolite repression (CR) in the Gram-positive genus, Bacillus, is mediated by a cis-acting element (CRE). We use here a consensus sequence to identify such elements in sequenced genes of Gram-positive bacteria. These are analysed with respect to position and type of gene in which they occur. CRE sequences near the promoter region are mainly identified in genes encoding carbon catabolic enzymes, which are thus likely to be subject to CR by a global mechanism. Functional aspects of CREs are evaluated.

In vivo gene silencing identifies the Mycobacterium tuberculosis proteasome as essential for the bacteria to persist in mice

The success of Mycobacterium tuberculosis (Mtb) as a human pathogen relies on its ability to resist eradication by the immune system. The identification of mechanisms that enable Mtb to persist is key for finding ways to limit latent tuberculosis, which affects one-third of the worlds population. Here we show that conditional gene silencing can be used to determine whether an Mtb gene required for optimal growth in vitro is also important for virulence and, if so, during which phase of an infection it is required. Application of this approach to the prcBA genes, which encode the core of the mycobacterial proteasome, revealed an unpredicted requirement of the core proteasome for the persistence of Mtb during the chronic phase of infection in mice. Proteasome depletion also attenuated Mtb in interferon-γ–deficient mice, pointing to a function of the proteasome beyond defense against the adaptive immune response. Genes that are essential for growth in vitro, in vivo or both account for approximately 20% of Mtbs genome. Conditional gene silencing could therefore facilitate the validation of up to 800 potential Mtb drug targets and improve our understanding of host-pathogen dynamics.

Stringent doxycycline-dependent control of gene activities using an episomal one-vector system

Conditional expression systems are of pivotal importance for the dissection of complex biological phenomena. Here, we describe a novel EBV-derived episomally replicating plasmid (pRTS-1) that carries all the elements for conditional expression of a gene of interest via Tet regulation. The vector is characterized by (i) low background activity, (ii) high inducibility in the presence of doxycycline (Dox) and (iii) graded response to increasing concentrations of the inducer. The chicken beta actin promoter and an element of the murine immunoglobin heavy chain intron enhancer drive constitutive expression of a bicistronic expression cassette that encodes the highly Dox-sensitive reverse tetracycline controlled transactivator rtTA2S-M2 and a Tet repressor-KRAB fusion protein (tTSKRAB) (silencer) placed downstream of an internal ribosomal entry site. The gene of interest is expressed from the bidirectional promoter Ptetbi-1 that allows simultaneous expression of two genes, of which one may be used as surrogate marker for the expression of the gene of interest. Tight down regulation is achieved through binding of the silencer tTSKRAB to Ptetbi-1 in the absence of Dox. Addition of Dox releases repression and via binding of rtTA2S-M2 activates Ptetbi-1.