HflX protein protects Escherichia coli from manganese stress.

The ribosome-binding GTPase HflX is required for manganese homeostasis in E. coli. While under normal conditions ΔhflX cells behave like wild type E. coli with respect to growth pattern and morphology, deletion of hflX makes E. coli cells extremely sensitive to manganese, characterized by arrested cell growth and filamentation. Here we demonstrate that upon complementation by hflX, manganese stress is relieved. In phenotypic studies done in a manganese-rich environment, ΔhflX cells were highly sensitive to antibiotics that bind the penicillin binding protein 3 (PBP3), suggesting that the manganese stress led to impaired peptidoglycan biosynthesis. An irregular distribution of dark bands of constriction along filaments, delocalization of the dark bands from midcell towards poles and subpoles, lack of septum formation and arrested cell division were observed in ΔhflX cells under manganese stress. However, chromosome replication and segregation of nucleoids were unaffected under these conditions, as observed from confocal microscopy imaging and FACS studies. We conclude that absence of HflX leads to manganese accumulation in E. coli cells, affecting cell septum formation, probably by modulating the activity of the cell division protein PBP3 (FtsI), a major component of the divisome apparatus. We propose that HflX acts as a gatekeeper, regulating the influx of manganese into the cell.

Characterization of the autophosphorylation property of HflX, a ribosome-binding GTPase from Escherichia coli.

Escherichia coli HflX belongs to the widely distributed but poorly characterized HflX family of translation factor-related GTPases that is conserved from bacteria to humans. A 426-residue polypeptide that binds 50S ribosomes and has both GTPase and ATPase activities, HflX also exhibits autophosphorylation activity. We show that HflX(C), a C-terminal fragment of HflX, has an enhanced autophosphorylation activity compared to the full-length protein. Using a chemical stability assay and thin layer chromatography, we have determined that phosphorylation occurs at a serine residue. Each of the nine serine residues of HflX(C) was mutated to alanine. It was found that all but S211A retained autophosphorylation activity, suggesting that S211, located in the P-loop, was the likely site for autophosphorylation. While the S211A mutant lacked the autophosphorylation site, it possessed strong GTP binding and GTPase activities.

Universal minicircle sequence binding protein of Leishmania donovani regulates pathogenicity by controlling expression of cytochrome-b.

BACKGROUND: Leishmania contains a concatenated mitochondrial DNA, kDNA. Universal minicircle sequence binding protein (UMSBP), a mitochondrial protein, initiates kDNA replication by binding with a conserved universal minicircle sequence (UMS) of kDNA. Here, we describe first time in L. donovani the regulation of DNA binding activity of UMSBP and the role of UMSBP in virulence. METHODS: Insilco and EMSA study were performed to show UMS-binding activity of UMSBP. Tryparedoxin(TXN)-tryparedoxin peroxidase(TXNPx) assay as well as co-overexpression of cytochrome-b5 reductase-like protein (CBRL) and tryparedoxin in L. donovani were done to know the regulation of DNA binding activity of UMSBP. Knockout and episomal-expression constructs of UMSBP were transfected in L. donovani. The cell viability assay and immunofluorescence study to know the status of kDNA were performed. Macrophages were infected with transfected parasites. mRNA level of cytochrome b, activity of complex-III, intracellular ATP level of both transfected promastigotes and amastigotes as well as ROS concentration and the level of apoptosis of transfected promastigotes were measured. Level of oxidative phosphorylation of both transfected and un-transfected amastigotes were compared. Burden of transfected amastigotes in both macrophages and BALB/c mice were measured. RESULTS: L. donovani UMSBP is capable of binding with UMS, regulated by redox through mitochondrial enzymes, TXN, TXNPx and CBRL. Depletion of UMSBP (LdU(-/-)) caused kDNA loss, which decreased cytochrome-b expression [component of complex-III of electron transport chain (ETC)] and leads to the disruption of complex-III activity, decreased ATP generation, increased ROS level and promastigotes exhibited apoptosis like death. Interestingly, single knockout of UMSBP (LdU(-/+)) has no effect on promastigotes survival. However, single knockout in intracellular amastigotes demonstrate loss of mRNA level of cytochrome-b, disruption in the activity of complex-III and reduced production of ATP in amastigotes than wild type. This process interfere with the oxidative-phosphorylation and thereby completely inhibit the intracellular proliferation of LdU(-/+) amastigotes in human macrophages and in BALB/c mice. Amastigotes proliferation was restored as wild type after episomal expression of LdUMSBP in LdU(-/+) parasites (LdU(-/+)AB). CONCLUSION: The LdUMSBP regulates leishmanial mitochondrial respiration and pathogenesis. So, LdUMSBP may be an attractive target for rational drug designing and LdU(-/+) parasites could be considered as a live attenuated vaccine candidate against visceral leishmaniasis.

Crystallization and X-ray analysis of the transcription-activator protein C1 of bacteriophage P22 in complex with the PRE promoter element.

The transcription-activator protein C1 of the temperate phage P22 of Salmonella typhimurium plays a key role in the lytic versus lysogenic switch of the phage. A homotetramer of 92-residue polypeptides, C1 binds to an approximate direct repeat similar to the transcription activator CII of coliphage λ. Despite this and several other similarities, including 57% sequence identity to coliphage CII, many biochemical observations on P22 C1 cannot be explained based on the structure of CII. To understand the molecular basis of these differences, C1 was overexpressed and purified and subjected to crystallization trials. Although no successful hits were obtained for the apoprotein, crystals could be obtained when the protein was subjected to crystallization trials in complex with a 23-mer promoter DNA fragment (PRE). These crystals diffracted very well at the home source, allowing the collection of a 2.2 Šresolution data set. The C1-DNA crystals belonged to space group P21, with unit-cell parameters a = 87.27, b = 93.58, c = 111.16 Å, ß = 94.51°. Solvent-content analysis suggests that the asymmetric unit contains three tetramer-DNA complexes. The three-dimensional structure is expected to shed light on the mechanism of activation by C1 and the molecular basis of its specificity.

Revisiting the mechanism of activation of cyclic AMP receptor protein (CRP) by cAMP in Escherichia coli: lessons from a subunit-crosslinked form of CRP.

Cyclic AMP receptor protein (CRP), the global transcription regulator in prokaryotes, is active only as a cAMP-CRP complex. Binding of cAMP changes the conformation of CRP, transforming it from a transcriptionally 'inactive' to an 'active' molecule. These conformers are also characterized by distinct biochemical properties including the ability to form an S-S crosslink between the C178 residues of its two monomeric subunits. We studied a CRP variant (CRP(cl)), in which the subunits are crosslinked. We demonstrate that CRP(cl) can activate transcription even in the absence of cAMP. Implications of these results for the crystallographically-determined structure of cAMP-CRP are discussed.

Novel MntR-independent mechanism of manganese homeostasis in Escherichia coli by the ribosome-associated protein HflX.

Manganese is a micronutrient required for activities of several important enzymes under conditions of oxidative stress and iron starvation. In Escherichia coli, the manganese homeostasis network primarily constitutes a manganese importer (MntH) and an exporter (MntP), which are regulated by the MntR dual regulator. In this study, we find that deletion of E. coli hflX, which encodes a ribosome-associated GTPase with unknown function, renders extreme manganese sensitivity characterized by arrested cell growth, filamentation, lower rate of replication, and DNA damage. We demonstrate that perturbation by manganese induces unprecedented influx of manganese in ΔhflX cells compared to that in the wild-type E. coli strain. Interestingly, our study indicates that the imbalance in manganese homeostasis in the ΔhflX strain is independent of the MntR regulon. Moreover, the influx of manganese leads to a simultaneous influx of zinc and inhibition of iron import in ΔhflX cells. In order to review a possible link of HflX with the λ phage life cycle, we performed a lysis-lysogeny assay to show that the Mn-perturbed ΔhflX strain reduces the frequency of lysogenization of the phage. This observation raises the possibility that the induced zinc influx in the manganese-perturbed ΔhflX strain stimulates the activity of the zinc-metalloprotease HflB, the key determinant of the lysis-lysogeny switch. Finally, we propose that manganese-mediated autophosphorylation of HflX plays a central role in manganese, zinc, and iron homeostasis in E. coli cells.

Studies on Escherichia coli HflKC suggest the presence of an unidentified λ factor that influences the lysis-lysogeny switch.

BACKGROUND: The lysis-lysogeny decision in the temperate coliphage λ is influenced by a number of phage proteins (CII and CIII) as well as host factors, viz. Escherichia coli HflB, HflKC and HflD. Prominent among these are the transcription factor CII and HflB, an ATP-dependent protease that degrades CII. Stabilization of CII promotes lysogeny, while its destabilization induces the lytic mode of development. All other factors that influence the lytic/lysogenic decision are known to act by their effects on the stability of CII. Deletion of hflKC has no effect on the stability of CII. However, when λ infects ΔhflKC cells, turbid plaques are produced, indicating stabilization of CII under these conditions. RESULTS: We find that CII is stabilized in ΔhflKC cells even without infection by λ, if CIII is present. Nevertheless, we also obtained turbid plaques when a ΔhflKC host was infected by a cIII-defective phage (λcIII67). This observation raises a fundamental question: does lysogeny necessarily correlate with the stabilization of CII? Our experiments indicate that CII is indeed stabilized under these conditions, implying that stabilization of CII is possible in ΔhflKC cells even in the absence of CIII, leading to lysogeny. CONCLUSION: We propose that a yet unidentified CII-stabilizing factor in λ may influence the lysis-lysogeny decision in ΔhflKC cells.

Escherichia coli HflK and HflC can individually inhibit the HflB (FtsH)-mediated proteolysis of lambdaCII in vitro.

LambdaCII is the key protein that influences the lysis/lysogeny decision of lambda by activating several phage promoters. The effect of CII is modulated by a number of phage and host proteins including Escherichia coli HflK and HflC. These membrane proteins copurify as a tightly bound complex 'HflKC' that inhibits the HflB (FtsH)-mediated proteolysis of CII both in vitro and in vivo. Individual purification of HflK and HflC has not been possible so far, since each requires the presence of the other for proper folding. We report the first purification of HflK and HflC separately as active and functional proteins and show that each can interact with HflB on its own and each inhibits the proteolysis of CII. They also inhibit the proteolysis of E. coli sigma(32) by HflB. We show that at low concentrations each protein is dimeric, based on which we propose a scheme for the mutual interactions of HflB, HflK and HflC in a supramolecular HflBKC protease complex.

HflD, an Escherichia coli protein involved in the lambda lysis-lysogeny switch, impairs transcription activation by lambdaCII.

The CII protein of bacteriophage lambda is the key regulator for the lytic-lysogenic choice of the viral lifecycle. An unstable homotetrameric transcription activator of the three phage promoters p(E), p(I) and p(aQ), lambdaCII is stabilized by lambdaCIII and destabilized by the host protease, Escherichia coli HflB (FtsH). In addition, other E. coli proteins HflK, HflC and HflD also influence lysogeny by acting upon CII. Among these, HflD (22.9kDa), a peripheral membrane protein that is exposed towards the cytoplasm, interacts with CII and decreases the frequency of lysogenization of lambda by stimulating the degradation of CII. In this study, we show that in addition to helping CII degradation, HflD inhibits the DNA binding by CII, thereby inhibiting CII-dependent transcription activation. From biochemical, biophysical and modelling studies we also suggest that HflD-CII interaction takes place through the Cys31-accessible surface area of monomeric HflD, which binds to tetrameric CII as a 1:1 complex.

Specific hydrophobic residues in the alpha4 helix of lambdaCII are crucial for maintaining its tetrameric structure and directing the lysogenic choice.

The CII protein of the temperate bacteriophage lambda is the decision-making factor that determines the viral lytic/lysogenic choice. It is a homotetrameric transcription activator that recognizes and binds specific direct repeat sequences TTGCN(6)TTGC in the lambda genome. The quaternary structure of CII is held by a four-helix bundle. It is known that the tetrameric organization of CII is necessary for its activity, but the molecular mechanism behind this requirement is not known. By specific site-directed mutagenesis of hydrophobic residues in the alpha4 helix of CII that constitutes the four-helix bundle, we found that residues leu70, val74 and leu78 were crucial for maintaining the tetrameric structure of the protein. When any of these residues was substituted by a polar one, CII lost its activity and failed to promote lysogeny. This loss of activity was accompanied by the inability of CII to form tetramers, to bind DNA or to activate transcription.

Properties of HflX, an enigmatic protein from Escherichia coli.

The Escherichia coli gene hflX was first identified as part of the hflA operon, mutations in which led to an increased frequency of lysogenization upon infection of the bacterium by the temperate coliphage lambda. Independent mutational studies have also indicated that the HflX protein has a role in transposition. Based on the sequence of its gene, HflX is predicted to be a GTP-binding protein, very likely a GTPase. We report here purification and characterization of the HflX protein. We also specifically examined its suggested functional roles mentioned above. Our results show that HflX is a monomeric protein with a high (30% to 40%) content of helices. It exhibits GTPase as well as ATPase activities, but it has no role in lambda lysogeny or in transposition.

E. coli HflX interacts with 50S ribosomal subunits in presence of nucleotides.

HflX is a GTP binding protein of unknown function. Based on the presence of the hflX gene in hflA operon, HflX was believed to be involved in the lytic-lysogenic decision during phage infection in Escherichia coli. We find that E. coli HflX binds 16S and 23S rRNA - the RNA components of 30S and 50S ribosomal subunits. Here, using purified ribosomal subunits, we show that HflX specifically interacts with the 50S. This finding is in line with the homology of HflX to GTPases involved in ribosome biogenesis. However, HflX-50S interaction is not limited to a specific nucleotide-bound state of the protein, and the presence of any of the nucleotides GTP/GDP/ATP/ADP is sufficient. In this respect, HflX is different from other GTPases. While E. coli HflX binds and hydrolyses both ATP and GTP, only the GTP hydrolysis activity is stimulated by 50S binding. This work uncovers interesting attributes of HflX in ribosome binding.

Direct CIII-HflB interaction is responsible for the inhibition of the HflB (FtsH)-mediated proteolysis of Escherichia coli sigma(32) by lambdaCIII.

The CIII protein of bacteriophage lambda exhibits antiproteolytic activity against the ubiquitous metalloprotease HflB (FtsH) of Escherichia coli, thereby stabilizing the lambdaCII protein and promoting lysogenic development of the phage. CIII also protects E.coli sigma(32), another substrate of HflB. We have recently shown that the protection of CII from HflB by CIII involves direct CIII-HflB binding, without any interaction between CII and CIII [HalderS, DattaAB & Parrack P (2007) J Bacteriol189, 8130-8138]. Such a mode of action for lambdaCIII would be independent of the HflB substrate. In this study, we tested the ability of CIII to protect sigma(32) from HflB digestion. The inhibition of HflB-mediated proteolysis of sigma(32) by CIII is very similar to that of lambdaCII, characterized by an enhanced protection by the core CIII peptide CIIIC (amino acids 14-41 of lambdaCIII) and a lack of interaction between sigma(32) and CIII.

Probing the antiprotease activity of lambdaCIII, an inhibitor of the Escherichia coli metalloprotease HflB (FtsH).

The CIII protein encoded by the temperate coliphage lambda acts as an inhibitor of the ubiquitous Escherichia coli metalloprotease HflB (FtsH). This inhibition results in the stabilization of transcription factor lambdaCII, thereby helping the phage to lysogenize the host bacterium. LambdaCIII, a small (54-residue) protein of unknown structure, also protects sigma(32), another specific substrate of HflB. In order to understand the details of the inhibitory mechanism of CIII, we cloned and expressed the protein with an N-terminal six-histidine tag. We also synthesized and studied a 28-amino-acid peptide, CIIIC, encompassing the central 14 to 41 residues of CIII that exhibited antiproteolytic activity. Our studies show that CIII exists as a dimer under native conditions, aided by an intersubunit disulfide bond, which is dispensable for dimerization. Unlike CIII, CIIIC resists digestion by HflB. While CIII binds to HflB, it does not bind to CII. On the basis of these results, we discuss various mechanisms for the antiproteolytic activity of CIII.

Cyclic AMP-dependent functional forms of cyclic AMP receptor protein from Vibrio cholerae.

The cyclic AMP receptor protein (CRP) from Escherichia coli, involved in the transcriptional regulation of a number of genes and operons, works by binding to specific sites upstream of promoters. CRP also binds cyclic AMP (cAMP), and this binding, which causes conformational changes in CRP, is mandatory for its activity. A cAMP-dependent variation in the conformation as well as biological activity of E. coli CRP has been reported, with the cAMP-CRP complex formed at high cAMP concentrations resembling the uncomplexed apoprotein CRP. CRP from Vibrio cholerae, which plays an important role in the regulation of virulence gene expression, has a 95% sequence identity with the E. coli protein. We have purified and characterized V. cholerae CRP and studied its transcription activation properties as a function of increasing cAMP concentrations. A biphasic dependence on cAMP levels was observed, similar to that found for E. coli CRP. The implications of these results on regulation of cAMP-CRP dependent promoters in V. cholerae has been discussed.

Structure of lambda CII: implications for recognition of direct-repeat DNA by an unusual tetrameric organization.

The temperate coliphage lambda, after infecting its host bacterium Escherichia coli, can develop either along the lytic or the lysogenic pathway. Crucial to the lysis/lysogeny decision is the homotetrameric transcription-activator protein CII (4 x 11 kDa) of the phage that binds to a unique direct-repeat sequence T-T-G-C-N6-T-T-G-C at each of the three phage promoters it activates: p(E), p(I), and p(aQ). Several regions of CII have been identified for its various functions (DNA binding, oligomerization, and susceptibility to host protease), but the crystal structure of the protein long remained elusive. Here, we present the three-dimensional structure of CII at 2.6-angstroms resolution. The CII monomer is comprised of four alpha helices and a disordered C terminus. The first three helices (alpha1-alpha3) form a compact domain, whereas the fourth helix (alpha4) protrudes in different orientations in each subunit. A four-helix bundle, formed by alpha4 from each subunit, holds the tetramer. The quaternary structure can be described as a dimer of dimers, but the tetramer does not exhibit a closed symmetry. This unusual quaternary arrangement allows the placement of the helix-turn-helix motifs of two of the four CII subunits for interaction with successive major grooves of B-DNA, from one face of DNA. This structure provides a simple explanation for how a homotetrameric protein may recognize a direct-repeat DNA sequence rather than the inverted-repeat sequences of most prokaryotic activators.

Role of C-terminal residues in oligomerization and stability of lambda CII: implications for lysis-lysogeny decision of the phage.

A crucial element in the lysis-lysogeny decision of the temperate coliphage lambda is the phage protein CII, which has several interesting properties. It promotes lysogeny through activation of three phage promoters p(E), p(I) and p(aQ), recognizing a direct repeat sequence TTGCN6TTGC at each. The three-dimensional structure of CII, a homo-tetramer of 97 residue subunits, is unknown. It is an unstable protein in vivo, being rapidly degraded by the host protease HflB (FtsH). This instability is essential for the function of CII in the lysis-lysogeny switch. From NMR and limited proteolysis we show that about 15 C-terminal residues of CII are highly flexible, and may act as a target for proteolysis in vivo. From in vitro transcription, isothermal calorimetry and gel chromatography of CII (1-97) and its truncated fragments CIIA (4-81/82) and CIIB (4-69), we find that residues 70-81/82 are essential for (a) tetramer formation, (b) operator binding and (c) transcription activation. Presumably, tetramerization is necessary for the latter functions. Based on these results, we propose a model for CII structure, in which protein-protein contacts for dimer and tetramer formation are different. The implications of tetrameric organization, essential for CII activity, on the recognition of the direct repeat sequence is discussed.

Disorder-order transition of lambda CII promoted by low concentrations of guanidine hydrochloride suggests a stable core and a flexible C-terminus.

The CII protein of bacteriophage lambda, which activates the synthesis of the lambda repressor, plays a key role in the lysis-lysogeny switch. CII has a small in vivo half-life due to its proteolytic susceptibility, and this instability is a key component for its regulatory role. The structural basis of this instability is not known. While studying guanidine hydrochloride-assisted unfolding of CII, we found that low concentrations of the chaotrope (50-500 microM) have a considerable effect on the structure of this protein. This effect is manifest in an increase in molar ellipticity, an enhancement of intrinsic tryptophan fluorescence intensity and a reduction in ANS binding. At low concentrations of guanidine hydrochloride CII is stabilized, as reflected in a significant decrease in the rate of proteolysis by trypsin and resistance to thermal aggregation, while the tetrameric nature of the protein is retained. Thus low concentrations of guanidine hydrochloride promote a more structured conformation of the CII protein. On the basis of these observations, a model has been proposed for the structure of CII wherein the protein equilibrates between a compact form and a proteolytically accessible form, in which the C-terminal region assumes different structures.