A putative twin-arginine translocation pathway in Legionella pneumophila

Legionella pneumophila is a facultative intracellular human pathogen causing Legionnaires' disease, a severe form of pneumonia. Because of the importance of secretion pathways in virulence, we were interested in the possible presence of the twin-arginine translocation (Tat) pathway in L. pneumophila. This secretion pathway is used to transport folded proteins, characterized by two arginines in their signal peptide, across the cytoplasmic membrane. We describe here the presence of a putative Tat pathway in L. pneumophila. Three genes encoding Escherichia coli TatA, TatB, and TatC homologues were identified. The tatA and tatB genes were shown to constitute an operon while tatC is monocistronic. RT-PCR analysis revealed expression of the tat genes during both exponential and stationary growth as well as during intracellular growth in Acanthamoeba castellanii. A search for the conserved twin-arginine motif in predicted signal peptides resulted in a list of putative Tat substrates.     

Development and validation of ELISA for detection of antibodies to Legionella pneumophila serogroup 1, 3 and 6 in human sera

The aim of this study was to determine whether separate measurement of immunoglobulin (Ig) M and G antibodies to Legionella (L.) pneumophila serogroups (sg) 1, 3 and 6 as single antigens can facilitate an early diagnosis of Legionnaires' disease. The developed ELISA was evaluated and compared with an in-house indirect Legionella immunofluorescence antibody test (IFAT) measuring Total Ig. A total of 193 sera from 128 patients with confirmed L. pneumophila infections were used to assess the sensitivity of the developed ELISA. The sensitivity was assessed in different time-periods after onset of symptoms. It was found that the sensitivity of the test increased during the first month of infection, IgM being the most sensitive; ranging from 13% in the first week after onset of symptoms, 45% in the second week to 84% in the third week; in the fourth (and beyond) week a drop to 67% was observed. The IFAT detecting L. pneumophila sg 1-6 had a sensitivity of 11%, 27%, 80% and 59%, respectively, during these time-periods. The test with the lowest sensitivity was the IgG ELISA (0%, 21%, 36% and 52%), but by combining the IgG results with the IgM results, the overall sensitivity of the assay was improved (13%, 48%, 88% and 70%).This study confirms that detection of IgG and IgM antibodies by ELISA is an important diagnostic tool especially during the initial phase of the disease, when supported by other tests like the urinary antigen test, PCR or culture.Furthermore, we showed that the ELISA is suitable for the detection of significant changes in antibody levels in paired serum samples. It was found that the sensitivity was higher for the ELISA assays than for the IFAT. Both the in-house IgM ELISA and the IFAT had a low false positive rate, while a 14% false positive rate was found for the IgG ELISA among serum samples from patients with other infections.     

The structure of the soluble domain of an archaeal Rieske iron-sulfur protein at 1.1 A resolution

The first crystal structure of an archaeal Rieske iron-sulfur protein, the soluble domain of Rieske iron-sulfur protein II (soxF) from the hyperthermo-acidophile Sulfolobus acidocaldarius, has been solved by multiple wavelength anomalous dispersion (MAD) and has been refined to 1.1 A resolution. SoxF is a subunit of the terminal oxidase supercomplex SoxM in the plasma membrane of S. acidocaldarius that combines features of a cytochrome bc(1) complex and a cytochrome c oxidase. The [2Fe-2S] cluster of soxF is most likely the primary electron acceptor during the oxidation of caldariella quinone by the cytochrome a(587)/Rieske subcomplex. The geometry of the [2Fe-2S] cluster and the structure of the cluster-binding site are almost identical in soxF and the Rieske proteins from eucaryal cytochrome bc(1) and b(6)f complexes, suggesting a strict conservation of the catalytic mechanism. The main domain of soxF and part of the cluster-binding domain, though structurally related, show a significantly divergent structure with respect to topology, non-covalent interactions and surface charges. The divergent structure of soxF reflects a different topology of the soxM complex compared to eucaryal bc complexes and the adaptation of the protein to the extreme ambient conditions on the outer membrane surface of a hyperthermo-acidophilic organism.     

Mitochondrial tRNA import in Trypanosoma brucei is independent of thiolation and the Rieske protein

Due to a complete lack of the tRNA genes in the mitochondrial genome of Trypanosoma brucei, all tRNAs needed for mitochondrial translation have to be imported into the organelle from the cytosol. A previous study showed that the modified nucleotide s²U could act as a negative determinant for mitochondrial tRNA import in another kinetoplastid, Leishmania tarentolae. We have investigated whether the same type of cytosolic control for tRNA retention exists in T. brucei. Based on Northern analysis with subcellular RNA fractions and in vitro import assays, we demonstrate that silencing of the cysteine desulfurase, TbNfs (TbIscS), the key enzyme in tRNA thiolation (s²U) and Fe-S cluster formation in vivo, has no effect on tRNA partitioning. This observation is especially surprising in light of a recent report suggesting that in L. tropica the Rieske Fe-S protein is an essential component of the RNA import complex (RIC). In line with the above observation, we also show that down-regulation of the Rieske protein by RNA interference, similar to the TbNfs knockdowns, has no effect on import. The data presented here supports the view that in T. brucei: (1) s²U is not a negative determinant for tRNA import; (2) the Rieske protein is not an essential component of the import machinery, and (3) since the Rieske protein is essential for respiration and maintenance of inner mitochondrial membrane potential, neither process plays a critical role in tRNA import. We therefore suggest that the T. brucei import machinery differs substantially from what has been described in Leishmania.     

Quantitative export of a reporter protein,GFP, by the twin-arginine translocation pathway in Escherichia coli

The Tat system mediates the transport of folded proteins across the bacterial cytoplasmic membrane. To study the properties of the Escherichia coli Tat-system, we used green fluorescent protein (GFP) fused to the twin-arginine signal peptide of TMAO reductase (TorA). In the presence of arabinose, low levels of this protein rapidly saturate the translocase and cause the accumulation of inactive, membrane-bound TorA-GFP; fluorescence microscopy also showed active TorA-GFP to be distributed throughout the cytoplasm. However, the efficiency of export can be massively increased by alteration of the growth conditions, and further increased by overexpression of the tatABC genes. Under these conditions, the levels of GFP in the periplasm are raised over 20-fold and the export efficiency nears 100%. These results show that the Tat-system is relatively inactive under some growth conditions and the data suggest that the system may be applicable for the larger-scale export of heterologous proteins.     

Resonance Raman characterization of archaeal and bacterial Rieske protein variants with modified hydrogen bond network around the [2Fe-2S] center

The rate of quinol oxidation by cytochrome bc(1)/b(6)f complex is in part associated with the redox potential (E(m)) of its Rieske [2Fe-2S] center, for which an approximate correlation with the number of hydrogen bonds to the cluster has been proposed. Here we report comparative resonance Raman (RR) characterization of bacterial and archaeal high-potential Rieske proteins and their site-directed variants with a modified hydrogen bond network around the cluster. Major differences among their RR spectra appear to be associated in part with the presence or absence of Tyr-156 (in the Rhodobacter sphaeroides numbering) near one of the Cys ligands to the cluster. Elimination of the hydrogen bond between the terminal cysteinyl sulfur ligand (S(t)) and Tyr-Oeta (as with the Y156W variant, which has a modified histidine N(epsilon) pK(a,ox)) induces a small structural bias of the geometry of the cluster and the surrounding protein in the normal coordinate system, and significantly affects some Fe-S(b/t) stretching vibrations. This is not observed in the case of the hydrogen bond between the bridging sulfide ligand (S(b)) and Ser-Ogamma, which is weak and/or unfavorably oriented for extensive coupling with the Fe-S(b/t) stretching vibrations.     

The presequence pathway is involved in protein sorting to the mitochondrial outer membrane

The mitochondrial outer membrane contains integral α-helical and β-barrel proteins that are imported from the cytosol. The machineries importing β-barrel proteins have been identified, however, different views exist on the import of α-helical proteins. It has been reported that the biogenesis of Om45, the most abundant signal-anchored protein, does not depend on proteinaceous components, but involves direct insertion into the outer membrane. We show that import of Om45 occurs via the translocase of the outer membrane and the presequence translocase of the inner membrane. Assembly of Om45 in the outer membrane involves the MIM machinery. Om45 thus follows a new mitochondrial biogenesis pathway that uses elements of the presequence import pathway to direct a protein to the outer membrane.     

An essential role for the DnaK molecular chaperone in stabilizing over-expressed substrate proteins of the bacterial twin-arginine translocation pathway

All secreted proteins in Escherichia coli must be maintained in an export-competent state before translocation across the inner membrane. In the case of the Sec pathway, this function is carried out by the dedicated SecB chaperone and the general chaperones DnaK-DnaJ-GrpE and GroEL-GroES, whose job collectively is to render substrate proteins partially or entirely unfolded before engagement of the translocon. To determine whether these or other general molecular chaperones are similarly involved in the translocation of folded proteins through the twin-arginine translocation (Tat) system, we screened a collection of E. coli mutant strains for their ability to transport a green fluorescent protein (GFP) reporter through the Tat pathway. We found that the molecular chaperone DnaK was essential for cytoplasmic stability of GFP bearing an N-terminal Tat signal peptide, as well as for numerous other recombinantly expressed endogenous and heterologous Tat substrates. Interestingly, the stability conferred by DnaK did not require a fully functional Tat signal as substrates bearing translocation defective twin lysine substitutions in the consensus Tat motif were equally unstable in the absence of DnaK. These findings were corroborated by crosslinking experiments that revealed an in vivo association between DnaK and a truncated version of the Tat substrate trimethylamine N-oxide reductase (TorA502) bearing an RR or a KK signal peptide. Since TorA502 lacks nine molybdo-cofactor ligands essential for cofactor attachment, the involvement of DnaK is apparently independent of cofactor acquisition. Finally, we show that the stabilizing effects of DnaK can be exploited to increase the expression and translocation of Tat substrates under conditions where the substrate production level exceeds the capacity of the Tat translocase. This latter observation is expected to have important consequences for the use of the Tat system in biotechnology applications where high levels of periplasmic expression are desirable.     

Construction of an Escherichia coli K-12 strain deleted for manganese and iron superoxide dismutase genes and its use in cloning the iron superoxide dismutase gene of Legionella pneumophila.

Summary An Escherichia coli K-12 strain deleted for sodA and sodB (manganese and iron superoxide dismulases) was constructed and characterized by Southern blotting, enzyme assays, and physiological analyses. The sod deletion strain was used to clone the iron superoxide dismutase gene of Legionella pneumophila by complementation to paraquat resistance.     

Dephosphorylation of the Rieske iron-sulfur protein after induction of the mitochondrial permeability transition

In the mitochondrial permeability transition (MPT), MPT pores open to cause the mitochondrial inner membrane to become non-selectively permeable to molecules of mass up to 1500 Da. In this study, we used proteomics to investigate protein changes after MPT induction. Isolated rat liver mitochondria were incubated with various MPT inducers, including CaCl2, tert-butylhydroperoxide, and phenylarsine oxide, in the presence and absence of the MPT inhibitor, cyclosporin A. MPT induction was confirmed by an absorbance swelling assay. Mitochondrial membrane proteins prepared from control and treated mitochondria were separated by two-dimensional (2D) gel electrophoresis and stained with SyproRuby or Coomassie blue. Proteins of interest were further identified by mass spectrometry. 2D gel electrophoresis by isoelectric focusing and SDS-PAGE consistently showed a protein spot that shifted to a more basic isoelectric point after the MPT. This shift was prevented by CsA but did not occur after protonophoric uncoupling. Mass spectrometry identified this protein as the Rieske iron-sulfur protein (RISP) of ubiquinol-cytochrome c reductase (Complex III). Phosphatase treatment of sonicated mitochondria caused the same shift in RISP as occurred in MPT inducer-treated mitochondria. 2D gel electrophoresis by blue-native-PAGE and SDS-PAGE showed that RISP existed as an apparent monomer in mitochondrial membranes in addition to forming a complex with ubiquinol-cytochrome c reductase. These findings suggest that RISP may be part of MPT pores and that dephosphorylation of RISP may play a role in regulation of the MPT.     

ShyA, a membrane protein for proper septation of hyphae in Streptomyces

The life cycle of Streptomyces involves the formation of filamentous substrate and aerial hyphae. Following cessation of growth of an aerial hypha, multiple septation occurs at the tip to produce a chain of unigenomic spores. A gene, shyA, which influences several aspects of this growth, was isolated and partially characterized in Streptomyces coelicolor. The gene product is a representative of a well-conserved family of small actinomycete proteins. The shyA mutant sporulates normally but displays hyper septum formation and altered spore-chain morphology. Biochemical separation experiments and immunofluorescence staining demonstrated that the shyA gene product locates at cell membranes. Moreover, yeast two-hybrid screen and GST-pull-down assay showed that ShyA can interact with itself. Altogether, ShyA belongs to a new family of membrane-associated proteins which plays a role in morphological differentiation in actinomycetes.     

Identification of a 40S Ribosomal protein (S17) that is differentially expressed between the macroschizont and piroplasm stages of Theileria annulata.

The nucleotide and protein sequence of the 40S ribosomal protein S17 (RibS17) of the protozoan parasite Theileria annulata has been determined. Southern blot analysis showed the gene was single copy and comparative sequence analysis revealed that the predicted polypeptide had high sequence homology with the RibS17 from other organisms. Northern blot analysis showed that there was a 3-fold increase in the level of RibS17 RNA between the macroschizont and the piroplasm stage of the lifecycle, whereas, there was no difference in expression between the sporozoite and the macroschizont stages. Antisera to the purified fusion protein, corresponding to the terminal 50 amino acids of the protein sequence, were raised in rabbits. Western analysis detected a polypeptide of the predicted size that was more abundant in the piroplasm stage compared with the macroschizont stage. Immunofluorescence analysis with the same antisera revealed a strong signal in the macroschizont and piroplasm stages, but the antiserum did not cross-react with the bovine host cells. The antisera did, however, cross-react with Toxoplasma gondii tachyzoites and Plasmodium falciparum merozoites. The possible functional significance of the stage related increase in abundance of a ribosomal protein is discussed.     

The sulfhydryl oxidase Erv1 is a substrate of the Mia40-dependent protein translocation pathway

The thiol oxidase Erv1 and the redox-regulated receptor Mia40/Tim40 are components of a disulfide relay system which mediates import of proteins into the intermembrane space (IMS) of mitochondria. Here we report that Erv1 requires Mia40 for its import into mitochondria. After passage across the translocase of the mitochondrial outer membrane Erv1 interacts via disulfide bonds with Mia40. Erv1 does not contain twin “CX₃C” or twin “CX₉C” motifs which are crucial for import of typical substrates of this pathway and it does not need two “CX₂C” motifs for import into mitochondria. Thus, Erv1 represents an unusual type of substrate of the Mia40-dependent import pathway.     

A role for mrgA, a DPS family protein, in the internal transport of Fe in the cyanobacterium Synechocystis sp. PCC6803

The mrgA protein of the cyanobacterium Synechocystis sp. PCC6803 is a member of the DPS Fe storage protein family. The physiological role of this protein was studied using a disruption mutant in the mrgA gene (slr1894) and by measuring intracellular Fe quotas, 77K chlorophyll fluorescence and growth rates. It was found that the deletion of the mrgA gene did not impair the Fe storage capacity, as the intracellular Fe quotas of the ΔmrgA cells were comparable to those of the wild type. Furthermore, the cellular response to decreasing external Fe concentrations, as detected by the emergence of the CP43′ 77K fluorescence band, was similar in wild type and mutant cultures. On the other hand, a considerable slow down in the growth rate of ΔmrgA cultures was observed upon transfer from Fe replete to Fe depleted medium, indicating impeded utilization of the plentiful intracellular Fe. Based on these results, we suggest that mrgA plays an important role in the transport of intracellular Fe from storage (within bacterioferritins) to biosynthesis of metal cofactors throughout the cell's growth.     

The mechanosensitive channel protein MscL is targeted by the SRP to the novel YidC membrane insertion pathway of Escherichia coli

The mechanosensitive channel MscL in the inner membrane of Escherichia coli is a homopentameric complex involved in homeostasis when cells are exposed to hypo-osmotic conditions. The E. coli MscL protein is synthesized as a polypeptide of 136 amino acid residues and uses the bacterial signal recognition particle (SRP) for membrane targeting. The protein is inserted into the membrane independently of the Sec translocon. Mutants affected in the Sec-components are competent for MscL assembly. Translocation of the periplasmic domain was detected using a membrane-impermeant, sulfhydryl-specific gel-shift reagent. The modification of a single cysteine residue at position 68 indicated its translocation across the inner membrane. From these in vivo experiments, it is concluded that the electrical chemical membrane potential is not necessary for membrane insertion of MscL. However, depletion of the membrane insertase YidC inhibits translocation of the protein across the membrane. We show here that YidC is essential for efficient membrane insertion of the MscL protein. YidC is a component of a recently identified membrane insertion pathway that is evolutionarily conserved in bacteria, mitochondria and chloroplasts.     

The TatAd component of the Bacillus subtilis twin-arginine protein transport system forms homo-multimeric complexes in its cytosolic and membrane embedded localisation

The twin arginine translocation (Tat) system has the capacity to transfer completely folded proteins across the bacterial cytoplasmic membrane and the thylakoid membrane of plant chloroplasts. The most abundant TatA protein of this system has been suggested to form the protein conducting channel. Here, the molecular organisation of soluble and membrane embedded Bacillus subtilis TatA_d was analysed using negative contrast and freeze-fractured electron microscopy. In both compartments, the protein showed homo-oligomerisation. In aqueous solution, TatA_d formed homo-multimeric micelle-like complexes. Freeze-fracture analysis of proteoliposomes revealed self association of membrane-integrated TatA_d independent from TatC_d, the second component of this transport system. Immunogold labelling demonstrated that the substrate prePhoD was co-localised with membrane-integrated TatA_d complexes.     

The association of small heat shock protein Hsp16.3 with the plasma membrane of Mycobacterium tuberculosis: dissociation of oligomers is a prerequisite

Hsp16.3, a small heat shock protein from Mycobacterium tuberculosis (MTB), was originally identified as an immuno-dominant antigen and later found to be a major membrane protein. In vitro studies show that Hsp16.3 exists as nonamers and undergoes dynamic dissociation/re-association equilibrium in solutions. Nevertheless, neither the details nor the physiological implications of the presence of Hsp16.3 in the plasma membrane have been studied. In this study, we demonstrated that the purified Hsp16.3 proteins were able to interact with the MTB plasma membrane in a specific and reversible manner, suggesting that there might be subunit exchange between membrane-bound Hsp16.3 and soluble Hsp16.3 oligomers. The dissociation of Hsp16.3 oligomers appears to be a prerequisite for its membrane binding, which is interesting in view that the dissociation of small heat shock protein oligomers was also found to be necessary for it to bind denaturing substrate proteins. Furthermore, the oligomeric structure of Hsp16.3 seems to be more dynamic and flexible when incubating with the mycobacterium lipids. The physiological implications of these observations for Hsp16.3, and small heat shock proteins in general, are discussed.     

The NMR solution structure of the 30S ribosomal protein S27e encoded in gene RS27_ARCFU of Archaeoglobus fulgidis reveals a novel protein fold

The Archaeoglobus fulgidis gene RS27_ARCFU encodes the 30S ribosomal protein S27e. Here, we present the high-quality NMR solution structure of this archaeal protein, which comprises a C4 zinc finger motif of the CX(2)CX(14-16)CX(2)C class. S27e was selected as a target of the Northeast Structural Genomics Consortium (target ID: GR2), and its three-dimensional structure is the first representative of a family of more than 116 homologous proteins occurring in eukaryotic and archaeal cells. As a salient feature of its molecular architecture, S27e exhibits a beta-sandwich consisting of two three-stranded sheets with topology B(decreasing), A(increasing), F(decreasing), and C(increasing), D(decreasing), E(increasing). Due to the uniqueness of the arrangement of the strands, the resulting fold was found to be novel. Residues that are highly conserved among the S27 proteins allowed identification of a structural motif of putative functional importance; a conserved hydrophobic patch may well play a pivotal role for functioning of S27 proteins, be it in archaeal or eukaryotic cells. The structure of human S27, which possesses a 26-residue amino-terminal extension when compared with the archaeal S27e, was modeled on the basis of two structural templates, S27e for the carboxy-terminal core and the amino-terminal segment of the archaeal ribosomal protein L37Ae for the extension. Remarkably, the electrostatic surface properties of archaeal and human proteins are predicted to be entirely different, pointing at either functional variations among archaeal and eukaryotic S27 proteins, or, assuming that the function remained invariant, to a concerted evolutionary change of the surface potential of proteins interacting with S27.     

The Klebsiella pneumoniae nitrogenase Fe protein gene (nifH) functionally substitutes for the chlL gene in Chlamydomonas reinhardtii

The entire coding region of chlL, an essential chloroplast gene required for chlorophyll biosynthesis in the dark in Chlamydomonas reinhardtii, was precisely replaced by either the Klebsiella pneumoniae nifH (encoding the structural component of nitrogenase Fe protein) or the Escherichia coli uidA reporter gene encoding beta-glucuronidase. Homoplasmic nifH or uidA transformants were identified by Southern blots after selection on minimal medium plates for several generations. All the uidA transformants had the "yellow-in-the-dark" phenotype characteristic of chlL mutants, whereas homoplasmic nifH transformants exhibited a partial "green-in-the-dark" phenotype. NifH protein was detected in the nifH transformants but not in the wild-type strain by Western blotting. Fluorescence emission measurements also showed the existence of chlorophyll in the dark-grown nifH transformants, but not in the dark-grown uidA transformants. The nifH transplastomic form of C. reinhardtii that lacks the chlL gene can still produce chlorophyll in the dark, suggesting that the nifH product can at least partially substitute for the function of the putative "chlorophyll iron protein" encoded by chlL. Thus, introducing nitrogen fixation gene directly into a chloroplast genome is likely to be feasible and providing a possible way of engineering chloroplasts with functional nitrogenase. Notably, to introduce foreign genes without also introducing selective marker genes, a novel two-step chloroplast transformation strategy has been developed.