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.     

Characterization and membrane assembly of the TatA component of the Escherichia coli twin-arginine protein transport system

Proteins bearing a signal peptide with a consensus twin-arginine motif are translocated via the Tat pathway, a multiprotein system consisting minimally of the integral inner membrane proteins TatA, TatB, and TatC. On a molar basis, TatA is the major pathway component. Here we show that TatA can be purified independently of the other Tat proteins as a 460 kDa homooligomeric complex. Homooligomer formation requires the amino-terminal membrane-anchoring domain of TatA. According to circular dichroism spectroscopy, approximately half of the TatA polypeptide forms alpha-helical secondary structure in both detergent solution and proteoliposomes. An expressed construct without the transmembrane segment is largely unstructured in aqueous solution but is able to insert into phospholipid monolayers and interacts with membrane bilayers. Protease accessibility experiments indicate that the extramembranous region of TatA is located at the cytoplasmic face of the cell membrane.     

The twin-arginine signal peptide of PhoD and the TatAd/Cd proteins of Bacillus subtilis form an autonomous Tat translocation system.

The bacterial twin-arginine translocation (Tat) pathway has been recently described for PhoD of Bacillus subtilis, a phosphodiesterase containing a twin-arginine signal peptide. The expression of phoD is co-regulated with the expression of tatA(d) and tatC(d) genes localized downstream of phoD. To characterize the specificity of PhoD transport further, translocation of PhoD was investigated in Escherichia coli. By using gene fusions, we analyzed the particular role of the signal peptide and the mature region of PhoD in canalizing the transport route. A hybrid protein consisting of the signal peptide of beta-lactamase and mature PhoD was transported in a Sec-dependent manner indicating that the mature part of PhoD does not contain information canalizing the selected translocation route. Pre-PhoD, as well as a fusion protein consisting of the signal peptide of PhoD (SP(PhoD)) and beta-galactosidase (LacZ), remained cytosolic in the E. coli. Thus, SP(PhoD) is not recognized by E. coli transport systems. Co-expression of B. subtilis tatA(d)/C(d) genes resulted in the processing of SP(PhoD)-LacZ and periplasmic localization of LacZ illustrating a close substrate specificity of the TatA(d)/C(d) transport system. While blockage of the Sec-dependent transport did not affect the localization of SP(PhoD)-LacZ, translocation and processing was dependent on the pH gradient of the cytosolic membrane. Thus, the minimal requirement of a functional Tat-dependent protein translocation system consists of a twin-arginine signal peptide-containing Tat substrate, its specific TatA/C proteins, and the pH gradient across the cytosolic membrane.     

Selective contribution of the twin-arginine translocation pathway to protein secretion in Bacillus subtilis

The availability of the complete genome sequence of Bacillus subtilis has allowed the prediction of all exported proteins of this Gram-positive eubacterium. Recently, approximately 180 secretory and 114 lipoprotein signal peptides were predicted to direct protein export from the cytoplasm. Whereas most exported proteins appear to use the Sec pathway, 69 of these proteins could potentially use the Tat pathway, as their signal peptides contain RR- or KR-motifs. In the present studies, proteomic techniques were applied to verify how many extracellular B. subtilis proteins follow the Tat pathway. Strikingly, the extracellular accumulation of 13 proteins with potential RR/KR-signal peptides was Tat-independent, showing that their RR/KR-motifs are not recognized by the Tat machinery. In fact, only the phosphodiesterase PhoD was shown to be secreted in a strictly Tat-dependent manner. Sodium azide-inhibition of SecA strongly affected the extracellular appearance of de novo synthesized proteins, including the lipase LipA and two other proteins with predicted RR/KR-signal peptides. The SecA-dependent export of pre-LipA is particularly remarkable, because its RR-signal peptide conforms well to stringent criteria for the prediction of Tat-dependent export in Escherichia coli. Taken together, our observations show that the Tat pathway makes a highly selective contribution to the extracellular proteome of B. subtilis.     

Affinity of TatCd for TatAd elucidates its receptor function in the Bacillus subtilis twin arginine translocation (Tat) translocase system

Twin arginine translocation (Tat) systems catalyze the transport of folded proteins across the bacterial cytosolic membrane or the chloroplast thylakoid membrane. In the Tat systems of Escherichia coli and many other species TatA-, TatB-, and TatC-like proteins have been identified as essential translocase components. In contrast, the Bacillus subtilis phosphodiesterase PhoD-specific system consists only of a pair of TatA(d)/TatC(d) proteins and involves a TatA(d) protein engaged in a cytosolic and a membrane-embedded localization. Because soluble TatA(d) was able to bind the twin arginine signal peptide of prePhoD prior to membrane integration it could serve to recruit its substrate to the membrane via the interaction with TatC(d). By analyzing the distribution of TatA(d) and studying the mutual affinity with TatC(d) we have shown here that TatC(d) assists the membrane localization of TatA(d). Besides detergent-solubilized TatC(d), membrane-integrated TatC(d) showed affinity for soluble TatA(d). By using a peptide library-specific binding of TatA(d) to cytosolic loops of membrane protein TatC(d) was demonstrated. Depletion of TatC(d) in B. subtilis resulted in a drastic reduction of TatA(d), indicating a stabilizing effect of TatC(d) for TatA(d). In addition, the presence of the substrate prePhoD was the prerequisite for appropriate localization in the cytosolic membrane of B. subtilis as demonstrated by freeze-fracture experiments.     

Calcium transport in membrane vesicles of Bacillus subtilis.

Right-side-out membrane vesicles of Bacillus subtilis W23 grown on tryptone-citrate medium accumulated Ca2+ under aerobic conditions in the presence of a suitable electron donor. Ca2+ uptake was an electrogenic process which was completely inhibited by carbonyl cyanide m-chlorophenylhydrazone or valinomycin and not by nigericin. This electrogenic uptake of calcium was strongly dependent on the presence of phosphate and magnesium ions. The system had a low affinity for Ca2+. The kinetic constants in membrane vesicles were Km = 310 microM Ca2+ and Vmax = 16 nmol/mg of protein per min. B. subtilis also possesses a Ca2+ extrusion system. Right-side-out-oriented membrane vesicles accumulated Ca2+ upon the artificial imposition of a pH-gradient, inside acid. This system had a high affinity for Ca2+; Km = 17 microM Ca2+ and Vmax = 3.3 nmol/mg of protein per min. Also, a membrane potential, inside positive, drove Ca2+ transport via this Ca2+ extrusion system. Evidence for a Ca2+ extrusion system was also supplied by studies of inside-out-oriented membrane vesicles in which Ca2+ uptake was energized by respiratory chain-linked oxidation of NADH or ascorbate-phenazine methosulfate. Both components of the proton motive force, the pH gradient and the membrane potential, drove Ca2+ transport via the Ca2+ extrusion system, indicating a proton-calcium antiport system with a H+ to Ca2+ stoichiometry larger than 2. The kinetic parameters of this Ca2+ extrusion system in inside-out-oriented membranes were Km = 25 microM and Vmax = 0.7 nmol/mg of protein per min.     

Specificity of the tyrosine-phenylalanine transport system in Bacillus subtilis

l-Tyrosine and l-phenylalanine enter cells of Bacillus subtilis via a system of active transport that exhibits complex kinetic behavior. The specificity of the transport system was characterized both at low concentrations of transport substrate (where affinity for l-tyrosine or l-phenylalanine is high but capacity is low) and at high concentrations (where affinity is low but capacity is high). Specificity was not found to differ significantly as a function of either l-tyrosine or l-phenylalanine concentration. Kinetic analysis showed that the relationship between the uptake of l-phenylalanine and l-tyrosine is strictly competitive. Neither l-tyrosine nor l-phenylalanine uptake was competitively inhibited by other naturally occurring l-amino acids, indicating the importance of the phenyl side chain to uptake specificity. Hence, it is concluded that l-tyrosine and l-phenylalanine are transported by a common system that is specific for these two amino acids. The abilities of analogue derivatives of l-tyrosine and l-phenylalanine to inhibit the uptake of l-[(14)C]tyrosine and l-[(14)C]phenylalanine competitively were determined throughout a wide range of substrate and inhibitor concentrations. In this manner, the contributions of the side chain, the alpha-amino group and the carboxyl group to uptake specificity were established. It is concluded that the positively charged alpha-amino group contributes more significantly to uptake specificity than does the negatively charged carboxyl group. The recognition of a phenyl ring is an essential feature of specificity; other amino acids with aromatic side chains, such as the indole and imidazole rings of l-tryptophan and l-histidine, do not compete with l-tyrosine and l-phenylalanine for uptake. The presence of the p-hydroxy substitutent in the side chain (as in l-tyrosine) enhances the uptake of the aryl amino acid analogues investigated.     

A major protein component of the Bacillus subtilis biofilm matrix

Microbes construct structurally complex multicellular communities (biofilms) through production of an extracellular matrix. Here we present evidence from scanning electron microscopy showing that a wild strain of the Gram positive bacterium Bacillus subtilis builds such a matrix. Genetic, biochemical and cytological evidence indicates that the matrix is composed predominantly of a protein component, TasA, and an exopolysaccharide component. The absence of TasA or the exopolysaccharide resulted in a residual matrix, while the absence of both components led to complete failure to form complex multicellular communities. Extracellular complementation experiments revealed that a functional matrix can be assembled even when TasA and the exopolysaccharide are produced by different cells, reinforcing the view that the components contribute to matrix formation in an extracellular manner. Having defined the major components of the biofilm matrix and the control of their synthesis by the global regulator SinR, we present a working model for how B. subtilis switches between nomadic and sedentary lifestyles.     

The Escherichia coli TatABC system and a Bacillus subtilis TatAC-type system recognise three distinct targeting determinants in twin-arginine signal peptides

The Tat system transports folded proteins across bacterial and thylakoid membranes. In Gram-negative organisms, it is encoded by tatABC genes and the system recognizes substrates bearing signal peptides with a conserved twin-arginine motif. Most Gram-positive organisms lack a tatB gene, indicating major differences in organisation and/or mechanism. Here, we have characterized the essential targeting determinants that are recognized by a Bacillus subtilis TatAC-type system, TatAdCd. Substitution by lysine of either of the twin-arginine residues in the TorA signal peptide can be tolerated, but the presence of twin-lysine residues blocks export completely. We show that additional determinants can be as important as the twin-arginine motif. Replacement of the −1 serine by alanine, in either the TorA or DmsA signal peptide, almost blocks export by either the B. subtilis TatAdCd or Escherichia coli TatABC systems, firmly establishing the importance of this −1 residue in these signal peptides. Surprisingly, the +2 leucine in the DmsA signal peptide (sequence SRRGLV) appears to play an equally important role and substitution by alanine or phenylalanine blocks export by both the B. subtilis and E. coli systems. These data identify three distinct determinants, whose importance varies depending on the signal peptide in question. The data also show that the B. subtilis TatAdCd and E. coli TatABC systems recognize very similar determinants within their target peptides, and exhibit surprisingly similar responses to mutations within these determinants.     

The twin-arginine translocation system and its capability for protein secretion in biotechnological protein production

The biotechnological production of recombinant proteins is challenged by processes that decrease the yield, such as protease action, aggregation, or misfolding. Today, the variation of strains and vector systems or the modulation of inducible promoter activities is commonly used to optimize expression systems. Alternatively, aggregation to inclusion bodies may be a desired starting point for protein isolation and refolding. The discovery of the twin-arginine translocation (Tat) system for folded proteins now opens new perspectives because in most cases, the Tat machinery does not allow the passage of unfolded proteins. This feature of the Tat system can be exploited for biotechnological purposes, as expression systems may be developed that ensure a virtually complete folding of a recombinant protein before purification. This review focuses on the characteristics that make recombinant Tat systems attractive for biotechnology and discusses problems and possible solutions for an efficient translocation of folded proteins.     

Structural characterization of the pore forming protein TatAd of the twin-arginine translocase in membranes by solid-state 15N-NMR

The transmembrane protein TatA is the pore forming unit of the twin-arginine translocase (Tat), which has the unique ability of transporting folded proteins across the cell membrane. This ATP-independent protein export pathway is a recently discovered alternative to the general secretory (Sec) system of bacteria. To obtain insight in the translocation mechanism, the structure and alignment in the membrane of the well-folded segments 2-45 of TatAd from Bacillus subtilis was studied here. Using solid-state NMR in bicelles containing anionic lipids, the topology and orientation of TatAd was determined in an environment mimicking the bacterial membrane. A wheel-like pattern, characteristic for a tilted transmembrane helix, was observed in 15N chemical shift /15N-1H dipolar coupling correlation NMR spectra. Analysis of this PISA wheel revealed a 14-16 residue long N-terminal membrane-spanning helix which is tilted by 17 degrees with respect to the membrane normal. In addition, comparison of uniformly and selectively 15N-labeled TatA2-45 samples allowed determination of the helix polarity angle.     

Characterization of glutamate transport system in hydrophobic protein (H protein) of Bacillus subtilis.

Hydrophobic protein (H protein) was isolated from membrane fractions of Bacillus subtilis and constituted into artificial membrane vesicles with lipid of B. substilis. Glutamate was accumulated into the vesicle when a Na+ gradient across the membrane was imposed. The maximum effect of Na+ on the transport was achieved at a concentration of about 40 mM, while the apparent Km for Na+ was approximately 8 mM. On the other hand, Km for glutamate in the presence of 50 mM Na+ was about 8 micro M. Increasing the concentration of Na+ resulted in a decrease in Km for glutamate, maximum velocity was not affected. The transport was sensitive to monensin (Na+ ionophore). Glutamate was also accumulated when pH gradient (interior alkaline) across the membrane was imposed or a membrane potential was induced with K+-diffusion potential. The pH gradient-driven glutamate transport was sensitive to carbonylcyanide m-chlorophenylhydrazone and the apparent Km for glutamate was approximately 25 microM. These results indicate that two kinds of glutamate transport system were present in H protein: one is Na+ dependent and the other is H+ dependent.     

The conformations of the manganese transport regulator of Bacillus subtilis in its metal-free state

The manganese transport regulator (MntR) from Bacillus subtilis binds cognate DNA sequences in response to elevated manganese concentrations. MntR functions as a homodimer that binds two manganese ions per subunit. Metal binding takes place at the interface of the two domains that comprise each MntR subunit: an N-terminal DNA-binding domain and a C-terminal dimerization domain. In order to elucidate the link between metal binding and activation, a crystallographic study of MntR in its metal-free state has been undertaken. Here we describe the structures of the native protein and a selenomethionine-containing variant, solved to 2.8 A. The two structures contain five crystallographically unique subunits of MntR, providing diverse views of the metal-free protein. In apo-MntR, as in the manganese complex, the dimer is formed by dyad-related C-terminal domains that provide a conserved structural core. Similarly, each DNA-binding domain largely retains the folded conformation found in metal bound forms of MntR. However, compared to metal-activated MntR, the DNA-binding domains move substantially with respect to the dimer interface in apo-MntR. Overlays of multiple apo-MntR structures indicate that there is a greater range of positioning allowed between N and C-terminal domains in the metal-free state and that the DNA-binding domains of the dimer are farther apart than in the activated complex. To further investigate the conformation of the DNA-binding domain of apo-MntR, a site-directed spin labeling experiment was performed on a mutant of MntR containing cysteine at residue 6. Consistent with the crystallographic results, EPR spectra of the spin-labeled mutant indicate that tertiary structure is conserved in the presence or absence of bound metals, though slightly greater flexibility is present in inactive forms of MntR.     

Active transport of manganese in isolated membrane vesicles of Bacillus subtilis.

Membrane vesicles isolated from cells of bacillus subtilis W23 accumulate manganese in the presence of an energy source. The artificial electron donor system ascorbate and phenazine methosulfate or reduced nicotinamide adenine dinucleotide and phenazine methosulfate can supply the energy for the uptake. D-Lactate in the presence or absence of phenazine methosulfate would not support manganese accumulation. Anaerobiosis, cyanide, m-chlorophenyl carbonylcyanide hydrozone, valinomycin, gramicidin, and p-hydroxy-mercuribenzoate inhibit the uptake. The inhibition by p-hydroxymercuribenzoate is prevented by excess dithiothreitol. Potassium fluoride or sodium arsenate has no effect on the uptake. The manganese transport system in the B. subtilis vesicles exhibits Michaelis-Menten kinetics with a Km of 13 muM and a Vmax of 1.7 nmol/min per mg (dry weight) of membranes. The uptake of manganese is specific and is not inhibited by 0.1 mM CaCL2 or Mgcl2.     

Sequence-specific binding of prePhoD to soluble TatAd indicates protein-mediated targeting of the Tat export in Bacillus subtilis

The Tat (twin-arginine protein translocation) system initially discovered in the thylakoid membrane of chloroplasts has been described recently for a variety of eubacterial organisms. Although in Escherichia coli four Tat proteins with calculated membrane spanning domains have been demonstrated to mediate Tat-dependent transport, a specific transport system for twin-arginine signal peptide containing phosphodiesterase PhoD of Bacillus subtilis consists of one TatA/TatC (TatAd/TatCd) pair of proteins. Here, we show that TatAd was found beside its membrane-integrated localization in the cytosol were it interacted with prePhoD. prePhoD was efficiently co-immunoprecipitated by TatAd. Inefficient co-immunoprecipitation of mature PhoD and missing interaction to Sec-dependent and cytosolic peptides by TatAd demonstrated a particular role of the twin-arginine signal peptide for this interaction. Affinity of prePhoD to TatAd was interfered by peptides containing the twin-arginine motif but remained active when the arginine residues were substituted. The selective binding of TatAd to peptides derived from the signal peptide of PhoD elucidated the function of the twin-arginine motif as a target site for pre-protein TatAd interaction. Substitution of the binding motif demonstrated the pivotal role of basic amino acid residues for TatA binding. These features suggest that TatA interacts prior to membrane integration with its pre-protein substrate and could therefore assist targeting of twin-arginine pre-proteins.     

Identification of a second oligopeptide transport system in Bacillus subtilis and determination of its role in sporulation

Summary
Sporulation in Bacillus subtilis depends on an intact oligopeptide transport system, the Opp system. Mutants in opp sporulate poorly but second-site revertants can be found that restore sporulation and pep-tide transport. These second-site mutations were found in a second oligopeptide transport system, app , in which the peptide-binding protein, AppA, is mutant owing to a frame-shift mutation, and the revertants restore the original frame. The AppA mutation is present in the 168 strain of B. subtilis. The app operon consists of five genes in the order appD-appF-appA-appB-appC , with the locus designations corresponding to their homologue in the opp operon. Homology between the app and opp proteins ranges from 54% identity for AppF and OppF, to 22% identity for AppA and OppA. Both the App and Opp permease systems can transport tetra- and pentapeptides, but tripeptides are not transported by the App system. Strains of the genotype app ⁺opp⁻ are resistant to the tripeptide antibiotic bialaphos. The repaired App system can substitute completely for the Opp system in both sporulation and competence for genetic transformation. The pheno-types raised some speculation about the subunit configuration of the Opp system.     

The energized membrane and cellular autolysis in Bacillus subtilis

Lysis of exponential cultures of B. subtilis follows the addition of reagents that dissipate either the electrical or pH gradients of cellular membranes. Stationary-phase cells or cultures that have been inhibited in division by macromolecular-synthesis inhibitors also lyse when uncoupling agents or ionophores are added to the growth medium. Autolysis occurs after brief starvation for a carbon source. Protoplasts are unaffected by azide or other lysis-inducing agents. Electron-donating agents, such as phenazine methosulfate and ascorbate, are effective in retarding autolysis. The addition of an oxidizable carbon source to starved and lysing cultures prevents their autolysis. These results suggest that cellular lysis in B. subtilis and energized membrane are tightly coupled. The fluorescence intensity and the wavelength of maximal fluorescence of 8-anilino-1-naphthalene sulfonic acid, when added to bacterial suspensions, appear to be qualitatively related to the rate of cell lysis. Analyses show that ATP limitations are probably not involved in the elicitation of lysis by ionophores, uncoupling agents or starvation. Measurements of protonmotive forces in the lysis-prone cells suggest that a threshold force of more than 85 mV may be required to maintain cellular integrity. Lipoteichoic acids, polyelectrolytes such as dextran sulfate or phospholipids do not modify the rate of cellular lysis when added to suspensions containing azide or other reagents that eliminate transmembrane protonmotive forces. We interpret the results to suggest that the in vivo control of autolysin activity in B. subtilis is related to the energized membrane     

The oligopeptide transport system of Bacillus subtilis plays a role in the initiation of sporulation

Bacillus subtilis spo0K mutants are blocked at the first step in sporulation. The spo0K strain was found to contain two mutations: one was linked to the trpS locus, and the other was elsewhere on the chromosome. The mutation linked to trpS was responsible for the sporulation defect (spo-). The unlinked mutation enhanced this sporulation deficiency but had no phenotype on its own. The spo- mutation was located in an operon of five genes highly homologous to the oligopeptide transport (Opp) system of Gram-negative species. Studies with toxic peptide analogues showed that this operon does indeed encode a peptide-transport system. However, unlike the Opp system of Salmonella typhimurium, one of the two ATP-binding proteins, OppF, was not required for peptide transport or for sporulation. The OppA peptide-binding protein, which is periplasmically located in Gram-negative species, has a signal sequence characteristic of lipoproteins with an amino-terminal lipo-amino acid anchor. Cellular location studies revealed that OppA was associated with the cell during exponential growth, but was released into the medium in stationary phase. A major role of the Opp system in Gram-negative bacteria is the recycling of cell-wall peptides as they are released from the growing peptidoglycan. We postulate that the accumulation of such peptides may play a signalling role in the initiation of sporulation, and that the sporulation defect in opp mutants results from an inability to transport these peptides.