FAD binding by ApbE protein from Salmonella enterica: a new class of FAD-binding proteins

The periplasmic protein ApbE was identified through the analysis of several mutants defective in thiamine biosynthesis and was implicated as having a role in iron-sulfur cluster biosynthesis or repair. While mutations in apbE cause decreased activity of several iron-sulfur enzymes in vivo, the specific role of ApbE remains unknown. Members of the AbpE family include NosX and RnfF, which have been implicated in oxidation-reduction associated with nitrous oxide and nitrogen metabolism, respectively. In this work, we show that ApbE binds one FAD molecule per monomeric unit. The structure of ApbE in the presence of bound FAD reveals a new FAD-binding motif. Protein variants that are nonfunctional in vivo were generated by random and targeted mutagenesis. Each variant was substituted in the environment of the FAD and analyzed for FAD binding after reconstitution. The variant that altered a key tyrosine residue involved in FAD binding prevented reconstitution of the protein.     

The apbE Gene Encodes a Lipoprotein Involved in Thiamine Synthesis in Salmonella typhimurium

Thiamine pyrophosphate is an essential cofactor that is synthesized de novo in Salmonella typhimurium. The biochemical steps and gene products involved in the conversion of aminoimidazole ribotide (AIR), a purine intermediate, to the 4-amino-5-hydroxymethyl-2-methyl pyrimidine (HMP) moiety of thiamine have yet to be elucidated. We have isolated mutations in a new locus (Escherichia coli open reading frame designation yojK) at 49 min on the S. typhimurium chromosome. Two significant phenotypes associated with lesions in this locus (apbE) were identified. First, apbE purF double mutants require thiamine, specifically the HMP moiety. Second, in the presence of adenine, apbE single mutants require thiamine, specifically both the HMP and the thiazole moieties. Together, the phenotypes associated with apbE mutants suggest that flux through the purine pathway has a role in regulating synthesis of the thiazole moiety of thiamine and are consistent with ApbE being involved in the conversion of AIR to HMP. The product of the apbE gene was found to be a 36-kDa membrane-associated lipoprotein, making it the second membrane protein implicated in thiamine synthesis.     

Function of YidC for the insertion of M13 procoat protein in Escherichia coli: translocation of mutants that show differences in their membrane potential dependence and Sec requirement.

The membrane insertion of the Sec-independent M13 Procoat protein in bacteria requires the membrane electrochemical potential and the integral membrane protein YidC. We show here that YidC is involved in the translocation but not in the targeting of the Procoat protein, because we found the protein was partitioned into the membrane in the absence of YidC. YidC can function also to promote membrane insertion of Procoat mutants that insert independently of the membrane potential, proving that the effect of YidC depletion is not due to a dissipation of the membrane potential. We also found that YidC is absolutely required for Sec-dependent translocation of a long periplasmic loop of a mutant Procoat in which the periplasmic region has been extended from 20 to 194 residues. Furthermore, when Sec-dependent membrane proteins with large periplasmic domains were overproduced under YidC-limited conditions, we found that the exported proteins pro-OmpA and pre-peptidoglycan-associated lipoprotein accumulated in the cytoplasm. This suggests for Sec-dependent proteins that YidC functions at a late stage in membrane insertion, after the Sec translocase interacts with the translocating membrane protein. These studies are consistent with the understanding that YidC cooperates with the Sec translocase for membrane translocation and that YidC is required for clearing the protein-conducting channel.     

Deletion of lolB, Encoding an Outer Membrane Lipoprotein, Is Lethal for Escherichia coli and Causes Accumulation of Lipoprotein Localization Intermediates in the Periplasm

Outer membrane lipoproteins of Escherichia coli are released from the inner membrane upon the formation of a complex with a periplasmic chaperone, LolA, followed by localization to the outer membrane. In vitro biochemical analyses revealed that the localization of lipoproteins to the outer membrane generally requires an outer membrane lipoprotein, LolB, and occurs via transient formation of a LolB-lipoprotein complex. On the other hand, a mutant carrying the chromosomal lolB gene under the control of the lac promoter-operator grew normally in the absence of LolB induction if the mutant did not possess the major outer membrane lipoprotein Lpp, suggesting that LolB is only important for the localization of Lpp in vivo. To examine the in vivo function of LolB, we constructed a chromosomal lolB null mutant harboring a temperature-sensitive helper plasmid carrying the lolB gene. At a nonpermissive temperature, depletion of the LolB protein due to loss of the lolB gene caused cessation of growth and a decrease in the number of viable cells irrespective of the presence or absence of Lpp. LolB-depleted cells accumulated the LolA-lipoprotein complex in the periplasm and the mature form of lipoproteins in the inner membrane. Taken together, these results indicate that LolB is the first example of an essential lipoprotein for E. coli and that its depletion inhibits the upstream reactions of lipoprotein trafficking.     

Testing the '+2 rule' for lipoprotein sorting in the Escherichia coli cell envelope with a new genetic selection

We report a novel strategy for selecting mutations that mislocalize lipoproteins within the Escherichia coli cell envelope and describe the mutants obtained. A strain carrying a deletion of the chromosomal malE gene, coding for the periplasmic maltose-binding protein (MalE), cannot use maltose unless a wild-type copy of malE is present in trans. Replacement of the natural signal peptide of preMalE by the signal peptide and the first four amino acids of a cytoplasmic membrane-anchored lipoprotein resulted in N-terminal fatty acylation of MalE (lipoMalE) and anchoring to the periplasmic face of the cytoplasmic membrane, where it could still function. When the aspartate at position +2 of this protein was replaced by a serine, lipoMalE was sorted to the outer membrane, where it could not function. Chemical mutagenesis followed by selection for maltose-using mutants resulted in the identification of two classes of mutations. The single class I mutant carried a plasmid-borne mutation that replaced the serine at position +2 by phenylalanine. Systematic substitutions of the amino acid at position +2 revealed that, besides phenylalanine, tryptophan, tyrosine, glycine and proline could all replace classical cytoplasmic membrane lipoprotein sorting signal (aspartate +2). Analysis of known and putative lipoproteins encoded by the E. coli K-12 genome indicated that these amino acids are rarely found at position +2. In the class II mutants, a chromosomal mutation caused small and variable amounts of lipoMalE to remain associated with the cytoplasmic membrane. Similar amounts of another, endogenous outer membrane lipoprotein, NlpD, were also present in the cytoplasmic membrane in these mutants, indicating a minor, general defect in the sorting of outer membrane lipoproteins. Four representative class II mutants analysed were shown not to carry mutations in the lolA or lolB genes, known to be involved in the sorting of lipoproteins to the outer membrane.     

Lack of the ApbC or ApbE protein results in a defect in Fe-S cluster metabolism in Salmonella enterica serovar Typhimurium

The isc genes function in the assembly of Fe-S clusters and are conserved in many prokaryotic and eukaryotic organisms. In most bacteria studied, the isc operon can be deleted without loss of cell viability, indicating that additional systems for Fe-S cluster assembly must exist. Several laboratories have described nutritional and biochemical defects resulting from mutations in the isc operon. Here we demonstrate that null mutations in two genes of unknown function, apbC and apbE, result in similar cellular deficiencies. Exogenous ferric chloride suppressed these deficiencies in the apbC and apbE mutants, distinguishing them from previously described isc mutants. The deficiencies caused by the apbC and isc mutations were additive, which is consistent with Isc and ApbC's having redundant functions or with Isc and ApbC's functioning in different areas of Fe-S cluster metabolism (e.g., Fe-S cluster assembly and Fe-S cluster repair). Both the ApbC and ApbE proteins are similar in sequence to proteins that function in metal cofactor assembly. Like the enzymes with sequence similarity to ApbC, purified ApbC protein was able to hydrolyze ATP. The data herein are consistent with the hypothesis that the ApbC and ApbE proteins function in Fe-S cluster metabolism in vivo.     

Localization-controlled specificity of FAD:threonine flavin transferases in Klebsiella pneumoniae and its implications for the mechanism of Na-translocating NADH:quinone oxidoreductase

The Klebsiella pneumoniae genome contains genes for two putative flavin transferase enzymes (ApbE1 and ApbE2) that add FMN to protein Thr residues. ApbE1, but not ApbE2, has a periplasm-addressing signal sequence. The genome also contains genes for three target proteins with the Dxx(s/t)gAT flavinylation motif: two subunits of Na⁺-translocating NADH:quinone oxidoreductase (Na⁺-NQR), and a 99.5 kDa protein, KPK_2907, with a previously unknown function. We show here that KPK_2907 is an active cytoplasmically-localized fumarate reductase. K. pneumoniae cells with an inactivated kpk_2907 gene lack cytoplasmic fumarate reductase activity, while retaining this activity in the membrane fraction. Complementation of the mutant strain with a kpk_2907-containing plasmid resulted in a complete recovery of cytoplasmic fumarate reductase activity. KPK_2907 produced in Escherichia coli cells contains 1 mol/mol each of covalently bound FMN, noncovalently bound FMN and noncovalently bound FAD. Lesion in the ApbE1 gene in K. pneumoniae resulted in inactive Na⁺-NQR, but cytoplasmic fumarate reductase activity remained unchanged. On the contrary, lesion in the ApbE2 gene abolished the fumarate reductase but not the Na⁺-NQR activity. Both activities could be restored by transformation of the ApbE1- or ApbE2-deficient K. pneumoniae strains with plasmids containing the Vibrio cholerae apbE gene with or without the periplasm-directing signal sequence, respectively. Our data thus indicate that ApbE1 and ApbE2 bind FMN to Na⁺-NQR and fumarate reductase, respectively, and that, contrary to the presently accepted view, the FMN residues are on the periplasmic side of Na⁺-NQR. A new, “electron loop” mechanism is proposed for Na⁺-NQR, involving an electroneutral Na⁺/electron symport. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.     

The secretin-specific, chaperone-like protein of the general secretory pathway: separation of proteolytic protection and piloting functions

The chaperone-like protein of the main terminal branch of the general secretory pathway from Klebsiella oxytoca , the outer membrane lipoprotein PulS, protects the multimeric secretin PulD from degradation and promotes its correct localization to the outer membrane. To determine whether these are separable functions, or whether resistance to proteolysis results simply from correct localization of PulD, we replaced the lipoprotein-type signal peptide of PulS by the signal peptide of periplasmic maltose-binding protein. The resulting periplasmic PulS retained its ability to protect PulD, but not its ability to localize PulD to the outer membrane and to function in pullulanase secretion. Periplasmic PulS competed with wild-type PulS to prevent pullulanase secretion, presumably again by causing mislocalization of PulD. A hybrid protein comprising the mature part of PulS fused to the C-terminus of full-length maltose-binding protein (MalE–PulS) had similar properties to the periplasmic PulS protein. Moreover, MalE–PulS was shown to associate with PulD by amylose-affinity chromatography. The MalE–PulS hybrid was rendered completely functional (i.e. it restored pullulanase secretion in a pulS mutant) by replacing its signal peptide with a lipoprotein-type signal peptide. However, this fatty-acylated hybrid protein was only functional if it also carried a lipoprotein sorting signal that targeted it to the outer membrane. Thus, the two functions of PulS are separate and fully dissociable. Incorrect localization, rather than proteolysis, of PulD in the absence of PulS was shown to be the factor that causes high-level induction of the phage shock response. The Erwinia chrysanthemi PulS homologue, OutS, can substitute for PulS, and PulS can protect the secretin OutD from proteolysis in Escherichia coli , indicating the possible existence of a family of PulS-like chaperone proteins.     

Growth Inhibition by Hexoses of a Temperature-Sensitive Thiazoleless Mutant of Salmonella typhimurium

A Salmonella typhimurium mutant showing impairment in the utilization of hexoses was isolated after treatment with N-methyl-N'-nitro-N-nitrosoguanidine. At 30 C, it grew with hexoses (glucose, fructose, galactose, mannitol), glycerol, succinate, or acid-hydrolyzed casein. At 37 C, it failed to grow with any of the hexoses. Enzymatic determinations demonstrated, however, that the enzymes of the glycolytic pathway (up to the formation of triose phosphates) were present and active at 25 and 32 C. At 42 C, the mutant did not grow with any of the carbon sources used. At both 37 and 42 C, the mutant grew perfectly well with hexoses if yeast extract was present. The metabolite required for growth was thiamine or, specifically, its thiazole moiety. If glucose was added to a culture growing in glycerol, at 37 C, growth was inhibited. This inhibition was relieved by the addition of thiamine or thiazole. Thus, at 37 C and only in the presence of hexoses, the mutant manifests a requirement for thiazole. This auxotrophy is absolute at 42 C. These data indicate that, in this mutant, some derivative of hexoses inhibits the synthesis of thiazole, and that this inhibition is also dependent on the temperature of incubation. The position in the bacterial chromosome of the genetic locus of this lesion (thz(-)) was determined by conjugation and found to coincide with the only thiamine (thi) locus so far reported.     

Structural determinants required for apical sorting of an intestinal brush-border membrane protein

The distinct protein and lipid constituents of the apical and basolateral membranes in polarized cells are sorted by specific signals. O-Glycosylation of a highly polarized intestinal brush-border protein sucrase isomaltase is implicated in its apical sorting through interaction with sphingolipid-cholesterol microdomains. We characterized the structural determinants required for this mechanism by focusing on two major domains in pro-SI, the membrane anchor and the Ser/Thr-rich stalk domain. Deletion mutants lacking either domain, pro-SI(DeltaST) (stalk-free) and pro-SI(DeltaMA) (membrane anchor-free), were constructed and expressed in polarized Madin-Darby canine kidney cells. In the absence of the membrane anchoring domain, pro-SI(DeltaMA) does not associate with lipid rafts and the mutant is randomly delivered to both membranes. Therefore, the O-glycosylated stalk region is not sufficient per se for the high fidelity of apical sorting of pro-SI. Pro-SI(DeltaST) does not associate either with lipid rafts and its targeting behavior is similar to that of pro-SI(DeltaMA). Only wild type pro-SI containing both determinants, the stalk region and membrane anchor, associates with lipid microdomains and is targeted correctly to the apical membrane. However, not all sequences in the stalk region are required for apical sorting. Only O-glycosylation of a stretch of 12 amino acids (Ala(37)-Pro(48)) juxtapose the membrane anchor is required in conjunction with the membrane anchoring domain for correct targeting of pro-SI to the apical membrane. Other O-glycosylated domains within the stalk (Ala(49)-Pro(57)) are not sufficient for apical sorting. We conclude that the recognition signal for apical sorting of pro-SI comprises O-glycosylation of the Ala(37)-Pro(48) stretch and requires the presence of the membrane anchoring domain.     

Skp, a molecular chaperone of gram-negative bacteria, is required for the formation of soluble periplasmic intermediates of outer membrane proteins.

Using a cross-linking approach, we have analyzed the function of Skp, a presumed molecular chaperone of the periplasmic space of Escherichia coli, during the biogenesis of an outer membrane protein (OmpA). Following its transmembrane translocation, OmpA interacts with Skp in close vicinity to the plasma membrane. In vitro, Skp was also found to bind strongly and specifically to pOmpA nascent chains after their release from the ribosome suggesting the ability of Skp to recognize early folding intermediates of outer membrane proteins. Pulse labeling of OmpA in spheroplasts prepared from an skp null mutant revealed a specific requirement of Skp for the release of newly translocated outer membrane proteins from the plasma membrane. Deltaskp mutant cells are viable and show only slight changes in the physiology of their outer membranes. In contrast, double mutants deficient both in Skp and the periplasmic protease DegP (HtrA) do not grow at 37 degrees C in rich medium. We show that in the absence of an active DegP, a lack of Skp leads to the accumulation of protein aggregates in the periplasm. Collectively, our data demonstrate that Skp is a molecular chaperone involved in generating and maintaining the solubility of early folding intermediates of outer membrane proteins in the periplasmic space of Gram-negative bacteria.     

Internal deletions in the gene for an Escherichia coli outer membrane protein define an area possibly important for recognition of the outer membrane by this polypeptide

A series of overlapping deletions has been constructed in the ompA gene which encodes the 325-residue Escherichia coli outer membrane protein OmpA. Immunoelectron microscopy showed that the OmpA fragments were either located in the periplasmic space or were associated with the outer membrane. Apparently an area between residues 154 and 180 is required for this association; all proteins missing this area were found to be periplasmic. The nature of this association remained unknown; no membrane-protected tryptic fragments could be identified for any of these polypeptides. Hybrid genes were constructed encoding parts of the periplasmic maltose binding protein and an area of the ompA gene coding for residues 154-274. The corresponding proteins were not localized to the outer membrane but remained attached to the outer face of the plasma membrane, possibly because the normal mechanism of release from this membrane was impaired. In the OmpA protein the conspicuous sequence Ala180-Pro-Ala-Pro-Ala-Pro-Ala-Pro187 exists. Frameshift mutants were constructed to eliminate this sequence. There was no effect on the incorporation of the mutant proteins into the outer membrane. Thus, this "hinge" region is not involved in sorting. A proposal suggesting the existence of a sorting signal common to several outer membrane proteins (Benson, S. A., Bremer, E., and Silhavy, T. J. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 3830-3834) was subsequently rejected (Bosch, D., Leunissen, J., Verbakel, J., de Jong, M., van Erp, H., and Tommassen, J. (1986) J. Mol. Biol. 189, 449-455; Freudl, R., Schwarz, H., Klose, M., Movva, N. R., and Henning, U. (1985) EMBO J. 4, 3593-3598). Although it is not known whether or not the outer membrane association observed represents a step in the normal sorting mechanism, it is concluded that it remains an open question whether or not a sorting signal, as proposed originally, exists in outer membrane proteins.     

Insertion of an outer membrane protein in Escherichia coli requires a chaperone-like protein.

Only one of the characterized components of the main terminal branch of the general secretory pathway (GSP) in Gram-negative bacteria, GspD, is an integral outer membrane protein that could conceivably form a channel to permit protein transport across this membrane. PulD, a member of the GspD protein family required for pullulanase secretion by Klebsiella oxytoca, is shown here to form outer membrane-associated complexes which are not readily dissociated by SDS treatment. The outer membrane association of PulD is absolutely dependent on another component of the GSP, the outer membrane-anchored lipoprotein PulS. Furthermore, the absence of PulS resulted in limited proteolysis of PulD and caused induction of the so-called phage shock response, as measured by increased expression of the pspA gene. We propose that PulS may be the first member of a new family of periplasmic chaperones that are specifically required for the insertion of a group of outer membrane proteins into this membrane. PulS is only the second component of the main terminal branch of the GSP for which a precise function can be proposed.     

Localization of the outer membrane subunit OprM of resistance-nodulation-cell division family multicomponent efflux pump in Pseudomonas aeruginosa

The outer membrane subunit OprM of the multicomponent efflux pump of Pseudomonas aeruginosa has been assumed to form a transmembrane xenobiotic exit channel across the outer membrane. We challenged this hypothesis to clarify the underlying ambiguity by manipulating the amino-terminal signal sequence of the OprM protein of the MexAB-OprM efflux pump in P. aeruginosa. [(3)H]Palmitate uptake experiments revealed that OprM is a lipoprotein. The following lines of evidence unequivocally established that the OprM protein functioned at the periplasmic space. (i) The OprM protein, in which a signal sequence including Cys-18 was replaced with that of periplasmic azurin, appeared in the periplasmic space but not in the outer membrane fraction, and the protein fully functioned as the pump subunit. (ii) The hybrid OprM containing the N-terminal transmembrane segment of the inner membrane protein, MexF, appeared exclusively in the inner membrane fraction. The hybrid protein containing 186 or 331 amino acid residues of MexF was fully active for the antibiotic extrusion, but a 42-residue protein was totally inactive. (iii) The mutant OprM, in which the N-terminal cysteine residue was replaced with another amino acid, appeared unmodified with fatty acid and was fractionated in both the periplasmic space and the inner membrane fraction but not in the outer membrane fraction. The Cys-18-modified OprM functioned for the antibiotic extrusion indistinguishably from that in the wild-type strain. We concluded, based on these results, that the OprM protein was anchored in the outer membrane via fatty acid(s) attached to the N-terminal cysteine residue and that the entire polypeptide moiety was exposed to the periplasmic space.     

The same periplasmic ExbD residues mediate in vivo interactions between ExbD homodimers and ExbD-TonB heterodimers

The TonB system couples cytoplasmic membrane proton motive force to TonB-gated outer membrane transporters for active transport of nutrients into the periplasm. In Escherichia coli, cytoplasmic membrane proteins ExbB and ExbD promote conformational changes in TonB, which transmits this energy to the transporters. The only known energy-dependent interaction occurs between the periplasmic domains of TonB and ExbD. This study identified sites of in vivo homodimeric interactions within ExbD periplasmic domain residues 92 to 121. ExbD was active as a homodimer (ExbD(2)) but not through all Cys substitution sites, suggesting the existence of conformationally dynamic regions in the ExbD periplasmic domain. A subset of homodimeric interactions could not be modeled on the nuclear magnetic resonance (NMR) structure without significant distortion. Most importantly, the majority of ExbD Cys substitutions that mediated homodimer formation also mediated ExbD-TonB heterodimer formation with TonB A150C. Consistent with the implied competition, ExbD homodimer formation increased in the absence of TonB. Although ExbD D25 was not required for their formation, ExbD dimers interacted in vivo with ExbB. ExbD-TonB interactions required ExbD transmembrane domain residue D25. These results suggested a model where ExbD(2) assembled with ExbB undergoes a transmembrane domain-dependent transition and exchanges partners in localized homodimeric interfaces to form an ExbD(2)-TonB heterotrimer. The findings here were also consistent with our previous hypothesis that ExbD guides the conformation of the TonB periplasmic domain, which itself is conformationally dynamic.     

Identification of a TcpC-TcpQ outer membrane complex involved in the biogenesis of the toxin-coregulated pilus of Vibrio cholerae

The toxin-coregulated pilus (TCP) of Vibrio cholerae and the soluble TcpF protein that is secreted via the TCP biogenesis apparatus are essential for intestinal colonization. The TCP biogenesis apparatus is composed of at least nine proteins but is largely uncharacterized. TcpC is an outer membrane lipoprotein required for TCP biogenesis that is a member of the secretin protein superfamily. In the present study, analysis of TcpC in a series of strains deficient in each of the TCP biogenesis proteins revealed that TcpC was absent specifically in a tcpQ mutant. TcpQ is a predicted periplasmic protein required for TCP biogenesis. Fractionation studies revealed that the protein is not localized to the periplasm but is associated predominantly with the outer membrane fraction. An analysis of the amount of TcpQ present in the series of tcp mutants demonstrated the inverse of the TcpC result (absence of TcpQ in a tcpC deletion strain). Complementation of the tcpQ deletion restored TcpC levels and TCP formation, and similarly, complementation of tcpC restored TcpQ. Metal affinity pull-down experiments performed using His-tagged TcpC or TcpQ demonstrated a direct interaction between TcpC and TcpQ. In the presence of TcpQ, TcpC was found to form a high-molecular-weight complex that is stable in 2% sodium dodecyl sulfate and at temperatures below 65°C, a characteristic of secretin complexes. Fractionation studies in which TcpC was overexpressed in the absence of TcpQ showed that TcpQ is also required for proper localization of TcpC to the outer membrane.     

Three homologues, including two membrane-bound proteins, of the disulfide oxidoreductase DsbA in Neisseria meningitidis: effects on bacterial growth and biogenesis of functional type IV pili

Many proteins, especially membrane and exported proteins, are stabilized by intramolecular disulfide bridges between cysteine residues without which they fail to attain their native functional conformation. The formation of these bonds is catalyzed in Gram-negative bacteria by enzymes of the Dsb system. Thus, the activity of DsbA has been shown to be necessary for many phenotypes dependent on exported proteins, including adhesion, invasion, and intracellular survival of various pathogens. The Dsb system in Neisseria meningitidis, the causative agent of cerebrospinal meningitis, has not, however, been studied. In a previous work where genes specific to N. meningitidis and not present in the other pathogenic Neisseria were isolated, a meningococcus-specific dsbA gene was brought to light (Tinsley, C. R., and Nassif, X. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 11109-11114). Inactivation of this gene, however, did not result in deficits in the phenotypes commonly associated with DsbA. A search of available genome data revealed that the meningococcus contains three dsbA genes encoding proteins with different predicted subcellular locations, i.e. a soluble periplasmic enzyme and two membrane-bound lipoproteins. Cell fractionation experiments confirmed the localization in the inner membrane of the latter two, which include the previously identified meningococcus-specific enzyme. Mutational analysis demonstrated that the deletion of any single enzyme was compensated by the action of the remaining two on bacterial growth, whereas the triple mutant was unable to grow at 37 degrees C. Remarkably, however, the combined absence of the two membrane-bound enzymes led to a phenotype of sensitivity to reducing agents and loss of functionality of the pili. Although in many species a single periplasmic DsbA is sufficient for the correct folding of various proteins, in the meningococcus a membrane-associated DsbA is required for a wild type DsbA+ phenotype even in the presence of a functional periplasmic DsbA.     

Biosynthesis of the pyrimidine moiety of thiamine. A new route of pyrimidine biosynthesis involving purine intermediates

1. The pattern of distribution on the purine pathway of mutants of Salmonella typhimurium LT2 that had the double growth requirement for a purine plus the pyrimidine moiety of thiamine (ath mutants) indicated that purines and the pyrimidine moiety of thiamine share the early part of their biosynthetic pathways, and that 4-aminoimidazole ribonucleotide (AIR) is the last common intermediate. Two mutants that at first appeared anomalous were further investigated and found not to affect this deduction. 2. The ribonucleoside form of AIR (AIR(s)) satisfied the requirements both for a purine and for the pyrimidine moiety of thiamine of an ath mutant. 3. Methionine was required for the conversion of AIR into the pyrimidine moiety. 4. Radioactive AIR(s) was converted into radioactive pyrimidine moiety by an ath mutant without significant dilution of specific radioactivity. 5. Possible mechanisms for pyrimidine-moiety biosynthesis from AIR are discussed.     

Probing the Borrelia burgdorferi surface lipoprotein secretion pathway using a conditionally folding protein domain

Surface lipoproteins of Borrelia spirochetes are important virulence determinants in the transmission and pathogenesis of Lyme disease and relapsing fever. To further define the conformational secretion requirements and to identify potential lipoprotein translocation intermediates associated with the bacterial outer membrane (OM), we generated constructs in which Borrelia burgdorferi outer surface lipoprotein A (OspA) was fused to calmodulin (CaM), a conserved eukaryotic protein undergoing calcium-dependent folding. Protein localization assays showed that constructs in which CaM was fused to full-length wild-type (wt) OspA or to an intact OspA N-terminal "tether" peptide retained their competence for OM translocation even in the presence of calcium. In contrast, constructs in which CaM was fused to truncated or mutant OspA N-terminal tether peptides were targeted to the periplasmic leaflet of the OM in the presence of calcium but could be flipped to the bacterial surface upon calcium chelation. This indicated that in the absence of an intact tether peptide, unfolding of the CaM moiety was required in order to facilitate OM traversal. Together, these data further support a periplasmic tether peptide-mediated mechanism to prevent premature folding of B. burgdorferi surface lipoproteins. The specific shift in the OM topology of sequence-identical lipopeptides due to a single-variable change in environmental conditions also indicates that surface-bound Borrelia lipoproteins can localize transiently to the periplasmic leaflet of the OM.     

IcsA surface presentation in Shigella flexneri requires the periplasmic chaperones DegP, Skp, and SurA

A Shigella flexneri degP mutant, which was defective for plaque formation in Henle cell monolayers, had a reduced amount of IcsA detectable on the bacterial surface with antibody. However, the mutant secreted IcsA to the outer membrane at wild-type levels. This suggests that IcsA adopts an altered conformation in the outer membrane of the degP mutant with reduced exposure on the cell surface. IcsA is, therefore, unlikely to be accessible to actin-nucleating proteins within the eukaryotic cell cytoplasm, which is required for bacterial movement within the host cell and cell-to-cell spread. The degP mutant was somewhat more sensitive to detergents, antibiotics, and the antimicrobial peptide magainin, indicating that the degP phenotype was not limited to IcsA surface presentation. The plaque defect of the degP mutant, which is independent of DegP protease activity, was suppressed by overexpression of the periplasmic chaperone Skp but not by SurA. S. flexneri skp and surA mutants failed to form plaques in Henle cell monolayers and were defective in cell surface presentation and polar localization of IcsA. Therefore, the three periplasmic folding factors DegP, Skp, and SurA were all required for IcsA localization and plaque formation by S. flexneri.