Characterization of a periplasmic protein involved in iron utilization of Actinobacillus actinomycetemcomitans.

The periodontopathic bacterium Actinobacillus actinomycetemcomitans possesses a 35-kDa periplasmic iron-repressible protein. Its regulation is mediated by the Fur protein, as was inferred from the Fur-binding consensus sequence at the -35 position of the gene for the 35-kDa protein and from the relaxed expression of the gene in a mutant with an altered Fur-binding sequence. The 35-kDa protein, designated AfuA, has strong homology to HitA and FbpA of Haemophilus influenzae and Neisseria meningitidis, respectively, which serve as periplasmic iron transport proteins.     

Two regions of mature periplasmic maltose-binding protein of Escherichia coli involved in secretion.

Six mutations in malE, the structural gene for the periplasmic maltose-binding protein (MBP) from Escherichia coli, prevent growth on maltose as a carbon source, as well as release of the mutant proteins by the cold osmotic-shock procedure. These mutations correspond to insertion of an oligonucleotide linker, concomitant with a deletion. One of the mutations (malE127) affects the N-terminal extension (the signal peptide), whereas the five others lie within the mature protein. As expected, the export of protein MalE127 is blocked at an early stage. This protein is neither processed to maturity nor sensitive to proteinase K in spheroplasts. In contrast, in the five other mutants, the signal peptide is cleaved and the protein is accessible to proteinase K added to spheroplasts. This indicates that the five mutant proteins are, at least in part, exported through the inner membrane. We propose that the corresponding mutations define two regions of the mature protein (between residues 18 and 42 and between residues 280 and 306), which are important for release of the protein from the inner membrane into the periplasm. We discuss the results in terms of possible conformational changes at this late step of export to the periplasm.     

The periplasmic binding protein of a tripartite tricarboxylate transporter is involved in signal transduction

A new type of solute importer has been identified recently in various bacterial genera and called the tripartite tricarboxylate transporter (TTT). TTTs consist of two cytoplasmic membrane proteins and a periplasmic solute-binding protein. In the whooping cough agent Bordetella pertussis, a TTT system that has been called BctCBA mediates the uptake of citrate, with BctA and BctB being the membrane components and BctC, the periplasmic protein. Here, we describe that the expression of the bctCBA operon is induced by the presence of citrate in the milieu. The signalling cascade involves both BctC and the signal transduction two-component system BctDE, encoded by an operon adjacent to bctCBA. Furthermore, two-hybrid analyses and affinity chromatography experiments indicated that citrate-liganded BctC interacts with the periplasmic domain of the sensor protein, BctE. Thus, BctC is part of the signalling cascade leading to upregulation of the transporter operon in the presence of its solute, a new function for periplasmic binding proteins of TT transporters.     

The Tmc complex from Desulfovibrio vulgaris hildenborough is involved in transmembrane electron transfer from periplasmic hydrogen oxidation

Three membrane-bound redox complexes have been reported in Desulfovibrio spp., whose genes are not found in the genomes of other sulfate reducers such as Desulfotalea psycrophila and Archaeoglobus fulgidus. These complexes contain a periplasmic cytochrome c subunit of the cytochrome c(3) family, and their presence in these organisms probably correlates with the presence of a pool of periplasmic cytochromes c(3), also absent in the two other sulfate reducers. In this work we report the isolation and characterization of the first of such complexes, Tmc from D. vulgaris Hildenborough, which is associated with the tetraheme type II cytochrome c(3). The isolated Tmc complex contains four subunits, including the TpIIc(3) (TmcA), an integral membrane cytochrome b (TmcC), and two cytoplasmically predicted proteins, an iron-sulfur protein (TmcB) and a tryptophan-rich protein (TmcD). Spectroscopic studies indicate the presence of eight hemes c and two hemes b in the complex pointing to an alpha(2)betagammadelta composition (TmcA(2)BCD). EPR analysis reveals the presence of a [4Fe4S](3+) center and up to three other iron-sulfur centers in the cytoplasmic subunit. Nearly full reduction of the redox centers in the Tmc complex could be obtained upon incubation with hydrogenase/TpIc(3), supporting the role of this complex in transmembrane transfer of electrons resulting from periplasmic oxidation of hydrogen.     

The LptA protein of Escherichia coli is a periplasmic lipid A-binding protein involved in the lipopolysaccharide export pathway

The LptA protein of Escherichia coli has been implicated in the transport of lipopolysaccharide (LPS) from the inner membrane to the outer membrane. Here we provide evidence that LptA binds structurally diverse LPS substrates in vitro and demonstrate that it interacts specifically with the lipid A domain of LPS. These results are consistent with LptA playing a chaperone role in the transport of LPS across the periplasm and have implications for possible assembly models.     

Anaerobic arsenite oxidation by novel denitrifying isolates

Autotrophic microorganisms have been isolated that are able to derive energy from the oxidation of arsenite [As(III)] to arsenate [As(V)] under aerobic conditions. Based on chemical energetics, microbial oxidation of As(III) can occur in the absence of oxygen, and may be relevant in some environments. Enrichment cultures were established from an arsenic contaminated industrial soil amended with As(III) as the electron donor, inorganic C as the carbon source and nitrate as the electron acceptor. In the active enrichment cultures, oxidation of As(III) was stoichiometrically coupled to the reduction of NO(3) (-). Two autotrophic As(III)-oxidizing strains were isolated that completely oxidized 5 mM As(III) within 7 days under denitrifying conditions. Based on 16S rRNA gene sequencing results, strain DAO1 was 99% related to Azoarcus and strain DAO10 was most closely related to a Sinorhizobium. The nitrous oxide reductase (nosZ) and the RuBisCO Type II (cbbM) genes were successfully amplified from both isolates underscoring their ability to denitrify and fix CO(2) while coupled to As(III) oxidation. Although limited work has been done to examine the diversity of anaerobic autotrophic oxidizers of As(III), this process may be an important component in the biological cycling of arsenic within the environment.     

Microbial oxidation of arsenite in a subarctic environment: diversity of arsenite oxidase genes and identification of a psychrotolerant arsenite oxidiser

Background  

Arsenic is toxic to most living cells. The two soluble inorganic forms of arsenic are arsenite (+3) and arsenate (+5), with arsenite the more toxic. Prokaryotic metabolism of arsenic has been reported in both thermal and moderate environments and has been shown to be involved in the redox cycling of arsenic. No arsenic metabolism (either dissimilatory arsenate reduction or arsenite oxidation) has ever been reported in cold environments (i.e. < 10°C).     

A periplasmic iron-binding protein contributes toward inward copper supply

Periplasmic substrate binding proteins are known for iron, zinc, manganese, nickel, and molybdenum but not copper. Synechocystis PCC 6803 requires copper for thylakoid-localized plastocyanin and cytochrome oxidase. Here we show that mutants deficient in a periplasmic substrate binding protein FutA2 have low cytochrome oxidase activity and produce cytochrome c6 when grown under copper conditions (150 nm) in which wild-type cells use plastocyanin rather than cytochrome c6. Anaerobic separation of extracts by two-dimensional native liquid chromatography followed by metal analysis and peptide mass-fingerprinting establish that accumulation of copper-plastocyanin is impaired, but iron-ferredoxin is unaffected in DeltafutA2 grown in 150 nm copper. However, recombinant FutA2 binds iron in preference to copper in vitro with an apparent Fe(III) affinity similar to that of its paralog FutA1, the principal substrate binding protein for iron import. FutA2 is also associated with iron and not copper in periplasm extracts, and this Fe(III)-protein complex is absent in DeltafutA2. There are differences in the soluble protein and small-molecule complexes of copper and iron, and the total amount of both elements increases in periplasm extracts of DeltafutA2 relative to wild type. Changes in periplasm protein and small-molecule complexes for other metals are also observed in DeltafutA2. It is proposed that FutA2 contributes to metal partitioning in the periplasm by sequestering Fe(III), which limits aberrant Fe(III) associations with vital binding sites for other metals, including copper.     

A novel periplasmic carrier protein involved in the sorting and transport of Escherichia coli lipoproteins destined for the outer membrane.

Lipoproteins are localized in the outer or inner membrane of Escherichia coli, depending on the species of amino acid located next to the N-terminal fatty acylated Cys. The major outer membrane lipoprotein (Lpp) expressed in spheroplasts was, however, retained in the inner membrane as a mature form. A novel protein that is essential for the release of Lpp from the inner membrane was discovered in the periplasm and purified. The partial amino acid sequence of this 20 kDa protein (p20) was determined and used to clone a gene for p20. Sequencing of the gene revealed that p20 is synthesized as a precursor with a signal sequence. p20 formed a soluble complex only with outer membrane-directed lipoproteins such as Lpp, indicating that p20 plays a critical role in the sorting of lipoproteins. Lpp released from the inner membrane in the presence of p20 was specifically assembled into the outer membrane in vitro. These results indicate that p20 is a periplasmic carrier protein involved in the translocation of lipoproteins from the inner to the outer membrane.     

Alix, a protein regulating endosomal trafficking, is involved in neuronal death

Alix/AIP1 is a cytoplasmic protein, which was first characterized as an interactor of ALG-2, a calcium-binding protein necessary for cell death. Alix has also recently been defined as a regulator of the endo-lysosomal system. Here we have used post-mitotic cerebellar neurons to test Alix function in caspase-dependent and -independent cell death. Indeed, these neurons survived when cultured in 25 mm potassium-containing medium but underwent apoptosis soon after the extracellular potassium was lowered to 5 mm. In agreement with other studies, we show that caspases are activated after K+ deprivation, but that inhibition of these proteases, using the pancaspase inhibitor boc-aspartyl(OMe)-fluoromethylketone, has no effect on cell survival. Transfection experiments demonstrated that Alix overexpression is sufficient to induce caspase activation, whereas overexpression of its C-terminal half, Alix-CT, blocks caspase activation and cell death after K+ deprivation. We also define a 12-amino acid PXY repeat of the C-terminal proline-rich domain necessary for binding ALG-2. Deletion of this domain in Alix or in Alix-CT abolished the effects of the overexpressed proteins on neuronal survival, demonstrating that the ALG-2-binding region is crucial for the death-modulating function of Alix. Overall, these findings define the Alix/ALG-2 complex as a regulator of cell death controlling both caspase-dependent and -independent pathways. They also suggest a molecular link between the endo-lysosomal system and the effectors of the cell death machinery.     

Evidence that protein kinase C is involved in regulating glucose transport in the adipocyte

• 1.1. The role of protein kinase C in the mechanism of stimulation of glucose transport in rat adipocytes was investigated.
• 2.2. Glucose transprt was stimulated by dioleoylglycerol (DOG), tetradecanoyl phorbol acetate (TPA) and phospholipase C (PLC).
• 3.3. Agents that inhibit protein kinase C (polymyxin B, gossypol and quercitin) also inhibited glucose transport that had been stimulated by DOG, TPA, PLC and insulin.

     

A novel sec-independent periplasmic protein translocation pathway in Escherichia coli.

The trimethylamine N-oxide (TMAO) reductase of Escherichia coli is a soluble periplasmic molybdoenzyme. The precursor of this enzyme possesses a cleavable N-terminal signal sequence which contains a twin-arginine motif. By using various moa, mob and mod mutants defective in different steps of molybdocofactor biosynthesis, we demonstrate that acquisition of the molybdocofactor in the cytoplasm is a prerequisite for the translocation of the TMAO reductase. The activation and translocation of the TMAO reductase precursor are post-translational processes, and activation is dissociable from translocation. The export of the TMAO reductase is driven mainly by the proton motive force, whereas sodium azide exhibits a limited effect on the export. The most intriguing observation is that translocation of the TMAO reductase across the cytoplasmic membrane is independent of the SecY, SecE, SecA and SecB proteins. Depletion of Ffh, a core component of the signal recognition particle of E. coli, appears to have a slight effect on the export of the TMAO reductase. These results strongly suggest that the translocation of the molybdoenzyme TMAO reductase into the periplasm uses a mechanism fundamentally different from general protein translocation.     

A periplasmic fluorescent reporter protein and its application in high-throughput membrane protein topology analysis

We have developed a periplasmic fluorescent reporter protein suitable for high-throughput membrane protein topology analysis in Escherichia coli. The reporter protein consists of a single chain (scFv) antibody fragment that binds to a fluorescent hapten conjugate with high affinity. Fusion of the scFv to membrane protein sites that are normally exposed in the periplasmic space tethers the scFv onto the inner membrane. Following permealization of the outer membrane to allow diffusion of the fluorescent hapten into the periplasm, binding to the anchored scFv renders the cells fluorescent. We show that cell fluorescence is an accurate and sensitive reporter of the location of residues within periplasmic loops. For topological analysis, a set of nested deletions in the membrane protein gene is employed to construct two libraries of gene fusions, one to the scFvand one to the cytoplasmic reporter green fluorescent protein (GFP). Fluorescent clones are isolated by flow cytometry and the sequence of the fusion junctions is determined to identify amino acid residues within periplasmic and cytoplasmic loops, respectively. We applied this methodology to the topology analysis of E. coli TatC protein for which previous studies had led to conflicting results. The ease of screening libraries of fusions by flow cytometry enabled the rapid identification of almost 90 highly fluorescent scFv and GFP fusions, which, in turn, allowed the fine mapping of TatC membrane topology.     

The genetic basis of anoxygenic photosynthetic arsenite oxidation

‘Photoarsenotrophy’, the use of arsenite as an electron donor for anoxygenic photosynthesis, is thought to be an ancient form of phototrophy along with the photosynthetic oxidation of Fe(II), H₂S, H₂ and . Photoarsenotrophy was recently identified from Paoha Island's (Mono Lake, CA) arsenic‐rich hot springs. The genomes of several photoarsenotrophs revealed a gene cluster, arxB2AB1CD, where arxA is predicted to encode for the sole arsenite oxidase. The role of arxA in photosynthetic arsenite oxidation was confirmed by disrupting the gene in a representative photoarsenotrophic bacterium, resulting in the loss of light‐dependent arsenite oxidation. In situ evidence of active photoarsenotrophic microbes was supported by arxA mRNA detection for the first time, in red‐pigmented microbial mats within the hot springs of Paoha Island. This work expands on the genetics for photosynthesis coupled to new electron donors and elaborates on known mechanisms for arsenic metabolism, thereby highlighting the complexities of arsenic biogeochemical cycling.