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1.
Nat Commun ; 12(1): 4687, 2021 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-34344901

RESUMO

Lipoproteins are important for bacterial growth and antibiotic resistance. These proteins use lipid acyl chains attached to the N-terminal cysteine residue to anchor on the outer surface of cytoplasmic membrane. In Gram-negative bacteria, many lipoproteins are transported to the outer membrane (OM), a process dependent on the ATP-binding cassette (ABC) transporter LolCDE which extracts the OM-targeted lipoproteins from the cytoplasmic membrane. Lipid-anchored proteins pose a unique challenge for transport machinery as they have both hydrophobic lipid moieties and soluble protein component, and the underlying mechanism is poorly understood. Here we determined the cryo-EM structures of nanodisc-embedded LolCDE in the nucleotide-free and nucleotide-bound states at 3.8-Å and 3.5-Å resolution, respectively. The structural analyses, together with biochemical and mutagenesis studies, uncover how LolCDE recognizes its substrate by interacting with the lipid and N-terminal peptide moieties of the lipoprotein, and identify the amide-linked acyl chain as the key element for LolCDE interaction. Upon nucleotide binding, the transmembrane helices and the periplasmic domains of LolCDE undergo large-scale, asymmetric movements, resulting in extrusion of the captured lipoprotein. Comparison of LolCDE and MacB reveals the conserved mechanism of type VII ABC transporters and emphasizes the unique properties of LolCDE as a molecule extruder of triacylated lipoproteins.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Lipoproteínas/metabolismo , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/genética , Acilação , Trifosfato de Adenosina/metabolismo , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Sítios de Ligação , Membrana Celular/metabolismo , Microscopia Crioeletrônica , Escherichia coli/genética , Escherichia coli/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Mutação , Periplasma/metabolismo , Conformação Proteica , Transporte Proteico
2.
Nat Commun ; 12(1): 4349, 2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34272394

RESUMO

Bacterial extracellular polysaccharides (EPSs) play critical roles in virulence. Many bacteria assemble EPSs via a multi-protein "Wzx-Wzy" system, involving glycan polymerization at the outer face of the cytoplasmic/inner membrane. Gram-negative species couple polymerization with translocation across the periplasm and outer membrane and the master regulator of the system is the tyrosine autokinase, Wzc. This near atomic cryo-EM structure of dephosphorylated Wzc from E. coli shows an octameric assembly with a large central cavity formed by transmembrane helices. The tyrosine autokinase domain forms the cytoplasm region, while the periplasmic region contains small folded motifs and helical bundles. The helical bundles are essential for function, most likely through interaction with the outer membrane translocon, Wza. Autophosphorylation of the tyrosine-rich C-terminus of Wzc results in disassembly of the octamer into multiply phosphorylated monomers. We propose that the cycling between phosphorylated monomer and dephosphorylated octamer regulates glycan polymerization and translocation.


Assuntos
Cápsulas Bacterianas/química , Cápsulas Bacterianas/metabolismo , Proteínas de Escherichia coli/química , Escherichia coli/metabolismo , Proteínas de Membrana/química , Periplasma/metabolismo , Polissacarídeos Bacterianos/metabolismo , Proteínas Tirosina Quinases/química , Motivos de Aminoácidos , Domínio Catalítico , Microscopia Crioeletrônica , Citoplasma/metabolismo , Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Espectrometria de Massas , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Modelos Moleculares , Periplasma/química , Fosforilação , Conformação Proteica em alfa-Hélice , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Tirosina/química , Tirosina/metabolismo
3.
Mol Cell ; 81(15): 3145-3159.e7, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34214465

RESUMO

Hershey and Chase used bacteriophage T2 genome delivery inside Escherichia coli to demonstrate that DNA, not protein, is the genetic material. Seventy years later, our understanding of viral genome delivery in prokaryotes remains limited, especially for short-tailed phages of the Podoviridae family. These viruses expel mysterious ejection proteins found inside the capsid to form a DNA-ejectosome for genome delivery into bacteria. Here, we reconstitute the phage T7 DNA-ejectosome components gp14, gp15, and gp16 and solve the periplasmic tunnel structure at 2.7 Å resolution. We find that gp14 forms an outer membrane pore, gp15 assembles into a 210 Å hexameric DNA tube spanning the host periplasm, and gp16 extends into the host cytoplasm forming a ∼4,200 residue hub. Gp16 promotes gp15 oligomerization, coordinating peptidoglycan hydrolysis, DNA binding, and lipid insertion. The reconstituted gp15:gp16 complex lacks channel-forming activity, suggesting that the pore for DNA passage forms only transiently during genome ejection.


Assuntos
Bacteriófago T7/genética , DNA Viral/química , Periplasma/química , Proteínas do Core Viral/química , Biologia Computacional , Microscopia Crioeletrônica , Citoplasma/química , DNA Viral/metabolismo , Bicamadas Lipídicas/metabolismo , Periplasma/genética , Periplasma/metabolismo , Podoviridae/química , Podoviridae/genética , Proteínas do Core Viral/metabolismo
4.
Nat Microbiol ; 6(7): 910-920, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34183815

RESUMO

Bacterial species have diverse cell shapes that enable motility, colonization and virulence. The cell wall defines bacterial shape and is primarily built by two cytoskeleton-guided synthesis machines, the elongasome and the divisome. However, the mechanisms producing complex shapes, like the curved-rod shape of Vibrio cholerae, are incompletely defined. Previous studies have reported that species-specific regulation of cytoskeleton-guided machines enables formation of complex bacterial shapes such as cell curvature and cellular appendages. In contrast, we report that CrvA and CrvB are sufficient to induce complex cell shape autonomously of the cytoskeleton in V. cholerae. The autonomy of the CrvAB module also enables it to induce curvature in the Gram-negative species Escherichia coli, Pseudomonas aeruginosa, Caulobacter crescentus and Agrobacterium tumefaciens. Using inducible gene expression, quantitative microscopy and biochemistry, we show that CrvA and CrvB circumvent the need for patterning via cytoskeletal elements by regulating each other to form an asymmetrically localized, periplasmic structure that binds directly to the cell wall. The assembly and disassembly of this periplasmic structure enables dynamic changes in cell shape. Bioinformatics indicate that CrvA and CrvB may have diverged from a single ancestral hybrid protein. Using fusion experiments in V. cholerae, we find that a synthetic CrvA/B hybrid protein is sufficient to induce curvature on its own, but that expression of two distinct proteins, CrvA and CrvB, promotes more rapid curvature induction. We conclude that morphological complexity can arise independently of cell-shape specification by the core cytoskeleton-guided synthesis machines.


Assuntos
Proteínas de Bactérias/metabolismo , Bactérias Gram-Negativas/citologia , Proteínas de Bactérias/genética , Parede Celular/metabolismo , Citoesqueleto/metabolismo , Evolução Molecular , Bactérias Gram-Negativas/crescimento & desenvolvimento , Bactérias Gram-Negativas/metabolismo , Peptidoglicano/metabolismo , Periplasma/metabolismo , Vibrio cholerae/citologia , Vibrio cholerae/crescimento & desenvolvimento , Vibrio cholerae/metabolismo
5.
Appl Microbiol Biotechnol ; 105(11): 4609-4620, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34043081

RESUMO

Escherichia coli represents one of the most widely used hosts for recombinant protein production, but its limited capacity for producing extracellular proteins is often cited as a drawback. NJ7G_0991 is an extracellular protein of the haloarchaeon Natrinema sp. J7-2 and comprises a signal peptide, a putative LolA-like domain, and a C-terminal domain of unknown function. Here, we found that the full-length (0991) and the C-terminal domain-deletion variant (0991ΔC) of NJ7G_0991, but not its signal peptide-deletion variant (0991ΔS), were efficiently released into the culture supernatant of E. coli without extensive cell lysis as determined by ß-galactosidase activity assay. After lysozyme treatment, E. coli cells producing 0991 or 0991ΔC, but not 0991ΔS, were converted from rod-shaped forms to spheres, suggesting that the secretion of 0991 or 0991ΔC into the periplasm leads to an increase of outer membrane permeability of E. coli. A pelB signal peptide was fused to the N-terminus of the LolA-like domain, and the resulting variant PelB-0991ΔC could be released into the culture supernatant of E. coli more efficiently than 0991ΔC. By using PelB-0991ΔC as a co-expression partner, the extracellular production level of a recombinant thermostable subtilase WF146 could be enhanced by up to 14-fold, and the extracellular concentration of an active site variant of WF146 (WF146-SA) reached up to 129 mg/l. To the best of our knowledge, this is the first report on archaeal protein-based co-expression system for extracellular production of recombinant proteins in E. coli. KEY POINTS: • The haloarchaeal protein NJ7G_0991 can be efficiently released into the culture supernatant of E. coli. • The recombinant NJ7G_0991 increases the outer membrane permeability of E. coli. • The LolA-like domain of NJ7G_0991 can be used as a co-expression partner to improve extracellular production of recombinant proteins in E. coli.


Assuntos
Proteínas de Escherichia coli , Proteínas Periplásmicas de Ligação , Permeabilidade da Membrana Celular , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Periplasma/metabolismo , Sinais Direcionadores de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
6.
Appl Environ Microbiol ; 87(13): e0314420, 2021 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-33893117

RESUMO

Recent work with Methylorubrum extorquens AM1 identified intracellular, cytoplasmic lanthanide storage in an organism that harnesses these metals for its metabolism. Here, we describe the extracellular and intracellular accumulation of lanthanides in the Beijerinckiaceae bacterium RH AL1, a newly isolated and recently characterized methylotroph. Using ultrathin-section transmission electron microscopy (TEM), freeze fracture TEM (FFTEM), and energy-dispersive X-ray spectroscopy, we demonstrated that strain RH AL1 accumulates lanthanides extracellularly at outer membrane vesicles (OMVs) and stores them in the periplasm. High-resolution elemental analyses of biomass samples revealed that strain RH AL1 can accumulate ions of different lanthanide species, with a preference for heavier lanthanides. Its methanol oxidation machinery is supposedly adapted to light lanthanides, and their selective uptake is mediated by dedicated uptake mechanisms. Based on transcriptome sequencing (RNA-seq) analysis, these presumably include the previously characterized TonB-ABC transport system encoded by the lut cluster but potentially also a type VI secretion system. A high level of constitutive expression of genes coding for lanthanide-dependent enzymes suggested that strain RH AL1 maintains a stable transcript pool to flexibly respond to changing lanthanide availability. Genes coding for lanthanide-dependent enzymes are broadly distributed taxonomically. Our results support the hypothesis that central aspects of lanthanide-dependent metabolism partially differ between the various taxa. IMPORTANCE Although multiple pieces of evidence have been added to the puzzle of lanthanide-dependent metabolism, we are still far from understanding the physiological role of lanthanides. Given how widespread lanthanide-dependent enzymes are, only limited information is available with respect to how lanthanides are taken up and stored in an organism. Our research complements work with commonly studied model organisms and showed the localized storage of lanthanides in the periplasm. This storage occurred at comparably low concentrations. Strain RH AL1 is able to accumulate lanthanide ions extracellularly and to selectively utilize lighter lanthanides. The Beijerinckiaceae bacterium RH AL1 might be an attractive target for developing biorecovery strategies to obtain these economically highly demanded metals in environmentally friendly ways.


Assuntos
Beijerinckiaceae/metabolismo , Lantânio/metabolismo , Membrana Externa Bacteriana/metabolismo , Proteínas de Bactérias/genética , Beijerinckiaceae/genética , Beijerinckiaceae/ultraestrutura , Regulação Bacteriana da Expressão Gênica , Metanol/metabolismo , Microscopia Eletrônica de Transmissão , Periplasma/metabolismo
7.
Commun Biol ; 4(1): 448, 2021 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-33837253

RESUMO

In Pseudomonas aeruginosa, Ttg2D is the soluble periplasmic phospholipid-binding component of an ABC transport system thought to be involved in maintaining the asymmetry of the outer membrane. Here we use the crystallographic structure of Ttg2D at 2.5 Å resolution to reveal that this protein can accommodate four acyl chains. Analysis of the available structures of Ttg2D orthologs shows that they conform a new substrate-binding-protein structural cluster. Native and denaturing mass spectrometry experiments confirm that Ttg2D, produced both heterologously and homologously and isolated from the periplasm, can carry two diacyl glycerophospholipids as well as one cardiolipin. Binding is notably promiscuous, allowing the transport of various molecular species. In vitro binding assays coupled to native mass spectrometry show that binding of cardiolipin is spontaneous. Gene knockout experiments in P. aeruginosa multidrug-resistant strains reveal that the Ttg2 system is involved in low-level intrinsic resistance against certain antibiotics that use a lipid-mediated pathway to permeate through membranes.


Assuntos
Proteínas de Bactérias/genética , Glicerofosfolipídeos/metabolismo , Proteínas de Membrana Transportadoras/genética , Periplasma/metabolismo , Pseudomonas aeruginosa/genética , Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Farmacorresistência Bacteriana/genética , Proteínas de Membrana Transportadoras/metabolismo , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/metabolismo
8.
Methods Mol Biol ; 2302: 81-99, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33877624

RESUMO

Atomic force microscopy has emerged as a valuable complementary technique in membrane structural biology. The apparatus is capable of probing individual membrane proteins in fluid lipid bilayers at room temperature with spatial resolution at the molecular length scale. Protein conformational dynamics are accessible over a range of biologically relevant timescales. This chapter presents methodology our group uses to achieve robust AFM image data of the General Secretory system, the primary pathway of protein export from the cytoplasm to the periplasm of E. coli. Emphasis is given to measuring and maintaining biochemical activity and to objective AFM image processing methods. For example, the biochemical assays can be used to determine chemomechanical coupling efficiency of surface adsorbed translocases. The Hessian blob algorithm and its extension to nonlocalized linear features, the line detection algorithm, provide automated feature delineations. Many of the methods discussed here can be applied to other membrane protein systems of interest.


Assuntos
Escherichia coli/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Imagem Individual de Molécula/métodos , Algoritmos , Citoplasma/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Microscopia de Força Atômica , Periplasma/metabolismo , Conformação Proteica , Transporte Proteico
9.
J Biol Chem ; 296: 100699, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33895133

RESUMO

N-acetylneuraminate (Neu5Ac), an abundant sugar present in glycans in vertebrates and some bacteria, can be used as an energy source by several prokaryotes, including Escherichia coli. In solution, more than 99% of Neu5Ac is in cyclic form (≈92% beta-anomer and ≈7% alpha-anomer), whereas <0.5% is in the open form. The aldolase that initiates Neu5Ac metabolism in E. coli, NanA, has been reported to act on the alpha-anomer. Surprisingly, when we performed this reaction at pH 6 to minimize spontaneous anomerization, we found NanA and its human homolog NPL preferentially metabolize the open form of this substrate. We tested whether the E. coli Neu5Ac anomerase NanM could promote turnover, finding it stimulated the utilization of both beta and alpha-anomers by NanA in vitro. However, NanM is localized in the periplasmic space and cannot facilitate Neu5Ac metabolism by NanA in the cytoplasm in vivo. We discovered that YhcH, a cytoplasmic protein encoded by many Neu5Ac catabolic operons and belonging to a protein family of unknown function (DUF386), also facilitated Neu5Ac utilization by NanA and NPL and displayed Neu5Ac anomerase activity in vitro. YhcH contains Zn, and its accelerating effect on the aldolase reaction was inhibited by metal chelators. Remarkably, several transition metals accelerated Neu5Ac anomerization in the absence of enzyme. Experiments with E. coli mutants indicated that YhcH expression provides a selective advantage for growth on Neu5Ac. In conclusion, YhcH plays the unprecedented role of providing an aldolase with the preferred unstable open form of its substrate.


Assuntos
Frutose-Bifosfato Aldolase/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Escherichia coli/enzimologia , Frutose-Bifosfato Aldolase/química , Modelos Moleculares , Ácido N-Acetilneuramínico/química , Periplasma/metabolismo , Conformação Proteica , Transporte Proteico , Estereoisomerismo
10.
Biomolecules ; 11(2)2021 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-33669653

RESUMO

The type III secretion system (T3SS) is a virulence apparatus used by many Gram-negative pathogenic bacteria to cause infections. Pathogens utilizing a T3SS are responsible for millions of infections yearly. Since many T3SS knockout strains are incapable of causing systemic infection, the T3SS has emerged as an attractive anti-virulence target for therapeutic design. The T3SS is a multiprotein molecular syringe that enables pathogens to inject effector proteins into host cells. These effectors modify host cell mechanisms in a variety of ways beneficial to the pathogen. Due to the T3SS's complex nature, there are numerous ways in which it can be targeted. This review will be focused on the direct targeting of components of the T3SS, including the needle, translocon, basal body, sorting platform, and effector proteins. Inhibitors will be considered a direct inhibitor if they have a binding partner that is a T3SS component, regardless of the inhibitory effect being structural or functional.


Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias/genética , Bactérias Gram-Negativas/genética , Sistemas de Secreção Tipo III/metabolismo , Virulência , Adenosina Trifosfatases/química , Animais , Antibacterianos/farmacologia , Citoplasma/metabolismo , Ilhas Genômicas , Humanos , Chaperonas Moleculares/química , Peptídeos/química , Periplasma/metabolismo , Ligação Proteica , Domínios Proteicos , Transporte Proteico , Salmonella
11.
Appl Environ Microbiol ; 87(10)2021 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-33741623

RESUMO

Extracellular electron transfer (EET) is an important biological process in microbial physiology as found in dissimilatory metal oxidation/reduction and interspecies electron transfer in syntrophy in natural environments. EET also plays a critical role in microorganisms relevant to environmental biotechnology in metal-contaminated areas, metal corrosion, bioelectrochemical systems, and anaerobic digesters. Geobacter species exist in a diversity of natural and artificial environments. One of the outstanding features of Geobacter species is the capability of direct EET with solid electron donors and acceptors, including metals, electrodes, and other cells. Therefore, Geobacter species are pivotal in environmental biogeochemical cycles and biotechnology applications. Geobacter sulfurreducens, a representative Geobacter species, has been studied for direct EET as a model microorganism. G. sulfurreducens employs electrically conductive pili (e-pili) and c-type cytochromes for the direct EET. The biological function and electronics applications of the e-pili have been reviewed recently, and this review focuses on the cytochromes. Geobacter species have an unusually large number of cytochromes encoded in their genomes. Unlike most other microorganisms, Geobacter species localize multiple cytochromes in each subcellular fraction, outer membrane, periplasm, and inner membrane, as well as in the extracellular space, and differentially utilize these cytochromes for EET with various electron donors and acceptors. Some of the cytochromes are functionally redundant. Thus, the EET in Geobacter is complicated. Geobacter coordinates the cytochromes with other cellular components in the elaborate EET system to flourish in the environment.


Assuntos
Citocromos/metabolismo , Geobacter/metabolismo , Membrana Externa Bacteriana/metabolismo , Transporte de Elétrons , Membranas Intracelulares/metabolismo , Periplasma/metabolismo
12.
Biochemistry ; 60(6): 465-476, 2021 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-33538578

RESUMO

The anaerobic bacterium Chrysiogenes arsenatis respires using the oxyanion arsenate (AsO43-) as the terminal electron acceptor, where it is reduced to arsenite (AsO33-) while concomitantly oxidizing various organic (e.g., acetate) electron donors. This respiratory activity is catalyzed in the periplasm of the bacterium by the enzyme arsenate reductase (Arr), with expression of the enzyme controlled by a sensor histidine kinase (ArrS) and a periplasmic-binding protein (PBP), ArrX. Here, we report for the first time, the molecular structure of ArrX in the absence and presence of bound ligand arsenate. Comparison of the ligand-bound structure of ArrX with other PBPs shows a high level of conservation of critical residues for ligand binding by these proteins; however, this suite of PBPs shows different structural alterations upon ligand binding. For ArrX and its homologue AioX (from Rhizobium sp. str. NT-26), which specifically binds arsenite, the structures of the substrate-binding sites in the vicinity of a conserved and critical cysteine residue contribute to the discrimination of binding for these chemically similar ligands.


Assuntos
Arseniato Redutases/química , Bactérias/metabolismo , Sequência de Aminoácidos/genética , Arseniato Redutases/metabolismo , Arseniatos/química , Arseniatos/metabolismo , Bactérias/química , Composição de Bases/genética , Sítios de Ligação , Catálise , Cristalografia por Raios X/métodos , Histidina Quinase/metabolismo , Oxirredutases/metabolismo , Periplasma/metabolismo , Proteínas Periplásmicas de Ligação/química , Proteínas Periplásmicas de Ligação/metabolismo , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA/métodos
13.
Microbiology (Reading) ; 167(3)2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33620307

RESUMO

Rhodobacter sphaeroides can use C4-dicarboxylic acids to grow heterotrophically or photoheterotropically, and it was previously demonstrated in Rhodobacter capsulatus that the DctPQM transporter system is essential to support growth using these organic acids under heterotrophic but not under photoheterotrophic conditions. In this work we show that in R. sphaeroides this transporter system is essential for photoheterotrophic and heterotrophic growth, when C4-dicarboxylic acids are used as a carbon source. We also found that over-expression of dctPQM is detrimental for photoheterotrophic growth in the presence of succinic acid in the culture medium. In agreement with this, we observed a reduction of the dctPQM promoter activity in cells growing under these conditions, indicating that the amount of DctPQM needs to be reduced under photoheterotrophic growth. It has been reported that the two-component system DctS and DctR activates the expression of dctPQM. Our results demonstrate that in the absence of DctR, dctPQM is still expressed albeit at a low level. In this work, we have found that the periplasmic component of the transporter system, DctP, has a role in both transport and in signalling the DctS/DctR two-component system.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Periplasma/metabolismo , Rhodobacter sphaeroides/metabolismo , Proteínas de Bactérias/genética , Transporte Biológico , Ácidos Dicarboxílicos/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos da radiação , Processos Heterotróficos , Luz , Proteínas de Membrana Transportadoras/genética , Periplasma/genética , Processos Fototróficos , Regiões Promotoras Genéticas , Rhodobacter sphaeroides/genética , Rhodobacter sphaeroides/crescimento & desenvolvimento , Rhodobacter sphaeroides/efeitos da radiação , Ácido Succínico/metabolismo
14.
Proc Natl Acad Sci U S A ; 118(1)2021 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-33443152

RESUMO

O antigens are important cell surface polysaccharides in gram-negative bacteria where they extend core lipopolysaccharides in the extracellular leaflet of the outer membrane. O antigen structures are serotype specific and form extended cell surface barriers endowing many pathogens with survival benefits. In the ABC transporter-dependent biosynthesis pathway, O antigens are assembled on the cytosolic side of the inner membrane on a lipid anchor and reoriented to the periplasmic leaflet by the channel-forming WzmWzt ABC transporter for ligation to the core lipopolysaccharides. In many cases, this process depends on the chemical modification of the O antigen's nonreducing terminus, sensed by WzmWzt via a carbohydrate-binding domain (CBD) that extends its nucleotide-binding domain (NBD). Here, we provide the cryo-electron microscopy structure of the full-length WzmWzt transporter from Aquifex aeolicus bound to adenosine triphosphate (ATP) and in a lipid environment, revealing a highly asymmetric transporter organization. The CBDs dimerize and associate with only one NBD. Conserved loops at the CBD dimer interface straddle a conserved peripheral NBD helix. The CBD dimer is oriented perpendicularly to the NBDs and its putative ligand-binding sites face the transporter to likely modulate ATPase activity upon O antigen binding. Further, our structure reveals a closed WzmWzt conformation in which an aromatic belt near the periplasmic channel exit seals the transporter in a resting, ATP-bound state. The sealed transmembrane channel is asymmetric, with one open and one closed cytosolic and periplasmic portal. The structure provides important insights into O antigen recruitment to and translocation by WzmWzt and related ABC transporters.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Bactérias/metabolismo , Antígenos O/biossíntese , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , Transporte Biológico , Membrana Celular/metabolismo , Microscopia Crioeletrônica/métodos , Hidrólise , Lipopolissacarídeos/metabolismo , Antígenos O/metabolismo , Periplasma/metabolismo , Domínios Proteicos
15.
Nat Microbiol ; 6(2): 221-233, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33432152

RESUMO

Three classes of ion-driven protein motors have been identified to date: ATP synthase, the bacterial flagellar motor and a proton-driven motor that powers gliding motility and the type 9 protein secretion system in Bacteroidetes bacteria. Here, we present cryo-electron microscopy structures of the gliding motility/type 9 protein secretion system motors GldLM from Flavobacterium johnsoniae and PorLM from Porphyromonas gingivalis. The motor is an asymmetric inner membrane protein complex in which the single transmembrane helices of two periplasm-spanning GldM/PorM proteins are positioned inside a ring of five GldL/PorL proteins. Mutagenesis and single-molecule tracking identify protonatable amino acid residues in the transmembrane domain of the complex that are important for motor function. Our data provide evidence for a mechanism in which proton flow results in rotation of the periplasm-spanning GldM/PorM dimer inside the intra-membrane GldL/PorL ring to drive processes at the bacterial outer membrane.


Assuntos
Proteínas de Bactérias/química , Sistemas de Secreção Bacterianos/química , Flavobacterium/fisiologia , Porphyromonas gingivalis/fisiologia , Microscopia Crioeletrônica , Flavobacterium/metabolismo , Movimento , Periplasma/metabolismo , Porphyromonas gingivalis/metabolismo , Domínios Proteicos , Multimerização Proteica , Prótons , Imagem Individual de Molécula
16.
Microbiology (Reading) ; 167(3)2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33502310

RESUMO

Biofilm formation in the human intestinal pathogen Vibrio cholerae is in part regulated by norspermidine, spermidine and spermine. V. cholerae senses these polyamines through a signalling pathway consisting of the periplasmic protein, NspS, and the integral membrane c-di-GMP phosphodiesterase MbaA. NspS and MbaA belong to a proposed class of novel signalling systems composed of periplasmic ligand-binding proteins and membrane-bound c-di-GMP phosphodiesterases containing both GGDEF and EAL domains. In this signal transduction pathway, NspS is hypothesized to interact with MbaA in the periplasm to regulate its phosphodiesterase activity. Polyamine binding to NspS likely alters this interaction, leading to the activation or inhibition of biofilm formation depending on the polyamine. The purpose of this study was to determine the amino acids important for NspS function. We performed random mutagenesis of the nspS gene, identified mutant clones deficient in biofilm formation, determined their responsiveness to norspermidine and mapped the location of these residues onto NspS homology models. Single mutants clustered on two lobes of the NspS model, but the majority were found on a single lobe that appeared to be more mobile upon norspermidine binding. We also identified residues in the putative ligand-binding site that may be important for norspermidine binding and interactions with MbaA. Ultimately, our results provide new insights into this novel signalling pathway in V. cholerae and highlight differences between periplasmic binding proteins involved in transport versus signal transduction.


Assuntos
Proteínas de Bactérias/genética , Biofilmes , Vibrio cholerae/genética , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Mutagênese , Periplasma/genética , Periplasma/metabolismo , Domínios Proteicos , Alinhamento de Sequência , Transdução de Sinais , Vibrio cholerae/química , Vibrio cholerae/fisiologia
17.
J Biosci Bioeng ; 131(1): 47-52, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-32994133

RESUMO

Aconitic acid, an unsaturated tricarboxylic acid, is used in the chemical industry as raw materials for organic synthesis, especially as a specific substrate for a flavoring agent. trans-Aconitic acid (tAA) is a trans-isomer of cis-aconitic acid and detected in some plants and bacteria. However, biosynthetic route and metabolism of tAA in relation to assimilation have been unknown. Aconitate isomerase (AI; EC 5.3.3.7) catalyzes the reversible isomerization between cis-aconitic acid and tAA. Pseudomonas sp. WU-0701 was isolated as a bacterium assimilating tAA as sole carbon source, and characterization and gene identification of AI were already reported. Here, we describe that Pseudomonas sp. WU-0701 exhibited growth in each synthetic medium containing glucose, citric acid, isocitric acid, or tAA as sole carbon source. AI was intracellularly detected all the time during the cultivation of the strain WU-0701 cells, irrespective of the carbon sources; AI activity was detected even in the glucose-grown cells. Through the subcellular fractionation experiments, AI was detected in the periplasmic fraction. This is the first report indicating that a bacterium belonging to the genus Pseudomonas is constitutive for the AI production.


Assuntos
Ácido Aconítico/metabolismo , Isomerases/biossíntese , Pseudomonas/metabolismo , Periplasma/metabolismo , Pseudomonas/citologia
18.
Appl Environ Microbiol ; 87(2)2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33127815

RESUMO

Gluconobacter oxydans has the unique property of a glucose oxidation system in the periplasmic space, where glucose is oxidized incompletely to ketogluconic acids in a nicotinamide cofactor-independent manner. Elimination of the gdhM gene for membrane-bound glucose dehydrogenase, the first enzyme for the periplasmic glucose oxidation system, induces a metabolic change whereby glucose is oxidized in the cytoplasm to acetic acid. G. oxydans strain NBRC3293 possesses two molecular species of type II NADH dehydrogenase (NDH), the primary and auxiliary NDHs that oxidize NAD(P)H by reducing ubiquinone in the cell membrane. The substrate specificities of the two NDHs are different from each other: primary NDH (p-NDH) oxidizes NADH specifically but auxiliary NDH (a-NDH) oxidizes both NADH and NADPH. We constructed G. oxydans NBRC3293 derivatives defective in the ndhA gene for a-NDH, in the gdhM gene, and in both. Our ΔgdhM derivative yielded higher cell biomass on glucose, as reported previously, but grew at a lower rate than the wild-type strain. The ΔndhA derivative showed growth behavior on glucose similar to that of the wild type. The ΔgdhM ΔndhA double mutant showed greatly delayed growth on glucose, but its cell biomass was similar to that of the ΔgdhM strain. The double mutant accumulated intracellular levels of NAD(P)H and thus shifted the redox balance to reduction. Accumulated NAD(P)H levels might repress growth on glucose by limiting oxidative metabolisms in the cytoplasm. We suggest that a-NDH plays a crucial role in redox homeostasis of nicotinamide cofactors in the absence of the periplasmic oxidation system in G. oxydans IMPORTANCE Nicotinamide cofactors NAD+ and NADP+ mediate redox reactions in metabolism. Gluconobacter oxydans, a member of the acetic acid bacteria, oxidizes glucose incompletely in the periplasmic space-outside the cell. This incomplete oxidation of glucose is independent of nicotinamide cofactors. However, if the periplasmic oxidation of glucose is abolished, the cells oxidize glucose in the cytoplasm by reducing nicotinamide cofactors. Reduced forms of nicotinamide cofactors are reoxidized by NADH dehydrogenase (NDH) on the cell membrane. We found that two kinds of NDH in G. oxydans have different substrate specificities: the primary enzyme is NADH specific, and the auxiliary one oxidizes both NADH and NADPH. Inactivation of the latter enzyme in G. oxydans cells in which we had induced cytoplasmic glucose oxidation resulted in elevated intracellular levels of NAD(P)H, limiting cell growth on glucose. We suggest that the auxiliary enzyme is important if G. oxydans grows independently of the periplasmic oxidation system.


Assuntos
Gluconobacter oxydans/enzimologia , NADH Desidrogenase/metabolismo , NADP/metabolismo , NAD/metabolismo , Gluconobacter oxydans/genética , Gluconobacter oxydans/metabolismo , Homeostase , Niacinamida/metabolismo , Oxirredução , Periplasma/metabolismo
19.
J Phycol ; 57(1): 295-310, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33128798

RESUMO

Periplasmic or external carbonic anhydrases (CAs) have been well accepted as playing a crucial role in the acquisition of dissolved inorganic carbon; however, no cytological evidence or molecular information on these enzymes has been reported in seaweeds to date. In this study, the full-length cDNA sequence coding for a putative periplasmic Sjα-CA2 was cloned from the gametophytes of Saccharina japonica, an industrial brown seaweed. It was 1,728 bp in length and included a 263-bp 5'-untranslated region (UTR), a 577-bp 3'-UTR, and an 888-bp open reading frame encoding a protein precursor consisting of 295 amino acids. The mature protein, after removal of a predicted 28-residue signal peptide, was composed of 267 amino acids with a relative molecular weight of 29.27 kDa. Multisequence alignment and phylogenetic analysis indicated that it was a member of the α-CA family. Enzyme activity assays showed that the recombinant Sjα-CA2 in Escherichia coli possessed CO2 hydration and esterase activities, thus identifying this gene Sjα-CA2 in function. Immunogold electron microscopic observations with the prepared anti-Sjα-CA2 polyclonal antibody illustrated that Sjα-CA2 was located in periplasmic space of the kelp gametophyte cells. Quantitative real-time PCR results revealed that the transcription of Sjα-CA2 was induced by elevated HCO 3 - levels, but it was little changed while the kelp gametophytes were subjected to elevated CO2 concentrations. This study suggests that the periplasmic Sjα-CA2 might play a role in adapting to elevated environmental levels of HCO 3 - by dehydration of HCO 3 - to generate CO2 , which could be readily taken up by S. japonica gametophytes.


Assuntos
Anidrases Carbônicas , Feófitas , Sequência de Aminoácidos , Anidrases Carbônicas/genética , Anidrases Carbônicas/metabolismo , Células Germinativas Vegetais/metabolismo , Periplasma/metabolismo , Feófitas/genética , Feófitas/metabolismo , Filogenia
20.
Biochim Biophys Acta Biomembr ; 1863(2): 183502, 2021 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-33130098

RESUMO

The YidC insertase of Escherichia coli inserts membrane proteins with small periplasmic loops (~20 residues). However, it has difficulty transporting loops that contain positively charged residues compared to negatively charged residues and, as a result, increasing the positive charge has an increased requirement for the Sec machinery as compared to negatively charged loops (Zhu et al., 2013; Soman et al., 2014). This suggested that the polarity and charge of the periplasmic regions of membrane proteins determine the YidC and Sec translocase requirements for insertion. Here we tested this polarity/charge hypothesis by showing that insertion of our model substrate protein procoat-Lep can become YidC/Sec dependent when the periplasmic loop was converted to highly polar even in the absence of any charged residues. Moreover, adding a number of hydrophobic amino acids to a highly polar loop can decrease the Sec-dependence of the otherwise strictly Sec-dependent membrane proteins. We also demonstrate that the length of the procoat-Lep loop is indeed a determinant for Sec-dependence by inserting alanine residues that do not markedly change the overall hydrophilicity of the periplasmic loop. Taken together, the results support the polarity/charge hypothesis as a determinant for the translocase requirement for procoat insertion.


Assuntos
Membrana Celular/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Periplasma/metabolismo , Canais de Translocação SEC/metabolismo , Animais , Linhagem Celular , Membrana Celular/química , Membrana Celular/genética , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Camundongos , Periplasma/química , Periplasma/genética , Estrutura Secundária de Proteína , Canais de Translocação SEC/química , Canais de Translocação SEC/genética
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