Two Regions of EpsL Involved in Species-Specific Protein-Protein Interactions with EpsE and EpsM of the General Secretion Pathway inVibrio cholerae

ABSTRACT Extracellular secretion of proteins via the type II or general secretion pathway in gram-negative bacteria requires the assistance of at least 12 gene products that are thought to form a complex apparatus through which secreted proteins are translocated. Although this apparatus is specifically required only for the outer membrane translocation step during transport across the bacterial cell envelope, it is believed to span both membranes. The EpsE, EpsL, and EpsM proteins of the type II apparatus in Vibrio cholerae are thought to form a trimolecular complex that is required to either control the opening and closing of the secretion pore or to transduce energy to the site of outer membrane translocation. EpsL is likely to play an important role in this relay by interacting with both the cytoplasmic EpsE protein and the cytoplasmic membrane protein EpsM, which is predominantly exposed on the periplasmic side of the membrane. We have now extended this model and mapped the separate regions within EpsL that contain the EpsE and EpsM binding domains. By taking advantage of the species specificity of the type II pathway, we have used chimeric proteins composed of EpsL and its homologue, ExeL, fromAeromonas hydrophila together with either EpsE or itsAeromonas homologue, ExeE, to complement the secretion defect in both epsL and exeL mutant strains. These studies have mapped the species-specific EpsE binding site to the N-terminal cytoplasmic region between residues 57 and 216 of EpsL. In addition, the species-specific EpsM binding site was mapped to the C-terminal half of EpsL by coimmunoprecipitation of EpsM with different EpsL-ExeL chimeras. This site is present in the region between amino acids 216 and 296, which contains the predicted membrane-spanning segment of EpsL.

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Soluble versions of outer membrane cytochromes function as exporters for heterologously produced cargo proteins

Microbial Cell Factories ◽

10.1186/s12934-019-1270-2 ◽

2019 ◽

Vol 18 (1) ◽

Author(s):

Helge M. Dietrich ◽

Miriam Edel ◽

Thea Bursac ◽

Manfred Meier ◽

Katrin Sturm-Richter ◽

...

Keyword(s):

Outer Membrane ◽

Culture Supernatant ◽

Cellulase Activity ◽

Type Ii Secretion ◽

Type Ii ◽

Recognition Sequence ◽

Secretion Pathway ◽

N Terminus ◽

Cargo Proteins ◽

Lipid Anchor

AbstractThis study reveals that it is possible to secrete truncated versions of outer membrane cytochromes into the culture supernatant and that these proteins can provide a basis for the export of heterologously produced proteins. Different soluble and truncated versions of the outer membrane cytochrome MtrF were analyzed for their suitability to be secreted. A protein version with a very short truncation of the N-terminus to remove the recognition sequence for the addition of a lipid anchor is secreted efficiently to the culture supernatant, and moreover this protein could be further truncated by a deletion of 160 amino acid and still is detectable in the supernatant. By coupling a cellulase to this soluble outer membrane cytochrome, the export efficiency was measured by means of relative cellulase activity. We conclude that outer membrane cytochromes of S. oneidensis can be applied as transporters for the export of target proteins into the medium using the type II secretion pathway.

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Pairing your Sox: Identification of Sox11 partner proteins and interaction domains in the developing neural plate

10.1101/2020.04.23.057919 ◽

2020 ◽

Author(s):

Kaela S. Singleton ◽

Pablo Silva-Rodriguez ◽

Elena M. Silva

Keyword(s):

Protein Interactions ◽

Neural Development ◽

Specific Protein ◽

Neural Plate ◽

Neural Fate ◽

Mouse Cortex ◽

Interaction Domains ◽

Species Specific ◽

And Function ◽

Partner Proteins

AbstractSox11, a member of the SoxC family of transcription factors, has distinct functions at different times in neural development. Studies in mouse, frog, chick and zebrafish show that Sox11 promotes neural fate, neural differentiation, and neuron maturation in the central nervous system. These diverse roles are controlled in part by spatial and temporal-specific protein interactions. However, the partner proteins and Sox11-interaction domains underlying these diverse functions are not well defined. Here, we identify partner proteins and the domains of Xenopus Sox11(xSox11) required for protein interaction and function during neurogenesis. Our data show that Sox11 co-localizes and interacts with Pou3f2 and Ngn2 in the anterior neural plate and in early neurons, respectively. We also demonstrate that xSox11 does not interact with Ngn1, a high affinity partner of Sox11 in the mouse cortex, suggesting that Sox11 has species-specific partner proteins. Additionally, we determined that the N-terminus including the HMG domain of xSox11 is necessary for interaction with Pou3f2 and Ngn2, and established a novel role for the N-terminal 46 amino acids in the establishment of placodal progenitors. This is the first identification of partner proteins for Xenopus Sox11 and of domains required for partner protein interactions and distinct roles in neurogenesis.

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Core architecture of a bacterial type II secretion system

Nature Communications ◽

10.1038/s41467-019-13301-3 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 13

Author(s):

Anastasia A. Chernyatina ◽

Harry H. Low

Keyword(s):

Outer Membrane ◽

Cell Envelope ◽

Type Ii Secretion ◽

Stoichiometric Ratio ◽

Type Ii ◽

Secretion Systems ◽

Modular Architecture ◽

Membrane Complex ◽

Negative Stain ◽

Bacterial Type

AbstractBacterial type II secretion systems (T2SSs) translocate virulence factors, toxins and enzymes across the cell outer membrane. Here we use negative stain and cryo-electron microscopy to reveal the core architecture of an assembled T2SS from the pathogen Klebsiella pneumoniae. We show that 7 proteins form a ~2.4 MDa complex that spans the cell envelope. The outer membrane complex includes the secretin PulD, with all domains modelled, and the pilotin PulS. The inner membrane assembly platform components PulC, PulE, PulL, PulM and PulN have a relative stoichiometric ratio of 2:1:1:1:1. The PulE ATPase, PulL and PulM combine to form a flexible hexameric hub. Symmetry mismatch between the outer membrane complex and assembly platform is overcome by PulC linkers spanning the periplasm, with PulC HR domains binding independently at the secretin base. Our results show that the T2SS has a highly dynamic modular architecture, with implication for pseudo-pilus assembly and substrate loading.

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Roles of pgaABCD Genes in Synthesis, Modification, and Export of the Escherichia coli Biofilm Adhesin Poly-β-1,6-N-Acetyl-d-Glucosamine

Journal of Bacteriology ◽

10.1128/jb.01920-07 ◽

2008 ◽

Vol 190 (10) ◽

pp. 3670-3680 ◽

Cited By ~ 165

Author(s):

Yoshikane Itoh ◽

John D. Rice ◽

Carlos Goller ◽

Archana Pannuri ◽

Jeannette Taylor ◽

...

Keyword(s):

Escherichia Coli ◽

Outer Membrane ◽

Protein Interactions ◽

Biofilm Formation ◽

Cell Envelope ◽

Outer Membrane Proteins ◽

Attachment Site ◽

Covalent Modification ◽

Carbohydrate Binding ◽

K 12

ABSTRACT The linear homopolymer poly-β-1,6-N-acetyl-d-glucosamine (β-1,6-GlcNAc; PGA) serves as an adhesin for the maintenance of biofilm structural stability in diverse eubacteria. Its function in Escherichia coli K-12 requires the gene products of the pgaABCD operon, all of which are necessary for biofilm formation. PgaC is an apparent glycosyltransferase that is required for PGA synthesis. Using a monoclonal antibody directed against E. coli PGA, we now demonstrate that PgaD is also needed for PGA formation. The deletion of genes for the predicted outer membrane proteins PgaA and PgaB did not prevent PGA synthesis but did block its export, as shown by the results of immunoelectron microscopy (IEM) and antibody adsorption assays. IEM also revealed a conditional localization of PGA at the cell poles, the initial attachment site for biofilm formation. PgaA contains a predicted β-barrel porin and a superhelical domain containing tetratricopeptide repeats, which may mediate protein-protein interactions, implying that it forms the outer membrane secretin for PGA. PgaB contains predicted carbohydrate binding and polysaccharide N-deacetylase domains. The overexpression of pgaB increased the primary amine content (glucosamine) of PGA. Site-directed mutations targeting the N-deacetylase catalytic activity of PgaB blocked PGA export and biofilm formation, implying that N-deacetylation promotes PGA export through the PgaA porin. The results of previous studies indicated that N-deacetylation of β-1,6-GlcNAc in Staphylococcus epidermidis by the PgaB homolog, IcaB, anchors it to the cell surface. The deletion of icaB resulted in release of β-1,6-GlcNAc into the growth medium. Thus, covalent modification of β-1,6-GlcNAc by N-deacetylation serves distinct biological functions in gram-negative and gram-positive species, dictated by cell envelope differences.

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Exchange of Xcp (Gsp) Secretion Machineries betweenPseudomonas aeruginosa and Pseudomonas alcaligenes: Species Specificity Unrelated to Substrate Recognition

Journal of Bacteriology ◽

10.1128/jb.183.3.959-967.2001 ◽

2001 ◽

Vol 183 (3) ◽

pp. 959-967 ◽

Cited By ~ 27

Author(s):

Arjan de Groot ◽

Margot Koster ◽

Manon Gérard-Vincent ◽

Gijs Gerritse ◽

Andrée Lazdunski ◽

...

Keyword(s):

Secretory Pathway ◽

Gram Negative Bacteria ◽

Gene Products ◽

Type Ii ◽

Gram Negative ◽

Secretion Pathway ◽

Heat Stable ◽

Pseudomonas Alcaligenes ◽

Mutant Complementation ◽

Species Specific

ABSTRACT Pseudomonas aeruginosa and Pseudomonas alcaligenes are gram-negative bacteria that secrete proteins using the type II or general secretory pathway, which requires at least 12 xcp gene products (XcpA and XcpP to -Z). Despite strong conservation of this secretion pathway, gram-negative bacteria usually cannot secrete exoproteins from other species. Based on results obtained with Erwinia, it has been proposed that the XcpP and/or XcpQ homologs determine this secretion specificity (M. Linderberg, G. P. Salmond, and A. Collmer, Mol. Microbiol. 20:175–190, 1996). In the present study, we report that XcpP and XcpQ of P. alcaligenes could not substitute for their respectiveP. aeruginosa counterparts. However, these complementation failures could not be correlated to species-specific recognition of exoproteins, since these bacteria could secrete exoproteins of each other. Moreover, when P. alcaligenes xcpP andxcpQ were expressed simultaneously in a P. aeruginosa xcpPQ deletion mutant, complementation was observed, albeit only on agar plates and not in liquid cultures. After growth in liquid culture the heat-stable P. alcaligenes XcpQ multimers were not detected, whereas monomers were clearly visible. Together, our results indicate that the assembly of a functional Xcp machinery requires species-specific interactions between XcpP and XcpQ and between XcpP or XcpQ and another, as yet uncharacterized component(s).

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LIM domains of cysteine-rich protein 1 (CRP1) are essential for its zyxin-binding function

Biochemical Journal ◽

10.1042/bj3310885 ◽

1998 ◽

Vol 331 (3) ◽

pp. 885-892 ◽

Cited By ~ 20

Author(s):

Karen L. SCHMEICHEL ◽

Mary C. BECKERLE

Keyword(s):

Binding Site ◽

Protein Interactions ◽

Specific Protein ◽

Binding Activity ◽

Structural Motif ◽

Lim Domain ◽

Lim Domains ◽

Cysteine Rich Protein ◽

Binding Function ◽

Domain Mapping

Previous studies have demonstrated that the adhesion-plaque protein, zyxin, interacts specifically with a 23 kDa protein, called the cysteine-rich protein 1 (CRP1), which has been implicated in myogenesis. Primary sequence analyses have revealed that both zyxin and CRP1 exhibit multiple copies of a structural motif called the LIM domain. LIM domains, which are defined by the consensus CX2CX16–23HX2CX2CX2CX16–23CX2–3(C,H,D), are found in a variety of proteins that are involved in cell growth and differentiation. Recent studies have established that LIM domains are zinc-binding structures that mediate specific protein–protein interactions. For example, in the case of the zyxin–CRP1 interaction, one of zyxin's three LIM domains is necessary and sufficient for binding to CRP1. Because the CRP1 molecule is comprised primarily of two LIM domains, we were interested in the possibility that the binding site for zyxin on CRP1 might also be contained within a single LIM domain. Consistent with the hypothesis that the LIM domains of CRP1 are critical for the protein's zyxin-binding function, zinc-depleted CRP1 displays a reduced zyxin-binding activity. However, domain mapping analyses have revealed that neither of the two individual LIM domains of CRP1 can support a wild-type interaction with zyxin. Collectively, our results suggest that the binding site for zyxin on CRP1 is not contained within a single contiguous sequence of amino acids. Instead, the interaction appears to rely on the co-ordinate action of a number of residues that are displayed in both of CRP1's LIM domains.

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Cardiolipin aids in lipopolysaccharide transport to the gram-negative outer membrane

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2018329118 ◽

2021 ◽

Vol 118 (15) ◽

pp. e2018329118

Author(s):

Martin V. Douglass ◽

François Cléon ◽

M. Stephen Trent

Keyword(s):

Outer Membrane ◽

Protein Interactions ◽

Lipid A ◽

Cell Envelope ◽

Transport Systems ◽

Gram Negative ◽

Membrane Asymmetry ◽

Synthetic Lethal ◽

Bacterial Fitness ◽

Synthetically Lethal

In Escherichia coli, cardiolipin (CL) is the least abundant of the three major glycerophospholipids in the gram-negative cell envelope. However, E. coli harbors three distinct enzymes that synthesize CL: ClsA, ClsB, and ClsC. This redundancy suggests that CL is essential for bacterial fitness, yet CL-deficient bacteria are viable. Although multiple CL–protein interactions have been identified, the role of CL still remains unclear. To identify genes that impact fitness in the absence of CL, we analyzed high-density transposon (Tn) mutant libraries in combinatorial CL synthase mutant backgrounds. We found LpxM, which is the last enzyme in lipid A biosynthesis, the membrane anchor of lipopolysaccharide (LPS), to be critical for viability in the absence of clsA. Here, we demonstrate that CL produced by ClsA enhances LPS transport. Suppressors of clsA and lpxM essentiality were identified in msbA, a gene that encodes the indispensable LPS ABC transporter. Depletion of ClsA in ∆lpxM mutants increased accumulation of LPS in the inner membrane, demonstrating that the synthetic lethal phenotype arises from improper LPS transport. Additionally, overexpression of ClsA alleviated ΔlpxM defects associated with impaired outer membrane asymmetry. Mutations that lower LPS levels, such as a YejM truncation or alteration in the fatty acid pool, were sufficient in overcoming the synthetically lethal ΔclsA ΔlpxM phenotype. Our results support a model in which CL aids in the transportation of LPS, a unique glycolipid, and adds to the growing repertoire of CL–protein interactions important for bacterial transport systems.

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Isolation of hMRE11B: failure to complement yeast mre11 defects due to species-specific protein interactions

Gene ◽

10.1016/s0378-1119(98)00530-7 ◽

1998 ◽

Vol 225 (1-2) ◽

pp. 107-116 ◽

Cited By ~ 8

Author(s):

Mahmood Chamankhah ◽

Ying-Fei Wei ◽

Wei Xiao

Keyword(s):

Protein Interactions ◽

Specific Protein ◽

Species Specific

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Direct Interaction of the EpsL and EpsM Proteins of the General Secretion Apparatus in Vibrio cholerae

Journal of Bacteriology ◽

10.1128/jb.181.10.3129-3135.1999 ◽

1999 ◽

Vol 181 (10) ◽

pp. 3129-3135 ◽

Cited By ~ 72

Author(s):

Maria Sandkvist ◽

Lloyd P. Hough ◽

Mira M. Bagdasarian ◽

Michael Bagdasarian

Keyword(s):

Outer Membrane ◽

Vibrio Cholerae ◽

Cytoplasmic Membrane ◽

Cell Envelope ◽

Gel Filtration ◽

Direct Interaction ◽

Stable Complex ◽

Extracellular Secretion ◽

Triton X

ABSTRACT The general secretion pathway of gram-negative bacteria is responsible for extracellular secretion of a number of different proteins, including proteases and toxins. This pathway supports secretion of proteins across the cell envelope in two distinct steps, in which the second step, involving translocation through the outer membrane, is assisted by at least 13 different gene products. Two of these components, the cytoplasmic membrane proteins EpsL and EpsM ofVibrio cholerae, have been purified and characterized. Based on gel filtration analysis, both purified EpsM(His)6 and wild-type EpsL present in anEscherichia coli Triton X-100 extract are dimeric proteins. EpsL and EpsM were also found to interact directly and form a Triton X-100 stable complex that could be precipitated with either anti-EpsL or anti-EpsM antibodies. In addition, when the L and M proteins were coexpressed in E. coli, they formed a stable complex and protected each other from proteolytic degradation, indicating that these two proteins interact in vivo and that no other Eps protein is required for their association. Since EpsL is predicted to contain a large cytoplasmic domain, while EpsM is predominantly exposed on the periplasmic side, we speculate that these components might be part of a structure that is involved in bridging the inner and outer membranes. Furthermore, since EpsL has previously been shown to interact with the autophosphorylating cytoplasmic membrane protein EpsE, we hypothesize that this trimolecular complex might be involved in regulating the opening and closing of the secretion pore and/or transducing energy to the site of outer membrane translocation.

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Picornaviral polymerase domain exchanges reveal a modular basis for distinct biochemical activities of viral RNA-dependent RNA polymerases

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.013906 ◽

2020 ◽

Vol 295 (31) ◽

pp. 10624-10637 ◽

Cited By ~ 1

Author(s):

Colleen L. Watkins ◽

Brian J. Kempf ◽

Stéphanie Beaucourt ◽

David J. Barton ◽

Olve B. Peersen

Keyword(s):

Protein Interactions ◽

Active Sites ◽

Specific Protein ◽

Rna Polymerases ◽

Viral Fitness ◽

Structural Basis ◽

Protein Protein Interactions ◽

Elongation Complex ◽

Domain Interactions ◽

Species Specific

Picornaviral RNA-dependent RNA polymerases (RdRPs) have low replication fidelity that is essential for viral fitness and evolution. Their global fold consists of the classical “cupped right hand” structure with palm, fingers, and thumb domains, and these RdRPs also possess a unique contact between the fingers and thumb domains. This interaction restricts movements of the fingers, and RdRPs use a subtle conformational change within the palm domain to close their active sites for catalysis. We have previously shown that this core RdRP structure and mechanism provide a platform for polymerases to fine-tune replication rates and fidelity to optimize virus fitness. Here, we further elucidated the structural basis for differences in replication rates and fidelity among different viruses by generating chimeric RdRPs from poliovirus and coxsackievirus B3. We designed these chimeric polymerases by exchanging the fingers, pinky finger, or thumb domains. The results of biochemical, rapid-quench, and stopped-flow assays revealed that differences in biochemical activity map to individual modular domains of this polymerase. We found that the pinky finger subdomain is a major regulator of initiation and that the palm domain is the major determinant of catalytic rate and nucleotide discrimination. We further noted that thumb domain interactions with product RNA regulate translocation and that the palm and thumb domains coordinately control elongation complex stability. Several RdRP chimeras supported the growth of infectious poliovirus, providing insights into enterovirus species–specific protein–protein interactions required for virus replication.

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