What is the difference between transport proteins and effector proteins in gram-negative bacteria?

What is the difference between transport proteins and effector proteins in gram-negative bacteria?

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When considering pathogenic gram-negative bacteria, is there any difference between the function of transport proteins and effector proteins? Or are they of the same functionality? Any reference would be very helpful.

Is there any difference between the function of transport proteins and effector proteins?

Yes. In this context, the term "effector proteins" refers to proteins that are inserted into a host cell on infection to modulate host cell processes. Effector proteins are inserted into a host cell using a secretion system. The proteins involved in the secretion system are transport proteins, but the effector proteins themselves have a wide variety of functions. They include enzymes, transcription factors, and protein-protein interaction partners, and they have been shown to regulate many host cellular processes, from metabolism, to vesicular trafficking, cell adhesion, and apoptosis.

You can read more about this here. This review has an interesting perspective, and the Wikipedia page on Bacterial effector proteins isn't bad either.

One contribution of 12 to a theme issue ‘The bacterial cell envelope’.

Published by the Royal Society. All rights reserved.


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Porins are composed of beta sheets (β sheets) made up of beta strands (β strands) which are linked together by beta turns on the cytoplasmic side and long loops of amino acids on the other. The β strands lie in an antiparallel fashion and form a cylindrical tube, called a beta barrel (β barrel). [2] The amino acid composition of the porin β strands are unique in that polar and nonpolar residues alternate along them. This means that the nonpolar residues face outward so as to interact with the nonpolar lipids of outer membrane, whereas the polar residues face inwards into the center of the beta barrel to create the aqueous channel. The specific amino acids in the channel determine the specificity of the porin to different molecules.

The β barrels that make up a porin are composed of as few as eight β strands to as many as twenty-two β strands. The individual strands are joined together by loops and turns. [3] The majority of porins are monomers however, some dimeric porins have been discovered, as well as an octameric porin. [4] Depending on the size of the porin, the interior of the protein may either be filled with water, have up to two β strands folded back into the interior, or contain a "stopper" segment composed of β strands.

All porins form homotrimers in the outer membrane, meaning that three identical porin subunits associate together to form a porin super-structure with three channels. [5] Hydrogen bonding and dipole-dipole interactions between each monomer in the homotrimer ensure that they do not dissociate, and remain together in the outer membrane.

Several parameters have been used to describe the structure of a porin protein. They include the tilting angle (α), shear number (S), strand number (n), and barrel radius (R). [6] The tilting angle refers to the angle relative to the membrane. The shear number (S) is the number of amino acid residues found in each β strands. Strand number (n) is the amount of β strands in the porin, and barrel radius (R) refers to the radius of the opening of the porin. These parameters are related via the following formulas:

Using these formulas, the structure of a porin can be determined by knowing only a few of the available parameters. While the structure of many porins have been determined using X-ray crystallography, the alternative method of sequencing protein primary structure may also be used instead.

Porins are water-filled pores and channels found in the membranes of bacteria and eukaryotes. Porin-like channels have also been discovered in archaea. [7] Note that the term "nucleoporin" refers to unrelated proteins that facilitate transport through nuclear pores in the nuclear envelope.

Porins are primarily involved in passively transporting hydrophilic molecules of various sizes and charges across the membrane. [8] For survival, certain required nutrients and substrates must be transported into the cells. Likewise, toxins and wastes must be transported out to avoid toxic accumulation. [9] Additionally, porins can regulate permeability and prevent lysis by limiting the entry of detergents into the cell. [8]

Two types of porins exist to transport different materials– general and selective. General porins have no substrate specificities, though some exhibit slight preferences for anions or cations. [8] Selective porins are smaller than general porins, and have specificities for chemical species. These specificities are determined by the threshold sizes of the porins, and the amino acid residues lining them. [5]

In gram-negative bacteria, the inner membrane is the major permeability barrier. [10] The outer membrane is more permeable to hydrophilic substances, due to the presence of porins. [5] Porins have threshold sizes of transportable molecules that depend on the type of bacteria and porin. Generally, only substances less than 600 Daltons in size can diffuse through. [8]

Porins were first discovered in gram-negative bacteria, but gram-positive bacteria with both types of porins have been found. [9] They exhibit similar transport functions but have a more limited variety of porins, compared to the distribution found in gram-negative bacteria. [9] Gram-positive bacteria lack outer membranes, so these porin channels are instead bound to specific lipids within the cell walls. [7]

Porins are also found in eukaryotes, specifically in the outer membranes of mitochondria and chloroplasts. [9] [10] The organelles contain general porins that are structurally and functionally similar to bacterial ones. These similarities have supported the Endosymbiotic theory, through which eukaryotic organelles arose from gram-negative bacteria. [10] However, eukaryotic porins exhibit the same limited diversity as gram-positive porins, and also display a greater voltage-dependent role during metabolism. [9] [10]

Archaea also contain ion channels that have originated from general porins. [7] The channels are found in the cell envelope and help facilitate solute transfer. They have similar characteristics as bacterial and mitochondrial porins, indicating physiological overlaps over all three domains of life. [7]

Many porins are targets for host immune cells, resulting in signaling pathways that lead to bacterial degradation. Therapeutic treatments, like vaccinations and antibiotics, are used to supplement this immune response. [5] Specific antibiotics have been designed to travel through porins in order to inhibit cellular processes. [8]

However, due to selective pressure, bacteria can develop resistance through mutations in the porin gene. [5] The mutations may lead to a loss of porins, resulting in the antibiotics having a lower permeability or being completely excluded from transport. These changes have contributed to the global emergence of antibiotic resistance, and an increase in mortality rates from infections. [5]

The discovery of porins has been attributed to Hiroshi Nikaido, nicknamed "the porinologist." [11]

According to TCDB, there are five evolutionarily independent superfamilies of porins. Porin superfamily I includes 47 families of porins with a range of numbers of trans-membrane β-strands (β-TMS). These include the GBP, SP and RPP porin families. While PSF I includes 47 families, PSF II-V each contain only 2 families. While PSF I derives members from gram-negative bacteria primarily one family of eukaryotic mitochondrial porins, PSF II and V porins are derived from Actinobacteria. PSF III and V are derived from eukaryotic organelle. [12] [13]

Porin Superfamily I Edit

1.B.1 - The General bacterial porin family
1.B.2 - The Chlamydial Porin (CP) Family
1.B.3 - The Sugar porin (SP) Family
1.B.4 - The Brucella-Rhizobium porin (BRP) Family
1.B.5 - The Pseudomonas OprP Porin (POP) Family
1.B.6 - OmpA-OmpF porin (OOP) family
1.B.7 Rhodobacter PorCa porin (RPP) family
1.B.8 Mitochondrial and plastid porin (MPP) family
1.B.9 FadL outer membrane protein (FadL) family
1.B.10 Nucleoside-specific channel-forming outer membrane porin (Tsx) family
1.B.11 Outer membrane fimbrial usher porin (FUP) family
1.B.12 Autotransporter-1 (AT-1) family
1.B.13 Alginate export porin (AEP) family
1.B.14 Outer membrane receptor (OMR) family
1.B.15 Raffinose porin (RafY) family
1.B.16 Short chain amide and urea porin (SAP) family
1.B.17 Outer membrane factor (OMF) family
1.B.18 Outer membrane auxiliary (OMA) protein family
1.B.19 Glucose-selective OprB porin (OprB) family
1.B.20 Two-partner secretion (TPS) family
1.B.21 OmpG porin (OmpG) family
1.B.22 Outer bacterial membrane secretin (secretin) family
1.B.23 Cyanobacterial porin (CBP) family
1.B.24 Mycobacterial porin
1.B.25 Outer membrane porin (Opr) family
1.B.26 Cyclodextrin porin (CDP) family
1.B.31 Campylobacter jejuni major outer membrane porin (MomP) family
1.B.32 Fusobacterial outer membrane porin (FomP) family
1.B.33 Outer membrane protein insertion porin (Bam complex) (OmpIP) family
1.B.34 Corynebacterial porins
1.B.35 Oligogalacturonate-specific porin (KdgM) family
1.B.39 Bacterial porin, OmpW (OmpW) family
1.B.42 - The Outer Membrane Lipopolysaccharide Export Porin (LPS-EP) Family
1.B.43 - The Coxiella Porin P1 (CPP1) Family
1.B.44 - The Probable Protein Translocating Porphyromonas gingivalis Porin (PorT) Family
1.B.49 - The Anaplasma P44 (A-P44) Porin Family
1.B.54 - Intimin/Invasin (Int/Inv) or Autotransporter-3 family
1.B.55 - The Poly Acetyl Glucosamine Porin (PgaA) Family
1.B.57 - The Legionella Major-Outer Membrane Protein (LM-OMP) Family
1.B.60 - The Omp50 Porin (Omp50 Porin) Family
1.B.61 - The Delta-Proteobacterial Porin (Delta-Porin) Family
1.B.62 - The Putative Bacterial Porin (PBP) Family
1.B.66 - The Putative Beta-Barrel Porin-2 (BBP2) Family
1.B.67 - The Putative Beta Barrel Porin-4 (BBP4) Family
1.B.68 - The Putative Beta Barrel Porin-5 (BBP5) Superfamily
1.B.70 - The Outer Membrane Channel (OMC) Family
1.B.71 - The Proteobacterial/Verrucomicrobial Porin (PVP) Family
1.B.72 - The Protochlamydial Outer Membrane Porin (PomS/T) Family
1.B.73 - The Capsule Biogenesis/Assembly (CBA) Family
1.B.78 - The DUF3374 Electron Transport-associated Porin (ETPorin) Family

Porin Superfamily II (MspA Superfamily) Edit

1.B.24 - Mycobacterial porin
1.B.58 - Nocardial Hetero-oligomeric Cell Wall Channel (NfpA/B) Family

Porin Superfamily III Edit

1.B.28 - The Plastid Outer Envelope Porin of 24 kDa (OEP24) Family
1.B.47 - The Plastid Outer Envelope Porin of 37 kDa (OEP37) Family

Porin Superfamily IV (Tim17/OEP16/PxMPL (TOP) Superfamily) Edit

This superfamily includes protein that comprise pores in multicomponent protein translocases as follows: 3.A.8 - [Tim17 (P39515) Tim22 (Q12328) Tim23 (P32897)] 1.B.69 - [PXMP4 (Q9Y6I8) PMP24 (A2R8R0)] 3.D.9 - [NDH 21.3 kDa component (P25710)]

1.B.30 - The Plastid Outer Envelope Porin of 16 kDa (OEP16) Family
1.B.69 - The Peroxysomal Membrane Porin 4 (PxMP4) Family
3.A.8 - The Mitochondrial Protein Translocase (MPT) Family

Porin Superfamily V (Corynebacterial PorA/PorH Superfamily) Edit

1.B.34 - The Corynebacterial Porin A (PorA) Family 1.B.59 - The Outer Membrane Porin, PorH (PorH) Family


Gram-negative bacteria are surrounded by two membrane bilayers separated by a space termed the periplasm. The periplasm is a multipurpose compartment separate from the cytoplasm whose distinct reducing environment allows more efficient and diverse mechanisms of protein oxidation, folding, and quality control. The periplasm also contains structural elements and important environmental sensing modules, and it allows complex nanomachines to span the cell envelope. Recent work indicates that the size or intermembrane distance of the periplasm is controlled by periplasmic lipoproteins that anchor the outer membrane to the periplasmic peptidoglycan polymer. This periplasm intermembrane distance is critical for sensing outer membrane damage and dictates length of the flagellar periplasmic rotor, which controls motility. These exciting results resolve longstanding debates about whether the periplasmic distance has a biological function and raise the possibility that the mechanisms for maintenance of periplasmic size could be exploited for antibiotic development.

Citation: Miller SI, Salama NR (2018) The gram-negative bacterial periplasm: Size matters. PLoS Biol 16(1): e2004935.

Published: January 17, 2018

Copyright: © 2018 Miller, Salama. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: National Institutes of Health (grant number R01AI054423). Received by NRS. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. National Institutes of Health (grant number 5U19AI107775). Received by SIM. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Abbreviations: ABC, ATP-binding cassette IM, inner membrane Lpp, Braun’s lipoprotein LPS, lipopolysaccharide OM, outer membrane PG, peptidoglycan RcsF, Regulator of capsule synthesis F

Provenance: Commissioned externally peer reviewed.

Gram-negative bacteria, like the energy organelles of plants and animals (the chloroplast and mitochondria), have two membrane bilayers termed the outer and inner membranes. The space between these two membranes is termed the periplasm. Long before single-cell eukaryotes, the periplasm evolved as the first extracytoplasmic compartment to provide an important competitive adaption to gram-negative bacteria. Early knowledge and the discovery of the periplasm developed even before its morphological visualization. In the 1960s, scientists were trying to understand how toxic enzymes involved in degradation of important biological molecules, such as ribonucleases and phosphatases produced by the gram-negative bacteria Escherichia coli, were not toxic to the cell. Biochemical extraction methods suggested a separate compartment, because such extraction preserved the inner membrane-bound cytoplasm, and these spheroplasts could grow again and synthesize more enzymes [1]. The development of electron microscopy led to the visualization of the two membrane bilayers separated by the periplasm [2].

The additional membrane allows for the creation of the periplasm as a separate cellular compartment whose novel functions likely provided a significant and perhaps even more important selective advantage than toxin exclusion (Table 1). These novel functions include protein transport, folding, oxidation, and quality control similar to the eukaryotic cell endoplasmic reticulum. The periplasm also allows for the sequestration of enzymes that may be toxic in the cytoplasm, important signaling functions, and cell division regulation. Additionally, it contributes to the ability of the cell to withstand turgor pressure by providing structural systems that work in concert with the outer membrane, such as peptidoglycan and lipoproteins, multidrug efflux systems, and specific solutes that contribute to a Donnan or ionic potential across the outer membrane. The periplasm also contains the assembly platforms involved in secretion of uniquely structured beta-barrel proteins, lipoproteins, and glycerolphospholipids to the outer membrane (Fig 1).

Shown is the asymmetric bilayer of lipopolysaccharide and glycerolphospholipids that comprise the outer membrane. The inner membrane is a symmetric bilayer of glycerolphospholipids. The periplasmic space is the region between these membranes that includes a variety of enzymes and functions, including the oxidation and quality control of proteins. Also within the periplasmic space is a layer of crosslinked sugars and amino acids termed peptidoglycan, which surrounds the cell. The peptidoglycan is linked to the outer membrane in enteric bacteria through covalent transpeptidase linkages between an abundant outer membrane lipoprotein Lpp. A variety of sensors sit in the inner membrane with periplasmic domains sensing environmental change and, in the case of the Rcs system, a change in location of the RcsF outer membrane lipoprotein. Multicomponent protein complexes such as the flagellar machine span the two membranes. IM, inner membrane Lpp, Braun’s lipoprotein LPS, lipopolysaccharide RcsF, Regulator of capsule synthesis F.

The outer membrane is a unique organelle connected to other parts of the cell envelope via the periplasm. Gram-positive bacteria lack an outer membrane but have a more extensive peptidoglycan polymer protecting their surface. In contrast to the bacterial inner membrane—which is a bilayer of glycerolphospholipids similar to that of most mammalian membranes and which has specific flow characterized by lateral diffusion—the outer membrane has restricted flow [3]. It is a unique bilayer, with the inner leaflet having a typical glycerolphospholipid content of phosphotidylethanolamine, phosphatidylglycerol, and cardiolipin and the outer leaflet largely composed of a unique glycolipid, lipopolysaccharide (LPS) [4]. The LPS phosphates confer a negative charge to the surface, and a specific Donnan potential is created across the outer membrane into the periplasm [5]. The outer membrane functions as a selective barrier that allows the transport of valuable nutrients while providing a barrier against toxic compounds, such as cationic antimicrobial compounds produced by all organisms, including many gram-positive bacteria [6]. Another component of this barrier are outer membrane proteins with a unique beta-barrel structure that are inserted into the outer membrane through a specific periplasmic chaperone system [7]. These proteins assemble into the outer membrane as specific puncta, indicating the outer membrane likely assembles into specific discrete patches containing protein and the unique asymmetric lipid bilayer [8]. Included among these outer membrane proteins are the porins, which can act as selective channels that allow hydrophilic substrates of a specific size entrance to the periplasm. Luckily for humans, these porins transport hydrophilic beta-lactam antibiotics, which allows their penetration into the periplasm, where they target the synthesis of the important structural element of the cell wall—the polymeric peptidoglycan. The outer membrane in some bacteria is anchored to the peptidoglycan polymer through abundant lipoproteins, which are inserted into the inner leaflet of the outer membrane through specific secretion systems [9]. A variety of important protein complexes function as nanomachines and utilize ATP hydrolysis to secrete macromolecules or turn a motility organelle termed the flagella [10,11,12]. Therefore, the outer membrane and the inner membrane are also connected across the periplasm by membrane-spanning protein complexes. Hence, the outer membrane is composed of distinctly assembled patches that comprise a complex organelle that can be attached to the peptidoglycan layer and the inner membrane through covalent and noncovalent protein linkages. The assembly of the outer membrane and its link to the peptidoglycan and cytoplasm creates a space between the inner membrane and the outer membrane, which is the periplasm.

Despite the important functions contained within the periplasmic space, for many years there has been debate about the intermembrane distance or size of this compartment and whether there is uniformity of spacing between the inner and outer membranes throughout the cell. There was concern that many of the visualizations of this space as being of a specific size were artifacts of fixation for imaging by electron microscopy and that, in fact, the space was actually only a potential space. The early electron microscopic studies of Bayer demonstrated adhesions between the outer and inner membrane that obliterated part of these spaces he suggested that points of adhesion were areas where the major outer leaflet lipid, LPS, was delivered to the outer membrane from its site of synthesis at the inner membrane [13]. However, his work was subsequently discredited as being derived from observation of potential fixation artifacts, though many experts today think that there may be real protein-based adhesions between the membranes because some efflux and transport systems do not contain components of sufficient dimensions to span the visualized space. The presence of specific areas in which the membranes are close together would explain how some of these ATP-binding cassette (ABC) transport and efflux pumps could work these systems have periplasmic protein components that are essential for efflux, LPS, or other glycolipid transport but lack an intrinsic size or polymeric nature large enough to reach the outer membrane and thus provide a mechanism to promote transport. Furthermore, the periplasm contains many other components that necessitate at least some volume for the periplasmic space, most prominently the peptidoglycan polymeric layer surrounding the cell. At present, it is unclear how these transporters get around this polymer and the width of the periplasm to contact the membrane, though recent work demonstrating that outer membrane lipoproteins can coordinate peptidoglycan synthesis through direct contact indicates that at least some proteins may fit through pores in peptidoglycan to accomplish important functions [14]

In contrast, a variety of organelles, including the flagellum and the virulence-associated Type III secretion system needle complex, require the assembly of polymers within the periplasm that span the two membranes. In the case of the flagellum, its rod or driveshaft spans the periplasm, and its length is determined by the polymer contacting the outer membrane. Elegant recent work by the group of Kelly Hughes has shown that the size of the periplasm, or the distance between the two membranes, is controlled largely in enteric bacteria by a specific lipoprotein termed Braun’s lipoprotein (or Lpp), which covalently links the outer membrane to the peptidoglycan layer [15]. This is quite remarkable because Lpp is the most abundant protein present in enteric bacteria, described by Braun 48 years ago, and until this point no specific function had been ascribed to it. This alpha-helical protein is inserted through its lipid anchor into the inner leaflet of the outer membrane and covalently linked to the peptidoglycan polymer by a family of transpeptidases [16]. Lengthening these lipoproteins that allow expansion of the periplasm leads to a longer flagellar rod and more efficient swimming behavior. These authors interpreted this result as indicating that there must be other evolutionarily selected functions that limited the periplasmic size, forcing a reduction in swimming efficiency. In this issue of PLOS Biology, one of those important functions is revealed: a signaling function of envelope damage controlled by another outer membrane lipoprotein, Regulator of capsule synthesis F (RcsF), which senses disorder or damage of the envelope.

Gram-negative bacteria have a variety of important functions that sense membrane damage and toxic compounds, such as antimicrobial peptides, which damage the outer membrane [17,18]. These sensing systems include those that allow remodeling of the bacterial surface to be more resistant to toxic compounds—analogous to spaceships energizing their shields in science fiction stories [19]. Some of these sensing systems are receptors that function as sensor kinases with domains in the periplasm to sense specific molecules or damage. However, one of the more unique sensor kinase systems, termed the Rcs system—which on membrane damage activates synthesis of extracellular polysaccharide to provide cellular protection and biofilm formation—has an outer membrane lipoprotein RcsF, which interacts with signaling proteins with specific periplasmic domains on envelope damage and peptidoglycan stress to activate the synthesis of extracellular polysaccharide production and other stress-related coping pathways [20]. Thus, envelope damage in some way brings the RcsF lipoprotein in greater proximity to the inner membrane-sensing system, and thus it evolved to sense disorder in the outer membrane and/or peptidoglycan (Fig 2). In this issue of PLOS Biology, the authors conclusively demonstrate that this sensing requires the periplasm to be a specific size because mutations that lengthen the highly abundant Lpp lipoprotein anchor from the outer membrane to the peptidoglycan (resulting in an increased size of the periplasm) abolished signaling unless the sensing lipoprotein (which on membrane damage must reach to the inner membrane sensor) is also lengthened [21]. This work also clearly shows a very specific order and size to the periplasm the size of the periplasm is clearly seen as it exists in association with the changes in lipoprotein anchoring or length by cryo-electron microscopy. This technology and electron tomography used in the work of the Hughes group in relation to the flagellar rotor [15] are revolutionizing our view of the bacterial cell envelope and the protein complexes that span the periplasm to perform important functions [22].

Molecular Recombination

In each of the cases of HGT, the process is only successful if the genes can be expressed by the altered cell. In conjugation, the genes are located on a plasmid, under the control of promoters on the plasmid. In transformation and transduction, where naked DNA is gaining access to the cell, the DNA could easily be broken down by the cell with no genetic expression occurring. In order for the genes to be expressed, the DNA must be recombined with the recipient’s chromosome.

The most common mechanism of molecular recombination is homologous recombination, involving the RecA protein. In this process DNA from two sources are paired, based on similar nucleotide sequence in one area. An endonuclease nicks one strand, allowing RecA to pair up bases from different strands, a process known as strand invasion. The cross-over between DNA molecules is resolved with resolvase, which cuts and rejoins the DNA into two separate dsDNA molecules.

Recombination can also occur using site-specific recombination, a process often used by viruses to insert their genome into the chromosome of their host. This type of recombination is also used by transposable elements (see next section).

Membrane insertion

Historically, considerably more attention has been devoted to how proteins get across membranes than to how they insert into them. In part, this has been due to the experimental intractability of membrane insertion unlike protein translocation, one cannot simply look in a supernatant fraction to see how much protein has appeared there. The subject of membrane insertion was given a boost, however, with the discovery that bacteria possess an essential protein, YidC ( Samuelson et al., 2000 ), that is similar to Oxa1, a protein involved in the insertion of mitochondrial inner membrane proteins. As explained by Ross Dalbey from Ohio State University, Columbus, YidC depletion turns out to have quite pleiotropic effects on protein traffic in bacteria. Some of these effects are direct, while others are due to defects in the insertion of YidC-dependent proteins involved in energy transduction and translocation ( Yi et al., 2003 ). Interestingly, YidC seems to operate both in conjunction with ( Houben et al., 2002 ) and without ( van der Laan et al., 2004 ) the SRP-Sec system. Exactly how YidC operates remains a mystery.

The translocation role of the Sec machinery is now well established and the crystal structure of one such machinery has helped conceptualize models for how it works. Transmembrane segments of integral inner membrane proteins that use the Sec machinery in E. coli must leave the translocon and slip laterally into the membrane. This process presumably occurs through sideways opening of the translocation channel to the lipid bilayer ( Osborne et al., 2005 ). The orientation of successive transmembrane segments is determined by charged amino acids on either side of these hydrophobic anchors and, as explained by Mikhail Bogdanov from the University of Texas, Houston, by charged phospholipids. Incorrect topology results from phosphatidylethanolamine (PE) depletion in vivo (usually, one transmembrane segment loops out of the membrane and subsequent segments are in the wrong order). This can be mimicked by reconstituting membrane proteins into phospholipid vesicles in vitro. Furthermore, the incorrect topology can be corrected by adding back the missing PE, implying that, contrary to established dogma, topology is not irrevocably fixed in time and space ( Zhang et al., 2003 ).

Despite substantial effort by a number of groups, the mechanisms of outer membrane protein (OMP) insertion remained only vaguely understood until the breakthrough discovery of the role played by the essential Omp85/YaeT protein ( Voulhoux et al., 2003 Wu et al., 2005 ). In the talks by Tom Silhavy (Princeton University) and Jan Tommassen (University of Utrecht), we learned that Omp85/YaeT is required for the insertion of many, and possibly all β-barrel OMPs and, in E. coli at least, is associated with several lipoproteins, some of which are also essential cell components ( Malinverni et al., 2006 ). Jan Tommassen demonstrated that, in artificial lipid bilayers, E. coli YaeT forms channels whose activity can be modulated by the C-terminal amphipathic β strand from an outer membrane porin. This is a very significant finding because Tommassen's previous work showed the critical importance of this segment of OMPs for their insertion ( Struyve et al., 1991 ). Why YaeT should form a channel is not immediately clear, and neither is the way it promotes insertion. One intriguing possibility is that the POTRA repeat domains ( Sanchez-Pulido et al., 2003 ), of which there are five in the large predicted periplasmic domain of YaeT, interact with successive amphipathic β strands as they arrive at the outer membrane. As all OMPs characterized to date have less than 20 transmembrane segments, there would have to be four YaeT monomers per complex to accommodate a single OMP. According to Tommassen, Omp85/YaeT does appear to be tetrameric, although the number of protomers per complex might vary to accommodate proteins with different numbers of transmembrane segments.

Thus, the POTRA domains could form a periplasmic cavity in which the OMP β strands are correctly organized (note that, in contrast to membrane proteins with α-helical transmembrane segments, which are often arranged in non-linear fashion, transmembrane segments in OMPs are organized in numerical order around the walls of the barrel) prior to insertion. Once the complete barrel has assembled, it might slide perpendicularly into the membrane within the Omp85/YaeT superstructure, which would then dissociate to release the OMP into the membrane, where multimerization could occur.

Biogenesis of ATs

Transport Across Membranes and β-Barrel Insertion

Like most OM proteins, ATs follow a conserved pathway in their biogenesis.

Autotransporters are translated in the cytosol where the polypeptide chain is kept in an unfolded state by the help of chaperones and translocated across the inner membrane (IM) into the periplasm by the SecYEG translocon (Sijbrandi et al., 2003 Tsirigotaki et al., 2017). An N-terminal signal sequence ensures proper recognition of the AT as a Sec target, and targeting and secretion through the IM and signal peptide cleavage after transport works in the same way as for other Sec-secreted proteins (Papanikou et al., 2007). Some ATs, like Hbp and AIDA-I, show an extended Sec signal sequence which might aid in slowing down IM translocation and thus in prevention of premature folding and aggregation of the AT within the periplasm (Henderson et al., 1998 Szabady et al., 2005 Jacob-Dubuisson et al., 2013). For type Vb systems, it has been shown that some TpsA passengers aggregate much faster than others and therefore retaining the AT bound to the Sec is beneficial Otp is a protein which is not prone to aggregation and therefore does not require fast transport to the OM (Choi and Bernstein, 2010). In other systems like FHA, quick secretion is of importance as degradation of unfolded FHA by DegP is more likely due to the length of the FHA precursor (Baud et al., 2009).

In the periplasm, ATs are kept unfolded but in a folding-competent state, shielded from aggregation by periplasmic chaperones like SurA, Skp and DegP (Baud et al., 2009 Ieva and Bernstein, 2009 Oberhettinger et al., 2012 Pavlova et al., 2013 Weirich et al., 2017). Insertion of the β-barrel domain of ATs is then facilitated by the β-barrel assembly machinery (BAM) complex (Jain and Goldberg, 2007 Leo et al., 2012). In E. coli, it is composed of five subunits, BamA through BamE. This complex interacts with most if not all OM integral β-barrel proteins (Lee et al., 2018). The 16-stranded β-barrel integral membrane protein BamA helps in insertion of the substrate barrel into the OM by a not yet entirely understood mechanism (Schiffrin et al., 2017). For type Va ATs, it has been clearly shown by crosslinking experiments that the 12-stranded β-barrel membrane anchor folds and inserts into the OM aided directly by the BAM complex. The passenger of EspP, an E. coli AT, for example, can be crosslinked to periplasmic chaperones, as well as to its β-barrel domain and to BamA (Ieva and Bernstein, 2009 Pavlova et al., 2013). Similarly, type Vc and Ve ATs interact with the Bam complex, as shown for YadA and Invasin (Roggenkamp et al., 2003 Oberhettinger et al., 2015).

Passenger Secretion

While most other bacterial secretion systems have access to energy sources like proton gradients across the IM or are directly energized by cytoplasmic ATP, ATs only span the OM, which is too leaky for ion gradients, and the periplasm is devoid of ATP (Nikaido and Vaara, 1985 Silhavy et al., 2006). Various models for how the secretion and folding process of passengers is energized have been proposed. One plausible explanation is that the energy for transport comes from the intrinsic folding capacity of the AT itself, either directly driving export or leading to a Brownian ratchet model where, once secreted, the passenger cannot slide back into the periplasm and is therefore driven to move outside the cell and fold (Henderson et al., 2004 Choi and Bernstein, 2010). Furthermore, asymmetric charge distribution within the passenger has been put forward as a possible driving factor for passenger secretion (Kang𠆞the and Bernstein, 2013).

Passenger transport and secretion differ slightly between the various AT subclasses due to differences in domain organization. In type Va ATs, the passenger is transported via a C-terminus-first mechanism. According to the widely accepted hairpin-loop model of secretion, a hairpin-loop is formed at the C-terminus of the passenger in the interior of the β-barrel, followed by sequential folding of the passenger on the cell surface starting from the C-terminus (Junker et al., 2006). This was shown for multiple members of the type Va AT subclass, including Pertactin, Hbp and EspP (Junker et al., 2009 Peterson et al., 2010 Soprova et al., 2010).

For type Vb secretion, models are somewhat different since in the TPSSs the β-barrel domain is separated from the passenger domain. After the TpsB transporter is properly inserted into the OM by the BAM complex, recognition of TpsA by TpsB is provided by interaction of the TpsB POTRAs and the N-terminal TPS signal of TpsA (Baud et al., 2009). The TPS signal is a conserved stretch with an amphipathic character that remains unfolded in the periplasm. Association and dissociation rates of the TPS signal with the TpsB POTRA domains are high based on surface plasmon resonance experiments, making the interaction transient, and helping in later release of the TpsA substrate from its transporter (Delattre et al., 2010 Guérin et al., 2017). NMR experiments have shown similar highly dynamic interactions (Garnett et al., 2015). Crosslinking experiments have further shown that the TPS signal interacts with the TpsB POTRA domains, as well as some central amino acids within the barrel lumen (Baud et al., 2014). Similarly to all other Type V secretion systems, it is assumed that during transport, TpsA is unfolded as it passes through the central pore of the TpsB barrel and that folding of the substrate occurs during exit from the transporter barrel.

There are two different models for how the export of the TpsA is initiated: one is that, like in other ATs, a hairpin is formed within the barrel pore driving folding of the secreted substrate in a C-to-N direction. Release of the TpsB-bound TPS domain would then occur at the end of secretion, after major parts of TpsA have already folded (Pavlova et al., 2013 Norell et al., 2014). In this case, the high on/off rate between the PORTA domains and the TPS signal domain would facilitate the release that is based on the pulling forces generated by the folding process itself (Guérin et al., 2017). According to the second model, the N-terminal TPS domain nucleates folding, i.e., the TPS domain is exported first and the rest of the protein folds N-to-C (Hodak and Jacob-Dubuisson, 2007). The fact that the TpsA proteins’ N-terminal domain can also fold independently bolsters this argument (Clantin et al., 2004, 2007).

In type Vc ATs, passenger secretion is more intricate due to the trimeric nature of the proteins. Three passenger polypeptide chains have to be orchestrated through a comparatively narrow β-barrel domain. After formation of the 12-stranded β-barrel, the passenger is transported to the exterior of the cell starting with the formation of a hairpin loop of each of the three passenger domains followed by folding of the coiled coil stalk (Linke et al., 2006 Szczesny and Lupas, 2008 Mikula et al., 2012 Chauhan et al., 2019). Transport of three distinct polypeptide chains in a hairpin loop conformation across a comparably small barrel might be sterically challenging. The interior of type Vc β-barrels contains many glycine and alanine residues which have small side chains, and it has been suggested that this facilitates passage of multiple chains though the barrel interior (Mikula et al., 2012). Additionally, β-barrel proteins are not necessarily fully rigid pores. The capacity of 𠇋reathing” movement without breakage of the hydrogen bonding has already been shown for the usher protein FimD, which in its apo-structure is more narrow than when bound to a transport substrate (Phan et al., 2011). Similar breathing behavior would be necessary in type Vc autotransport to accommodate all chains simultaneously. An additional problem comes with the highly intertwined passenger structure in type Vc systems. Sequential folding after initial hairpin formation would build up mechanical strain. It has been shown for some examples that an YxD/RxD motif toward the C-terminus of the passenger helps in initiation of passenger folding and folding outside the membrane anchor, potentially by releasing mechanical strain. YxD motifs furthermore stabilize right-handed coiled-coils whereas RxD motifs support left-handed coiled-coils (Alvarez et al., 2010). In addition, while the core residues of coiled-coil proteins are generally hydrophobic, some trimeric AT passengers contain hydrophilic residues in these positions. These residues can coordinate anions, which might allow sequences that are otherwise not easily folded to interact and stabilize (Hartmann et al., 2009 Leo et al., 2011).

It is not yet entirely clear how passenger secretion works in type Vd systems, and what role the POTRA domain plays in this (Salacha et al., 2010). It might function either as a chaperone for the passenger, aiding in secretion, or aiding in the recruitment of proteases for passenger cleavage. In some strains of F. nucleatum the passenger domain of FplA seems to be cleaved off while in other strains this could not be shown (Casasanta et al., 2017). It is unclear whether proteolytic cleavage of the passenger of type Vd ATs is achieved via autoproteolysis, like in some type Va ATs, or via an independent protease, like in the example of the NalP cleaving the type Va AT IgA protease for release from its β-barrel domain (Salacha et al., 2010 Casasanta et al., 2017). However, the fact that type Vd passengers remain uncleaved in some strains and when heterologously expressed in E. coli supports the latter interpretation (Salacha et al., 2010).

The biogenesis of type Ve ATs is similar to the one of type Va ATs. Although the topology of type Ve ATs is inverted, the β-barrel functions as a transport pore in an analogous way via formation of a hairpin-loop, and the passenger is secreted in a very similar fashion to the passenger secretion of classical ATs, but in the opposite direction (N-to-C rather than C-to-N) (Oberhettinger et al., 2012, 2015). Folding is energized by sequential folding of the extracellular Ig-like domains, as shown for the example of Intimin (Leo et al., 2016).


Exotoxins are discharged from bacterial cells and known as the most poisonous substance able to cause disease. These are heat-sensitive proteins. Gram-positive and Gram-negative bacteria both have the ability to produce exotoxin. The exotoxin production is diversified in a few strains of bacterial species while mostly it is the same in all. In a few species, toxin production is associated with the lysogenic phase. There is a diverse range of exotoxin which are classified according to their site of action namely they are cytotoxin, a neurotoxin, enterotoxin, leukotoxin.


DMEM, Dulbecco’s Modified Eagle Medium DSS, dextran sulfate sodium DTT, dithiothreitol EcN, Escherichia coli Nissle 1917 FBS, fetal bovine serum LB, Luria-Bertani broth LPS, lipopolysaccharide MAMPs, microbial-associated molecular patterns NLR, NOD-like receptor NOD, nucleotide oligomerization domain OMVs, outer membrane vesicles PAGE, polyacrylamide gel electrophoresis PBS, phosphate buffer saline PG, peptidoglycan PRR, pattern recognition receptor RIP2, receptor-interacting protein 2 RT-qPCR, quantitative reverse transcription PCR SDS, sodium dodecyl sulfate TLR, Toll-like receptor ZOs, zonula occludens.

Abstract: The objective of these series of experiments was to identify two unknown bacteria’s. Broth culture #20 was selected and subjected to qualitative te.

The subunits are produced separately in the nucleolus, released by the nuclear envelope out into the cytosol, and then synthesized. They are made of rRNA, or.

Cell wall- Prokaryotic cells have a cell wall whereas eukaryotic cells don’t. The cell wall is what provides shape and protects the cell components. Nucleus-.

The terms prokaryotes and eukaryotes are in reference to where the DNA is actually housed. Eukaryotic cells are found within organism which are single-celled.

In prokaryotes, the DNA is in the cytoplasm, but specifically it is concentrated in the nucleoid region. In eukaryotes, the DNA is all contained and stored i.

Scheme 2: β-lactam antibiotic mechanism with DD-transpeptidase STAND IN. Therefore, the covalent bond between the β-lactam antibiotic and the DD-transpeptida.

The cell wall is composed of several layers of peptidoglycan which are held together by teichoic acids, which gives the cell wall a negative charge. Teichoic.

It 's often called the “protein factories” for the cell. Ribosomes join together with with amino acids and those two together is what makes proteins. They’re.

This test is done to determine whether a zone of inhibition is seen. The size of the zone of inhibition will be dependent on how effective the antibiotic is .

Gene expression by definition is involved with protein synthesis and RNA originates from DNA, and then along with ribosomes are able to transcribe and transl.