Porins Of Gram-negative Bacteria Research Articles

Zellwandkanäle von Corynebacteriales

In addition to a thick peptidoglycan layer, the cell wall of Corynebacteriales contains mycolic acids covalently linked to arabinogalactan. The mycolic acids are organized with extractable lipids in a permeability barrier. The cell wall of different families of Corynebacteriales contains channels responsible for its permeability. These channels have a structure completely different from porins of Gram-negative bacteria. Their structures depend on the length of the mycolic acids of the corresponding genera.

Porin Activity of Anaplasma phagocytophilum Outer Membrane Fraction and Purified P44

Anaplasma phagocytophilum, an obligatory intracellular bacterium that causes human granulocytic anaplasmosis, has significantly less coding capacity for biosynthesis and central intermediary metabolism than do free-living bacteria. Thus, A. phagocytophilum needs to usurp and acquire various compounds from its host. Here we demonstrate that the isolated outer membrane of A. phagocytophilum has porin activity, as measured by a liposome swelling assay. The activity allows the diffusion of L-glutamine, the monosaccharides arabinose and glucose, the disaccharide sucrose, and even the tetrasaccharide stachyose, and this diffusion could be inhibited with an anti-P44 monoclonal antibody. P44s are the most abundant outer membrane proteins and neutralizing targets of A. phagocytophilum. The P44 protein demonstrates characteristics consistent with porins of gram-negative bacteria, including detergent solubility, heat modifiability, a predicted structure of amphipathic and antiparallel beta-strands, an abundance of polar residues, and a C-terminal phenylalanine. We purified native P44s under two different nondenaturing conditions. When reconstituted into proteoliposomes, both purified P44s exhibited porin activity. P44s are encoded by approximately 100 p44 paralogs and go through extensive antigenic variation. The 16-transmembrane-domain beta-strands consist of conserved P44 N- and C-terminal regions. By looping out the hypervariable region, the porin structure is conserved among diverse P44 proteins yet enables antigenic variation for immunoevasion. The tricarboxylic acid (TCA) cycle of A. phagocytophilum is incomplete and requires the exogenous acquisition of L-glutamine or L-glutamate for function. Efficient diffusion of L-glutamine across the outer membrane suggests that the porin feeds the Anaplasma TCA cycle and that the relatively large pore size provides Anaplasma with the necessary metabolic intermediates from the host cytoplasm.

Crystal Structure of the Monomeric Porin OmpG

The outer membrane (OM) of Gram-negative bacteria contains a large number of channel proteins that mediate the uptake of ions and nutrients necessary for growth and functioning of the cell. An important group of OM channel proteins are the porins, which mediate the non-specific, diffusion-based passage of small (<600 Da) polar molecules. All porins of Gram-negative bacteria that have been crystallized to date form stable trimers, with each monomer composed of a 16-stranded β-barrel with a relatively narrow central pore. In contrast, the OmpG porin is unique, as it appears to function as a monomer. We have determined the X-ray crystal structure of OmpG from Escherichia coli to a resolution of 2.3 Å. The structure shows a 14-stranded β-barrel with a relatively simple architecture. Due to the absence of loops that fold back into the channel, OmpG has a large (∼13 Å) central pore that is considerably wider than those of other E. coli porins, and very similar in size to that of the toxin α-hemolysin. The architecture of the channel, together with previous biochemical and other data, suggests that OmpG may form a non-specific channel for the transport of larger oligosaccharides. The structure of OmpG provides the starting point for engineering studies aiming to generate selective channels and for the development of biosensors.

Topology of the Porin MspA in the Outer Membrane of Mycobacterium smegmatis

MspA is the major porin of Mycobacterium smegmatis mediating the exchange of hydrophilic solutes across the outer membrane (OM). It is the prototype of a new family of octameric porins with a single central channel of 9.6 nm in length and consists of two hydrophobic beta-barrels of 3.7 nm in length and a more hydrophilic, globular rim domain. The length of the hydrophobic domain of MspA does not match the thicknesses of mycobacterial OMs of 5-12 nm as derived from electron micrographs. Further, the membrane topology of MspA is unknown as it is for any other mycobacterial OM protein. We used MspA as a molecular ruler to define the boundaries of the OM of M. smegmatis by surface labeling of single cysteine mutants. Seventeen mutants covered the surface of the rim domain and were biotinylated with a membrane-impermeable reagent. The label efficiencies in vitro were remarkably similar to the predicted accessibilities of the cysteines. By contrast, six of these mutants were protected from biotinylation in M. smegmatis cells. Tryptophan 21 defines a horizontal plane that dissects the surface-exposed versus the membrane-protected residues of MspA. The 8 phenylalanines at position 99 form a ring at the periplasmic end of the hydrophobic beta-barrel domain. These results indicated that (i) the membrane boundaries of MspA are defined by aromatic girdles as in porins of Gram-negative bacteria and (ii) loops and a 3.4-nm long part of the hydrophilic rim domain are embedded into the OM of M. smegmatis. This is the first report suggesting that elements other than hydrophobic alpha-helices or beta-sheets are integrated into a lipid membrane.

Isolation and partial characterization of the major protein of the outer membrane of Pasteurella haemolytica and Pasteurella multocida

The N-terminal amino acid sequence of the 35 kDa (p35) major outer membrane protein (MOMP) of P. multocida shared a strong homology with those of homotrimeric nonspecific porins of gram-negative bacteria. The capacity of outer membrane protein (OMP) preparations of P. multocida to bind to respiratory mucosal surface preparations was inhibited significantly by using a polyclonal anti-p35 antiserum in an adhesion ELISA. Anti-p35 antiserum cross-reacted with a 44 kDa (p44) MOMP of P. haemolytica. N-terminal sequencing of MOMP p44 revealed a homology of 81% with the putative porin MOMP p35 of P. multocida.

The bacterial porin superfamily: sequence alignment and structure prediction.

The porins of Gram-negative bacteria are responsible for the 'molecular sieve' properties of the outer membrane. They form large water-filled channels which allow the diffusion of hydrophilic molecules into the periplasmic space. Owing to the strong hydrophilicity of their amino acid sequence and the nature of their secondary structure (beta strands), conventional hydropathy methods for predicting membrane topology are useless for this class of protein. The large number of available porin amino acid sequences was exploited to improve the accuracy of the prediction in combination with tools detecting amphipathicity of secondary structure. Using the constraints of beta-sheet structure these porins are predicted to contain 16 membrane-spanning strands, 14 of which are common to the two (enteric and the neisserial) porin subfamilies.