Topology of the Porin MspA in the Outer Membrane of Mycobacterium smegmatis (original) (raw)
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Expression and regulation of the porin gene mspA of Mycobacterium smegmatis
2007
under the control of the mspA promoter at pH 4.5. These results demonstrated that the pH sensitivity is specific for the mspA promoter and is mediated by the untranslated region upstream of mspA. This mechanism can be exploited to subject other genes to pH dependent regulation in fusion with the mspA promoter. The antisense RNA was regulated by pH in the same manner as mspA. This indicated a stabilizing or activating role for the antisense RNA by hybridizing to mspA transcripts and either inducing ribosome binding or preventing RNA degradation. This work revealed first insights into both transcriptional and post-transcriptional mechanisms of regulation of porin gene expression in M. smegmatis. 110 species (Hartmans et al., 2004) are phylogeneticly separated in slow-and fast-growing mycobacteria on the basis of 16S rRNA sequence comparisons (Rogall et al., 1990). The fast-growing species with generation times of less than 5 hours are mostly non-pathogenic and saprophytic soil or water dwellers such as Mycobacterium smegmatis, M. phlei or M. chelonae. The majority of slow-growing strains is supposed to represent the most recently evolved organisms of this genus (Pitulle et al., 1992). Slow-growing species have generation times of 20 hours and longer and include many pathogens known to cause serious diseases in mammals, especially tuberculosis (TB) by M. tuberculosis and leprosy by M. leprae. 2.1.2 Medical relevance of mycobacteria Since long before their discovery and characterization in 1882 (Koch, 1882) mycobacteria pose a major health burden to mankind. There were estimated 9 million incidences of TB in 2004 with two million people dying due to M. tuberculosis infections per year (WHO, 2006a). Overall one-third of the world's population is infected with M. tuberculosis and every second one person gets newly infected (WHO, 2006b). However, infections often remain al., 2000; Lee et al., 1996). The IM is followed by the mycolyl-arabinogalactan-peptidoglycan complex (Brennan, 2003). A multi-layer of peptidoglycan (PG), a polysaccharide composed of β-1,4-glucosidic linked N-acetylglucosamine and N-glycolylmuramic acid is attached to the arabinogalactan (AG) via phosphodiester linkage (Dmitriev et al., 2000). The mycolic acids are covalently bound to the PG-AG co-polymer and form the inner leaflet of the asymmetric lipid bilayer of the OM (Niederweis, 2003). This covalent fusion results in lateral immobility of the inner leaflet of the cell wall (Barry et al., 1998) and in a very restricted fluidity of the outer hydrophilic compounds can cross the OM by diffusion through porins, hydrophobic can traverse the bilayer directly. chains of the monomers provide for the high stability of the trimeric assembly (Delcour, 2003). OmpF, OmpC and PhoE of E. coli belong to the group of general porins with no particular substrate specificity. OmpF builds moderately larger pores than OmpC. OmpF and OmpC prefer cations slightly over anions whereas PhoE favours anions (Nikaido, 2003). LamB or ScrY for example are specific porins for maltooligosaccharides or sucrose, respectively (Gehring et al., 1991; Hardesty et al., 1991). The OM embedded β-barrel part of the porins is conserved, whereas the variability in the connecting loops could be very high (Nikaido, 2003). Based on these differences, porins can fulfill a wide variety of tasks. The most prominent is to mediate nutrient uptake, but the extracellular loop structure is a potential site for adhesion to other cells and an important antibody target. In pathogenic common interaction of the PorB family with the mitochondrial voltage-dependent anionic channel (VADC) was demonstrated. Also the N. gonorrhoeae porin PorB1A contributes to increased invasiveness and improved survival against human serum (Massari et al., 2003). Porins from the opportunistic pathogen Pseudomonas aeruginosa were reported to induce apoptosis in epithelial cells, to damage the human spermatozoa and to provoke reduced Although classified as gram-positive bacteria, the OM of the mycolata, a group of actinomycetes characterized by their OM consisting of mycolic acids and free lipids, is functionally analogous to the OM of gram-negative bacteria. Since this OM builds an efficient permeability barrier, it is not surprising that porins occur all over this group. Porins were discovered in species of Nocardia, Mycobacterium, Corynebacterium, Rhodococcus and Tsukamurellae (Dörner et al., 2004; Nikaido, 2003). The existence of porins in mycobacteria was postulated in 1990 (Jarlier & Nikaido, 1990). The first mycobacterial porin was isolated two years later from M. chelonae (Trias et al., 1992), a 59 kDa protein with a single channel conductance of 2.7 nS in 1 M KCl in lipid bilayer measurements. Similar, the existence of porins of M. tuberculosis and M. bovis BCG was discovered. Detergent solubilized cell wall proteins formed channels in lipid bilayer experiments with conductances of 0.7 and 3 nS with Cell wall extracts of this mutant contained lower amounts of porins than the wild-type (Stephan et al., 2005) and thus had a decreased channel activity in lipid bilayer experiments. Furthermore, the uptake of glucose and cephaloridine was reduced four-and nine-fold respectively (Stahl et al., 2001), and the growth was slowed down as reflected by an increase of the generation time in 7H9 medium from 3.3 to 4.3 hours (Stephan et al., 2005), indicating that MspA is the major porin of M. smegmatis. MspA has three paralogues in M. smegmatis, named MspB, MspC and MspD and differing from the mature MspA protein in only 2, 4 and 18 amino acids, respectively. Their expression and functionality as porins was demonstrated recently, whereas mspB and mspD seem to act as backup porins for their expression is activated upon deletion of mspA and mspC (Stephan et al., 2005). Consecutive deletions of the corresponding porin genes decreased the permeability of the cell wall and increased the resistance towards hydrophilic antibiotics (Stephan et al., 2004b; Stephan et al., 2004c; Stephan et al., 2005). Furthermore, sequence comparisons with mspA identified one orthologue in M. chelonae and four in M. phlei (Niederweis, 2003). 2.5 Expression of mspA of M. smegmatis Earlier studies revealed first insights in the expression mechanisms of mspA of M. smegmatis. Primer extension analyses revealed two transcriptional start points (TSP) in the 5' upstream sequence of mspA (Thiel, 1999). One strong signal for G at the position-135 bp upstream of mspA, one weak signal for G at position-153 bp. Corresponding potential promoters were identified, but not yet characterized (Thiel, 1999). The dependence of mspA expression on environmental factors was investigated with Northern blot analyses. Amounts of mspA transcripts were elevated under nitrogen limitation, while carbon and phosphate limitation decreased mspA levels (Kaps, 2004). Growth at 28°C did not result in an altered mspA expression, however heat shock conditions at 42°C repressed the mspA expression more than 10-fold. 1.4-fold, 4-fold and 50-fold repression occurred in the presence of 10% glucose, 18 mM hydrogen peroxide and 0.5 M sodium chloride, respectively. No mspA mRNA was detectable when M. smegmatis was exposed to 10% ethanole or growing in a lowered medium pH of 3 (Kaps, 2004). Thus, the expression of the mycobacterial porin mspA is affected by environmental conditions as known from gram-negative bacteria as E. coli. The mechanism of porin regulation is unknown for M. smegmatis and for mycobacteria in general.
Cloning of the mspA gene encoding a porin from Mycobacterium smegmatis
Molecular Microbiology, 1999
Porins form channels in the mycolic acid layer of mycobacteria and thereby control access of hydrophilic molecules to the cell. We puri®ed a 100 kDa protein from Mycobacterium smegmatis and demonstrated its channel-forming activity by reconstitution in planar lipid bilayers. The mspA gene encodes a mature protein of 184 amino acids and an N-terminal signal sequence. MALDI mass spectrometry of the puri®ed porin revealed a mass of 19 406 Da, in agreement with the predicted mass of mature MspA. Dissociation of the porin by boiling in 80% dimethyl sulphoxide yielded the MspA monomer, which did not form channels any more. Escherichia coli cells expressing the mspA gene produced the MspA monomer and a 100 kDa protein, which had the same channel-forming activity as whole-cell extracts of M. smegmatis with organic solvents. These proteins were speci®cally detected by a polyclonal antiserum that was raised to puri®ed MspA of M. smegmatis. These results demonstrate that the mspA gene encodes a protein of M. smegmatis, which assembles to an extremely stable oligomer with high channel-forming activity. Database searches did not reveal signi®cant similarities to any other known protein. Southern blots showed that the chromosomes of fast-growing mycobacterial species contain homologous sequences to mspA, whereas no hybridization could be detected with DNA from slow growing mycobacteria. These results suggest that MspA is the prototype of a new class of channel-forming proteins.
Collection and characterisation of bacterial membrane proteins
Febs Letters, 2003
A general strategy for the amplified expression in Escherichia coli of membrane transport and receptor proteins from other bacteria is described. As an illustration we report the cloning of the putative α-ketoglutarate membrane transport gene from the genome of Helicobacter pylori, overexpression of the protein tagged with RGS(His)6 at the C-terminus, and its purification in mg quantities. The retention of structural and functional integrity was verified by circular dichroism spectroscopy and reconstitution of transport activity. This strategy for overexpression and purification is extended to additional membrane proteins from H. pylori and from other bacteria.
Protein-translocating outer membrane porins of Gram-negative bacteria
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2002
Five families of outer membrane porins that function in protein secretion in Gram-negative bacteria are currently recognized. In this report, these five porin families are analyzed from structural and phylogenetic standpoints. They are the fimbrial usher protein (FUP), outer membrane factor (OMF), autotransporter (AT), two-partner secretion (TPS) and outer membrane secretin (Secretin) families. All members of these families in the current databases were identified, and all full-length homologues were multiply aligned for structural and phylogenetic analyses. The organismal distribution of homologues in each family proved to be unique with some families being restricted to proteobacteria and others being widespread in other bacterial kingdoms as well as eukaryotes. The compositions of and size differences between subfamilies provide evidence for specific orthologous relationships, which agree with available functional information and intra-subfamily phylogeny. The results reveal that horizontal transfer of genes encoding these proteins between phylogenetically distant organisms has been exceptionally rare although transfer within select bacterial kingdoms may have occurred. The resultant in silico analyses are correlated with available experimental evidence to formulate models relevant to the structures and evolutionary origins of these proteins. D in Gram-negative bacteria. Three of these four families have been analyzed previously, the fimbrial usher protein (FUP) family [12 -14], the outer membrane factor (OMF) family [15 -17] and the autotransporter (AT) family . The fourth family, the two-partner secretion (TPS) family, has not, to our knowledge, been carefully examined from a phylogenetic standpoint . In this communication, we provide updates of the five families of outer membrane porins that are believed to function in the export of proteins via homooligomeric structures.
Molecular organization in bacterial cell membranes
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1972
A method is presented for the solubilization and fractionation of Micrococcus lysodeikticus membrane after it has been subjected to mild washing procedures (i.e. depleted membrane). The method involves the solubilization of the membrane, treatment with iodoacetamide and sodium dithionite and the action of sodium dodecyl sulphate on the membrane extracted with n-butanol. Polyacrylamide electrophoresis and Sephadex G-2oo filtration in the presence of dodecyl sulphate result in the separation of at least eleven protein components whose molecular weights range from 17 ooo to 75 ooo. Residual lipid and carbohydrate are found as components of the depleted membrane, and fractionated as a glycolipoprotein complex. A component tentatively identified as ribonucleic acid is also found associated with the membrane although its association with other membrane components appears to be broken by the detergent.
The EMBO Journal, 1986
The amino acid distribution in membrane spanning segments and connecting loops in bacterial inner membrane proteins was analysed. The basic residues Arg and Lys are four times less prevalent in periplasmic as compared to cytosolic connecting loops, whereas no comparable effect is observed for the acidic residues Asp and Glu. Also, Pro is shown to be tolerated to a much larger extent in membrane spanning segments with their N-terminus pointing towards the cytosol than in those with the opposite orientation. The significance of these findings with regard to the mechanism of biogenesis of bacterial inner membrane proteins is discussed.