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BMC Microbiology Feb 2009Highly pathogenic mycobacteria like Mycobacterium tuberculosis are characterised by their slow growth and their ability to reside and multiply in the very hostile...
BACKGROUND
Highly pathogenic mycobacteria like Mycobacterium tuberculosis are characterised by their slow growth and their ability to reside and multiply in the very hostile phagosomal environment and a correlation between the growth rate of mycobacteria and their pathogenicity has been hypothesised. Here, porin genes from M. fortuitum were cloned and characterised to address their impact on the growth rate of fast-growing and pathogenic mycobacteria.
RESULTS
Two genes encoding porins orthologous to MspA from M. smegmatis, porM1 and porM2, were cloned from M. fortuitum strains, which were originally isolated from human patients. Both porin genes were at least partially able to complement the mutations of a M. smegmatis mutant strain lacking the genes mspA and mspC with respect to the growth rate. PorM1 and porM2 were present in different strains of M. fortuitum including the type strain. Comparative expression analysis of porM genes revealed divergent porin expression among analysed M. fortuitum strains. Repression of the expression of porins by antisense technique decreased the growth rates of different M. fortuitum. The effects of over-expression of porM1 as well as porM2 varied depending on the strain and the concentration of antibiotic added to the medium and indicated that PorM1 and PorM2 enhance the growth of M. fortuitum strains, but also the diffusion of the antibiotic kanamycin into the cells.
CONCLUSION
This study demonstrates the important role of porin expression in growth as well as antibiotic susceptibility of the opportunistic bacterium M. fortuitum.
Topics: Amino Acid Sequence; Anti-Bacterial Agents; Cloning, Molecular; DNA, Bacterial; Gene Expression Profiling; Gene Knockdown Techniques; Gene Order; Genetic Complementation Test; Humans; Kanamycin; Molecular Sequence Data; Mutation; Mycobacterium Infections, Nontuberculous; Mycobacterium fortuitum; Mycobacterium smegmatis; Porins; Sequence Alignment; Sequence Analysis, DNA
PubMed: 19203364
DOI: 10.1186/1471-2180-9-31 -
Microbiology Spectrum Dec 2022and like organisms (BALOs) are a unique bacterial group that live by predating on other bacteria, consuming them from within to grow and replicate before the progeny...
and like organisms (BALOs) are a unique bacterial group that live by predating on other bacteria, consuming them from within to grow and replicate before the progeny come out to complete the life cycle. The mechanisms by which these predators recognize their prey and differentiate them from nonprey bacteria, however, are still not clear. Through genetic knockout and complementation studies in different Escherichia coli strains, we found that Bdellovibrio bacteriovorus strain 109J recognizes outer membrane porin F (OmpF) on the E. coli surface and that the activity of the E. coli EnvZ-OmpR regulatory system significantly impacts predation kinetics. OmpF is not the only signal by which BALOs recognize their prey, however, as B. bacteriovorus could eventually predate on the E. coli mutant after prolonged incubation. Furthermore, recognizing OmpF as a prey surface structure was dependent on the prey strain, as knocking out OmpF protein homologues in other prey species, including Escherichia fergusonii, Klebsiella pneumoniae, and Salmonella enterica, did not always reduce the predation rate. Consequently, although OmpF was found to be an important surface component used by Bdellovibrio to efficiently recognize and attack E. coli, future work is needed to determine what other prey surface structures are recognized by these predators. Bdellovibrio bacteriovorus and like organisms (BALOs) are Gram-negative predatory bacteria that attack other Gram-negative bacteria by penetrating their periplasm and consuming them from within to obtain the nutrients necessary for the predator's growth and replication. How these predators recognize their prey, however, has remained a mystery. Here, we show that the outer membrane porin F (OmpF) in E. coli is recognized by B. bacteriovorus strain 109J and that the loss of this protein leads to severely delayed predation. However, predation of several other prey species was not dependent on the recognition of this protein or its homologues, indicating that there are other structures recognized by the predators on the prey surface that are yet to be discovered.
Topics: Bdellovibrio bacteriovorus; Escherichia coli; Porins
PubMed: 36445149
DOI: 10.1128/spectrum.03094-22 -
Methods in Molecular Biology (Clifton,... 2021Bacterial porins often exhibit ion conductance and gating behavior which can be modulated by pH. However, the underlying control mechanism of gating is often complex,...
Bacterial porins often exhibit ion conductance and gating behavior which can be modulated by pH. However, the underlying control mechanism of gating is often complex, and direct inspection of the protein structure is generally insufficient for full mechanistic understanding. Here we describe Pretzel, a computational framework that can effectively model loop-based gating events in membrane proteins. Our method combines Monte Carlo conformational sampling, structure clustering, ensemble energy evaluation, and a topological gating criterion to model the equilibrium gating state under the pH environment of interest. We discuss details of applying Pretzel to the porin outer membrane protein G (OmpG).
Topics: Bacterial Outer Membrane Proteins; Escherichia coli Proteins; Hydrogen-Ion Concentration; Ion Channel Gating; Molecular Dynamics Simulation; Monte Carlo Method; Porins; Protein Domains
PubMed: 32918736
DOI: 10.1007/978-1-0716-0806-7_12 -
Journal of Bacteriology Jul 1998The genomic DNA of the BE strain of Escherichia coli has been scrutinized to detect porin genes that have not been identified so far. Southern blot analysis yielded two... (Comparative Study)
Comparative Study
The genomic DNA of the BE strain of Escherichia coli has been scrutinized to detect porin genes that have not been identified so far. Southern blot analysis yielded two DNA segments which proved highly homologous to, yet distinct from, the ompC, ompF, and phoE porin genes. The two genes were cloned and sequenced. One of them, designated ompN, encodes a porin which, due to low levels of expression, has eluded prior identification. The functional properties (single-channel conductance) of the OmpN porin, purified to homogeneity, closely resemble those of the OmpC porin from E. coli K-12. The second DNA fragment detected corresponds to the nmpC gene, which, due to an insertion of an IS1 element in its coding region, is not expressed in E. coli BE.
Topics: Amino Acid Sequence; Blotting, Southern; Carbohydrate Metabolism; Cloning, Molecular; Epitopes; Escherichia coli; Escherichia coli Proteins; Genes, Bacterial; Genotype; Lipid Bilayers; Molecular Sequence Data; Plasmids; Porins; Recombinant Proteins; Sequence Alignment; Sequence Homology, Amino Acid
PubMed: 9642192
DOI: 10.1128/JB.180.13.3388-3392.1998 -
Antimicrobial Agents and Chemotherapy Dec 2002The relationships between porin deficiency, active efflux of fluoroquinolones, and extended-spectrum beta-lactamase (ESBL) production were determined for 53 clinical...
Energy-dependent accumulation of norfloxacin and porin expression in clinical isolates of Klebsiella pneumoniae and relationship to extended-spectrum beta-lactamase production.
The relationships between porin deficiency, active efflux of fluoroquinolones, and extended-spectrum beta-lactamase (ESBL) production were determined for 53 clinical isolates of Klebsiella pneumoniae. Thirty-two ESBL-positive strains (including 22 strains expressing porins and 10 strains lacking porins) and 21 ESBL-negative strains were evaluated. Active efflux of norfloxacin was defined as a >/=50% increase in the accumulation of norfloxacin in the presence of carbonyl cyanide m-chlorophenylhydrazone (CCCP) in comparison with the corresponding basal value in the absence of CCCP. The quinolone resistance-determining regions of both gyrA and parC from 13 strains, representing all isolates with different porin profiles and with or without active efflux, were determined. Porin loss was significantly more common among ESBL-positive strains (10 of 32 [31.2%]) than among ESBL-negative strains (0 of 2 [0%]) (P < 0.01). Active efflux was observed in 7 of 10 (70%) strains lacking porins and in 4 of 43 (9.3%) strains producing porins (P < 0.001). The 11 strains showing active efflux corresponded to 3 of 21 (14.3%) ESBL-negative strains and 8 of 32 (25.5%) ESBL-positive strains (P > 0.05). Basal values of norfloxacin accumulation were higher in strains lacking active efflux than in those that had this mechanism (P < 0.05). In the absence of topoisomerase changes, the contribution of either porin loss or active efflux to fluoroquinolone resistance in K. pneumoniae was negligible. It is concluded that among K. pneumoniae strains of clinical origin, porin loss was observed only in those producing ESBL, and that a significant number of porin-deficient strains also expressed active efflux of norfloxacin. In terms of fluoroquinolone resistance, both mechanisms are significant only in the presence of topoisomerase modifications.
Topics: Anti-Infective Agents; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Drug Interactions; Klebsiella pneumoniae; Microbial Sensitivity Tests; Nalidixic Acid; Norfloxacin; Porins; beta-Lactamases
PubMed: 12435697
DOI: 10.1128/AAC.46.12.3926-3932.2002 -
Journal of Bacteriology Aug 2001Omp2a and Omp2b are highly homologous porins present in the outer membrane of the bacteria from the genus Brucella, a facultative intracellular pathogen. The genes...
Omp2a and Omp2b are highly homologous porins present in the outer membrane of the bacteria from the genus Brucella, a facultative intracellular pathogen. The genes coding for these proteins are closely linked in the Brucella genome and oriented in opposite directions. In this work, we present the cloning, purification, and characterization of four Omp2b size variants found in various Brucella species, and we compare their antigenic and functional properties to the Omp2a and Omp2b porins of Brucella melitensis reference strain 16M. The variation of the Omp2a and Omp2b porin sequences among the various strains of the genus Brucella seems to result mostly from multiple gene conversions between the two highly homologous genes. As shown in this study, this phenomenon has led to the creation of natural Omp2a and Omp2b chimeric proteins in Omp2b porin size variants. The comparison by liposome swelling assay of the porins sugar permeability suggested a possible functional differences between Omp2a and Omp2b, with Omp2a showing a more efficient pore in sugar diffusion. The sequence variability in the Omp2b size variants was located in the predicted external loops of the porin. Several epitopes recognized by anti-Omp2b monoclonal antibodies were mapped by comparison of the Omp2b size variants antigenicity, and two of them were located in the most exposed surface loops. However, since variations are mostly driven by simple exchanges of conserved motifs between the two genes (except for an Omp2b version from an atypical strain of Brucella suis biovar 3), the porin variability does not result in major antigenic variability of the Brucella surface that could help the bacteria during the reinfection of a host. Porin variation in Brucella seems to result mainly in porin conductivity modifications.
Topics: Amino Acid Sequence; Bacterial Proteins; Base Sequence; Brucella; Brucella melitensis; Chromosome Mapping; Circular Dichroism; Cloning, Molecular; Genetic Variation; Molecular Sequence Data; Plasmids; Porins; Protein Conformation; Sequence Alignment; Sequence Analysis, Protein; Sequence Homology, Amino Acid; Sequence Homology, Nucleic Acid; Species Specificity
PubMed: 11466287
DOI: 10.1128/JB.183.16.4839-4847.2001 -
Journal of Bacteriology May 1999Klebsiella pneumoniae porin genes were analyzed to detect mutations accounting for the porin deficiency observed in many beta-lactam-resistant strains. PCR and Southern...
Klebsiella pneumoniae porin genes were analyzed to detect mutations accounting for the porin deficiency observed in many beta-lactam-resistant strains. PCR and Southern blot analysis revealed the existence of a third porin gene in addition to the OmpK36 and OmpK35 porin genes previously described. This new porin gene was designated ompK37 and is present in all of the clinical isolates tested. The OmpK37 porin gene was cloned, sequenced, and overexpressed in Escherichia coli. In contrast to that of the major porins, OmpK37 porin expression was only detectable by Western blot analysis in porin-deficient beta-lactam-resistant strains, suggesting strong down regulation under standard laboratory conditions. Functional characterization suggested a narrower pore for the OmpK37 porin than for K. pneumoniae porins OmpK36 and OmpK35. This correlated with the susceptibility to certain beta-lactam antibiotics, since a K. pneumoniae strain expressing porin OmpK37, but not porin OmpK36 or OmpK35, was less susceptible to beta-lactam antibiotics than the same strain expressing either porin OmpK36 or OmpK35.
Topics: Amino Acid Sequence; Bacterial Proteins; Biological Transport; Carbohydrate Metabolism; Cloning, Molecular; Genes, Bacterial; Klebsiella pneumoniae; Microbial Sensitivity Tests; Molecular Sequence Data; Permeability; Porins; Sequence Analysis, DNA; Sequence Homology, Amino Acid; beta-Lactam Resistance
PubMed: 10217760
DOI: 10.1128/JB.181.9.2726-2732.1999 -
FEBS Letters Sep 2003Cell infection by picornaviruses leads to membrane permeabilization. Recent evidence suggests the involvement of the non-structural protein 2B in this process. We have... (Review)
Review
Cell infection by picornaviruses leads to membrane permeabilization. Recent evidence suggests the involvement of the non-structural protein 2B in this process. We have recently reported the detection of 2B porin-like activity in isolated membrane-protein systems that lack other cell components. According to data derived from these model membranes, four self-aggregated 2B monomers (i.e. tetramers) would be sufficient to permeabilize a single lipid vesicle, allowing the free diffusion of solutes under ca. 1000 Da. Our findings also support a role for lipids in protein oligomerization and subsequent pore opening. The lipid dependence of these processes points to negatively charged cytofacial surfaces as 2B cell membrane targets.
Topics: Amino Acid Sequence; Cell Membrane; Cell Membrane Permeability; Ion Channels; Kinetics; Lipid Metabolism; Models, Statistical; Molecular Sequence Data; Phosphatidylinositols; Picornaviridae; Porins; Protein Structure, Quaternary; Sequence Homology, Amino Acid
PubMed: 12972154
DOI: 10.1016/s0014-5793(03)00852-4 -
Biochimica Et Biophysica Acta.... Jun 2021Gram-negative bacteria cause the majority of highly drug-resistant bacterial infections. To cross the outer membrane of the complex Gram-negative cell envelope,...
Gram-negative bacteria cause the majority of highly drug-resistant bacterial infections. To cross the outer membrane of the complex Gram-negative cell envelope, antibiotics permeate through porins, trimeric channel proteins that enable the exchange of small polar molecules. Mutations in porins contribute to the development of drug-resistant phenotypes. In this work, we show that a single point mutation in the porin PorB from Neisseria meningitidis, the causative agent of bacterial meningitis, can strongly affect the binding and permeation of beta-lactam antibiotics. Using X-ray crystallography, high-resolution electrophysiology, atomistic biomolecular simulation, and liposome swelling experiments, we demonstrate differences in drug binding affinity, ion selectivity and drug permeability of PorB. Our work further reveals distinct interactions between the transversal electric field in the porin eyelet and the zwitterionic drugs, which manifest themselves under applied electric fields in electrophysiology and are altered by the mutation. These observations may apply more broadly to drug-porin interactions in other channels. Our results improve the molecular understanding of porin-based drug-resistance in Gram-negative bacteria.
Topics: Ampicillin; Anti-Bacterial Agents; Bacterial Proteins; Binding Sites; Crystallography, X-Ray; Drug Resistance, Bacterial; Liposomes; Molecular Dynamics Simulation; Mutagenesis, Site-Directed; Neisseria meningitidis; Permeability; Porins; Protein Binding; Protein Structure, Tertiary; Recombinant Proteins
PubMed: 33675718
DOI: 10.1016/j.bbamem.2021.183601 -
Biochimica Et Biophysica Acta May 2002Five families of outer membrane porins that function in protein secretion in Gram-negative bacteria are currently recognized. In this report, these five porin families... (Review)
Review
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.
Topics: Consensus Sequence; Gram-Negative Bacteria; Multigene Family; Phylogeny; Porins; Protein Transport; Sequence Homology, Nucleic Acid; Software
PubMed: 11988218
DOI: 10.1016/s0005-2736(02)00359-0