Recurrent emergence of Klebsiella pneumoniae carbapenem resistance mediated by an inhibitory ompK36 mRNA secondary structure.

Outer membrane porins in Gram-negative bacteria facilitate antibiotic influx. In Klebsiella pneumoniae, modifications in the porin OmpK36 are implicated in increasing resistance to carbapenems. An analysis of large K. pneumoniae genome collections, encompassing major healthcare-associated clones, revealed the recurrent emergence of a synonymous cytosine-to-thymine transition at position 25 (25c > t) in ompK36. We show that the 25c > t transition increases carbapenem resistance through depletion of OmpK36 from the outer membrane. The mutation attenuates K. pneumoniae in a murine pneumonia model, which accounts for its limited clonal expansion observed by phylogenetic analysis. However, in the context of carbapenem treatment, the 25c > t transition tips the balance toward treatment failure, thus accounting for its recurrent emergence. Mechanistically, the 25c > t transition mediates an intramolecular messenger RNA (mRNA) interaction between a uracil encoded by 25t and the first adenine within the Shine-Dalgarno sequence. This specific interaction leads to the formation of an RNA stem structure, which obscures the ribosomal binding site thus disrupting translation. While mutations reducing OmpK36 expression via transcriptional silencing are known, we uniquely demonstrate the repeated selection of a synonymous ompK36 mutation mediating translational suppression in response to antibiotic pressure.

Getting Drugs through Small Pores: Exploiting the Porins Pathway in Pseudomonas aeruginosa.

Understanding molecular properties of outer membrane channels of Gram-negative bacteria is of fundamental significance as they are the entry point of polar antibiotics into bacteria. Outer membrane proteomics revealed OccK8 (OprE) to be among the five most expressed substrate specific channels of the clinically important Pseudomonas aeruginosa. The high-resolution X-ray structure and electrophysiology highlighted a very narrow pore. However, experimental in vitro methods showed the transport of natural amino acids and antibiotics, among them ceftazidime. We used molecular dynamics simulations to reveal the importance of the physicochemical properties of ceftazidime in modulating the translocation through OccK8, proposing a structure-function relationship. As in general porins, the internal electric field favors the translocation of polar molecules by gainful energy compensation in the central constriction region. Importantly, the comparatively narrow OccK8 pore can undergo a substrate-induced expansion to accommodate relatively large-sized substrates.

Multi-drug resistant Pseudomonas aeruginosa nosocomial strains: Molecular epidemiology and evolution.

Pseudomonas aeruginosa causes a wide variety of nosocomial infections. In the study, phylogenetic, selective pressure analysis and homology modelling were applied to oprD efflux pump gene with the aim to characterize multi-drug resistant strains circulating in the nosocomial setting, their transmission dynamics and ongoing evolution. One hundred ninety-three consecutive inpatients with Pseudomonas aeruginosa infection were enrolled at the University Campus Bio-Medico of Rome, between January 2015 and December 2016. oprD gene was sequenced in 20 nosocomial multi-drug resistant P. aeruginosa strains. Phylogeographic, selective pressure, residue conservation analysis and homology modelling were performed. Clinical epidemiological data were extracted from patient medical records. Multi-drug resistant strains accounted for the 36% of total strains and were responsible of 20 cases of nosocomial infections. P. aeruginosa infections occurred prevalently in the West area, especially at the location IIIW and in the Geriatric ward. The time of the most recent common ancestor indicated that strains could have been introduced in the hospital since the end of the year 2009 with the most probable location in general surgery ward. By selective pressure analysis, 29 positions under diversifying selection have been identified and mapped onto the OprD model. Most of the observed residue substitutions are predicted to be destabilizing and some of them occurred in the Loops 2 and 3 that are involved in solute selection and carbapenem susceptibility. The molecular and evolutionary analysis of Multi-drug resistant strains circulating in the nosocomial setting may provide useful insights into the epidemiology and the mechanisms leading to resistance, contributing to infection control improvement.

Oral treponeme major surface protein: Sequence diversity and distributions within periodontal niches.

Treponema denticola and other species (phylotypes) of oral spirochetes are widely considered to play important etiological roles in periodontitis and other oral infections. The major surface protein (Msp) of T. denticola is directly implicated in several pathological mechanisms. Here, we have analyzed msp sequence diversity across 68 strains of oral phylogroup 1 and 2 treponemes; including reference strains of T. denticola, Treponema putidum, Treponema medium, 'Treponema vincentii', and 'Treponema sinensis'. All encoded Msp proteins contained highly conserved, taxon-specific signal peptides, and shared a predicted 'three-domain' structure. A clone-based strategy employing 'msp-specific' polymerase chain reaction primers was used to analyze msp gene sequence diversity present in subgingival plaque samples collected from a group of individuals with chronic periodontitis (n=10), vs periodontitis-free controls (n=10). We obtained 626 clinical msp gene sequences, which were assigned to 21 distinct 'clinical msp genotypes' (95% sequence identity cut-off). The most frequently detected clinical msp genotype corresponded to T. denticola ATCC 35405T , but this was not correlated to disease status. UniFrac and libshuff analysis revealed that individuals with periodontitis and periodontitis-free controls harbored significantly different communities of treponeme clinical msp genotypes (P<.001). Patients with periodontitis had higher levels of clinical msp genotype diversity than periodontitis-free controls (Mann-Whitney U-test, P<.05). The relative proportions of 'T. vincentii' clinical msp genotypes were significantly higher in the control group than in the periodontitis group (P=.018). In conclusion, our data clearly show that both healthy and diseased individuals commonly harbor a wide diversity of Treponema clinical msp genotypes within their subgingival niches.

Carbapenem-resistant Acinetobacter baumannii from Serbia: revision of CarO classification.

Carbapenem-resistant A. baumannii present a significant therapeutic challenge for the treatment of nosocomial infections in many European countries. Although it is known that the gradient of A. baumannii prevalence increases from northern to southern Europe, this study provides the first data from Serbia. Twenty-eight carbapenem-resistant A. baumannii clinical isolates were collected at a Serbian pediatric hospital during a 2-year period. The majority of isolates (67.68%) belonged to the sequence type Group 1, European clonal complex II. All isolates harbored intrinsic OXA-51 and AmpC cephalosporinase. OXA-23 was detected in 16 isolates (57.14%), OXA-24 in 23 isolates (82.14%) and OXA-58 in 11 isolates (39.29%). Six of the isolates (21.43%) harbored all of the analyzed oxacillinases, except OXA-143 and OXA-235 that were not detected in this study. Production of oxacillinases was detected in different pulsotypes indicating the presence of horizontal gene transfer. NDM-1, VIM and IMP were not detected in analyzed clinical A. baumannii isolates. ISAba1 insertion sequence was present upstream of OXA-51 in one isolate, upstream of AmpC in 13 isolates and upstream of OXA-23 in 10 isolates. In silico analysis of carO sequences from analyzed A. baumannii isolates revealed the existence of two out of six highly polymorphic CarO variants. The phylogenetic analysis of CarO protein among Acinetobacter species revised the previous classification CarO variants into three groups based on strong bootstraps scores in the tree analysis. Group I comprises four variants (I-IV) while Groups II and III contain only one variant each. One half of the Serbian clinical isolates belong to Group I variant I, while the other half belongs to Group I variant III.

Trends of the major porin gene (ompF) evolution: insight from the genus Yersinia.

OmpF is one of the major general porins of Enterobacteriaceae that belongs to the first line of bacterial defense and interactions with the biotic as well as abiotic environments. Porins are surface exposed and their structures strongly reflect the history of multiple interactions with the environmental challenges. Unfortunately, little is known on diversity of porin genes of Enterobacteriaceae and the genus Yersinia especially. We analyzed the sequences of the ompF gene from 73 Yersinia strains covering 14 known species. The phylogenetic analysis placed most of the Yersinia strains in the same line assigned by 16S rDNA-gyrB tree. Very high congruence in the tree topologies was observed for Y. enterocolitica, Y. kristensenii, Y. ruckeri, indicating that intragenic recombination in these species had no effect on the ompF gene. A significant level of intra- and interspecies recombination was found for Y. aleksiciae, Y. intermedia and Y. mollaretii. Our analysis shows that the ompF gene of Yersinia has evolved with nonrandom mutational rate under purifying selection. However, several surface loops in the OmpF porin contain positively selected sites, which very likely reflect adaptive diversification Yersinia to their ecological niches. To our knowledge, this is a first investigation of diversity of the porin gene covering the whole genus of the family Enterobacteriaceae. This study demonstrates that recombination and positive selection both contribute to evolution of ompF, but the relative contribution of these evolutionary forces are different among Yersinia species.

A novel OmpY porin from Yersinia pseudotuberculosis: structure, channel-forming activity and trimer thermal stability.

A novel OmpY porin was predicted based on the Yersinia pseudotuberculosis genome analysis. Whereas it has the different genomic annotation such as "outer membrane protein N" (ABS46310.1) in str. IP 31758 or "outer membrane protein C2, porin" (YP_070481.1) in str. IP32953, it might be warranted to rename the OmpN/OmpC2 to OmpY, "outer membrane protein Y", where letter "Y" pertained to Yersinia. Both phylogenetic analysis and genomic localization clearly support that the OmpY porin belongs to a new group of general bacterial porins. The recombinant OmpY protein with its signal sequence was overexpressed in porin-deficient Escherichia coli strain. The mature rOmpY was shown to insert into outer membrane as a trimer. The OmpY porin, isolated from the outer membrane, was studied employing spectroscopic, electrophoretic and bilayer lipid membranes techniques. The far UV CD spectrum of rOmpY was essentially identical to that of Y. pseudotuberculosis OmpF. The near UV CD spectrum of rOmpY was weaker and smoother than that of OmpF. The rOmpY single-channel conductance was 180 ± 20 pS in 0.1 M NaCl and was lower than that of the OmpF porin. As was shown by electrophoretic and bilayer lipid membrane experiments, the rOmpY trimers were less thermostable than the OmpF trimers. The porins differed in the trimer-monomer transition temperature by about 20°C. The three-dimensional structural models of the Y. pseudotuberculosis OmpY and OmpF trimers were generated and the intra- and intermonomeric interactions stabilizing the porins were investigated. The difference in the thermal stability of OmpY and OmpF trimers was established to correlate with the difference in intermonomeric polar contacts.

PorA types in Neisseria meningitidis serogroup B isolated in Argentina from 2001 to 2003: implications for the design of an outer membrane protein-based vaccine.

Identification of Neisseria meningitidis PorA types remains important, as the PorA protein is a major immunogenic component of several meningococcal vaccines under development. In this study, 191 N. meningitidis serogroup B isolates collected in Argentina through active laboratory-based surveillance from 2001 to 2003 were serosubtyped. Nucleotide sequences of the porA variable region 1 (VR1) and VR2 regions were determined in 52 non-serosubtypeable isolates. A substantial number of distinct VR types were identified, and a new VR2 variant from the P1.16 family was described. This is the first report describing PorA types in N. meningitidis serogroup B isolates in Argentina. Furthermore, the wide diversity of subtypes detected by serosubtyping and genosubtyping reveals the difficulty in designing a useful outer-membrane vaccine applicable in this country. A possible mechanism responsible for altered PorA expression was analysed in two PorA types.

Evaluation of PorB variable region typing of Neisseria gonorrhoeae using PCR-ELISA in samples collected from men who have sex with men.

A polymerase chain reaction (PCR)-based enzyme-linked immunosorbent assay (ELISA) methodology was developed to characterize Neisseria gonorrhoeae porB gene variable regions (VR); the methodology was evaluated in comparison to porB VR typing by checkerboard hybridization. Clinical noncultured samples from 35 men who have sex with men (MSM), positive by nucleic amplification assays for N. gonorrhoeae, were typed using a panel of 40 oligonucleotide probes to porB VRs and compared to checkerboard hybridization. Complete concordance was observed between the two methods at PIB VRs 1, 3, and 7. At the more degenerate VRs 5 and 6, PCR ELISA resulted in obtaining more typeable VRs than checkerboard hybridization due to single nucleotide mismatches. By PCR ELISA, two predominant PIB porB types were identified in 58% of the samples and the remaining 16 samples had one of six other porB types. Both PCR ELISA and checkerboard hybridization methods of porB VR typing allowed characterization of N. gonorrhoeae from noncultured clinical samples including throat and rectal swabs and discriminated N. gonorrhoeae from N. meningitidis present in some of the samples. PCR ELISA is a rapid, relatively inexpensive and alternative molecular typing method for N. gonorrhoeae, suitable for use in conjunction with molecular diagnostic tests.

Phylogenetic relationships of Campylobacter jejuni based on porA sequences.

Campylobacter porins are the dominant major outer membrane protein (MOMP) of these bacteria. They are composed of hypervariable, surface-exposed, peptide loops and membrane-embedded, conserved peptide regions. Porins are functionally important and may also be useful for molecular subtyping methods but have not yet been well characterized. We therefore sequenced the porA gene from 39 Campylobacter isolates, including multilocus sequence type (MLST) reference strains, isolates from patients with the Guillain-Barré syndrome, other clinical isolates, and serotyping reference strains. These were compared with additional sequences available from GenBank. Three distinct porA lineages were observed after phylogenetic analysis. Both Campylobacter coli and Campylobacter jejuni were found with group 3 porA sequences, and this was the only group showing any evidence of recombination among porA genes. There was no recombination between porA genes from C. jejuni groups 1 and 2, suggesting there may be functional constraints on changes at this locus. Most of the amino acid differences among the three groups were present in surface-exposed loops, and dissimilar substitutions were found when groups 1 and 2 MOMP were compared. Different MOMP sequence groups may have different biological or antigenic properties, which in turn may be associated with survival in different environments, host adaptation, or virulence.

Phenotypic assessment of Neisseria meningitidis isolates obtained from patients with invasive meningococcal disease in Greece, 1993-2003: implications for serogroup B vaccines based on PorA serosubtype antigens.

Serogroup B is the major isolate from patients with invasive meningococcal disease (IMD) in Greece. This study used the whole cell enzyme-linked immuosorbent assay (ELISA) with monoclonal antibodies to screen Neisseria meningitidis isolates obtained from patients with IMD between 1993 and 2003 to determine if serosubtypes included in the hexavalent Por A OMP vaccines being tested in northern Europe were prevalent in Greece. During this period there were significant changes in the proportions of serogroups B and C isolated from patients. Serogroup C was predominant in 1996-1997 but fell sharply with corresponding increases in serogroup B. Of the 591 isolates sent to the National Meningitis Reference Laboratory in Athens during this period, 325 (55%) were serogroup B. Among those tested for serosubtype, porA proteins used for the vaccine being tested in Britain were detected on 85/284 (30%) strains and for the vaccine being tested in the Netherlands 175/284 (62%). P1.14 (58/284, 20%) the predominant serosubtype among the Greek isolates, is not present in either vaccine formulation; 23/284 (8%) strains did not react with any of the monoclonal antibodies. Our results indicate that introduction of the vaccines currently being evaluated in northern Europe would not be warranted in the Greek population.

Molecular tests can allow confirmation of invasive meningococcal disease when isolates yield atypical maltose, glucose or gamma-glutamyl peptidase test results.

Analysis of atypical meningococci from invasive disease that either (i) did not produce acid from maltose and glucose or (ii) were gamma-glutamyl peptidase test negative for porA and porB DNA variable region (VR) type, multilocus sequence type, and for presence of capsule transport gene ctrA, conclusively demonstrated that these are Neisseria meningitidis.

Genetic typing of the porin protein of Neisseria gonorrhoeae from clinical noncultured samples for strain characterization and identification of mixed gonococcal infections.

Molecular methods that characterize the Neisseria gonorrhoeae porin protein Por are needed to study gonococcal pathogenesis in the natural host and to classify strains from direct clinical samples used with nucleic acid amplification-based tests. We have defined the capabilities of por variable region (VR) typing and determined suitable conditions to apply the method to direct clinical specimens. Nested PCR from spiked urine samples detected 1 to 10 copies of template DNA; freezing spiked whole urine greatly reduced the ability to amplify porB. In a laboratory model of mixed gonococcal infections, the por type of one strain could be determined in the presence of a 100-fold excess of another. por VR typing was used to examine clinical samples from women enrolled in studies conducted in Baltimore, Md., and Madagascar. por type was determined from 100% of paired cervical swab and wick samples from 20 culture-positive women from Baltimore; results for eight individuals (40%) suggested infection with more than one strain. In frozen urine samples from Madagascar, porB was amplified and typed from 60 of 126 samples from ligase chain reaction (LCR)-positive women and 3 samples from LCR-negative women. The por VR types of 13 samples (21%) suggested the presence of more than one gonococcal strain. Five por types, identified in >45% of women with typed samples, were common to both geographic areas. Molecular typing is an important adjunct to nucleic acid amplification-based diagnostics. Methods that utilize direct clinical samples and can identify mixed infections may contribute significantly to studies of host immunity, gonococcal epidemiology, and pathogenesis.

Arsenic trioxide uptake by human and rat aquaglyceroporins.

Aquaglyceroporins are channels that allow downhill movement of uncharged solutes such as glycerol and urea. Arsenic trioxide has recently been shown to be translocated by mouse mAQP7 and rat rAQP9. In this study we examined the ability of the four known human members of the aquaglyceroporin family, hAQP3, hAQP7, hAQP9, and hAQP10, to facilitate As(OH)(3) movement in Xenopus oocytes. The order of effectiveness as an As(III) transporter was found to be hAQP9 > hAQP7, with little or no transport by hAQP3 or hAQP10. From comparison with the crystal structure of the bacterial homologue GlpF and the bovine erythrocyte water channel bAQP1, AQP9 residues Phe-64 and Arg-219 are predicted to serve as part of the selectivity filter. The requirement for Phe-64 and Arg-219 in arsenic trioxide translocation was examined by site-directed mutagenesis of rAQP9, taking advantage of the fact that rat AQP9 catalyzes (73)As(OH)(3) uptake in Saccharomyces cerevisiae and in oocytes. R219A, R219K, F64A, F64T, and F64W were expressed in both yeast and oocytes, and permeability of arsenic trioxide and glycerol was measured. A lysine but not an alanine residue could substitute for the highly conserved Arg-219, indicating that a positive charge is required at the entry to the channel. In contrast, the phenylalanine residue, which is believed to position substrates near the conserved arginine, was not required for either arsenic trioxide or glycerol uptake. The results support the hypothesis that arsenic trioxide and glycerol use the same translocation pathway in AQP9.

The physiological properties of a novel family of VDAC-like proteins from Drosophila melanogaster.

VDAC, a major protein of the mitochondrial outer membrane, forms voltage-dependent, anion-selective channels permeable to most metabolites. Although multiple isoforms of VDAC have been found in different organisms, only one isoform (porin/DVDAC) has been previously reported for Drosophila melanogaster. We have examined the physiological properties of three other Drosophila proteins (CG17137, CG17139, and CG17140) whose primary sequences have significant homology to DVDAC. A comparison of their hydropathy profiles (beta-pattern) with known VDAC sequences indicates the same fundamental folding pattern but with major insertions and deletions. The ability of these proteins to form channels was tested on planar membranes and liposomes. Channel activity was observed with varying degrees of similarity to VDAC. Two of these proteins (CG17137 and CG17140) produced channels with anionic selectivity in the open state. Sometimes channels exhibited closure and voltage gating, but for CG17140 this occurred at much higher voltages than is typical for VDAC. CG17139 was not able to form channels. DVDAC and CG17137 were able to rescue the temperature-sensitive conditional-lethal phenotype of VDAC-deficient yeast, whereas CG17139 and CG17140 demonstrated no complementation. Similar structure and channel formation indicate that VDAC-like proteins are part of the larger VDAC family but the modifications are indicative of specialized functions.

Annotation and evolutionary relationships of a small regulatory RNA gene micF and its target ompF in Yersinia species.

BACKGROUND: micF RNA, a small regulatory RNA found in bacteria, post-transcriptionally regulates expression of outer membrane protein F (OmpF) by interaction with the ompF mRNA 5'UTR. Phylogenetic data can be useful for RNA/RNA duplex structure analyses and aid in elucidation of mechanism of regulation. However micF and associated genes, ompF and ompC are difficult to annotate because of either similarities or divergences in nucleotide sequence. We report by using sequences that represent "gene signatures" as probes, e.g., mRNA 5'UTR sequences, closely related genes can be accurately located in genomic sequences. RESULTS: Alignment and search methods using NCBI BLAST programs have been used to identify micF, ompF and ompC in Yersinia pestis and Yersinia enterocolitica. By alignment with DNA sequences from other bacterial species, 5' start sites of genes and upstream transcriptional regulatory sites in promoter regions were predicted. Annotated genes from Yersinia species provide phylogenetic information on the micF regulatory system. High sequence conservation in binding sites of transcriptional regulatory factors are found in the promoter region upstream of micF and conservation in blocks of sequences as well as marked sequence variation is seen in segments of the micF RNA gene. Unexpected large differences in rates of evolution were found between the interacting RNA transcripts, micF RNA and the 5' UTR of the ompF mRNA. micF RNA/ompF mRNA 5' UTR duplex structures were modeled by the mfold program. Functional domains such as RNA/RNA interacting sites appear to display a minimum of evolutionary drift in sequence with the exception of a significant change in Y. enterocolitica micF RNA. CONCLUSIONS: Newly annotated Yersinia micF and ompF genes and the resultant RNA/RNA duplex structures add strong phylogenetic support for a generalized duplex model. The alignment and search approach using 5' UTR signatures may be a model to help define other genes and their start sites when annotated genes are available in well-defined reference organisms.

Biophysics of gating phenomena in voltage-dependent OmpC mutant porin channels (R74C and R37C) of Escherichia coli outer membranes.

The mechanism by which the membrane potential closes and opens voltage-dependent beta-barrel membrane channels is not fully understood. OmpC porins form trimeric water-filled channels when incorporated into artificial bilayers, each monomer having a conductance of approximately 510 pS in 1 M KCl. These channels are relatively insensitive to membrane potential difference (pd) and close only when the pd exceeds +/-250 mV. Another well-known trimer, OmpF, has a monomer conductance of approximately 780 pS in 1 M NaCl, is more sensitive to pd, and can be closed reversibly when a pd of more than +/-150 mV is applied to the channel-containing membranes. With the aid of the 3D atomic structure of these channels determined by X-ray crystallography, and using site-directed mutagenesis, specific amino acids can be substituted in desired locations in the channel lumen. In this study we have used mutants 37C and 74C and attached fluorescence probes to them to monitor polarity changes in the channel lumen during gating. From the observed changes in polarity, we conclude that conformational changes occur in the channel which interrupt the electrolyte conducting pathway.

Site-directed mutagenesis within the central constriction site of ScrY (sucroseporin): effect on ion transport and comparison of maltooligosaccharide binding to LamB of Escherichia coli.

The 3-D structures of the maltooligosaccharide-specific LamB-channel of Escherichia coli (also called maltoporin) and sucrose-specific ScrY (sucroseporin) are known from X-ray crystallography. The central constriction of the channels formed by the external loop 3 is controlled by a number of different amino acids. The most prominent one of these, N192, D201 and F204, were replaced by site-directed mutagenesis into those of LamB, which, according to the 3-D model of both channels are localized at similar places. The ScrY single mutants ScrYN192R, ScrYD201Y and ScrYF204D and the ScrY triple mutant ScrY3113 (N192R + D201Y + F204D) were created together with the triple mutant ScrY3213, which lacks also amino acids 1 to 61 from the N-terminal end. The mutant proteins were purified to homogeneity and were reconstituted into lipid bilayer membranes. In these experiments, the single-channel conductance of the mutants in different salt solutions and the stability constants for binding of different maltooligosaccharides to the mutant channels was measured using titration experiments with carbohydrates. The carbohydrate-induced block of the channel function could also be used for the study of current noise through the different mutant ScrY-channels. The analysis of the power density spectra allowed the evaluation of the on- and off-rate constants (k1 and k-1) of carbohydrate-binding to the binding site inside the channels. The results suggest that both on- and off-rate constants were affected by the mutations. Most of them showed a substantial effect on carbohydrate binding kinetics. Nevertheless, single-channel conductance and carbohydrate binding of ScrY3113 mutant were still different from that of LamB, suggesting that not only the amino acids of the central constriction but also the general architecture of both channels have a substantial influence on channel properties.

Function of pseudomonas porins in uptake and efflux.

Porins are proteins that form water-filled channels across the outer membranes of Gram-negative bacteria and thus make this membrane semipermeable. There are four types of porins: general/nonspecific porins, substrate-specific porins, gated porins, and efflux porins (also called channel-tunnels). The recent publication of the genomic sequence of Pseudomonas aeruginosa PAO1 has dramatically increased our understanding of the porins of this organism. In particular this organism has 3 large families of porins: the OprD family of specific porins (19 members), the OprM family of efflux porins (18 members), and the TonB-interacting family of gated porins (35 members). These familial relationships underlie functional similarities such that well-studied members of these families become prototypes for other members. We summarize here the latest information on these porins.

Protein-translocating outer membrane porins of Gram-negative bacteria.

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.