scholarly journals Localized Hypermutation is the Major Driver of Meningococcal Genetic Variability during Persistent Asymptomatic Carriage

mBio ◽  
2020 ◽  
Vol 11 (2) ◽  
Author(s):  
Luke R. Green ◽  
Ali A. Al-Rubaiawi ◽  
Mohammad A. R. M. Al-Maeni ◽  
Odile B. Harrison ◽  
Matthew Blades ◽  
...  
Keyword(s):  
Genetic Variation ◽  
Gene Transfer ◽  
Outer Membrane ◽  
Antigenic Variation ◽  
Allelic Variation ◽  
Host Responses ◽  
Content Type ◽  
Type Iv ◽  
Asymptomatic Carriage ◽  
Genes Encoding

ABSTRACT Host persistence of bacteria is facilitated by mutational and recombinatorial processes that counteract loss of genetic variation during transmission and selection from evolving host responses. Genetic variation was investigated during persistent asymptomatic carriage of Neisseria meningitidis. Interrogation of whole-genome sequences for paired isolates from 25 carriers showed that de novo mutations were infrequent, while horizontal gene transfer occurred in 16% of carriers. Examination of multiple isolates per time point enabled separation of sporadic and transient allelic variation from directional variation. A comprehensive comparative analysis of directional allelic variation with hypermutation of simple sequence repeats and hyperrecombination of class 1 type IV pilus genes detected an average of seven events per carrier and 2:1 bias for changes due to localized hypermutation. Directional genetic variation was focused on the outer membrane with 69% of events occurring in genes encoding enzymatic modifiers of surface structures or outer membrane proteins. Multiple carriers exhibited directional and opposed switching of allelic variants of the surface-located Opa proteins that enables continuous expression of these adhesins alongside antigenic variation. A trend for switching from PilC1 to PilC2 expression was detected, indicating selection for specific alterations in the activities of the type IV pilus, whereas phase variation of restriction modification (RM) systems, as well as associated phasevarions, was infrequent. We conclude that asymptomatic meningococcal carriage on mucosal surfaces is facilitated by frequent localized hypermutation and horizontal gene transfer affecting genes encoding surface modifiers such that optimization of adhesive functions occurs alongside escape of immune responses by antigenic variation. IMPORTANCE Many bacterial pathogens coexist with host organisms, rarely causing disease while adapting to host responses. Neisseria meningitidis, a major cause of meningitis and septicemia, is a frequent persistent colonizer of asymptomatic teenagers/young adults. To assess how genetic variation contributes to host persistence, whole-genome sequencing and hypermutable sequence analyses were performed on multiple isolates obtained from students naturally colonized with meningococci. High frequencies of gene transfer were observed, occurring in 16% of carriers and affecting 51% of all nonhypermutable variable genes. Comparative analyses showed that hypermutable sequences were the major mechanism of variation, causing 2-fold more changes in gene function than other mechanisms. Genetic variation was focused on genes affecting the outer membrane, with directional changes in proteins responsible for bacterial adhesion to host surfaces. This comprehensive examination of genetic plasticity in individual hosts provides a significant new platform for rationale design of approaches to prevent the spread of this pathogen.

scholarly journals Identification of a Novel Conjugative Plasmid in Mycobacteria That Requires Both Type IV and Type VII Secretion

mBio ◽  
2014 ◽  
Vol 5 (5) ◽  
Author(s):  
Roy Ummels ◽  
Abdallah M. Abdallah ◽  
Vincent Kuiper ◽  
Anouar Aâjoud ◽  
Marion Sparrius ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Gene Transfer ◽  
Horizontal Gene Transfer ◽  
Antibiotic Resistance Genes ◽  
Conjugative Plasmid ◽  
Content Type ◽  
Conjugative Plasmids ◽  
Type Vii Secretion ◽  
Type Iv ◽  
Slow Growing

ABSTRACTConjugative plasmids have been identified in a wide variety of different bacteria, ranging from proteobacteria to firmicutes, and conjugation is one of the most efficient routes for horizontal gene transfer. The most widespread mechanism of plasmid conjugation relies on different variants of the type IV secretion pathway. Here, we describe the identification of a novel type of conjugative plasmid that seems to be unique for mycobacteria. Interestingly, while this plasmid is efficiently exchanged between different species of slow-growing mycobacteria, includingMycobacterium tuberculosis, it could not be transferred to any of the fast-growing mycobacteria tested. Genetic analysis of the conjugative plasmid showed the presence of a locus containing homologues of three type IV secretion system components and a relaxase. In addition, a new type VII secretion locus was present. Using transposon insertion mutagenesis, we show that in fact both these secretion systems are essential for conjugation, indicating that this plasmid represents a new class of conjugative plasmids requiring two secretion machineries. This plasmid could form a useful new tool to exchange or introduce DNA in slow-growing mycobacteria.IMPORTANCEConjugative plasmids play an important role in horizontal gene transfer between different bacteria and, as such, in their adaptation and evolution. This effect is most obvious in the spread of antibiotic resistance genes. Thus far, conjugation of natural plasmids has been described only rarely for mycobacterial species. In fact, it is generally accepted thatM. tuberculosisdoes not show any recent sign of horizontal gene transfer. In this study, we describe the identification of a new widespread conjugative plasmid that can also be efficiently transferred toM. tuberculosis. This plasmid therefore poses both a threat and an opportunity. The threat is that, through the acquisition of antibiotic resistance markers, this plasmid could start a rapid spread of antibiotic resistance genes between pathogenic mycobacteria. The opportunity is that we could use this plasmid to generate new tools for the efficient introduction of foreign DNA in slow-growing mycobacteria.

scholarly journals SalmonellaTol-Pal Reduces Outer Membrane Glycerophospholipid Levels for Envelope Homeostasis and Survival during Bacteremia

2018 ◽  
Vol 86 (7) ◽  
Author(s):  
Revathi Masilamani ◽  
Melina B. Cian ◽  
Zachary D. Dalebroux
Keyword(s):  
Outer Membrane ◽  
Membrane Lipids ◽  
Lipid Homeostasis ◽  
Content Type ◽  
Bacterial Morphology ◽  
Serovar Typhimurium ◽  
Genes Encoding ◽  
Rifampin Resistance ◽  
Mutant Phenotypes ◽  
Acyl Coenzyme A

ABSTRACTSalmonellae regulate membrane lipids during infection, but the exact proteins and mechanisms that promote their survival during bacteremia remain largely unknown. Mutations in genes encoding the conservedSalmonella entericaserovar Typhimurium (S. Typhimurium) Tol-Pal apparatus caused the outer membrane (OM) sensor lipoprotein, RcsF, to become activated. The capsule activation phenotype for the mutants suggested that Tol-Pal might influence envelope lipid homeostasis. The mechanism involves reducing OM glycerophospholipid (GPL) levels, since the mutant salmonellae similarly accumulated phosphatidylglycerols (PGl) and phosphatidylethanolamines (PE) within the OM in comparison to the wild type. The data support theEscherichia colimodel, whereby Tol-Pal directs retrograde GPL translocation across the periplasm. TheS. Typhimurium mechanism involves contributions from YbgC, a cytoplasmic acyl coenzyme A (acyl-CoA) thioesterase, and CpoB, a periplasmic TolA-binding protein. The functional relationship between Tol-Pal and YbgC and CpoB was previously unresolved. TheS. Typhimurium Tol-Pal proteins contribute similarly toward promoting OM-GPL homeostasis and Rcs signaling inactivity but differently toward promoting bacterial morphology, rifampin resistance, survival in macrophages, and survival in mice. For example,tolQ,tolR,tolA, andcpoBmutants were significantly more attenuated thanybgC,tolB, andpalmutants in a systemic mouse model of disease. Therefore, key roles exist for TolQ, TolR, TolA, and CpoB during murine bacteremia, which are independent of maintaining GPL homeostasis. The ability of TolQR to channel protons across the inner membrane (IM) is necessary forS. Typhimurium TolQRA function, since mutating conserved channel-facing residues rendered TolQ ineffective at rescuing deletion mutant phenotypes. Therefore, Tol-Pal promotesS. Typhimurium survival during bacteremia, in part, by reducing OM GPL concentrations, while TolQRA and CpoB enhance systemic virulence by additional mechanisms.

scholarly journals Complete Nucleotide Sequence of ablaKPC-Harboring IncI2 Plasmid and Its Dissemination in New Jersey and New York Hospitals

2013 ◽  
Vol 57 (10) ◽  
pp. 5019-5025 ◽  
Author(s):  
Liang Chen ◽  
Kalyan D. Chavda ◽  
Nahed Al Laham ◽  
Roberto G. Melano ◽  
Michael R. Jacobs ◽  
...  
Keyword(s):  
New York ◽  
New Jersey ◽  
Nucleotide Sequence ◽  
Complete Nucleotide Sequence ◽  
Multidrug Resistant ◽  
Content Type ◽  
Type Iv ◽  
Genes Encoding ◽  
Significant Public Health ◽  
Integration Sites

ABSTRACTKlebsiella pneumoniaecarbapenemase (KPC)-producingK. pneumoniaestrains have spread worldwide and become a significant public health threat.blaKPC, the plasmid-borne KPC gene, was frequently identified on numerous transferable plasmids in different incompatibility replicon groups. Here we report the complete nucleotide sequence of a novelblaKPC-3-harboring IncI2 plasmid, pBK15692, isolated from a multidrug-resistantK. pneumoniaeST258 strain isolated from a New Jersey hospital in 2005. pBK15692 is 78 kb in length and carries a backbone that is similar to those of other IncI2 plasmids (pR721, pChi7122-3, pHN1122-1, and pSH146-65), including the genes encoding type IV pili and shufflon regions. Comparative genomics analysis of IncI2 plasmids reveals that they possess a conserved plasmid backbone but are divergent with respect to the integration sites of resistance genes. In pBK15692, theblaKPC-3-harboring Tn4401was inserted into a Tn1331element and formed a nested transposon. A PCR scheme was designed to detect the prevalence of IncI2 and pBK15692-like plasmids from a collection of clinical strains from six New Jersey and New York hospitals isolated between 2007 and 2011. IncI2 plasmids were found in 46.2% isolates from 318 clinicalK. pneumoniaestrains. Notably, 59 pBK15692-like plasmids (23%) have been identified in 256 KPC-bearingK. pneumoniaestrains, and all carried KPC-3 and belong to the epidemic ST258 clone. Our study revealed that the prevalence of IncI2 plasmids has been considerably underestimated. Further studies are needed to understand the distribution of this plasmid group in other health care regions and decipher the association between IncI2 plasmids andblaKPC-3-bearing ST258 strains.

scholarly journals Association and Evidence for Linked Recognition of Type IV Secretion System Proteins VirB9-1, VirB9-2, and VirB10 in Anaplasma marginale

2011 ◽  
Vol 80 (1) ◽  
pp. 215-227 ◽  
Author(s):  
Kaitlyn Morse ◽  
Junzo Norimine ◽  
Guy H. Palmer ◽  
Eric L. Sutten ◽  
Timothy V. Baszler ◽  
...  
Keyword(s):  
T Cell ◽  
Outer Membrane ◽  
Anaplasma Marginale ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Type Iv Secretion System ◽  
Interacting Protein ◽  
Content Type ◽  
Cell Responses ◽  
Type Iv

ABSTRACTLike several other bacterial pathogens,Anaplasma marginalehas an outer membrane that induces complete protection from infection and disease. However, the proteins that confer protective immunity and whether protection requires interacting proteins and/or linked T-cell and immunoglobulin G epitopes are not known. Our goal is to target the conserved type IV secretion system (T4SS) to identify conserved, immunogenic membrane proteins that are interacting and linked recognition candidates. Linked recognition is a process by which a B cell is optimally activated by a helper T cell that responds to the same, or physically associated, antigen.A. marginaleT4SS proteins VirB2, VirB4-1, VirB4-2, VirB6-1, VirB7, VirB8-2, VirB9-1, VirB9-2, VirB10, VirB11, and VirD4 were screened for their ability to induce IgG and to stimulate CD4+T cells from outer membrane-vaccinated cattle. VirB9-1, VirB9-2, and VirB10 induced the strongest IgG and T-cell responses in the majority of cattle, although three animals with major histocompatibility complex class II DRB3 restriction fragment length polymorphism types 8/23, 3/16, and 16/27 lacked T-cell responses to VirB9-1, VirB9-1 and VirB9-2, or VirB9-2 and VirB10, respectively. For these animals, VirB9-1-, VirB9-2-, and VirB10-specific IgG production may be associated with T-cell help provided by responses to an interacting protein partner(s). Interacting protein partners indicated by far-Western blotting were confirmed by immunoprecipitation assays and revealed, for the first time, specific interactions of VirB9-1 with VirB9-2 and VirB10. The immunogenicity and interactions of VirB9-1, VirB9-2, and VirB10 justify their testing as a linked protein vaccine againstA. marginale.

scholarly journals Lateral Gene Transfer Acts As an Evolutionary Shortcut to Efficient C4 Biochemistry

2020 ◽  
Vol 37 (11) ◽  
pp. 3094-3104
Author(s):  
Chatchawal Phansopa ◽  
Luke T Dunning ◽  
James D Reid ◽  
Pascal-Antoine Christin
Keyword(s):  
Genetic Variation ◽  
Amino Acid ◽  
Gene Transfer ◽  
Catalytic Efficiency ◽  
Distinct Species ◽  
Amino Acid Replacement ◽  
The Novel ◽  
Ideal System ◽  
Genes Encoding ◽  
Speed Up

Abstract The adaptation of proteins for novel functions often requires changes in their kinetics via amino acid replacement. This process can require multiple mutations, and therefore extended periods of selection. The transfer of genes among distinct species might speed up the process, by providing proteins already adapted for the novel function. However, this hypothesis remains untested in multicellular eukaryotes. The grass Alloteropsis is an ideal system to test this hypothesis due to its diversity of genes encoding phosphoenolpyruvate carboxylase, an enzyme that catalyzes one of the key reactions in the C4 pathway. Different accessions of Alloteropsis either use native isoforms relatively recently co-opted from other functions or isoforms that were laterally acquired from distantly related species that evolved the C4 trait much earlier. By comparing the enzyme kinetics, we show that native isoforms with few amino acid replacements have substrate KM values similar to the non-C4 ancestral form, but exhibit marked increases in catalytic efficiency. The co-option of native isoforms was therefore followed by rapid catalytic improvements, which appear to rely on standing genetic variation observed within one species. Native C4 isoforms with more amino acid replacements exhibit additional changes in affinities, suggesting that the initial catalytic improvements are followed by gradual modifications. Finally, laterally acquired genes show both strong increases in catalytic efficiency and important changes in substrate handling. We conclude that the transfer of genes among distant species sharing the same physiological novelty creates an evolutionary shortcut toward more efficient enzymes, effectively accelerating evolution.

scholarly journals Flagellar Mutants Have Reduced Pilus Synthesis in Caulobacter crescentus

2019 ◽  
Vol 201 (18) ◽  
Author(s):  
Courtney K. Ellison ◽  
Douglas B. Rusch ◽  
Yves V. Brun
Keyword(s):  
Cell Biology ◽  
Regulatory Networks ◽  
Caulobacter Crescentus ◽  
Single Cells ◽  
Bacterial Species ◽  
Type Iv Pili ◽  
Content Type ◽  
Transcription Profiles ◽  
Type Iv ◽  
Genes Encoding

ABSTRACT Surface appendages, such as flagella and type IV pili, mediate a broad range of bacterial behaviors, including motility, attachment, and surface sensing. While many species harbor both flagella and type IV pili, little is known about how or if their syntheses are coupled. Here, we show that deletions of genes encoding different flagellum machinery components result in a reduction of pilus synthesis in Caulobacter crescentus. First, we show that different flagellar mutants exhibit different levels of sensitivity to a pilus-dependent phage and that fewer cells within populations of flagellar mutants make pili. Furthermore, we find that single cells within flagellar mutant populations produce fewer pili per cell. We demonstrate that these gene deletions result in reduced transcription of pilus-associated genes and have a slight but significant effect on general transcription profiles. Finally, we show that the decrease in pilus production is due to a reduction in the pool of pilin subunits that are polymerized into pilus fibers. These data demonstrate that mutations in flagellar gene components not only affect motility but also can have considerable and unexpected consequences for other aspects of cell biology. IMPORTANCE Most bacterial species synthesize surface-exposed appendages that are important for environmental interactions and survival under diverse conditions. It is often assumed that these appendages act independently of each other and that mutations in either system can be used to assess functionality in specific processes. However, we show that mutations in flagellar genes can impact the production of type IV pili, as well as alter general RNA transcriptional profiles compared to a wild-type strain. These data demonstrate that seemingly simple mutations can broadly affect cell-regulatory networks.

scholarly journals Identification of Functions Affecting Predator-Prey Interactions between Myxococcus xanthus and Bacillus subtilis

2016 ◽  
Vol 198 (24) ◽  
pp. 3335-3344 ◽  
Author(s):  
Susanne Müller ◽  
Sarah N. Strack ◽  
Sarah E. Ryan ◽  
Mary Shawgo ◽  
Abigail Walling ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Soil Bacteria ◽  
Myxococcus Xanthus ◽  
Soil Organism ◽  
Multiple Traits ◽  
Content Type ◽  
Predator Prey ◽  
Type Iv ◽  
Genes Encoding ◽  
Predation Capacity

ABSTRACTSoil bacteria engage each other in competitive and cooperative ways to determine their microenvironments. In this study, we report the identification of a large number of genes required forMyxococcus xanthusto engageBacillus subtilisin a predator-prey relationship. We generated and tested over 6,000 individual transposon insertion mutants ofM. xanthusand found many new factors required to promote efficient predation, including the specialized metabolite myxoprincomide, an ATP-binding cassette (ABC) transporter permease, and a clustered regularly interspaced short palindromic repeat (CRISPR) locus encoding bacterial immunity. We also identified genes known to be involved in predation, including those required for the production of exopolysaccharides and type IV pilus (T4P)-dependent motility, as well as chemosensory and two-component systems. Furthermore, deletion of these genes confirmed their role during predation. Overall,M. xanthuspredation appears to be a multifactorial process, with multiple determinants enhancing predation capacity.IMPORTANCESoil bacteria engage each other in complex environments and utilize multiple traits to ensure survival. Here, we report the identification of multiple traits that enable a common soil organism,Myxococcus xanthus, to prey upon and utilize nutrients from another common soil organism,Bacillus subtilis. We mutagenized the predator and carried out a screen to identify genes that were required to either enhance or diminish capacity to consume prey. We identified dozens of genes encoding factors that contribute to the overall repertoire for the predator to successfully engage its prey in the natural environment.

scholarly journals Gene Transfer Potential of Outer Membrane Vesicles of Acinetobacter baylyi and Effects of Stress on Vesiculation

2014 ◽  
Vol 80 (11) ◽  
pp. 3469-3483 ◽  
Author(s):  
Shweta Fulsundar ◽  
Klaus Harms ◽  
Gøril E. Flaten ◽  
Pål J. Johnsen ◽  
Balu Ananda Chopade ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Zeta Potential ◽  
Outer Membrane ◽  
Bacterial Species ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Hydrodynamic Diameter ◽  
Acinetobacter Baylyi ◽  
Content Type ◽  
Dsdna Antibodies

ABSTRACTOuter membrane vesicles (OMVs) are continually released from a range of bacterial species. Numerous functions of OMVs, including the facilitation of horizontal gene transfer (HGT) processes, have been proposed. In this study, we investigated whether OMVs contribute to the transfer of plasmids between bacterial cells and species using Gram-negativeAcinetobacter baylyias a model system. OMVs were extracted from bacterial cultures and tested for the ability to vector gene transfer into populations ofEscherichia coliandA. baylyi, including naturally transformation-deficient mutants ofA. baylyi. Anti-double-stranded DNA (anti-dsDNA) antibodies were used to determine the movement of DNA into OMVs. We also determined how stress affected the level of vesiculation and the amount of DNA in vesicles. OMVs were further characterized by measuring particle size distribution (PSD) and zeta potential. Transmission electron microscopy (TEM) and immunogold labeling were performed using anti-fluorescein isothiocyanate (anti-FITC)-conjugated antibodies and anti-dsDNA antibodies to track the movement of FITC-labeled and DNA-containing OMVs. Exposure to OMVs isolated from plasmid-containing donor cells resulted in HGT toA. baylyiandE. coliat transfer frequencies ranging from 10−6to 10−8, with transfer efficiencies of approximately 103and 102per μg of vesicular DNA, respectively. Antibiotic stress was shown to affect the DNA content of OMVs as well as their hydrodynamic diameter and zeta potential. Morphological observations suggest that OMVs fromA. baylyiinteract with recipient cells in different ways, depending on the recipient species. Interestingly, the PSD measurements suggest that distinct size ranges of OMVs are released fromA. baylyi.

scholarly journals Long Persistence of a Streptococcus pneumoniae 23F Clone in a Cystic Fibrosis Patient

mSphere ◽  
2017 ◽  
Vol 2 (3) ◽  
Author(s):  
Martin Rieger ◽  
Harald Mauch ◽  
Regine Hakenbeck
Keyword(s):  
Cystic Fibrosis ◽  
Streptococcus Pneumoniae ◽  
Gene Transfer ◽  
Cystic Fibrosis Patient ◽  
Penicillin Resistance ◽  
Specific Gene ◽  
Gene Encoding ◽  
Content Type ◽  
Long Period ◽  
Genes Encoding

ABSTRACT Streptococcus pneumoniae is a common resident in the human nasopharynx. However, carriage can result in severe diseases due to a unique repertoire of pathogenicity factors that are rare in closely related commensal streptococci. We investigated a penicillin-resistant S. pneumoniae clone of serotype 23F isolated from a cystic fibrosis patient on multiple occasions over an unusually long period of over 3 years that was present without causing disease. Genome comparisons revealed an apparent nonfunctional pneumococcus-specific gene encoding a hyaluronidase, supporting the view that this enzyme adds to the virulence potential of the bacterium. The 23F clone harbored unique mosaic genes encoding penicillin resistance determinants, the product of horizontal gene transfer involving the commensal S. mitis as donor species. Sequences identical to one such mosaic gene were identified in an S. mitis strain from the same patient, suggesting that in this case S. pneumoniae played the role of donor. Streptococcus pneumoniae isolates of serotype 23F with intermediate penicillin resistance were recovered on seven occasions over a period of 37 months from a cystic fibrosis patient in Berlin. All isolates expressed the same multilocus sequence type (ST), ST10523. The genome sequences of the first and last isolates, D122 and D141, revealed the absence of two phage-related gene clusters compared to the genome of another ST10523 strain, D219, isolated earlier at a different place in Germany. Genomes of all three strains carried the same novel mosaic penicillin-binding protein (PBP) genes, pbp2x, pbp2b, and pbp1a; these genes were distinct from those of other penicillin-resistant S. pneumoniae strains except for pbp1a of a Romanian S. pneumoniae isolate. All PBPs contained mutations that have been associated with the penicillin resistance phenotype. Most interestingly, a mosaic block identical to an internal pbp2x sequence of ST10523 was present in pbp2x of Streptococcus mitis strain B93-4, which was isolated from the same patient. This suggests interspecies gene transfer from S. pneumoniae to S. mitis within the host. Nearly all genes expressing surface proteins, which represent major virulence factors of S. pneumoniae and are typical for this species, were present in the genome of ST10523. One exception was the hyaluronidase gene hlyA, which contained a 12-nucleotide deletion within the promoter region and an internal stop codon. The lack of a functional hyaluronidase might contribute to the ability to persist in the host for an unusually long period of time. IMPORTANCE Streptococcus pneumoniae is a common resident in the human nasopharynx. However, carriage can result in severe diseases due to a unique repertoire of pathogenicity factors that are rare in closely related commensal streptococci. We investigated a penicillin-resistant S. pneumoniae clone of serotype 23F isolated from a cystic fibrosis patient on multiple occasions over an unusually long period of over 3 years that was present without causing disease. Genome comparisons revealed an apparent nonfunctional pneumococcus-specific gene encoding a hyaluronidase, supporting the view that this enzyme adds to the virulence potential of the bacterium. The 23F clone harbored unique mosaic genes encoding penicillin resistance determinants, the product of horizontal gene transfer involving the commensal S. mitis as donor species. Sequences identical to one such mosaic gene were identified in an S. mitis strain from the same patient, suggesting that in this case S. pneumoniae played the role of donor.

scholarly journals Type IV Pilus Secretins Have Extracellular C Termini

mBio ◽  
2015 ◽  
Vol 6 (2) ◽  
Author(s):  
Joshua A. Lieberman ◽  
Courtney D. Petro ◽  
Stefani Thomas ◽  
Austin Yang ◽  
Michael S. Donnenberg
Keyword(s):  
Outer Membrane ◽  
Secondary Structure Prediction ◽  
Outer Membrane Proteins ◽  
Gram Negative Bacteria ◽  
Structural Constraints ◽  
Type Iv Pilus ◽  
Gram Negative ◽  
Content Type ◽  
Atomic Structures ◽  
Type Iv

ABSTRACTType IV pili (T4Ps) are surface appendages used by Gram-negative and Gram-positive pathogens for motility and attachment to epithelial surfaces. In Gram-negative bacteria, such as the important pediatric pathogen enteropathogenicEscherichia coli(EPEC), during extension and retraction, the pilus passes through an outer membrane (OM) pore formed by the multimeric secretin complex. The secretin is common to Gram-negative assemblies, including the related type 2 secretion (T2S) system and the type 3 secretion (T3S) system. The N termini of the secretin monomers are periplasmic and in some systems have been shown to mediate substrate specificity. In this study, we mapped the topology of BfpB, the T4P secretin from EPEC, using a combination of biochemical and biophysical techniques that allowed selective identification of periplasmic and extracellular residues. We applied rules based on solved atomic structures of outer membrane proteins (OMPs) to generate our topology model, combining the experimental results with secondary structure prediction algorithms and direct inspection of the primary sequence. Surprisingly, the C terminus of BfpB is extracellular, a result confirmed by flow cytometry for BfpB and a distantly related T4P secretin, PilQ, fromPseudomonas aeruginosa. Keeping with prior evidence, the C termini of two T2S secretins and one T3S secretin were not detected on the extracellular surface. On the basis of our data and structural constraints, we propose that BfpB forms a beta barrel with 16 transmembrane beta strands. We propose that the T4P secretins have a C-terminal segment that passes through the center of each monomer.IMPORTANCESecretins are multimeric proteins that allow the passage of secreted toxins and surface structures through the outer membranes (OMs) of Gram-negative bacteria. To date, there have been no atomic structures of the C-terminal region of a secretin, although electron microscopy (EM) structures of the complex are available. This work provides a detailed topology prediction of the membrane-spanning domain of a type IV pilus (T4P) secretin. Our study used innovative techniques to provide new and comprehensive information on secretin topology, highlighting similarities and differences among secretin subfamilies. Additionally, the techniques used in this study may prove useful for the study of other OM proteins.

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