Enhanced Binding of TonB to a Ligand-loaded Outer Membrane Receptor: ROLE OF THE OLIGOMERIC STATE OF TonB IN FORMATION OF A FUNCTIONAL FhuA·TonB COMPLEX

The ferric hydroxymate uptake (FhuA) receptor from Escherichia coli facilitates transport of siderophores ferricrocin and ferrichrome and siderophore-antibiotic conjugates such as albomycin and rifamycin CGP 4832. FhuA is also the receptor for phages T5, T1, 80, UC-1, for colicin M and for the antimicrobial peptide microcin MccJ21. Energy for transport is provided by the cytoplasmic membrane complex TonB·ExbB·ExbD, which uses the proton motive force of the cytoplasmic membrane to transduce energy to the outer membrane. To accomplish energy transfer, TonB contacts outer membrane receptors. However, the stoichiometry of TonB· receptor complexes and their sites of interaction remain uncertain. In this study, analyses of FhuA interactions with two recombinant TonB proteins by analytical ultracentrifugation revealed that TonB forms a 2:1 complex with FhuA. The presence of the FhuA-specific ligand ferricrocin enhanced the amounts of complex but is not essential for its formation. Surface plasmon resonance experiments demonstrated that FhuA·TonB interactions are multiple and have apparent affinities in the nanomolar range. TonB also possesses two distinct binding regions: one in the C terminus of the protein, for which binding to FhuA is ferricrocin-independent, and a higher affinity region outside the C terminus, for which ferricrocin enhances interactions with FhuA. Together these experiments establish that FhuA·TonB interactions are more intricate than originally predicted, that the TonB·FhuA stoichiometry is 2:1, and that ferricrocin modulates binding of FhuA to TonB at regions outside the C-terminal domain of TonB

Model Systems to Study the Chronic, Polymicrobial Infections in Cystic Fibrosis: Current Approaches and Exploring Future Directions

A recent workshop titled “Developing Models to Study Polymicrobial Infections,” sponsored by the Dartmouth Cystic Fibrosis Center (DartCF), explored the development of new models to study the polymicrobial infections associated with the airways of persons with cystic fibrosis (CF). The workshop gathered 351 investigators over two virtual sessions. Here, we present the findings of this workshop, summarize some of the challenges involved with developing such models, and suggest three frameworks to tackle this complex problem. The frameworks proposed here, we believe, could be generally useful in developing new model systems for other infectious diseases. Developing and validating new approaches to study the complex polymicrobial communities in the CF airway could open windows to new therapeutics to treat these recalcitrant infections, as well as uncovering organizing principles applicable to chronic polymicrobial infections more generally

Effects of N-Glycosylation Site Removal in Archaellins on the Assembly and Function of Archaella in Methanococcus maripaludis

In Methanococcus maripaludis S2, the swimming organelle, the archaellum, is composed of three archaellins, FlaB1S2, FlaB2S2 and FlaB3S2. All three are modified with an N-linked tetrasaccharide at multiple sites. Disruption of the N-linked glycosylation pathway is known to cause defects in archaella assembly or function. Here, we explored the potential requirement of N-glycosylation of archaellins on archaellation by investigating the effects of eliminating the 4 N-glycosylation sites in the wildtype FlaB2S2 protein in all possible combinations either by Asn to Glu (N to Q) substitution or Asn to Asp (N to D) substitutions of the N-glycosylation sequon asparagine. The ability of these mutant derivatives to complement a non-archaellated ΔflaB2S2 strain was examined by electron microscopy (for archaella assembly) and swarm plates (for analysis of swimming). Western blot results showed that all mutated FlaB2S2 proteins were expressed and of smaller apparent molecular mass compared to wildtype FlaB2S2, consistent with the loss of glycosylation sites. In the 8 single-site mutant complements, archaella were observed on the surface of Q2, D2 and D4 (numbers after N or Q refer to the 1st to 4th glycosylation site). Of the 6 double-site mutation complementations all were archaellated except D1,3. Of the 4 triple-site mutation complements, only D2,3,4 was archaellated. Elimination of all 4 N-glycosylation sites resulted in non-archaellated cells, indicating some minimum amount of archaellin glycosylation was necessary for their incorporation into stable archaella. All complementations that led to a return of archaella also resulted in motile cells with the exception of the D4 version. In addition, a series of FlaB2S2 scanning deletions each missing 10 amino acids was also generated and tested for their ability to complement the ΔflaB2S2 strain. While most variants were expressed, none of them restored archaellation, although FlaB2S2 harbouring a smaller 3-amino acid deletion was able to partially restore archaellation

Interactions between the energy-transducing protein TonB and the outer membrane receptor FhuA from Escherichia coli

Iron is an essential element required by most organisms. To acquire iron, Gram-negative bacteria utilize siderophores; compounds that bind iron and form soluble complexes. Escherichia coli imports siderophores via specialized transport systems. A model siderophore receptor system requires the integral outer membrane (OM) receptor FhuA, which facilitates transport of hydroxamate siderophores ferricrocin (Fc) and ferrichrome. Transport of siderophores by FhuA to the periplasm and into the cell requires energy provided by the proton motive force and delivered by the cytoplasmic membrane protein complex TonB-ExbB-ExbD. Energy is transduced from this complex by protein-protein interactions between TonB and FhuA. Although this system has been extensively studied, much remains unclear about interactions between TonB and FhuA.Sedimentation velocity analytical ultracentrifugation (AUC) experiments involving mixtures of recombinant TonB and FhuA were conducted to determine the oligomeric state of TonB-FhuA complexes. These experiments demonstrated that wild-type TonB-FhuA proteins form a 2:1 (TonB:FhuA) complex, and that this complex is more abundant when FhuA is pre-incubated with the siderophore Fc. Deletion of the N-terminal region of TonB also demonstrated a 2:1 complex stoichiometry with FhuA, but in much lower abundance. Deletion of a highly conserved proline-rich region within TonB resulted in the formation of a 1:1 complex, suggesting a novel role for this domain in the formation of a functional 2:1 complex.Affinities of interaction between wild-type and mutant TonB and FhuA proteins were measured by surface plasmon resonance (SPR). SPR data revealed that wild-type TonB-FhuA interactions are comprised of multiple interaction sites, and undergo an intermediate rearrangement event prior to the formation of the 2:1 complex. Deletions of TonB domains do not disrupt this rearrangement, but do decrease affinity. FhuA cork domain deletions also identified novel sites important for stable interactions.Information of TonB-FhuA interactions derived by AUC and SPR described herein has contributed greatly to the purification and crystallization of TonB-FhuA co-complexes. Overall, these complementary biophysical techniques have provided important mechanistic details into the protein-mediated transport of iron into Gram-negative bacteria, and will provide a conceptual framework of TonB-FhuA that will help in elucidating aspects of the high-resolution structure of the complex

The Role of Pseudomonas aeruginosa Lipopolysaccharide in Bacterial Pathogenesis and Physiology

The major constituent of the outer membrane of Gram-negative bacteria is lipopolysaccharide (LPS), which is comprised of lipid A, core oligosaccharide, and O antigen, which is a long polysaccharide chain extending into the extracellular environment. Due to the localization of LPS, it is a key molecule on the bacterial cell wall that is recognized by the host to deploy an immune defence in order to neutralize invading pathogens. However, LPS also promotes bacterial survival in a host environment by protecting the bacteria from these threats. This review explores the relationship between the different LPS glycoforms of the opportunistic pathogen Pseudomonas aeruginosa and the ability of this organism to cause persistent infections, especially in the genetic disease cystic fibrosis. We also discuss the role of LPS in facilitating biofilm formation, antibiotic resistance, and how LPS may be targeted by new antimicrobial therapies

Bypassing the Need for the Transcriptional Activator EarA through a Spontaneous Deletion in the BRE Portion of the fla Operon Promoter in Methanococcus maripaludis

In Methanococcus maripaludis, the euryarchaeal archaellum regulator A (EarA) is required for the transcription of the fla operon, which is comprised of a series of genes which encode most of the proteins needed for the formation of the archaeal swimming organelle, the archaellum. In mutants deleted for earA (ΔearA), there is almost undetectable transcription of the fla operon, Fla proteins are not synthesized and the cells are non-archaellated. In this study, we have isolated a spontaneous mutant of a ΔearA mutant in which the restoration of the transcription and translation of the fla operon (using flaB2, the second gene of the operon, as a reporter), archaella formation and swarming motility were all restored even in the absence of EarA. Analysis of the DNA sequence from the fla promoter of this spontaneous mutant revealed a deletion of three adenines within a string of seven adenines in the transcription factor B recognition element (BRE). When the three adenine deletion in the BRE was regenerated in a stock culture of the ΔearA mutant, very similar phenotypes to that of the spontaneous mutant were observed. Deletion of the three adenines in the fla promoter BRE resulted in the mutant BRE having high sequence identity to BREs from promoters that have strong basal transcription level in Mc. maripaludis and Methanocaldococcus jannaschii. These data suggest that EarA may help recruit transcription factor B to a weak BRE in the fla promoter of wild-type cells but is not required for transcription from the fla promoter with a strong BRE, as in the three adenine deletion version in the spontaneous mutant

Inroads through the bacterial cell envelope: seeing is believing

A singular feature of all prokaryotic cells in the presence of a cell envelope comprised of a cytoplasmic membrane and a cell wall. The introduction of bacterial cell fractionation techniques in the 1950's and 1960's along with developments in procedures for electron microscopy 'opened the window' towards an understanding of the chemical composition and architecture of the cell envelope. This review traces the contribution of Terry Beveridge in these endeavours, beginning with his doctoral studies in the 1970's on the structure of paracrystalline surface arrays (S-layers) followed by an exploration of cryogenic methods for preserving bacteria for ultrastructural analyses. His insights are reflected in a current example of the contribution of cryoelectron microscopy to S-layer studies - the structure and assembly of the surface array of Caulobacter crescentus. The review then focuses on Terry's contributions to imaging the ultrastructure of bacterial cell envelopes and to the development of cryoelectron microscopy techniques, including the use of CEMOVS (Cryoelectron Microscopy of Vitreous Sections) to 'see' the ultrastructure of the Gram-positive cell envelope - his last scientific endeavour.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author