About
59
Publications
59,825
Reads
How we measure 'reads'
A 'read' is counted each time someone views a publication summary (such as the title, abstract, and list of authors), clicks on a figure, or views or downloads the full-text. Learn more
1,422
Citations
Introduction
Bacteria can "glide" on hard surfaces (in the absence of flagella or type IV pili), but the mechanism responsible is poorly understood. Gliding in the Gram-negative bacterium Myxococcus xanthus involves a multiprotein complex moving internally along a helicoidal track and requires coupling to the substratum to produce propulsion. I am examining the structures, functions, and interactions of the coupling proteins using biochemical, biophysical, bioinformatic, molecular, and genetic approaches.
Current institution
Additional affiliations
July 2016 - present
November 2013 - July 2016
Position
- Canadian Institutes of Health Research Postdoctoral Fellow
Description
- Studying the mechanism of single-cell gliding motility in Myxococcus xanthus
July 2013 - July 2013
Position
- Visiting Scientist
Description
- Performed optical trap-based cell-surface fixation and tracking of beads on Myxococcus xanthus cells to study gliding motility motor properties
Education
September 2007 - June 2013
September 2001 - May 2006
Publications
Publications (59)
The surfaces of bacteria mediate a multitude of functions in the environment and in an infected host, including adhesion to both biotic and abiotic substrata, motility, immune system interaction and (or) activation, biofilm formation, and cell–cell communication, with many of these features directly influenced by cell-surface glycans. In both Gram-...
Wzx flippases are crucial for bacterial cell surface polysaccharide assembly as they transport undecaprenyl pyrophosphate-linked sugar repeat units from the cytoplasmic to the periplasmic leaflets of the inner membrane (IM) for final assembly. Our recently reported three-dimensional (3D) model structure of Wzx from Pseudomonas aeruginosa PAO1 (WzxP...
Heteropolymeric B-band O-antigen (O-Ag) biosynthesis in Pseudomonas aeruginosa PAO1 follows the Wzy-dependent pathway, beginning with translocation of undecaprenyl pyrophosphate-linked anionic O-Ag subunits (O units) from the inner to the outer leaflets of the inner membrane (IM). This translocation is mediated by the integral IM flippase Wzx. Thro...
IMPORTANCE
The opportunistic pathogen Pseudomonas aeruginosa synthesizes its virulence factor lipopolysaccharide via the Wzy-dependent pathway, requiring translocation, polymerization, and ligation of lipid-linked polysaccharide repeat units by the integral inner membrane proteins Wzx, Wzy, and WaaL, respectively. However, structural evidence to he...
Mutations on the Ras-family of small GTPases are among the most common molecular oncogenic drivers, with the HRas isoform being primarily associated with head-and-neck and genito-urinary cancers. Although once considered “undruggable,” recent efforts have identified a structurally conserved surface pocket in the Ras family, designated the SI/II poc...
Recent advances in iterative neural network analyses (e.g., AlphaFold2 and RoseTTA fold) have been revolutionary for protein 3D structure prediction, especially for difficult-to-manipulate α-helical/β-barrel integral membrane proteins. These model structures are calculated based on the coevolution of amino acids within the protein of interest and s...
A bstract
Integrins are crucial for eukaryotic cell attachment and motility within the extracellular matrix (ECM) via focal-adhesion formation, with their evolutionary emergence important for the development of multicellularity. Intriguingly, single gliding cells of the predatory deltaproteobacterium Myxococcus xanthus form bacterial focal-adhesion...
The predatory deltaproteobacterium Myxococcus xanthus uses a helically-trafficked motor at bacterial focal-adhesion (bFA) sites to power gliding motility. Using total internal reflection fluorescence and force microscopies, we identify the von Willebrand A domain-containing outer-membrane (OM) lipoprotein CglB as an essential substratum-coupling ad...
Secretion of high-molecular-weight polysaccharides across the bacterial envelope is ubiquitous, as it enhances prokaryotic survival in (a)biotic settings. Such polymers are often assembled by Wzx/Wzy- or ABC transporter-dependent schemes implicating outer membrane (OM) polysaccharide export (OPX) proteins in cell-surface polymer translocation. In t...
Metabolic labeling paired with click chemistry is a powerful approach for selectively imaging the surfaces of diverse bacteria. Herein, we explored the feasibility of labeling the lipopolysaccharide (LPS) of Myxococcus xanthus-a Gram-negative predatory social bacterium known to display complex outer membrane (OM) dynamics-via growth in the presence...
Lloyd and Tahon recently criticized proposed bacterial phylum nomenclature changes (K.G. Lloyd, G. Tahon, Nat Rev Microbiol 20:123-124, 2022, https://doi.org/10.1038/s41579-022-00684-2) precipitated by the International Committee on Systematics of Prokaryotes (ICSP)'s official recognition of phylum nomenclature rules. Here, we extend the critique....
The presence of an exopolysaccharide (EPS) layer surrounding bacterial cells, termed a “glycocalyx”, confers protection against toxic molecules. However, the effect of glycocalyx integrity on the tolerance to such agents is poorly understood. Using a modified disc-diffusion assay, we tested the susceptibility to a panel of antibiotics and oxidative...
Secretion of high-molecular-weight polysaccharides across the bacterial envelope is ubiquitous as it enhances prokaryotic survival in (a)biotic settings. Such polymers are often assembled by Wzx/Wzy- or ABC transporter-dependent schemes that implicate outer-membrane (OM) polysaccharide export (OPX) proteins in polymer translocation to the cell surf...
Exopolysaccharide (EPS) layers on the bacterial cell surface are key determinants of biofilm establishment and maintenance, leading to the formation of higher‐order 3D structures that confer numerous survival benefits to a cell community. In addition to a specific cell‐associated EPS glycocalyx, we recently revealed that the social δ‐proteobacteriu...
Bacterial surface exopolysaccharide (EPS) layers are key determinants of biofilm establishment and maintenance, leading to the formation of higher-order 3D structures conferring numerous survival benefits to a cell community. In addition to a specific EPS glycocalyx, we recently revealed that the social δ-proteobacterium Myxococcus xanthus secretes...
A bstract
The predatory deltaproteobacterium Myxococcus xanthus uses a helically-trafficked motor at bacterial focal adhesion (bFA) sites to power gliding motility. Using TIRF and force microscopy, we herein identify the integrin αI-domain-like outer-membrane (OM) lipoprotein CglB as an essential substratum-coupling protein of the gliding motility...
The development of multicellularity is a key evolutionary transition allowing for differentiation of physiological functions across a cell population that confers survival benefits; among unicellular bacteria, this can lead to complex developmental behaviors and the formation of higher-order community structures. Herein, we demonstrate that in the...
Single-cell microfluidics is a powerful method to study bacteria and determine their susceptibility to antibiotic treatment. Glass treatment by adhesive molecules is a potential solution to immobilize bacterial cells and perform microscopy, but traditional cationic polymers such as polylysine deeply affect bacterial physiology. In this work, we che...
Single cell microfluidics is powerful to study bacteria and determine their susceptibility to antibiotics treatment. Glass treatment by adhesive molecules is a potential solution to immobilize bacterial cells and perform microscopy but traditional cationic polymers such as poly-lysine deeply affect bacterial physiology. In this work, we chemically...
Various rod-shaped bacteria mysteriously glide on surfaces in the absence of appendages such as flagella or pili. In the deltaproteobacterium Myxococcus xanthus, a putative gliding motility machinery (Agl–Glt) localizes to so-called Focal Adhesion sites (FA) that form stationary contact points with the underlying surface. We discovered that the Agl...
O antigen (O-Ag) in many bacteria is synthesized via the Wzx/Wzy-dependent pathway in which Wzy polymerizes lipid-linked O-Ag subunits to modal lengths regulated by Wzz. Characterization of 83 site-directed mutants of Wzy from Pseudomonas aeruginosa PAO1 (WzyPa) in topologically-mapped periplasmic (PL) and cytoplasmic loops (CL) verified the functi...
Lysogenic bacteriophage D3 causes seroconversion of Pseudomonas aeruginosa PAO1 from serotype O5 to O16 by inverting the linkage between O-specific antigen (OSA) repeat units from α to β. The OSA
units are polymerized by Wzy to modal lengths regulated by Wzz1 and Wzz2. A key component of the D3 seroconversion machinery is the inhibitor of α-polymer...
Heteropolymeric O antigen (O-Ag)-capped lipopolysaccharide is the principal constituent of the Gram-negative bacterial cell surface. It is assembled via the integral inner membrane (IM) Wzx/Wzy-dependent pathway. In Pseudomonas aeruginosa, Wzx translocates lipid-linked anionic O-Ag subunits from the cytoplasmic to the periplasmic leaflets of the IM...
Bacterial cell surface polysaccharides confer resistance to external stress and promote survival in biotic and abiotic environments. Glycan assembly often occurs at the periplasmic leaflet of the inner membrane (IM) from undecaprenyl pyrophosphate (UndPP)-linked polysaccharide units via the Wzx/Wzy-dependent pathway. Wzx is an integral IM protein f...
Integral membrane proteins with α-helical transmembrane segments (TMS) are known to play important and diverse roles in prokaryotic cell physiology. The net hydrophobicity of TMS directly corresponds to the observed difficulties in expressing and purifying these proteins, let alone producing sufficient yields for structural studies using two-/three...
The processes responsible for the biosynthesis of antigenic cell-surface sugar polymers are applicable to a wide range of pathogenic bacteria, yet they remain poorly understood. Research in our laboratory focuses on the cell surface of Pseudomonas aeruginosa, an important opportunistic pathogen that infects a range of compromised patients, most not...
Heteropolymeric B-band lipopolysaccharide in \textit{Pseudomonas aeruginosa} PAO1 is synthesized via the Wzx/Wzy-dependent pathway, requiring a functional Wzx for flipping of anionic O-antigen (O-Ag) repeat units from the inner to the outer leaflet of the inner membrane (IM). Wzx is an integral IM protein for which the detailed topology has been ma...
Heteropolymeric B-band lipopolysaccharide in Pseudomonas aeruginosa PAO1 is synthesized via the Wzy-dependent pathway, requiring a functional Wzy for polymerization of O-antigen (O-Ag) repeat units in the periplasm. Wzy is an integral inner membrane protein for which the detailed topology has been mapped in a recent investigation by our group, reve...
The Wzy-dependent pathway for the biosynthesis of cell-surface polysaccharides is applicable to a wide range of Gram-negative bacteria, yet it remains poorly understood. In Pseudomonas aeruginosa, an often fatal opportunistic pathogen of compromised patients, this pathway is responsible for the synthesis of the immunodominant lipopolysaccharide gly...
Heteropolymeric B-band lipopolysaccharide in Pseudomonas aeruginosa PAO1 is synthesized via the so-called Wzy-dependent pathway, requiring a functional Wzy for polymerization of O-antigen repeat
units in the periplasm. Wzy is an integral inner membrane protein for which the detailed topology has been mapped in a recent
investigation (Islam, S. T.,...
Lipopolysccharide (LPS) is an integral component of the Pseudomonas aeruginosa cell envelope, occupying the outer leaflet of the outer membrane in this Gram-negative opportunistic pathogen. It is important for bacterium–host interactions and has been shown to be a major virulence factor for this organism. Structurally, P. aeruginosa LPS is composed...
The Wzy-dependent pathway for the biosynthesis of cell-surface polysaccharides is applicable to a wide range of Gram-negative bacteria, yet it remains poorly understood. In Pseudomonas aeruginosa, an often fatal opportunistic pathogen of compromised patients, this pathway is responsible for the synthesis of the immunodominant lipopolysaccharide gly...
FIG. S3 PSIPRED secondary-structure analysis of large cytoplasmic (CL) and periplasmic (PL) loop domains from Wzy (Y), with three residues from each flanking TMS included. CL2Y contains a tract of amino acids similar to the R/KXXXGXXXLhhhhD Walker B consensus motif (J. E. Walker, M. Saraste, M. J. Runswick, and N. J. Gay, EMBO J. 1:945–951, 1982)....
FIG. S2 PSIPRED secondary-structure analysis of large cytoplasmic (CL) and periplasmic (PL) loop domains from Wzx (X), with three residues from each flanking TMS included. As seen from the analysis of the potential secondary structure of CL1X, CL3X, and CL5X, an elaborate secondary structure is predicted to exist on the cytoplasmic face of the prot...
FIG. S1 LPS phenotype analysis. Chromosomal knockout mutants of wzx, wzy, and waaL in P. aeruginosa PAO1 are all unable to produce B-band LPS; however, the waaL mutant has been shown to produce full-length B-band O-Ag linked to Und-PP, as the biosynthetic machinery leading to the ligation step of the B-band LPS assembly pathway is still functional....
The Wzy-dependent pathway model for the biosynthesis and assembly of cell-surface polysaccharides is applicable to a wide range of Gram-negative bacteria, yet it remains poorly understood. Research in our laboratory focuses on the cell surface of Pseudomonas aeruginosa, an important opportunistic Gram-negative bacterial pathogen that can cause fata...
The Wzy-dependent pathway model for the biosynthesis and assembly of cell-surface polysaccharides is applicable to a wide range of both Gram-positive and Gram-negative bacteria, yet it remains poorly understood. One such cell-surface constituent is lipopolysaccharide (LPS), an integral component of the Gram-negative cell wall and major virulence fa...
Pseudomonas aeruginosa is an important opportunistic Gram-negative bacterial pathogen that can cause fatal infections in compromised patients including those with cancer, AIDS, and cystic fibrosis. This bacterium is also known to be intrinsically resistant to a wide range of antibiotics and endowed with inducible multiple-drug efflux pumps. A bette...
Infection by the opportunistic Gram-negative pathogen Pseudomonas aeruginosa is clinically prevalent in compromised patients suffering from such diseases as cancer, AIDS, and in particular, cystic fibrosis (CF). In the latter, over 80% of individuals are chronically infected, resulting in P. aeruginosa infection as the leading cause of death in CF-...
Safety of BCG is a major concern in countries with a high burden of HIV/AIDS. Current BCG vaccine comprises of a heterogeneous group of substrains showing genotypic differences. The impact of these differences on BCG efficacy and safety remains unknown. Here we show that three BCG substrains, BCG-Japan, -Moreau, and -Glaxo, do not produce phthiocer...
Lipooligosaccharides (LOSs) are antigenic glycolipids that are present in some species of Mycobacterium including the Canetti strain of M. tuberculosis. The core LOS structures from several mycobacterial organisms have been established, but the biosynthetic pathways of LOSs remain unknown. In this study, we describe two transposon insertion mutants...
Questions
Questions (5)
I have a proteolytic enzyme activity that is inhibited by EDTA, suggesting that the enzyme I'm investigating is a metalloprotease.
Unfortunately, EDTA can chelate several divalent cations, including Ca2+, Zn2+, Mg2+, and Mn2+, which are arguably the most common ion cofactors used by metalloproteases.
In the Sigma product literature, EDTA is listed as only targeting Ca2+ and Zn2+, and that to discriminate between Ca2+ and Zn2+ effects, to use EGTA and 1,10-phenanthroline (respectively). Nothing is mentioned about Mg2+ or Mn2+.
1) Does anybody agree that EGTA (Ca2+-"specific") and 1,10-phenanthroline (Zn2+-"specific") are the best chelators to use to distinguish between Ca2+ and Zn2+ effects?
2) At what concentrations?
3) Are there any better (more specific) chelators to use?
4) What about something specific for Mg2+ or Mn2+?
Thank you in advance to any fellow scientists that may have some helpful advice/tips to offer!
Cheers,
Salim
I have a protein that fractionates with the outer membrane sample of Gram-negative bacterial cells.
SignalP4.1 predicts that the native protein is processed between amino acids 19(Ala)—20(Cys).
LipoP1.0 predicts that there is indeed a lipid moiety attached to the +2 position, i.e. residue 21 (Glu)
What would be the most straight-forward way in which to experimentally determine the specific amino acid on the protein that is attached to the lipid?
Is there a way in which the lipid moiety can be removed to facilitate Edman degradation (i.e. N-terminal sequencing)?
Any insights or suggestions would be happily entertained.
Cheers,
Salim
I would like to grow my cells in broth at different pH values. However, over time, the pH of a culture will change as the cell density increases. What would be the best way in which to maintain an overnight shaking culture at a fixed pH?
Option 1: I would like to label an exposed His8 tag with a compound that will stay bound to the His8 tag during denaturing SDS-PAGE resolution of protein bands. In this way, non-labeled proteins should migrate faster (i.e. lower) in the gel, whereas proteins with a successfully-labeled His8 tag should be shifted higher in the gel relative to the unlabeled counterparts (as they are running more slowly).
Option 2: I would like to label an exposed His8 tag with a fluorescent compound (with an emission profile distinct from GFP) that will stay bound to the His8 tag during SDS-PAGE. Different amounts of His8 labelling could then be compared via densitometry.
Option 3: Options 1 and 2 COMBINED (this would be an ideal scenario)
NOTE: Ideally, the probes should be commercially available.
Any ideas?
