Outer membrane protein folding from an energy landscape perspective

The cell envelope is essential for the survival of Gram-negative bacteria. This specialised membrane is densely packed with outer membrane proteins (OMPs), which perform a variety of functions. How OMPs fold into this crowded environment remains an open question. Here, we review current knowledge about OFMP folding mechanisms in vitro and discuss how the need to fold to a stable native state has shaped their folding energy landscapes. We also highlight the role of chaperones and the β-barrel assembly machinery (BAM) in assisting OMP folding in vivo and discuss proposed mechanisms by which this fascinating machinery may catalyse OMP folding

Mechanistic Insights into the Capsule-Targeting Depolymerase from a Klebsiella pneumoniae Bacteriophage

The production of capsular polysaccharides by Klebsiella pneumoniae protects the bacterial cell from harmful environmental factors such as antimicrobial compounds and infection by bacteriophages (phages). To bypass this protective barrier, some phages encode polysaccharide-degrading enzymes referred to as depolymerases to provide access to cell surface receptors. Here, we characterized the phage RAD2, which infects K. pneumoniae strains that produce the widespread, hypervirulence-associated K2-type capsular polysaccharide. Using transposon-directed insertion sequencing, we have shown that the production of capsule is an absolute requirement for efficient RAD2 infection by serving as a first-stage receptor. We have identified the depolymerase responsible for recognition and degradation of the capsule, determined that the depolymerase forms globular appendages on the phage virion tail tip, and present the cryo-electron microscopy structure of the RAD2 capsule depolymerase at 2.7-Å resolution. A putative active site for the enzyme was identified, comprising clustered negatively charged residues that could facilitate the hydrolysis of target polysaccharides. Enzymatic assays coupled with mass spectrometric analyses of digested oligosaccharide products provided further mechanistic insight into the hydrolase activity of the enzyme, which, when incubated with K. pneumoniae, removes the capsule and sensitizes the cells to serum-induced killing. Overall, these findings expand our understanding of how phages target the Klebsiella capsule for infection, providing a framework for the use of depolymerases as antivirulence agents against this medically important pathogen. IMPORTANCE Klebsiella pneumoniae is a medically important pathogen that produces a thick protective capsule that is essential for pathogenicity. Phages are natural predators of bacteria, and many encode diverse "capsule depolymerases" which specifically degrade the capsule of their hosts, an exploitable trait for potential therapies. We have determined the first structure of a depolymerase that targets the clinically relevant K2 capsule and have identified its putative active site, providing hints to its mechanism of action. We also show that Klebsiella cells treated with a recombinant form of the depolymerase are stripped of capsule, inhibiting their ability to grow in the presence of serum, demonstrating the anti-infective potential of these robust and readily producible enzymes against encapsulated bacterial pathogens such as K. pneumoniae

Genetic variation in individuals from a population of the minimalist bacteriophage Merri-merri-uth nyilam marra-natj driving evolution of the virus

UNLABELLED: In a survey of a waterway on Wurundjeri land, two sub-populations of the bacteriophage Merri-merri-uth nyilam marra-natj (phage MMNM) were isolated on a permissive host, Klebsiella B5055 of capsule-type K2, but were distinguished by minor phenotypic differences. The variant phage MMNM(Ala134) showed an inhibited activity against Klebsiella AJ174-2, and this was used as a basis to select for further variation through experimental evolution. Over the course of an evolution experiment, 20 phages that evolved distinct phenotypes in terms of the morphologies of plaques formed when they infected host Klebsiella were subject to whole-genome sequencing. The evolved phages had mutations in a small set of proteins that contribute to the baseplate portion of the phage virion. Phages MMNM and MMNM(Ala134) are minimalist phages, with baseplates formed from only five predicted subunits, akin to other minimalist phages Pam3 and XM1. The homology between all three minimalist phages provided a structural framework to interpret the two classes of mutations derived through evolution in the presence of the semi-permissive host: those that affect the interfacial surfaces between baseplate subunits, and those in a base-plate associated tail-fiber. This study evidences that multiple small mutations can be fixed into a sub-population of phage to provide a basis for phenotypic variation that we suggest could ultimately provide for a shift of virus properties, as an alternative evolutionary scenario to the major genetic events that result in more well-studied evolutionary mechanism of phage mosaicism. IMPORTANCE: Bacteriophages (phages) are viruses that prey on bacteria. This study sampled natural phage populations to test the hypothesis that untapped genetic variation within a population can be the basis for the selection of phages to diversify their host-range. Sampling of a freshwater site revealed two populations of the phage Merri-merri-uth nyilam marra-natj (phage MMNM), differing by a variant residue (Val134Ala) in the baseplate protein MMNM_26. This sequence variation modulated bacterial killing in plaques, and further evolution of the phages on a semi-permissive bacterial host led to a new generation of phages with more diverse phenotypes in killing the bacterium Klebsiella pneumoniae

Conserved features in TamA enable interaction with TamB to drive the activity of the translocation and assembly module

The biogenesis of membranes from constituent proteins and lipids is a fundamental aspect of cell biology. In the case of proteins assembled into bacterial outer membranes, an overarching question concerns how the energy required for protein insertion and folding is accessed at this remote location of the cell. The translocation and assembly module (TAM) is a nanomachine that functions in outer membrane biogenesis and virulence in diverse bacterial pathogens. Here we demonstrate the interactions through which TamA and TamB subunits dock to bridge the periplasm, and unite the outer membrane aspects to the inner membrane of the bacterial cell. We show that specific functional features in TamA have been conserved through evolution, including residues surrounding the lateral gate and an extensive surface of the POTRA domains. Analysis by nuclear magnetic resonance spectroscopy and small angle X-ray scattering document the characteristic structural features of these POTRA domains and demonstrate rigidity in solution. Quartz crystal microbalance measurements pinpoint which POTRA domain specifically docks the TamB subunit of the nanomachine. We speculate that the POTRA domain of TamA functions as a lever arm in order to drive the activity of the TAM, assembling proteins into bacterial outer membranes