Biology subjects

Herfurth, M.

Publications and source records attributed to Herfurth, M..

5 recordsLinked to original sources

bioRxiv2024

A deterministic, c-di-GMP-dependent genetic program ensures the generation of phenotypically similar, symmetric daughter cells during cytokinesis

Phenotypic heterogeneity in bacteria results from stochastic processes or deterministic genetic programs. These deterministic programs often incorporate the versatile second messenger c-di-GMP, and by deploying c-di-GMP metabolizing enzyme(s) asymmetrically during cell division give rise to daughter cells with different c-di-GMP levels. By contrast, less is known about how phenotypic heterogeneity is kept to a minimum. Here, we identify a deterministic c-di-GMP-dependent genetic program that is hardwired into the cell cycle of Myxococcus xanthus to minimize phenotypic heterogeneity and guarantee the formation of phenotypically similar daughter cells during division. Cells lacking the diguanylate cyclase DmxA have an aberrant motility behaviour. DmxA is recruited to the cell division site and its activity switched on during cytokinesis, resulting in a dramatic but transient increase in the c-di-GMP concentration. During cytokinesis, this c-di-GMP burst ensures the symmetric incorporation and allocation of structural motility proteins and motility regulators at the new cell poles of the two daughters, thereby generating mirror-symmetric, phenotypically similar daughters with correct motility behaviours. These findings suggest a general c-di-GMP-dependent mechanism for minimizing phenotypic heterogeneity, and demonstrate that bacteria by deploying c-di-GMP metabolizing enzymes to distinct subcellular locations ensure the formation of dissimilar or similar daughter cells.

microbiology↗

bioRxiv2023

Large pilin subunits provide distinct structural and mechanical properties for the Myxococcus xanthus type IV pilus

Type IV pili (T4P) are ubiquitous bacterial cell surface filaments important for surface motility, adhesion to biotic and abiotic surfaces, DNA uptake, biofilm formation, and virulence. T4P are built from thousands of copies of the major pilin subunit and tipped by a complex composed of minor pilins and in some systems also the PilY1 adhesin. While the major pilins of structurally characterized T4P have lengths of up to 161 residues, the major pilin PilA of Myxococcus xanthus is unusually large with 208 residues. All major pilins have a highly conserved N-terminal domain and a highly variable C-terminal domain, and the additional residues in the M. xanthus PilA are due to a larger C-terminal domain. We solved the structure of the M. xanthus T4P (T4PMx) at a resolution of 3.0 [A] using cryo-electron microscopy (cryo-EM). The T4PMx follows the structural blueprint observed in other T4P with the pilus core comprised of the extensively interacting N-terminal 1-helices while the globular domains decorate the T4P surface. The atomic model of PilA built into this map shows that the large C-terminal domain has much more extensive intersubunit contacts than major pilins in other T4P. As expected from these greater contacts, the bending and axial stiffness of the T4PMx is significantly higher than that of other T4P and supports T4P-dependent motility on surfaces of different stiffnesses. Notably, T4PMx variants with interrupted intersubunit interfaces had decreased bending stiffness and strongly reduced motility on all surfaces. These observations support an evolutionary scenario whereby the large major pilin enables the formation of a rigid T4P that expands the environmental conditions in which the T4P system functions.

microbiology↗

bioRxiv2023

The mechanism for polar localization of the type IVa pilus machine

Type IVa pili (T4aP) are important for bacterial motility, adhesion, biofilm formation and virulence. This versatility is based on their cycles of extension, adhesion, and retraction. The conserved T4aP machine (T4aPM) drives these cycles, however the piliation pattern varies between species. To understand how these patterns are established, we focused on the T4aPM in Myxococcus xanthus that assembles following an outside-in pathway, starting with the polar incorporation of the PilQ secretin forming a multimeric T4aP conduit in the outer membrane. We demonstrate that PilQ recruitment to the nascent poles initiates during cytokinesis, but most is recruited to the new poles in the daughters after completion of cytokinesis. This recruitment depends on the peptidoglycan-binding AMIN domains in PilQ. Moreover, the pilotin Tgl stimulates PilQ multimerization in the outer membrane, is transiently recruited to the nascent and new poles in a PilQ-dependent manner, and dissociates after completion of secretin assembly. Altogether, our data support a model whereby PilQ polar recruitment and multimerization occur in two steps: The PilQ AMIN domains bind septal and polar peptidoglycan, thereby enabling polar Tgl localization, which then stimulates secretin multimerization in the outer membrane. Using computational analyses, we provide evidence for a conserved mechanism of T4aPM pilotins whereby the pilotin transiently interacts with the unfolded {beta}-lip, i.e. the region that eventually inserts into the outer membrane, of the secretin monomer. Finally, we suggest that the presence/absence of AMIN domain(s) in T4aPM secretins determines the different T4aPM localization patterns across bacteria. ImportanceType IVa pili (T4aP) are widespread bacterial cell surface structures with important functions in motility, surface adhesion, biofilm formation and virulence. Different bacteria have adapted different piliation patterns. To address how these patterns are established, we focused on the bipolar localization of the T4aP machine in the model organism M. xanthus by studying the localization of the PilQ secretin, the first component of this machine that assembles at the poles. Based on experiments using a combination of fluorescence microscopy, biochemistry and computational structural analysis, we propose that PilQ, and specifically its AMIN domains, binds septal and polar peptidoglycan, thereby enabling polar Tgl localization, which then stimulates PilQ multimerization in the outer membrane. We also propose that the presence and absence of AMIN domains in T4aP secretins determine the different piliation patterns across bacteria.

microbiology↗

bioRxiv2022

Evidence for a widespread third system for bacterial polysaccharide export across the outer membrane comprising a composite OPX/β-barrel translocon

In Gram-negative bacteria, secreted polysaccharides have multiple critical functions. In Wzx/Wzy- and ABC transporter-dependent pathways, an outer membrane (OM) polysaccharide export (OPX) type translocon exports the polysaccharide across the OM. The paradigm OPX protein WzaE. coli is an octamer, in which the eight C-terminal domains form an -helical OM pore, and the eight copies of the three N-terminal domains (D1-D3) a periplasmic cavity. In synthase-dependent pathways, the OM translocon is a 16- to 18- stranded {beta}-barrel protein. In Myxococcus xanthus, the secreted polysaccharide EPS is synthesized in a Wzx/Wzy-dependent pathway. Here, using experiments and computational structural biology, we characterize EpsX as an OM 18-stranded {beta}-barrel protein important for EPS synthesis and identify AlgE, a {beta}-barrel translocon of a synthase-dependent pathway, as its closest structural homolog. We also find that EpsY, the OPX protein of the EPS pathway, only consists of the periplasmic D1 and D2 domains and lacks the domain for spanning the OM (henceforth D1D2OPX protein). In vivo, EpsX and EpsY mutually stabilize each other, supporting their direct interaction. Based on these observations, we propose a model whereby EpsY and EpsX make up a novel type of translocon for polysaccharide export across the OM. Specifically, in this composite translocon, EpsX functions as the OM-spanning translocon together with the periplasmic D1D2OPX protein EpsY. Based on computational genomics, similar composite systems are present widespread in Gram-negative bacteria. This model provides a framework for these proteins future experimental characterization.

microbiology↗

bioRxiv2021

A repurposed, non-canonical cytochrome c, chaperones calcium binding by PilY1 for type IVa pili formation

Type IVa pili (T4aP) are versatile bacterial cell surface structures that undergo extension/adhesion/retraction cycles powered by the cell envelope-spanning T4aP machine. In this machine, a complex composed of four minor pilins and PilY1 primes T4aP extension and is also present at the pilus tip mediating adhesion. Similar to many other bacteria, Myxococcus xanthus contains multiple minor pilins/PilY1 sets that are incompletely understood. Here, we report that minor pilins and PilY1 (PilY1.1) of cluster_1 form priming and tip complexes contingent on a non-canonical cytochrome c (TfcP) with an unusual His/Cys heme ligation and calcium. We provide evidence that TfcP is unlikely to participate in electron transport and has been repurposed to promote calcium binding by PilY1.1 at low calcium concentrations, thereby stabilising PilY1.1 and enabling T4aP function in a broader range of calcium concentrations. These results identify a novel function of cytochromes c and illustrate how incorporating an accessory factor expands the environmental range under which the T4aP system functions.

microbiology↗