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PLoS Genetics Sep 2022The Ras-like GTPase MglA is a key regulator of front-rear polarity in the rod-shaped Myxococcus xanthus cells. MglA-GTP localizes to the leading cell pole and stimulates...
The Ras-like GTPase MglA is a key regulator of front-rear polarity in the rod-shaped Myxococcus xanthus cells. MglA-GTP localizes to the leading cell pole and stimulates assembly of the two machineries for type IV pili-dependent motility and gliding motility. MglA-GTP localization is spatially constrained by its cognate GEF, the RomR/RomX complex, and GAP, the MglB Roadblock-domain protein. Paradoxically, RomR/RomX and MglB localize similarly with low and high concentrations at the leading and lagging poles, respectively. Yet, GEF activity dominates at the leading and GAP activity at the lagging pole by unknown mechanisms. Here, we identify RomY and show that it stimulates MglB GAP activity. The MglB/RomY interaction is low affinity, restricting formation of the bipartite MglB/RomY GAP complex almost exclusively to the lagging pole with the high MglB concentration. Our data support a model wherein RomY, by forming a low-affinity complex with MglB, ensures that the high MglB/RomY GAP activity is confined to the lagging pole where it dominates and outcompetes the GEF activity of the RomR/RomX complex. Thereby, MglA-GTP localization is constrained to the leading pole establishing front-rear polarity.
Topics: Bacterial Proteins; Cell Polarity; GTP Phosphohydrolases; Guanosine Triphosphate; Myxococcus xanthus
PubMed: 36067225
DOI: 10.1371/journal.pgen.1010384 -
MBio Oct 2022In Gram-negative bacteria, secreted polysaccharides have multiple critical functions. In Wzx/Wzy- and ABC transporter-dependent pathways, an outer membrane (OM)...
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 Wza of Escherichia coli is an octamer in which the eight C-terminal domains form an α-helical OM pore and the eight copies of the three N-terminal domains (D1 to D3) form a periplasmic cavity. In synthase-dependent pathways, the OM translocon is a 16- to 18-stranded β-barrel protein. In Myxococcus xanthus, the secreted polysaccharide EPS (exopolysaccharide) is synthesized in a Wzx/Wzy-dependent pathway. Here, using experiments, phylogenomics, and computational structural biology, we identify and characterize EpsX as an OM 18-stranded β-barrel protein important for EPS synthesis and identify AlgE, a β-barrel translocon of a synthase-dependent pathway, as its closest structural homolog. We also find that EpsY, the OPX protein of the EPS pathway, consists only of the periplasmic D1 and D2 domains and completely lacks the domain for spanning the OM (herein termed a OPX protein). , EpsX and EpsY mutually stabilize each other and interact in pulldown experiments supporting their direct interaction. Based on these observations, we propose that EpsY and EpsX make up and represent a third type of translocon for polysaccharide export across the OM. Specifically, in this composite translocon, EpsX functions as the OM-spanning β-barrel translocon together with the periplasmic OPX protein EpsY. Based on computational genomics, similar composite systems are widespread in Gram-negative bacteria. Bacteria secrete a wide variety of polysaccharides that have critical functions in, e.g., fitness, surface colonization, and biofilm formation and in beneficial and pathogenic human-, animal-, and plant-microbe interactions. In Gram-negative bacteria, export of these chemically diverse polysaccharides across the outer membrane depends on two known translocons, i.e., an outer membrane OPX protein in Wzx/Wzy- and ABC transporter-dependent pathways and an outer membrane 16- to 18-stranded β-barrel protein in synthase-dependent pathways. Here, using a combination of experiments in Myxococcus xanthus, phylogenomics, and computational structural biology, we provide evidence supporting that a third type of translocon can export polysaccharides across the outer membrane. Specifically, in this translocon, an outer membrane-spanning β-barrel protein functions together with an entirely periplasmic OPX protein that completely lacks the domain for spanning the OM. Computational genomics support that similar composite systems are widespread in Gram-negative bacteria.
Topics: ATP-Binding Cassette Transporters; Bacterial Outer Membrane Proteins; Escherichia coli; Escherichia coli Proteins; Gram-Negative Bacteria; Periplasm; Polysaccharides, Bacterial
PubMed: 35972145
DOI: 10.1128/mbio.02032-22 -
International Journal of Molecular... Jul 2022Encapsulins are protein nanocages capable of harboring smaller proteins (cargo proteins) within their cavity. The function of the encapsulin systems is related to the...
Encapsulins are protein nanocages capable of harboring smaller proteins (cargo proteins) within their cavity. The function of the encapsulin systems is related to the encapsulated cargo proteins. The encapsulin (EncA) naturally encapsulates ferritin-like proteins EncB and EncC as cargo, resulting in a large iron storage nanocompartment, able to accommodate up to 30,000 iron atoms per shell. In the present manuscript we describe the binding and protection of circular double stranded DNA (pUC19) by EncA using electrophoretic mobility shift assays (EMSA), atomic force microscopy (AFM), and DNase protection assays. EncA binds pUC19 with an apparent dissociation constant of 0.3 ± 0.1 µM and a Hill coefficient of 1.4 ± 0.1, while EncC alone showed no interaction with DNA. Accordingly, the EncAC complex displayed a similar DNA binding capacity as the EncA protein. The data suggest that initially, EncA converts the plasmid DNA from a supercoiled to a more relaxed form with a beads-on-a-string morphology. At higher concentrations, EncA self-aggregates, condensing the DNA. This process physically protects DNA from enzymatic digestion by DNase I. The secondary structure and thermal stability of EncA and the EncA-pUC19 complex were evaluated using synchrotron radiation circular dichroism (SRCD) spectroscopy. The overall secondary structure of EncA is maintained upon interaction with pUC19 while the melting temperature of the protein () slightly increased from 76 ± 1 °C to 79 ± 1 °C. Our work reports, for the first time, the in vitro capacity of an encapsulin shell to interact and protect plasmid DNA similarly to other protein nanocages that may be relevant in vivo.
Topics: Bacterial Proteins; DNA; Ferritins; Iron; Myxococcus xanthus
PubMed: 35887179
DOI: 10.3390/ijms23147829 -
ChemCatChem Dec 2021Imine reductases (IREDs) offer biocatalytic routes to chiral amines and have a natural preference for the NADPH cofactor. In previous work, we reported enzyme...
Imine reductases (IREDs) offer biocatalytic routes to chiral amines and have a natural preference for the NADPH cofactor. In previous work, we reported enzyme engineering of the ()-selective IRED from (NADH-IRED-) yielding a NADH-dependent variant with high catalytic efficiency. However, no IRED with NADH specificity and ()-selectivity in asymmetric reductions has yet been reported. Herein, we applied semi-rational enzyme engineering to switch the selectivity of NADH-IRED-. The quintuple variant A241V/H242Y/N243D/V244Y/A245L showed reverse stereopreference in the reduction of the cyclic imine 2-methylpyrroline compared to the wild-type and afforded the ()-amine product with >99 % conversion and 91 % enantiomeric excess. We also report the crystal-structures of the NADPH-dependent ()-IRED- wild-type enzyme and the NADH-dependent NADH-IRED- variant and molecular dynamics (MD) simulations to rationalize the inverted stereoselectivity of the quintuple variant.
PubMed: 35873105
DOI: 10.1002/cctc.202101057 -
Frontiers in Cell and Developmental... 2022Plasmalogens are glycerophospholipids with a hallmark -1 vinyl ether bond that endows them with unique physical-chemical properties. They have proposed biological roles... (Review)
Review
Plasmalogens are glycerophospholipids with a hallmark -1 vinyl ether bond that endows them with unique physical-chemical properties. They have proposed biological roles in membrane organization, fluidity, signaling, and antioxidative functions, and abnormal plasmalogen levels correlate with various human pathologies, including cancer and Alzheimer's disease. The presence of plasmalogens in animals and in anaerobic bacteria, but not in plants and fungi, is well-documented. However, their occurrence in the obligately aerobic myxobacteria, exceptional among aerobic bacteria, is often overlooked. Tellingly, discovery of the key desaturase indispensable for vinyl ether bond formation, and therefore fundamental in plasmalogen biogenesis, emerged from delving into how the soil myxobacterium responds to light. A recent pioneering study unmasked myxobacterial CarF and its human ortholog TMEM189 as the long-sought plasmanylethanolamine desaturase (PEDS1), thus opening a crucial door to study plasmalogen biogenesis, functions, and roles in disease. The findings demonstrated the broad evolutionary sweep of the enzyme and also firmly established a specific signaling role for plasmalogens in a photooxidative stress response. Here, we will recount our take on this fascinating story and its implications, and review the current state of knowledge on plasmalogens, their biosynthesis and functions in the aerobic myxobacteria.
PubMed: 35646900
DOI: 10.3389/fcell.2022.884689 -
Frontiers in Microbiology 2022Many bacteria move on solid surfaces using gliding motility, without involvement of flagella or pili. Gliding of is powered by a proton channel homologous to the...
Many bacteria move on solid surfaces using gliding motility, without involvement of flagella or pili. Gliding of is powered by a proton channel homologous to the stators in the bacterial flagellar motor. Instead of being fixed in place and driving the rotation of a circular protein track like the flagellar basal body, the gliding machinery of travels the length of the cell along helical trajectories, while mechanically engaging with the substrate. Such movement entails a different molecular mechanism to generate propulsion on the cell. In this perspective, we will discuss the similarities and differences between the gliding machinery and bacterial flagellar motor, and use biophysical principles to generate hypotheses about the operating mechanism, efficiency, sensitivity to control, and mechanosensing of gliding.
PubMed: 35602090
DOI: 10.3389/fmicb.2022.891694 -
Frontiers in Microbiology 2022Myxobacteria exhibit a variety of complex social behaviors that all depend on coordinated movement of cells on solid surfaces. The cooperative nature of cell movements...
Myxobacteria exhibit a variety of complex social behaviors that all depend on coordinated movement of cells on solid surfaces. The cooperative nature of cell movements is known as social (S)-motility. This system is powered by cycles of type IV pili (Tfp) extension and retraction. Exopolysaccharide (EPS) also serves as a matrix to hold cells together. Here, we characterized a new S-motility gene in . This mutant is temperature-sensitive (Ts) for S-motility; however, Tfp and EPS are made. A 1 bp deletion was mapped to the MXAN_4099 locus and the gene was named . Null mutations in exhibit a synthetic enhanced phenotype with a null mutation, a previously characterized S-motility gene that exhibits a similar Ts phenotype. Our results suggest that SglS and SglT contribute toward Tfp function at high temperatures in redundant pathways. However, at low temperatures only one pathway is necessary for wild-type S-motility, while in the double mutant, motility is nearly abolished at low temperatures. Interestingly, the few cells that do move do so with a high reversal frequency. We suggest SglS and SglT play conditional roles facilitating Tfp retraction and hence motility in .
PubMed: 35586861
DOI: 10.3389/fmicb.2022.879090 -
Frontiers in Microbiology 2022The bacterium forms both developmental and vegetative types of biofilms. While the former has been studied on both agar plates and submerged surfaces, the latter has...
The bacterium forms both developmental and vegetative types of biofilms. While the former has been studied on both agar plates and submerged surfaces, the latter has been investigated predominantly on agar surfaces as swarming colonies. Here we describe the development of a microplate-based assay for the submerged biofilms of under vegetative conditions. We examined the impacts of inoculation, aeration, and temperature to optimize the conditions for the assay. Aeration was observed to be critical for the effective development of submerged biofilms by , an obligate aerobic bacterium. In addition, temperature plays an important role in the development of submerged biofilms. It is well established that the formation of submerged biofilms by many bacteria requires both exopolysaccharide (EPS) and the type IV pilus (T4P). EPS constitutes part of the biofilm matrix that maintains and organizes bacterial biofilms while the T4P facilitates surface attachment as adhesins. For validation, we used our biofilm assay to examine a multitude of strains with various EPS and T4P phenotypes. The results indicate that the levels of EPS, but not of piliation, positively correlate with submerged biofilm formation in .
PubMed: 35572678
DOI: 10.3389/fmicb.2022.894562 -
Frontiers in Microbiology 2022Extracellular DNA (eDNA) is a critical component in the extracellular matrix (ECM) of bacterial biofilms, while little is known about the mechanisms underlying how eDNA...
Extracellular DNA (eDNA) is a critical component in the extracellular matrix (ECM) of bacterial biofilms, while little is known about the mechanisms underlying how eDNA integrates into the ECM through potential macromolecular interactions. biofilm was employed as a suitable model for the investigation due to the co-distribution of eDNA and exopolysaccharides (EPS) owing to their direct interactions in the ECM. DNA is able to combine with EPS to form a macromolecular conjugate, which is dominated by the electrostatic forces participating in the polymer-polymer interactions. Without intercalation binding, DNA-EPS interactions exhibit a certain degree of reversibility. Acting as a strong extracellular framework during biofilm formation process, the eDNA-EPS complex not only facilitates the initial cell adhesion and subsequent establishment of ECM architecture, but also renders cells within biofilms stress resistances that are relevant to the survival of in some hostile environments. Furthermore, the EPS protects the conjugated DNA from the degradation by nucleic acid hydrolases, which leads to the continuous and stable existence of eDNA in the native ECM of biofilms. These results will shed light on developing prevention and treatment strategies against biofilm-related risks.
PubMed: 35531272
DOI: 10.3389/fmicb.2022.861865 -
PLoS Genetics Apr 2022Type Four Pili (T4P) are extracellular appendages mediating several bacterial functions such as motility, biofilm formation and infection. The ability to adhere to...
Type Four Pili (T4P) are extracellular appendages mediating several bacterial functions such as motility, biofilm formation and infection. The ability to adhere to substrates is essential for all these functions. In Myxococcus xanthus, during twitching motility, the binding of polar T4P to exopolysaccharides (EPS), induces pilus retraction and the forward cell movement. EPS are produced, secreted and weakly associated to the M. xanthus cell surface or deposited on the substrate. In this study, a genetic screen allowed us to identify two factors involved in EPS-independent T4P-dependent twitching motility: the PilY1.1 protein and the HsfBA phosphorelay. Transcriptomic analyses show that HsfBA differentially regulates the expression of PilY1 proteins and that the down-regulation of pilY1.1 together with the accumulation of its homologue pilY1.3, allows twitching motility in the absence of EPS. The genetic and bioinformatic dissection of the PilY1.1 domains shows that PilY1.1 might be a bi-functional protein with a role in priming T4P extension mediated by its conserved N-terminal domain and roles in EPS-dependent motility mediated by an N-terminal DUF4114 domain activated upon binding to Ca2+. We speculate that the differential transcriptional regulation of PilY1 homologs by HsfBA in response to unknown signals, might allow accessorizing T4P tips with different modules allowing twitching motility in the presence of alternative substrates and environmental conditions.
Topics: Bacterial Proteins; Cell Movement; Fimbriae Proteins; Fimbriae, Bacterial; Myxococcus xanthus
PubMed: 35486648
DOI: 10.1371/journal.pgen.1010188