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Methanogens are the main biological producers of methane on Earth. Methanosarcina acetivorans is one of the best characterized methanogens that has powerful genetic tools for genome editing. To study the physiology of this methanogen in further detail as well as to effectively balance the flux of their engineered metabolic pathways in expansive project undertakings, there is the need for controlled gene expression, which then requires the availability of well-characterized promoters and ribosome-binding sites (RBS). In this study, we constructed a library of 33 promoter-RBS combinations that includes 13 wild-type and 14 hybrid combinations, as well as six combination variants in which the 5'-untranslated region (5'UTR) was rationally engineered. The expression strength for each combination was calculated by inducing the expression of the β-glucuronidase reporter gene in M. acetivorans cells in the presence of the two most used growth substrates, either methanol (MeOH) or trimethyl amine (TMA). In this study, the constructed library covers a relatively wide range (140-fold) between the weakest and strongest promoter-RBS combination as well as shows a steady increase and allows different levels of gene expression. Effects on the gene expression strength were also assessed by making measurements at three distinct growth phases for all 33 promoter-RBS combinations. Our promoter-RBS library is effective in enabling the fine-tuning of gene expression in M. acetivorans for physiological studies and the design of metabolic engineering projects that, e.g., aim for the biotechnological valorization of one-carbon compounds., Importance: Methanogenic archaea are potent producers of the greenhouse gas methane and thus contribute substantially to global warming. Under controlled conditions, these microbes can catalyze the production of biogas, which is a renewable fuel, and might help counter global warming and its effects. Engineering the primary metabolism of Methanosarcina acetivorans to render it better and more useful requires controllable gene expression, yet only a few well-characterized promoters and RBSs are presently available. Our study rectifies this situation by providing a library of 33 different promoter-RBS combinations with a 140-fold dynamic range in expression strength. Future metabolic engineering projects can take advantage of this library by using these promoter-RBS combinations as an efficient and tunable gene expression system for M. acetivorans . Furthermore, the methodologies we developed in this study could also be utilized to construct promoter libraries for other types of methanogens., Competing Interests: The authors declare no conflict of interest.

Sortase-dependent pili are long surface appendages that mediate attachment, colonization and biofilm formation in certain genera and species of Gram-positive bacteria. Ligilactobacillus ruminis is an autochthonous gut commensal that relies on sortase-dependent LrpCBA pili for host adherence and persistence. X-ray crystal structure snapshots of the backbone pilin LrpA were captured in two atypical bent conformations leading to a zigzag morphology in the LrpCBA pilus structure. Small-angle X-ray scattering and structural analysis revealed that LrpA also adopts the typical linear conformation, resulting in an elongated pilus morphology. Various conformational analyses and biophysical experiments helped to demonstrate that a hinge region located at the end of the flexible N-terminal domain of LrpA facilitates a new closure-and-twist motion for assembling dynamic pili during the assembly process and host attachment. Further, the incongruent combination of flexible domain-driven conformational dynamics and rigid isopeptide bond-driven stability observed in the LrpCBA pilus might also extend to the sortase-dependent pili of other bacteria colonizing a host.

Surface pili (or fimbriae) are an important but conspicuous adaptation of several genera and species of Gram-negative and Gram-positive bacteria. These long and non-flagellar multi-subunit adhesins mediate the initial contact that a bacterium has with a host or environment, and thus have come to be regarded as a key colonization factor for virulence activity in pathogens or niche adaptation in commensals. Pili in pathogenic bacteria are well recognized for their roles in the adhesion to host cells, colonization of tissues, and establishment of infection. As an 'anti-adhesive' ploy, targeting pilus-mediated attachment for disruption has become a potentially effective alternative to using antibiotics. In this review, we give a description of the several structurally distinct bacterial pilus types thus far characterized, and as well offer details about the intricacy of their individual structure, assembly, and function. With a molecular understanding of pilus biogenesis and pilus-mediated host interactions also provided, we go on to describe some of the emerging new approaches and compounds that have been recently developed to prevent the adhesion, colonization, and infection of piliated bacterial pathogens., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Adhesion to host surfaces for bacterial survival and colonization involves a variety of molecular mechanisms. Ligilactobacillus ruminis, a strict anaerobe and gut autochthonous (indigenous) commensal, relies on sortase-dependent pili (LrpCBA) for adherence to the intestinal inner walls, thereby withstanding luminal content flow. Here, the LrpCBA pilus is a promiscuous binder to gut collagen, fibronectin and epithelial cells. Structurally, the LrpCBA pilus displays a representative hetero-oligomeric arrangement and consists of three types of pilin subunit, each with its own location and function, i.e. tip LrpC for adhesion, basal LrpB for anchoring and backbone LrpA for length. To provide further structural insights into the assembly, anchoring and functional mechanisms of sortase-dependent pili, each of the L. ruminis pilus proteins was produced recombinantly for crystallization and X-ray diffraction analysis. Crystals of LrpC, LrpB, LrpA and truncated LrpA generated by limited proteolysis were obtained and diffracted to resolutions of 3.0, 1.5, 2.2 and 1.4 Å, respectively. Anomalous data were also collected from crystals of selenomethionine-substituted LrpC and an iodide derivative of truncated LrpA. Successful strategies for protein production, crystallization and derivatization are reported.

To successfully colonize a host or environment, certain genera and species of Gram-positive bacteria have evolved to utilize the so-called sortase-dependent pilus, a long multi-subunit and non-flagellar surface adhesin. One example of this is Lactobacillus rhamnosus GG, a gut-adapted probiotic strain that produces SpaCBA pili. These structures are covalent hetero-oligomers built from three types of pilin subunit, each with a specific location and function (i.e., backbone SpaA for length, tip SpaC for adhesion, and basal SpaB for anchoring). Functionally, the SpaCBA pilus exhibits a promiscuous affinity for components on intestinal surfaces (e.g., mucus, collagen, and epithelial cells), which is largely attributed to the SpaC subunit. Then again, the basal SpaB pilin, in addition to acting as the terminal subunit during pilus assembly, displays an out of character mucoadhesive function. To address the structural basis of this unusual dual functionality, we reveal the 2.39 ​Å resolution crystal structure of SpaB. SpaB consists of one immunoglobulin-like CnaB domain and contains a putative intermolecular isopeptide bond-linking lysine and internal isopeptide bond-asparagine in an FPKN pilin motif within the C-terminal end. Remarkably, we found that a C-terminal stretch of positively charged lysine and arginine residues likely accounts for the atypical mucoadhesiveness of SpaB. Although harboring an autocatalytic triad of residues for a potential internal isopeptide interaction, the SpaB crystal structure lacked the visible electron density for intact bond formation, yet its presence was subsequently confirmed by mass spectral analysis. Finally, we propose a structural model that captures the exclusive basal positioning of SpaB in the SpaCBA pilus., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2020 The Author(s).)

Adhesion to cell surfaces is an essential and early prerequisite for successful host colonization by bacteria, and in most instances involves the specificities of various adhesins. Among bacterial Gram-positives, some genera and species mediate attachment to host cells by using long non-flagellar appendages called sortase-dependent pili. A case in point is the beneficial Lactobacillus rhamnosus GG gut-adapted strain that produces the so-called SpaCBA pilus, a structure noted for its promiscuous binding to intestinal mucus and collagen. Structurally, SpaCBA pili are heteropolymers of three different pilin-protein subunits, each with its own location and function in the pilus: backbone SpaA for length, basal SpaB for anchoring, and tip SpaC for adhesion. Previously, we solved the SpaA tertiary structure by X-ray crystallography and also reported on the crystallization of SpaB and SpaC. Here, we reveal the full-length high-resolution (1.9 Å) crystal structure of SpaC, a first for a sortase-dependent pilus-bearing commensal. The SpaC structure, unlike the representative four-domain architecture of other Gram-positive tip pilins, espouses an atypically longer five-domain arrangement that includes N-terminal 'binding' and C-terminal 'stalk' regions of two and three domains, respectively. With the prospect of establishing new mechanistic insights, we provide a structural basis for the multi-substrate binding nature of SpaC, as well as a structural model that reconciles its exclusive localization at the SpaCBA pilus tip., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The SpaB pilin is recognized as the basal subunit of the sortase-dependent SpaCBA pilus, which is known to be produced by the Gram-positive Lactobacillus rhamnosus GG, a gut-adapted commensal advocated to have health benefits. Despite seeming to function as an archetypal basal pilin by serving as the terminal subunit in pilus assembly, SpaB also assumes an atypical role as a mucoadhesive protein. To shed light on the structural factors that contribute to this dual functional behaviour, a recombinant form of the L. rhamnosus GG SpaB pilin was produced and purified for crystallization and X-ray diffraction experiments. The crystallization of SpaB remained particularly challenging until the implementation of a three-pronged crystallization approach involving C-terminal tail truncation, surface lysine methylation and magnesium additives. Ultimately, hexagonal crystals of SpaB were produced and were able to diffract to a resolution of 2.4 Å. This crystal form belonged to space group P6 5 22 or P6 1 22, with unit-cell parameters a = b = 51.53, c = 408.22 Å, α = β = 90.0, γ = 120.0°. Obtaining an interpretable electron-density map via single-wavelength anomalous diffraction (SAD) using iodide-derivative data sets did not succeed owing to the weak anomalous signal. As an alternative, attempts to provide phases by molecular replacement using the iodide-SAD data from SpaB and a collection of distant homology models (<28% sequence identity) are in progress.

For some Gram-positive genera and species, the long-extended and adhesive sortase-dependent pilus plays an essential role during host colonization, biofilm formation, and immune modulation. Lactobacillus rhamnosus GG is a gut-adapted commensal strain that harbors the operonic genes for the SpaCBA and SpaFED pili, both being comprised of three different protein subunits termed the backbone, tip, and basal pilins. Crystal structures of the backbone pilins (SpaA and SpaD) have recently been solved, and here we describe the high-resolution (1.5 Å) structural determination of the SpaE basal pilin. SpaE consists of two immunoglobulin-like CnaB domains, with each displaying a spontaneously formed internal isopeptide bond, though apparently slow forming in the N-terminal domain. Remarkably, SpaE contains an atypically lengthy unstructured C-terminal tail, along with an YPKN pilin motif peptide, which is normally reserved for backbone subunits. Based on our analysis of the crystal structure data, we provide a molecular model for the basal positioning of the SpaE pilin within the SpaFED pilus., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Effective colonization of host cells by some Gram-positive bacteria often involves using lengthy, adhesive macromolecular structures called sortase-dependent pili. Among commensals, the gut-adapted Lactobacillus rhamnosus GG strain encodes the operons for two varieties of these pili (SpaCBA and SpaFED), with each structure consisting of backbone, tip, and basal pilin subunits. Although the tertiary structure was recently solved for the backbone subunit (SpaA) of the SpaCBA pilus, no structural information exists for its counterpart in the SpaFED pilus. Here, we report several crystal structures for the SpaD backbone pilin, two of which capture the N-terminal domain in either the closed (linear) or open (bent) conformation. To our knowledge, this is the first observation of the bent conformation in Gram-positive pilin structures. Based on this bent conformation, we suggest a three-stage model, which we call the expose-ligate-seal mechanism, for the docking and assembly of backbone pilins into the sortase-dependent pilus., Competing Interests: The authors declare no competing interests.

One of the more conspicuous structural features that punctuate the outer cell surface of certain bacterial Gram-positive genera and species is the sortase-dependent pilus. As these adhesive and variable-length protrusions jut outward from the cell, they provide a physically expedient and useful means for the initial contact between a bacterium and its ecological milieu. The sortase-dependent pilus displays an elongated macromolecular architecture consisting of two to three types of monomeric protein subunits (pilins), each with their own specific function and location, and that are joined together covalently by the transpeptidyl activity of a pilus-specific C-type sortase enzyme. Sortase-dependent pili were first detected among the Gram-positive pathogens and subsequently categorized as an essential virulence factor for host colonization and tissue invasion by these harmful bacteria. However, the sortase-dependent pilus was rebranded as also a niche-adaptation factor after it was revealed that "friendly" Gram-positive commensals exhibit the same kind of pilus structures, which includes two contrasting gut-adapted species from the Lactobacillus genus, allochthonous Lactobacillus rhamnosus and autochthonous Lactobacillus ruminis . This review will highlight and discuss what has been learned from the latest research carried out and published on these lactobacillar pilus types., Competing Interests: The author declares no conflict of interest.

SpaE is the predicted basal pilin subunit in the sortase-dependent SpaFED pilus from the gut-adapted and commensal Lactobacillus rhamnosus GG. Thus far, structural characterization of the cell-wall-anchoring basal pilins has remained difficult and has been limited to only a few examples from pathogenic genera and species. To gain a further structural understanding of the molecular mechanisms that are involved in the anchoring and assembly of sortase-dependent pili in less harmful bacteria, L. rhamnosus GG SpaE for crystallization was produced by recombinant expression in Escherichia coli. Although several attempts to crystallize the SpaE protein were unsuccessful, trigonal crystals that diffracted to a resolution of 3.1 Å were eventually produced using PEG 3350 as a precipitant and high protein concentrations. Further optimization with a combination of additives led to the generation of SpaE crystals in an orthorhombic form that diffracted to a higher resolution of 1.5 Å. To expedite structure determination by SAD phasing, selenium-substituted (orthorhombic) SpaE crystals were grown and X-ray diffraction data were collected to 1.8 Å resolution.