Genetic code expansion

The Ca 2+ -release-activated Ca 2+ (CRAC) channel Orai1 is activated by interaction with the Ca 2+ sensor Stro­
mal Interaction Molecule 1 (STIM1). Owing to the lack of structurally resolved Orai1/STIM1 complexes, the
impact of their coupling on individual Orai1 transmembrane domain (TM) movements is unclear. This study
investigates STIM1-independent and STIM1-dependent Orai1-TM dynamics using photocrosslinking unnat­
ural amino acids (UAAs) at each individual TM position. We primarily identify CRAC-channel-like currents
directly after UAA incorporation or additional UV-light irradiation at TM3 sites that interface with non-pore-
lining TMs. Using UAAs combined with conventional site-directed mutagenesis and molecular dynamics sim­
ulations, we discover that pore opening involves a widening of interfaces formed by TM3 with non-pore-lining
TMs. Orai1 mutants with a UAA in TM3 exhibit weaker STIM1-induced activation after UV exposure, possibly
caused by a restricted widening of non-pore-lining TM interfaces. We demonstrate that photocrosslinking
UAAs are excellent tools for improving our understanding of key determinants and ion channel dynamics
modulating pore opening.

The genetic code is an indispensable system that translates nucleic acid sequences into proteins. This mechanism is not merely important; it is essential for the existence and propagation of all known life forms on Earth. The genetic code's universal nature across diverse organisms hints at its primordial origins, suggesting it was present from the very inception of life on our planet. This fundamental system enables the storage, transmission, and expression of genetic information, forming the basis for heredity and the diversity of life as we know it. The genetic code's role in the emergence of life on Earth cannot be overstated. It provides the blueprint for constructing proteins, the workhorses of cellular function. Without this code, the complex biochemical reactions necessary for life would be impossible to coordinate and execute. The precision and efficiency of this system are remarkable, allowing for the accurate production of thousands of different proteins that carry out a vast array of cellular functions. Interestingly, while the genetic code is nearly universal, some variations do exist in nature. The complexity and specificity of the genetic code pose significant challenges to explanations relying solely on unguided, naturalistic processes. The machinery required for translation, the precise matching of codons to amino acids, and the error-correction mechanisms all point to a level of sophistication that seems to defy random occurrence. The probability of such a system arising by chance is vanishingly small, leading some scientists to question whether undirected processes alone can account for its existence. Moreover, the genetic code exhibits characteristics of an optimized system. It shows a remarkable ability to minimize the impact of errors, a feature that appears finely tuned for biological function. The origin of the genetic code remains one of the most profound mysteries in biology. Its universality points to a single origin, yet its complexity challenges explanations based on gradual, step-wise emergence. The genetic code's essential role in life, combined with its apparent optimization and the existence of variants, presents a compelling case for reexamining our understanding of life's origins. These observations invite us to consider alternative explanations beyond the framework of unguided processes, opening new avenues for scientific inquiry and philosophical reflection on the nature of life itself.

Genetic code expansion technologies supplement the natural codon repertoire with assignable variants in vivo, but are often limited by heterologous translational components and low suppression efficiencies. Here, we explore engineered Escherichia coli tRNAs supporting quadruplet codon translation by first developing a library-cross-library selection to nominate quadruplet codon-anticodon pairs. We extend our findings using a phage-assisted continuous evolution strategy for quadruplet-decoding tRNA evolution (qtRNA-PACE) that improved quadruplet codon translation efficiencies up to 80-fold. Evolved qtRNAs appear to maintain codon-anticodon base pairing, are typically aminoacylated by their cognate tRNA synthetases, and enable processive translation of adjacent quadruplet codons. Using these components, we showcase the multiplexed decoding of up to four unique quadruplet codons by their corresponding qtRNAs in a single reporter. Cumulatively, our findings highlight how E. coli tRNAs can be engineered, evolved, and combined to decode quadruplet codons, portending future developments towards an exclusively quadruplet codon translation system.

Pseudomonas aeruginosa is a common opportunistic pathogen that can cause chronic infections in multiple disease states, including respiratory infections in patients with cystic fibrosis (CF) and non-CF bronchiectasis. Like many opportunists, P. aeruginosa forms multicellular biofilm communities that are widely thought to be an important determinant of bacterial persistence and resistance to antimicrobials and host immune effectors during chronic/recurrent infections. Poly (acetyl, arginyl) glucosamine (PAAG) is a glycopolymer which has antimicrobial activity against a broad range of bacterial species, and also has mucolytic activity which can normalize rheologic properties of cystic fibrosis mucus. In this study, we sought to evaluate the effect of PAAG on P. aeruginosa bacteria within biofilms in vitro, and in the context of experimental pulmonary infection in a rodent infection model. PAAG treatment caused significant bactericidal activity against P. aeruginosa biofilms, and a red...

ABSTRACTThe design of biomaterials to regenerate bone is likely to increasingly require modifications that reduce bacterial attachment and biofilm formation as infection during wound regeneration can significantly impede tissue repair and typically requires surgical intervention to restart the healing process. Here, we investigate the ability of a mineralized collagen biomaterial to natively resist infection as well as how the addition of manuka honey affects bacterial colonization and mesenchymal stem cell osteogenesis. We incorporate manuka honey into these scaffolds via either direct fabrication into the scaffold microarchitecture or via soaking the scaffold in a solution of Manuka honey after fabrication. Direct incorporation results in a change in the surface characteristics and porosity of mineralized collagen scaffolds. Soaking scaffolds in honey concentrations greater than 10% had significant negative effects on mesenchymal stem cell metabolic activity but soaking or incorpo...

Sequence homology in pre-divergence tRNA species revealed cofactor/adaptors cognate for 16 amino acids derived from oxaloacetate, pyruvate, phosphoglycerate, or phosphoenolpyruvate were related. Synthesis path-distances of these amino acids correlated with phylogenetic depth, reflecting relative residue frequency in pre-divergence sequences. Both metrics were thus aligned in the four sub-families of the Aspartate family, and misaligned in the small Glutamate family; a functional difference was noted and seen to parallel synthetase duality. Amino acid synthetic order, based on path-distances, indicate NH4+ fixer amino acids, Asp1, Asn2, and homologues, Glu1, Gln2, formed the first code. Together with a termination signal, they acquired all four triplet 4-sets in the XAN column (X, 5’ coding site; N, any 3’-base). An invariant mid-A conformed with pre-code translation on a poly(A) template by a ratchet-equipped ribosome resulting in random, polyanionic polypeptides. Code expansion occ...

Extracellular matrix plays a pivotal role in biofilm biology. Despite the importance of matrix proteins as potential targets for the development of antibacterial therapeutics little is known about matrix proteomes. As matrix responsible for some extracellular functions, it is expected that protein composition should be different compared to embedded in biofilm cells. This difference reflects possible active processes in the matrix. While P. aeruginosa is one of the most important pathogens with emerging antibiotic resistance only a few studies are devoted to matrix proteomes and there are no studies describing matrix proteome for any clinical isolates. Here we report the first matrix proteome in comparison with embedded cells for the clinical isolate of P. aeruginosa. We have identified the largest number of proteins in the matrix among all published studies devoted to P. aeruginosa biofilms. Functional classification of enriched in matrix proteins resulted in several bioprocess groups. Bioprocess groups with the largest number of overrepresented in matrix proteins were oxidation-reduction processes, proteolysis, and transmembrane transport. The top three represented in matrix bioprocesses concerning the size of the GO annotated database were: cell redox homeostasis, nucleoside metabolism, and fatty acid synthesis. Finally, we discuss obtained data in a prism of possible directions for antibiofilm therapeutics development.

Extracellular matrix plays a pivotal role in biofilm biology and proposed as a potential target for therapeutics development. As matrix is responsible for some extracellular functions and influence bacterial cytotoxicity against eukaryotic cells, it must have unique protein composition. P. aeruginosa is one of the most important pathogens with emerging antibiotic resistance, but only a few studies were devoted to matrix proteomes and there are no studies describing matrix proteome for any clinical isolates. Here we report the first biofilm matrix proteome of P. aeruginosa isolated from bronchoalveolar lavage of patient in intensive care unit. We have identified the largest number of proteins in the matrix among all published studies devoted to P. aeruginosa biofilms. Comparison of matrix proteome with proteome from embedded cells let us to identify several enriched bioprocess groups. Bioprocess groups with the largest number of overrepresented in matrix proteins were oxidation-reduc...

ABSTRACTBacillus subtilishas been extensively used to study the molecular mechanisms behind the development and dispersal of surface bacterial multicellular communities. Well-structured spatially organised communities (colony, pellicle, and submerged biofilm) share some similarities, but also display considerable differences at the structural, chemical and biological levels. To unveil the spatial transcriptional heterogeneity between the different communities, we analysed by RNA-seq nine spatio-physiological populations selected from planktonic and spatially organised communities. This led to a global landscape characterisation of gene expression profiles uncovering genes specifically expressed in each compartmental population. From this mesoscale analysis and using fluorescent transcriptional reporter fusions, 17 genes were selected and their patterns of expression reported at single cell scale with time-lapse confocal laser scanning microscopy (CLSM). Derived kymographs allowed to...

BackgroundBiofilm associated infections are the major contributor of mortality, morbidity and financial burden in patients with bacterial infection. Molecules with surfactant behaviour are known to show significant antibiofilm effect against these infections. Thus, newly discovered antibacterial Fmoc-phenylalanine (Fmoc-F) and other Fmoc-amino acids (Fmoc-AA) with surfactant properties, could have potential antibiofilm properties.ObjectivesTo evaluate and characterise the antibiofilm activity of Fmoc-F and some Fmoc-AA against various clinically relevant bacteria.MethodsBiofilm inhibition and eradication was evaluated by crystal violet staining procedure along with scanning electron microscopy (SEM). Attenuated Total Reflection - Fourier Transform Infrared Spectroscopy (ATR-FTIR), Biochemical assays and Congo red staining were employed to investigate mechanism of antibiofilm action.ResultsWe showed that Fmoc-F not only inhibits the biofilm formation in S. aureus and P. aeruginosa, b...

Competing Interests: Agrospheres Inc. is commercializing minicell technology for agricultural applications and MK is a consultant to, and co-founder of. Agrosheres. The University of Virginia Licensing and Ventures Group has filed a provisional patent application related to the work described in this report. Author contributions: H.Y., A.V.K., D.L.N.F.M, and V.R. designed and conducted the molecular biology, mutagenesis, bacterial transformation, recombinant protein expression, immunoblot, minicell production, and analysis experiments; C.C. conducted and analyzed image cytometry experiments; K.D. conducted and analyzed the cryo-EM experiments. M.C. provided statistical analyses; M.K. and S.L.Z. conceived, designed, and analyzed the experiments, drafted and revised the manuscript. All authors reviewed and approved the manuscript. .

SummaryIncorporation of more than one non-canonical amino acid (ncAA) within a single protein endows the resulting construct with multiple useful features such as augmented molecular recognition or covalent crosslinking capabilities. Herein, for the first time, we demonstrate the incorporation of two chemically distinct ncAAs into proteins biosynthesized inSaccharomyces cerevisiae. To complement ncAA incorporation in response to the amber (TAG) stop codon in yeast, we evaluated opal (TGA) stop codon suppression using three distinct orthogonal translation systems. We observed selective TGA readthrough without detectable cross-reactivity from host translation components. Readthrough efficiency at TGA was modulated by factors including the local nucleotide environment, gene deletions related to the translation process, and the identity of the suppressor tRNA. These observations facilitated systematic investigation of dual ncAA incorporation in both intracellular and yeast-displayed pro...

ABSTRACTBacillus subtilishas been extensively used to study the molecular mechanisms behind the development and dispersal of surface bacterial multicellular communities. Well-structured spatially organised communities (colony, pellicle, and submerged biofilm) share some similarities, but also display considerable differences at the structural, chemical and biological levels. To unveil the spatial transcriptional heterogeneity between the different communities, we analysed by RNA-seq nine spatio-physiological populations selected from planktonic and spatially organised communities. This led to a global landscape characterisation of gene expression profiles uncovering genes specifically expressed in each compartmental population. From this mesoscale analysis and using fluorescent transcriptional reporter fusions, 17 genes were selected and their patterns of expression reported at single cell scale with time-lapse confocal laser scanning microscopy (CLSM). Derived kymographs allowed to...

Biofilm is ubiquitous in natural environments, causing biofouling in industrial water systems and leading to liquidity and heat transfer efficiency decreases. In particular, multi-species coexistence in biofilms can provide the synergy needed to boost biomass production and enhance treatment resistance. In this study, a total of 37 bacterial strains were isolated from a cooling tower where acetic acid and propionic acid were used as the primary carbon sources. These isolates mainly belonged to Proteobacteria and Firmicutes, which occupied more than 80% of the total strains according to the 16S rRNA gene amplicon sequencing. Four species (Acinetobacter sp. CTS3, Corynebacterium sp. CTS5, Providencia sp. CTS12, and Pseudomonas sp. CTS17) were observed to co-exist in the synthetic medium, showing a synergistic effect towards biofilm formation. Three metabolic inhibitors (sulfathiazole, 3-Bromopyruvic acid, and 3-Nitropropionic acid) were employed as possible treatments against biofilm ...

ABSTRACTThe biofilm matrix is composed of exopolysaccharides, eDNA, membrane vesicles, and proteins. While proteomic analyses have identified numerous matrix proteins, their functions in the biofilm remain understudied compared to the other biofilm components. In thePseudomonas aeruginosabiofilm, several studies have identified OprF as an abundant matrix protein and, more specifically, as a component of biofilm membrane vesicles. OprF is a major outer membrane porin ofP. aeruginosacells. However, current data describing the effects of OprF in theP. aeruginosabiofilm is limited. Here we identify a nutrient-dependent effect of OprF in static biofilms, whereby ΔoprFcells form significantly less biofilm than wild type when grown in media containing glucose or low sodium chloride concentrations. Interestingly, this biofilm defect occurs during late static biofilm formation and is not dependent on the production of PQS, which is responsible for outer membrane vesicle production. Furthermo...

ABSTRACTBacillus subtilishas been extensively used to study the molecular mechanisms behind the development and dispersal of surface bacterial multicellular communities. Well-structured spatially organised communities (colony, pellicle, and submerged biofilm) share some similarities, but also display considerable differences at the structural, chemical and biological levels. To unveil the spatial transcriptional heterogeneity between the different communities, we analysed by RNA-seq nine spatio-physiological populations selected from planktonic and spatially organised communities. This led to a global landscape characterisation of gene expression profiles uncovering genes specifically expressed in each compartmental population. From this mesoscale analysis and using fluorescent transcriptional reporter fusions, 17 genes were selected and their patterns of expression reported at single cell scale with time-lapse confocal laser scanning microscopy (CLSM). Derived kymographs allowed to...

Biofilms represent a prevalent lifestyle of unicellular organism that confers protection to external challenges. The mechanisms by which archaea form biofilms are however not entirely clear.H. volcaniiis an extremely halophilic euryarchaeon that commonly colonizes salt crust surfaces.H. volcaniiproduces long and thin appendages called type IV pili that are known to play a function in surface attachment and biofilm formation in archaea and bacteria. Here, we used biophysical experiments to identify critical function of type IV pili in the mechanical integrity ofH. volcaniibiofilms. Using interferometric scattering microscopy (iSCAT) to non-invasively visualize T4P in live cells, we find that piliation varies across mutants expressing single pilin isoforms. Using microfluidic experiments, we found that the adhesive strength of these mutants correlates with their extent of piliation. We found that in flow,H. volcaniiforms clonal biofilms that extend in three dimensions. Expression of P...

ABSTRACTMicrobial biofilms are multicellular communities where bacteria produce an extracellular matrix mainly consisting of proteins and polysaccharides. These biofilms not only confer resistance against external stresses (e.g. antibiotics), but their physical and chemical properties can also adapt to environmental conditions (e.g. temperature, humidity, etc.). Gaining insight on how such cues affect the molecular structure of biofilms would greatly benefit to the emerging field of engineered living materials. In this work, we studied the physical-chemical properties of curli amyloid fibers extracted from biofilms grown on substrates with different water contents. Electron microscopy, FT-IR and fluorescence spectroscopy were used to characterize the conformation and physical-chemical properties of these fibers as a function of the biofilm growth conditions. The results show that varying the water content of the substrate leads to differences in the yield of curli fibers purified fr...

Cyanobacterial biofilms are ubiquitous and play important roles in diverse environments, yet, understanding of the processes underlying development of these aggregates is just emerging. Here we report cell specialization in formation of Synechococcus elongatus PCC 7942 biofilms - a hitherto unknown characteristic of cyanobacterial multicellularity. We show that only a quarter of the cell population expresses at high levels the four-gene ebfG-operon that is required for biofilm formation. Almost all cells, however, are assembled in the biofilm. Detailed characterization of EbfG4 encoded by this operon revealed cell-surface localization as well as its presence in the biofilm matrix. Moreover, EbfG1-3 were shown to form amyloid structures such as fibrils and are thus likely to contribute to the matrix structure. These data suggest a beneficial ‘division of labour’ during biofilm formation where only some of the cells allocate resources to produce matrix proteins – ‘public goods’ that s...

ABSTRACTRegardless of the site of infectivity, all pathogens require high energetic influxes. This energy is required to counterattack the host immune system and in the absence the bacterial infections are easily cleared by the immune system. This study is an investigation into one highly bioenergetic pathway in Pseudomonas aeruginosa involving the amino acid L-serine and the enzyme L-serine deaminase (L-SD). P. aeruginosa is an opportunistic pathogen causing infections in patients with compromised immune systems as well as patients with cystic fibrosis. L-SD has been linked directly to the pathogenicity of several organisms including but not limited to Campylobacter jejuni, Mycobacterium bovis, Streptococcus pyogenes, and Yersinia pestis. We hypothesized that P. aeruginosa L-SD is likely to be critical for its virulence. The genome sequence analysis revealed the presence of two L-SD homologs encoded by sdaA and sdaB. We analyzed the ability of P. aeruginosa to utilize serine and th...

Uropathogenic Escherichia coli (UPEC) is a major cause of urinary tract infections. Analysis of the innate immune response in immortalised urothelial cells suggests that the bacterial flagellar subunit, flagellin, is key in inducing host defences. A panel of 39 clinical uro-associated Escherichia coli isolates recovered from either asymptomatic bacteruria (ASB), cystitis or pyelonephritis patients, were characterised for motility and their ability to induce an innate response in urothelial cells stably transfected with a NFκB luciferase reporter. Twenty-four isolates (60%) were identified as motile with strains recovered from cystitis patients exhibiting a bipolar motility distribution pattern (P < 0.005) and associated with a 2-5 fold increase in NFκB signalling. Although two isolates were associated with swarm sizes of >7 cm and NFκB activities of >30 fold (P = 0.029), data overall suggested bacterial motility and the NFκB signalling response were not directly correlated....

Cyanobacterial biofilms are ubiquitous and play important roles in diverse environments, yet, understanding of the processes underlying development of these aggregates is just emerging. Here we report cell specialization in formation of Synechococcus elongatus PCC 7942 biofilms - a hitherto unknown characteristic of cyanobacterial multicellularity. We show that only a quarter of the cell population expresses at high levels the four-gene ebfG-operon that is required for biofilm formation. Almost all cells, however, are assembled in the biofilm. Detailed characterization of EbfG4 encoded by this operon revealed cell-surface localization as well as its presence in the biofilm matrix. Moreover, EbfG1-3 were shown to form amyloid structures such as fibrils and are thus likely to contribute to the matrix structure. These data suggest a beneficial ‘division of labour’ during biofilm formation where only some of the cells allocate resources to produce matrix proteins – ‘public goods’ that s...

Extracellular matrix plays a pivotal role in biofilm biology and proposed as a potential target for therapeutics development. As matrix is responsible for some extracellular functions and influence bacterial cytotoxicity against eukaryotic cells, it must have unique protein composition. P. aeruginosa is one of the most important pathogens with emerging antibiotic resistance, but only a few studies were devoted to matrix proteomes and there are no studies describing matrix proteome for any clinical isolates. Here we report the first biofilm matrix proteome of P. aeruginosa isolated from bronchoalveolar lavage of patient in intensive care unit. We have identified the largest number of proteins in the matrix among all published studies devoted to P. aeruginosa biofilms. Comparison of matrix proteome with proteome from embedded cells let us to identify several enriched bioprocess groups. Bioprocess groups with the largest number of overrepresented in matrix proteins were oxidation-reduc...

Kinetoplastids are single-celled parasites responsible for human and animal disease. For the vast majority of kinetoplastids, colonization of an insect host is required for transmission. Stable attachment to insect tissues via the single flagellum coincides with differentiation and morphological changes. Although this process is essential for the generation of infectious forms, the molecular mechanism driving differentiation following adherence is not well understood. To study this process, we elaborate upon an in vitro model in which flagellated swimming cells of the kinetoplastid Crithidia fasciculata rapidly differentiate following adhesion to artificial substrates. Manipulation of culture parameters revealed that growth phase and time had a strong influence on the proportion of adherent cells. Live imaging of cells transitioning from swimming to an attached cell fate show parasites undergoing a defined sequence of events including an initial adhesion near the base of the flagell...

ABSTRACTPoint accumulation for imaging in nanoscale topography (PAINT) is a single-molecule technique for super-resolution microscopy, achieving ∼5-25 nanometer resolution. Here we show that by transfecting the protein-of-interest with a docker-coil, rather than by adding the docker externally—as is the norm when using DNA tethers or antibodies as dockers—we can achieve similar localization, ∼10 nm. However, using a transfected docker has several experimental advances and simplifications. Most importantly, it allows Peptide-PAINT to be applied to transfected live cells, including surface proteins in mammalian cells and neurons under physiological conditions. The enhance resolution of Peptide-PAINT is also shown for organelles in fixed cells to unravel structural details including ≈40-nm and ≈60-nm axial repeats in vimentin filaments in the cytoplasm, and fiber shapes of sub-100-nm histone-rich regions in the nucleus.

Beyond their role in horizontal gene transfer, conjugative plasmids commonly encode homologues of bacterial regulators. Known plasmid regulator homologues have highly targeted effects upon the transcription of specific bacterial traits. Here, we characterise a plasmid translational regulator, RsmQ, capable of taking global regulatory control in Pseudomonas fluorescens and causing a behavioural switch from motile to sessile lifestyle. RsmQ acts as a global regulator controlling the host proteome through direct interaction with host mRNAs and interference with the host’s translational regulatory network. This mRNA interference leads to largescale proteomic changes in metabolic genes, key regulators and genes involved in chemotaxis, thus controlling bacterial metabolism and motility. Moreover, comparative analyses found RsmQ on a large number of divergent plasmids isolated from multiple bacterial host taxa, suggesting the widespread importance of RsmQ for manipulating bacterial behavio...

Codons expanded by a silent position (quadruplet or tetracodons) may solve the conundrum that at life's origins, the weak tricodon-anticodon interactions could not promote translation in the absence of complex ribosomes. Modern genomes have isolated tetracodons resulting from insertion mutations. Some bioinformatic analyses suggest that tetracoding stretches overlap with regular mitochondrial protein coding genes. These tetragenes are probably decoded by (antisense) tRNAs with expanded anticodons. They are GC-rich, which produce stronger basepairs than A:T interactions, suggesting expression at high temperatures. The hypothesis that tetracoding is an adaptation to high temperatures is tested here by comparing predicted mitochondrial tetracoding in Lepidosauria (lizards, amphisbaenia, and Sphenodon), in relation to body temperature, expecting more tetracoding in species with high body temperature. The association between tRNAs with expanded anticodons and tetracoding previously described for mammals and Drosophila is confirmed for Lepidosauria. Independent evidence indicates that tetracoding increases with body temperature, supporting the hypothesis that tetracoding is an adaptation for efficient translation when conditions (temperature) make triplet codon-anticodons too unstable to allow efficient protein elongation.

In rod-shaped bacteria morphological plasticity occurs in response to stress, which blocks cell division to promote filamentation. We demonstrate here that overexpression of the patatin-like phospholipase variant CapVQ329R but not CapV causes pronounced sulA-independent pyridoxine-inhibited cell filamentation and restriction of swimming and flagella production of Escherichia coli K-12 derivative MG1655. Mutational analyses of CapVQ329R indicated conserved amino acids in canonical patatin-like phospholipase A motifs, but not the nucleophilic serine to be required for the observed phenotypes. Furthermore, CapVQ329R alters rdar biofilm formation including expression of the biofilm activator CsgD. Moreover, commensal and pathogenic E. coli strains and Salmonella typhimurium also responded with cell filamentation and alteration in biofilm formation. In conclusion, this work identifies the CapV variant CapVQ329R as a pleiotropic regulator, emphasizes a scaffold function for patatin-like p...

Chromobacterium violaceum was subjected to sonic stimulation with 300 Hz sound, at five different levels of loudness in the range of 70 – 89.5 dB. Sonic stimulation was found to affect bacterial growth and quorum sensing regulated pigment (violacein) production significantly. Magnitude of this effect was found to be dependent on sound-level. The minimum critical difference required to cause any statistically significant change in bacterial response with respect to sound-level was found to be 13 dB. Growth of C. violaceum was affected more at lower sound intensity, whereas pigment production was affected more at higher sound intensity. Additional experiments with C. violaceum and Serratia marcescens indicated that even a silent speaker emitting no sound can alter bacterial growth and/or pigment production upto a minor extent. Size of the test tube in which bacteria are exposed to sonic stimulation, was not found to affect the results much.

Bacteria commonly protect themselves by forming multicellular structures called biofilms. The opportunistic pathogen Pseudomonas aeruginosa causes antibiotic-recalcitrant pneumonia by forming biofilms in the respiratory tract. Despite extensive in vitro experimentation, how P. aeruginosa forms biofilms at the airway mucosal surface is unresolved. We investigated P. aeruginosa biofilm biogenesis in optically-accessible tissue-engineered human lung models that emulate the airway mucosal environment. We found that P. aeruginosa forms mucus-associated biofilms within hours, much faster than previously observed in in vitro experiments. Early during colonization, P. aeruginosa induces contractions of luminal mucus which accelerates bacterial aggregation. We show that P. aeruginosa uses retractile type IV pili to actively compress mucus. Our results suggest that, while protecting epithelia, mucus constitutes a breeding ground for biofilms.

ABSTRACTP. aeruginosa produces serious chronic infections in hospitalized patients and immunocompromised individuals, including cystic fibrosis patients. The molecular mechanisms by which P. aeruginosa responds to antibiotics and other stresses to promote persistent infections may provide new avenues for therapeutic intervention. Azithromycin (AZM), an antibiotic frequently used in cystic fibrosis treatment, is thought to improve clinical outcomes through a number of mechanisms including impaired biofilm growth and QS. The mechanisms underlying the transcriptional response to AZM remain unclear. Here, we interrogated the P. aeruginosa transcriptional response to AZM using an improved genome-wide approach to quantitate RNA 3’-ends (3pMap). We also identified hundreds of P. aeruginosa genes subject to premature transcription termination in their transcript leaders using 3pMap. AZM treatment of planktonic and biofilm cultures alters the expression of hundreds of genes, including those ...

Codons expanded by a silent position (quadruplet or tetracodons) may solve the conundrum that at life's origins, the weak tricodon-anticodon interactions could not promote translation in the absence of complex ribosomes. Modern genomes have isolated tetracodons resulting from insertion mutations. Some bioinformatic analyses suggest that tetracoding stretches overlap with regular mitochondrial protein coding genes. These tetragenes are probably decoded by (antisense) tRNAs with expanded anticodons. They are GC-rich, which produce stronger basepairs than A:T interactions, suggesting expression at high temperatures. The hypothesis that tetracoding is an adaptation to high temperatures is tested here by comparing predicted mitochondrial tetracoding in Lepidosauria (lizards, amphisbaenia, and Sphenodon), in relation to body temperature, expecting more tetracoding in species with high body temperature. The association between tRNAs with expanded anticodons and tetracoding previously described for mammals and Drosophila is confirmed for Lepidosauria. Independent evidence indicates that tetracoding increases with body temperature, supporting the hypothesis that tetracoding is an adaptation for efficient translation when conditions (temperature) make triplet codon-anticodons too unstable to allow efficient protein elongation.

The shape and form of the flagellated eukaryotic parasite Leishmania is sculpted to its ecological niches and needs to be transmitted to each generation with great fidelity. The shape of the Leishmania cell is defined by the sub-pellicular microtubule array and the positioning of the nucleus, kinetoplast and the flagellum within this array. The flagellum emerges from the anterior end of the cell body through an invagination of the cell body membrane called the flagellar pocket. Within the flagellar pocket the flagellum is laterally attached to the side of the flagellar pocket by a cytoskeletal structure called the flagellum attachment zone (FAZ). During the cell cycle single copy organelles duplicate with a new flagellum assembling alongside the old flagellum and these are then segregated between the two daughter cells by cytokinesis, which initiates at the anterior cell tip. Here, we have investigated the role of the FAZ in the morphogenetic resolution of the anterior cell tip duri...

Bacterial populations frequently encounter potentially lethal environmental stress factors. Growing Bacillus subtilis populations are comprised of a mixture of “motile” and “sessile” cells but how this affects population-level fitness under stress is poorly understood. Here, we show that, unlike sessile cells, motile cells are readily killed by monovalent cations under conditions of nutrient deprivation – owing to elevated expression of the lytABC operon, which codes for a cell-wall lytic complex. Forced induction of the operon in sessile cells also causes lysis. We demonstrate that population composition is regulated by the quorum sensing regulator ComA, which can favor either the motile or the sessile state. Specifically social interactions by ComX-pheromone signaling enhance population-level fitness under stress. Our study highlights the importance of characterizing population composition and cellular properties for studies of bacterial physiology and functional genomics. Our fin...

We used single-molecule AFM force spectroscopy (AFM-SMFS) to screen residues along the backbone of a non-antibody protein binding scaffold (lipocalin/anticalin), and determine the optimal anchor point that maximizes binding strength of the interaction with its target (CTLA-4). By incorporating non-canonical amino acids into anticalin, and using click chemistry to attach an Fgβ peptide at internal sequence positions, we were able to mechanically dissociate anticalin from CTLA-4 by pulling from eight different anchoring residues using an AFM cantilever tip. We found that pulling on the anticalin from residue 60 or 87 resulted in significantly higher rupture forces and a decrease in koff by 2-3 orders of magnitude over a force range of 50-200 pN. Five of the six internal pulling points tested were significantly more stable than N- or C-terminal anchor points, rupturing at up to 250 pN at loading rates of 0.1-10 nN sec-1. Anisotropic network modelling and molecular dynamics simulations ...

Background: Majority of biliary tract cancer (BTC) patients have not access to surgery. They receive chemotherapy and/or palliative care (PC). We studied if early palliative care referral could influence overall survival (OS) and the aggressiveness of end-of-life care. Participants and Design: We conducted a retrospective cohort study in patients with non-curative BTC, diagnosed between 2013 and 2019 in 6 hospitals of Eastern France. PC was defined a specialist-delivered palliative care encounter.Results: 200 patients with BTC were included in our study, with 188 deaths. 87 (44%) had no PC, 30 (15%) had a very early PC (<3 months after diagnosis), 20 (10%) had an early PC (3-6 months), and 63 (32%) a late PC (>6 months). The median time between referral and death was 0.9 to 1.3 months (depending on groups). OS were 12.4 months (no PC), 3.0 months (PC<3 m), 6.4 months (PC 3-6 m), 16 months (PC>6 m). We had no evidence for survival improvement with early PC. PC seemed to r...

Most microorganisms exist in biofilms, which comprise aggregates of cells surrounded by an extracellular matrix that provides protection from external stresses. Based on the conditions under which they form, biofilm structures vary in significant ways. For instance, biofilms that develop when microbes are incubated under static conditions differ from those formed when microbes encounter the shear forces of a flowing liquid. Moreover, biofilms develop dynamically over time. Here, we describe a cost-effective, 3D-printed coverslip holder that facilitates surface adhesion assays under a broad range of standing and shaking culture conditions. This multi-panel adhesion (mPAD) mount further allows cultures to be sampled at multiple time points, ensuring consistency and comparability between samples and enabling analyses of the dynamics of biofilm formation. As a proof of principle, using the mPAD mount for shaking, oxic cultures, we confirm previous flow chamber experiments showing that P...

Multicellularity, the coordinated collective behaviour of cell populations, gives rise to the emergence of self-organized phenomena at many different spatio-temporal scales. At the genetic scale, oscillators are ubiquitous in regulation of multicellular systems, including during their development and regeneration. Synthetic biologists have successfully created simple synthetic genetic circuits that produce oscillations in single cells. Studying and engineering synthetic oscillators in a multicellular chassis can therefore give us valuable insights into how simple genetic circuits can encode complex multicellular behaviours at different scales. Here we develop a study of the coupling between the repressilator synthetic genetic ring oscillator and constraints on cell growth in colonies. We show in silico how mechanical constraints generate characteristic patterns of growth rate inhomogeneity in growing cell colonies. Next, we develop a simple one-dimensional model which predicts that coupling the repressilator to this pattern of growth rate via protein dilution generates travelling waves of gene expression. We show that the dynamics of these spatio-temporal patterns are determined by two parameters; the protein degradation and maximum expression rates of the repressors. We derive simple relations between these parameters and the key characteristics of the travelling wave patterns: firstly, wave speed is determined by protein degradation and secondly, wavelength is determined by maximum gene expression rate. Our analytical predictions and numerical results were in close quantitative agreement with detailed individual based simulations of growing cell colonies. Confirming published experimental results we also found that static ring patterns occur when protein stability is high. Our results show that this pattern can be induced simply by growth rate dilution and does not require transition to stationary phase as previously suggested. Our method generalizes easily to other genetic circuit architectures thus providing a framework for multi-scale rational design of spatio-temporal patterns from genetic circuits. We use this method to generate testable predictions for the synthetic biology design-build-test-learn cycle.

The budget is one aspect that determines the quality of the implementation of regional head elections (Pilkada). The budget problem that arose in the 2020 Pilkada Concurrent was the rationalization carried out by the DPRD on the Pilkada budget which was contained in the Regional Grant Agreement Text (NPHD) even though the NPHD was signed by the Regional Head and Pilkada Organizer (KPU and Bawaslu) as evidence of the budget allocation agreement for the activities of organizing and supervising the Pilkada. Examples of this rationalization practice occurred in 2 (two) regions, namely Ogan Komering Ulu Timur District, South Sumatra Province, and Tidore Islands City, North Maluku Province. There are 2 (two) factors that give rise to the rationalization problem. First, poor communication between the elements of Pilkada organizers (KPU and Bawaslu) together with the Government and the DPRD, which led to a deadlock and made the Central Government have to step in to resolve it. Second, the c...

The critical role of bacterial biofilms in chronic human infections calls for novel anti-biofilm strategies targeting the regulation of biofilm development. However, the regulation of biofilm development is very complex and can include multiple, highly interconnected signal transduction/response pathways, which are incompletely understood. We demonstrated previously that in the opportunistic, human pathogen P. aeruginosa, the PP2C-like protein phosphatase SiaA and the di-guanylate cyclase SiaD control the formation of macroscopic cellular aggregates, a type of suspended biofilms, in response to surfactant stress. In this study, we demonstrate that the SiaABC proteins represent a signal response pathway that functions through a partner switch mechanism to control biofilm formation. We also demonstrate that SiaABCD functionality is dependent on carbon substrate availability for a variety of substrates, and that upon carbon starvation, SiaB mutants show impaired dispersal, in particular with the primary fermentation product ethanol. This suggests that carbon availability is at least one of the key environmental cues integrated by the SiaABCD system. Further, our biochemical, physiological and crystallographic data reveals that the phosphatase SiaA and its kinase counterpart SiaB balance the phosphorylation status of their target protein SiaC at threonine 68 (T68). Crystallographic analysis of the SiaA-PP2C domain shows that SiaA is present as a dimer. Dynamic modelling of SiaA with SiaC suggested that SiaA interacts strongly with phosphorylated SiaC and dissociates rapidly upon dephosphorylation of SiaC. Further, we show that the known phosphatase inhibitor fumonisin inhibits SiaA mediated phosphatase activity in vitro. In conclusion, the present work improves our understanding of how P. aeuruginosa integrates specific environmental conditions, such as carbon availability and surfactant stress, to regulate cellular aggregation and biofilm formation. With the biochemical and structural characterization of SiaA, initial data on the catalytic inhibition of SiaA, and the interaction between SiaA and SiaC, our study identifies promising targets for the development of biofilm-interference drugs to combat infections of this aggressive opportunistic pathogen. .

Pseudomonas aeruginosa is the most prevalent bacterial species that contributes to cystic fibrosis (CF) respiratory failure. The impaired function of cystic fibrosis transmembrane conductance regulator leads to abnormal epithelial Cl− / HCO3− transport and acidification of airway surface liquid. However, it remains unclear why Pseudomonas aeruginosa preferentially colonizes in the CF lungs. In this study, we carried out studies to investigate if lower pH helps Pseudomonas aeruginosa adapt and thrive in the CF-like acidic lung environment. Our results reveal that Pseudomonas aeruginosa generally forms more biofilm and induces antibiotic resistance faster in acidic conditions and that this can be reversed by returning the acidic environment to physiologically neutral conditions. Pseudomonas aeruginosa appears to be highly adaptive to the CF-like acidic pH environment. By studying the effects of an acidic environment on bacterial response, we may provide a new therapeutic option in pre...

We have developed an efficient and inexpensive pipeline for streamlining large-scale collection and genome sequencing of bacterial isolates. Evaluation of this method involved a worldwide research collaboration focused on the model organism Salmonella enterica, the 10KSG consortium. By optimising a logistics pipeline that collected isolates as thermolysates, permitting shipment in ambient conditions, the project assembled a diverse collection of 10,419 clinical and environmental isolates from low- and middle-income countries in less than one year. The bacteria were obtained from 51 countries/territories dating from 1949 to 2017, with a focus on Africa and Latin-America. All isolates were collected in barcoded tubes and genome sequenced using an optimised DNA extraction method and the LITE pipeline for library construction. After Illumina sequencing, the total reagent cost was less than USD$10 per genome. Our method can be applied to genome-sequence other large bacterial collections ...

We used single-molecule AFM force spectroscopy (AFM-SMFS) to screen residues along the backbone of a non-antibody protein binding scaffold (lipocalin/anticalin), and determine the optimal anchor point that maximizes binding strength of the interaction with its target (CTLA-4). By incorporating non-canonical amino acids into anticalin, and using click chemistry to attach an Fgβ peptide at internal sequence positions, we were able to mechanically dissociate anticalin from CTLA-4 by pulling from eight different anchoring residues using an AFM cantilever tip. We found that pulling on the anticalin from residue 60 or 87 resulted in significantly higher rupture forces and a decrease in koff by 2-3 orders of magnitude over a force range of 50-200 pN. Five of the six internal pulling points tested were significantly more stable than N- or C-terminal anchor points, rupturing at up to 250 pN at loading rates of 0.1-10 nN sec-1. Anisotropic network modelling and molecular dynamics simulations ...

The quest for small molecules that avidly bind to G-quadruplex-DNA (G4-DNA, or G4), so called G4-ligands, has invigorated the G4 research field from its very inception. Massive efforts have been invested to i- screen or design G4-ligands, ii- evaluate their G4-interacting properties in vitro through a series of now widely accepted and routinely implemented assays, and iii- use them as unique chemical biology tools to interrogate cellular networks that might involve G4s. In sharp contrast, only uncoordinated efforts at developing small molecules aimed at destabilizing G4s have been invested to date, even though it is now recognized that such molecular tools would have tremendous application to neurobiology as many genetic and age-related diseases are caused by an over-representation of G4s, itself caused by a deficiency of G4-resolving enzymes, the G4-helicases. Herein, we report on our double effort to i- develop a reliable in vitro assay to identify molecules able to destabilize G4...

The critical role of bacterial biofilms in chronic human infections calls for novel anti-biofilm strategies targeting the regulation of biofilm development. However, the regulation of biofilm development is very complex and can include multiple, highly interconnected signal transduction/response pathways, which are incompletely understood. We demonstrated previously that in the opportunistic, human pathogen P. aeruginosa, the PP2C-like protein phosphatase SiaA and the di-guanylate cyclase SiaD control the formation of macroscopic cellular aggregates, a type of suspended biofilms, in response to surfactant stress. In this study, we demonstrate that the SiaABC proteins represent a signal response pathway that functions through a partner switch mechanism to control biofilm formation. We also demonstrate that SiaABCD functionality is dependent on carbon substrate availability for a variety of substrates, and that upon carbon starvation, SiaB mutants show impaired dispersal, in particular with the primary fermentation product ethanol. This suggests that carbon availability is at least one of the key environmental cues integrated by the SiaABCD system. Further, our biochemical, physiological and crystallographic data reveals that the phosphatase SiaA and its kinase counterpart SiaB balance the phosphorylation status of their target protein SiaC at threonine 68 (T68). Crystallographic analysis of the SiaA-PP2C domain shows that SiaA is present as a dimer. Dynamic modelling of SiaA with SiaC suggested that SiaA interacts strongly with phosphorylated SiaC and dissociates rapidly upon dephosphorylation of SiaC. Further, we show that the known phosphatase inhibitor fumonisin inhibits SiaA mediated phosphatase activity in vitro. In conclusion, the present work improves our understanding of how P. aeuruginosa integrates specific environmental conditions, such as carbon availability and surfactant stress, to regulate cellular aggregation and biofilm formation. With the biochemical and structural characterization of SiaA, initial data on the catalytic inhibition of SiaA, and the interaction between SiaA and SiaC, our study identifies promising targets for the development of biofilm-interference drugs to combat infections of this aggressive opportunistic pathogen. .

The quest for small molecules that avidly bind to G-quadruplex-DNA (G4-DNA, or G4), so called G4-ligands, has invigorated the G4 research field from its very inception. Massive efforts have been invested to i- screen or design G4-ligands, ii- evaluate their G4-interacting properties in vitro through a series of now widely accepted and routinely implemented assays, and iii- use them as unique chemical biology tools to interrogate cellular networks that might involve G4s. In sharp contrast, only uncoordinated efforts at developing small molecules aimed at destabilizing G4s have been invested to date, even though it is now recognized that such molecular tools would have tremendous application to neurobiology as many genetic and age-related diseases are caused by an over-representation of G4s, itself caused by a deficiency of G4-resolving enzymes, the G4-helicases. Herein, we report on our double effort to i- develop a reliable in vitro assay to identify molecules able to destabilize G4...