Curli Synthesis Research Articles

In vitro and in vivo enhancement effect of glabridin on the antibacterial activity of colistin, against multidrug resistant Escherichia coli strains

BackgroundThe increase in antimicrobial resistance leads to complications in treatments, prolonged hospitalization, and increased mortality. Glabridin (GLA) is a hydroxyisoflavan from Glycyrrhiza glabra L. that exhibits multiple pharmacological activities. Colistin (COL), a last-resort antibiotic, is increasingly being used in clinic against Gram-negative bacteria. Previous reports have shown that GLA is able to sensitize first line antibiotics such as norfloxacin and vancomycin on Staphylococcus aureus, implying that the use of GLA as an antibiotic adjuvant is a promising strategy for addressing the issue of drug resistance. However, the adjuvant effect on other antibiotics, especially COL, on Gram-negative bacteria such as Escherichia coli has not been studied. PurposeThe objective of our study was to investigate the targets of GLA and the synergistic effect of GLA and COL in E. coli, and to provide further evidence for the use of GLA as an antibiotic adjuvant to alleviate the problem of drug resistance. MethodsWe first investigated the interaction between GLA and enoyl-acyl carrier protein reductase, also called "FabI", through enzyme inhibition assay, differential scanning fluorimetry, isothermal titration calorimetry and molecular docking assay. We tested the transmembrane capacity of GLA on its own and combined it with several antibiotics. The antimicrobial activities of GLA and COL were evaluated against six different susceptible and resistant E. coli in vitro. Their interactions were analyzed using checkerboard assay, time-kill curve and CompuSyn software. A series of sensitivity tests was conducted in E. coli overexpressing the fabI gene. The development of COL resistance in the presence of GLA was tested. The antimicrobial efficacy of GLA and COL in a mouse model of urinary tract infection was assessed. The anti-biofilm effects of GLA and COL were investigated. ResultsIn this study, enzyme kinetic analysis and thermal analysis provided evidence for the interaction between GLA and FabI in E. coli. GLA enhanced the antimicrobial effect of COL and synergistically suppressed six different susceptible and resistant E. coli with COL. Overexpression experiments showed that targeted inhibition of FabI was a key mechanism by which GLA synergistically enhanced COL activity. The combination of GLA and COL slowed the development of COL resistance in E. coli. Combined GLA and COL treatment significantly reduced bacterial load and mitigated urinary tract injury in a mouse model of E. coli urinary tract infection. Additionally, GLA + COL inhibited the formation and eradication of biofilms and the synthesis of curli. ConclusionOur findings indicate that GLA synergistically enhances antimicrobial activities of COL by targeting inhibition of FabI in E. coli. GLA is expected to continue to be developed as an antibiotic adjuvant to address drug resistance issues.

Tannic acid inhibits Escherichia coli biofilm formation and underlying molecular mechanisms: Biofilm regulator CsgD

Biofilms often engender persistent infections, heightened antibiotic resistance, and the recurrence of infections. Therefor, infections related to bacterial biofilms are often chronic and pose challenges in terms of treatment. The main transcription regulatory factor, CsgD, activates csgABC-encoded curli to participate in the composition of extracellular matrix, which is an important skeleton for biofilm development in enterobacteriaceae. In our previous study, a wide range of natural bioactive compounds that exhibit strong affinity to CsgD were screened and identified via molecular docking. Tannic acid (TA) was subsequently chosen, based on its potent biofilm inhibition effect as observed in crystal violet staining. Therefore, the aim of this study was to investigate the specific effects of TA on the biofilm formation of clinically isolated Escherichia coli (E. coli). Results demonstrated a significant inhibition of E. coli Ec032 biofilm formation by TA, while not substantially affecting the biofilm of the ΔcsgD strain. Moreover, deletion of the csgD gene led to a reduction in Ec032 biofilm formation, alongside diminished bacterial motility and curli synthesis inhibition. Transcriptomic analysis and RT-qPCR revealed that TA repressed genes associated with the csg operon and other biofilm-related genes. In conclusion, our results suggest that CsgD is one of the key targets for TA to inhibit E. coli biofilm formation. This work preliminarily elucidates the molecular mechanisms of TA inhibiting E. coli biofilm formation, which could provide a lead structure for the development of future antibiofilm drugs.

Polyhexamethylene biguanide promotes adaptive cross-resistance to gentamicin in Escherichia coli biofilms.

Antimicrobial resistance is a critical public health issue that requires a thorough understanding of the factors that influence the selection and spread of antibiotic-resistant bacteria. Biocides, which are widely used in cleaning and disinfection procedures in a variety of settings, may contribute to this resistance by inducing similar defense mechanisms in bacteria against both biocides and antibiotics. However, the strategies used by bacteria to adapt and develop cross-resistance remain poorly understood, particularly within biofilms -a widespread bacterial habitat that significantly influences bacterial tolerance and adaptive strategies. Using a combination of adaptive laboratory evolution experiments, genomic and RT-qPCR analyses, and biofilm structural characterization using confocal microscopy, we investigated in this study how Escherichia coli biofilms adapted after 28 days of exposure to three biocidal active substances and the effects on cross-resistance to antibiotics. Interestingly, polyhexamethylene biguanide (PHMB) exposure led to an increase of gentamicin resistance (GenR) phenotypes in biofilms formed by most of the seven E. coli strains tested. Nevertheless, most variants that emerged under biocidal conditions did not retain the GenR phenotype after removal of antimicrobial stress, suggesting a transient adaptation (adaptive resistance). The whole genome sequencing of variants with stable GenR phenotypes revealed recurrent mutations in genes associated with cellular respiration, including cytochrome oxidase (cydA, cyoC) and ATP synthase (atpG). RT-qPCR analysis revealed an induction of gene expression associated with biofilm matrix production (especially curli synthesis), stress responses, active and passive transport and cell respiration during PHMB exposure, providing insight into potential physiological responses associated with adaptive crossresistance. In addition, confocal laser scanning microscopy (CLSM) observations demonstrated a global effect of PHMB on biofilm architectures and compositions formed by most E. coli strains, with the appearance of dense cellular clusters after a 24h-exposure. In conclusion, our results showed that the PHMB exposure stimulated the emergence of an adaptive cross-resistance to gentamicin in biofilms, likely induced through the activation of physiological responses and biofilm structural modulations altering gradients and microenvironmental conditions in the biological edifice.

A novel rspA gene regulates biofilm formation and virulence of Salmonella Typhimurium

Salmonella spp. are facultative anaerobic, Gram-negative, rod-shaped bacteria and belongs to the Enterobacteriaceae family. Although much has been known about Salmonella pathogenesis, the functional characterizations of certain genes are yet to be explored. The rspA (STM14_1818) is one such gene with putative dehydratase function, and its role in pathogenesis is unknown. The background information showed that rspA gene is upregulated in Salmonella when it resides inside macrophages, which led us to investigate its role in Salmonella pathogenesis. We generated the rspA knockout strain and complement strain in S. Typhimurium 14028. Ex-vivo and in-vivo infectivity was looked at macrophage and epithelial cell lines and Caenorhabditis elegans (C. elegans). The mutant strain differentially formed the biofilm at different temperatures by altering the expression of genes involved in the synthesis of cellulose and curli. Besides, the mutant strain is hyperproliferative intracellularly and showed increased bacterial burden in C. elegans. The mutant strain became more infectious and lethal, causing faster death of the worms than the wild type, and also modulates the worm's innate immunity. Thus, we found that the rspA deletion mutant was more pathogenic. In this study, we concluded that the rspA gene differentially regulates the biofilm formation in a temperature dependent manner by modulating the genes involved in the synthesis of cellulose and curli and negatively regulates the Salmonella virulence for longer persistence inside the host.

CsgI (YccT) Is a Novel Inhibitor of Curli Fimbriae Formation in Escherichia coli Preventing CsgA Polymerization and Curli Gene Expression.

Curli fimbriae are amyloids-found in bacteria (Escherichia coli)-that are involved in solid-surface adhesion and bacterial aggregation during biofilm formation. The curli protein CsgA is coded by a csgBAC operon gene, and the transcription factor CsgD is essential to induce its curli protein expression. However, the complete mechanism underlying curli fimbriae formation requires elucidation. Herein, we noted that curli fimbriae formation was inhibited by yccT-i.e., a gene that encodes a periplasmic protein of unknown function regulated by CsgD. Furthermore, curli fimbriae formation was strongly repressed by CsgD overexpression caused by a multicopy plasmid in BW25113-the non-cellulose-producing strain. YccT deficiency prevented these CsgD effects. YccT overexpression led to intracellular YccT accumulation and reduced CsgA expression. These effects were addressed by deleting the N-terminal signal peptide of YccT. Localization, gene expression, and phenotypic analyses revealed that YccT-dependent inhibition of curli fimbriae formation and curli protein expression was mediated by the two-component regulatory system EnvZ/OmpR. Purified YccT inhibited CsgA polymerization; however, no intracytoplasmic interaction between YccT and CsgA was detected. Thus, YccT-renamed CsgI (curli synthesis inhibitor)-is a novel inhibitor of curli fimbriae formation and has a dual role as an OmpR phosphorylation modulator and CsgA polymerization inhibitor.

CRISPRi-mediated suppression of E. coli Nissle 1917 virulence factors: A strategy for creating an engineered probiotic using csgD gene suppression.

BackgroundBiofilm formation is a complex phenomenon, and it is the causative agent of several human infections. Bacterial amyloids are involved in biofilm formation leading to infection persistence. Due to antibiotic resistance, their treatment is a great challenge for physicians. Probiotics, especially E. coli Nissle 1917 (EcN), are used to treat human intestinal disorders and ulcerative colitis. It also expresses virulence factors associated with biofilm and amyloid formation. EcN produces biofilm equivalent to the pathogenic UPEC strains.MethodsCRISPRi was used to create the knockdown mutants of the csgD gene (csgD-KD). The qRT-PCR was performed to assess the expression of the csgD gene in csgD-KD cells. The csgD-KD cells were also evaluated for the expression of csgA, csgB, fimA, fimH, ompR, luxS, and bolA genes. The gene expression data obtained was further confirmed by spectroscopic, microscopic, and other assays to validate our study.ResultsCRISPRi-mediated knockdown of csgD gene shows reduction in curli amyloid formation, biofilm formation, and suppression of genes (csgA, csgB, fimA, fimH, ompR, bolA, and luxS) involved in virulence factors production.ConclusionCurli amyloid fibers and fimbriae fibers play a critical role in biofilm formation leading to pathogenicity. CsgD protein is the master regulator of curli synthesis in E. coli. Hence, curli amyloid inhibition through the csgD gene may be used to improve the EcN and different probiotic strains by suppressing virulence factors.

Hormetic Promotion of Biofilm Growth by Polyvalent Bacteriophages at Low Concentrations.

Interactions between bacteriophages (phages) and biofilms are poorly understood despite their broad ecological and water quality implications. Here, we report that biofilm exposure to lytic polyvalent phages at low concentrations (i.e., 102-104 phages/mL) can counterintuitively promote biofilm growth and densification (corroborated by confocal laser scanning microscopy (CLSM)). Such exposure hormetically upregulated quorum sensing genes (by 4.1- to 24.9-fold), polysaccharide production genes (by 3.7- to 9.3-fold), and curli synthesis genes (by 4.5- to 6.5-fold) in the biofilm-dwelling bacterial hosts (i.e., Escherichia coli and Pseudomonas aeruginosa) relative to unexposed controls. Accordingly, the biofilm matrix increased its polysaccharide and extracellular DNA content relative to unexposed controls (by 41.8 ± 2.3 and 81.4 ± 2.2%, respectively), which decreased biofilm permeability and increased structural integrity. This contributed to enhanced resistance to disinfection with chlorine (bacteria half-lives were 6.08 ± 0.05 vs 3.91 ± 0.03 min for unexposed controls) and to subsequent phage infection (biomass removal was 18.2 ± 1.2 vs 32.3 ± 1.2% for unexposed controls), apparently by mitigating diffusion of these antibacterial agents through the biofilm. Overall, low concentrations of phages reaching a biofilm may result in unintended biofilm stimulation, which might accelerate biofouling, biocorrosion, or other biofilm-related water quality problems.

In vivo synthesis of bacterial amyloid curli contributes to joint inflammation during S. Typhimurium infection.

Reactive arthritis, an autoimmune disorder, occurs following gastrointestinal infection with invasive enteric pathogens, such as Salmonella enterica. Curli, an extracellular, bacterial amyloid with cross beta-sheet structure can trigger inflammatory responses by stimulating pattern recognition receptors. Here we show that S. Typhimurium produces curli amyloids in the cecum and colon of mice after natural oral infection, in both acute and chronic infection models. Production of curli was associated with an increase in anti-dsDNA autoantibodies and joint inflammation in infected mice. The negative impacts on the host appeared to be dependent on invasive systemic exposure of curli to immune cells. We hypothesize that in vivo synthesis of curli contributes to known complications of enteric infections and suggest that cross-seeding interactions can occur between pathogen-produced amyloids and amyloidogenic proteins of the host.

Genome rearrangements induce biofilm formation in Escherichia coli C \u2013 an old model organism with a new application in biofilm research

BackgroundEscherichia coli C forms more robust biofilms than other laboratory strains. Biofilm formation and cell aggregation under a high shear force depend on temperature and salt concentrations. It is the last of five E. coli strains (C, K12, B, W, Crooks) designated as safe for laboratory purposes whose genome has not been sequenced.ResultsHere we present the complete genomic sequence of this strain in which we utilized both long-read PacBio-based sequencing and high resolution optical mapping to confirm a large inversion in comparison to the other laboratory strains. Notably, DNA sequence comparison revealed the absence of several genes thought to be involved in biofilm formation, including antigen 43, waaSBOJYZUL for lipopolysaccharide (LPS) synthesis, and cpsB for curli synthesis. The first main difference we identified that likely affects biofilm formation is the presence of an IS3-like insertion sequence in front of the carbon storage regulator csrA gene. This insertion is located 86 bp upstream of the csrA start codon inside the − 35 region of P4 promoter and blocks the transcription from the sigma32 and sigma70 promoters P1-P3 located further upstream. The second is the presence of an IS5/IS1182 in front of the csgD gene. And finally, E. coli C encodes an additional sigma70 subunit driven by the same IS3-like insertion sequence. Promoter analyses using GFP gene fusions provided insights into understanding this regulatory pathway in E. coli.ConclusionsBiofilms are crucial for bacterial survival, adaptation, and dissemination in natural, industrial, and medical environments. Most laboratory strains of E. coli grown for decades in vitro have evolved and lost their ability to form biofilm, while environmental isolates that can cause infections and diseases are not safe to work with. Here, we show that the historic laboratory strain of E. coli C produces a robust biofilm and can be used as a model organism for multicellular bacterial research. Furthermore, we ascertained the full genomic sequence of this classic strain, which provides for a base level of characterization and makes it useful for many biofilm-based applications.

Comparative genomic analyses reveal diverse virulence factors and antimicrobial resistance mechanisms in clinical Elizabethkingia meningosepticastrains.

Three human clinical isolates of bacteria (designated strains Em1, Em2 and Em3) had high average nucleotide identity (ANI) to Elizabethkingia meningoseptica. Their genome sizes (3.89, 4.04 and 4.04 Mb) were comparable to those of other Elizabethkingia species and strains, and exhibited open pan-genome characteristics, with two strains being nearly identical and the third divergent. These strains were susceptible only to trimethoprim/sulfamethoxazole and ciprofloxacin amongst 16 antibiotics in minimum inhibitory tests. The resistome exhibited a high diversity of resistance genes, including 5 different lactamase- and 18 efflux protein- encoding genes. Forty-four genes encoding virulence factors were conserved among the strains. Sialic acid transporters and curli synthesis genes were well conserved in E. meningoseptica but absent in E. anophelis and E. miricola. E. meningoseptica carried several genes contributing to biofilm formation. 58 glycoside hydrolases (GH) and 25 putative polysaccharide utilization loci (PULs) were found. The strains carried numerous genes encoding two-component system proteins (56), transcription factor proteins (187~191), and DNA-binding proteins (6~7). Several prophages and CRISPR/Cas elements were uniquely present in the genomes.

Potential of Lactobacillus casei shirota’s strain against the biofilm-forming of Salmonella Spp

Biofilm of Salmonella spp. is formed through the expression of biofilm genes associated with proteins (bapA) regulated by curli synthesis genes (csg) which carry out adhesion, colonization, maturation, and dispersion on the surface of the intestinal epithelium. This study aimed to determine the antibiofilm activity of Lactobacillus casei Shirota’S strain (LcS) as an inhibitor of Salmonella spp. biofilm formation in vitro. This research was a true experimental study using Microtiter Plate 96 wells Biofilm Assay method. The sample used was the suspension of Salmonella spp. The treatment was in the form of adding a LcS suspension with a concentration series of 10-1;10-2; 10-3;10-4; and 10-5. Biofilm measurements were carried out using a microplate reader and obtained quantitative data in the form of Optical Density at a wavelength of 595nm. The results of this study showed that LcS suspension has antibiofilm activity ranging from 10-5 concentrations with a percentage of 36.58% (p<0.05). The results of exometabolism LcS can reduce Salmonella growth. Exopolysaccharide (EPS) and sortase-dependent proteins (SrtA) of LcS form barriers as competitive adhesion in inhibiting pathogenic biofilm formation.

Discovery of New Genes Involved in Curli Production by a Uropathogenic Escherichia coli Strain from the Highly Virulent O45:K1:H7 Lineage.

ABSTRACTCurli are bacterial surface-associated amyloid fibers that bind to the dye Congo red (CR) and facilitate uropathogenic Escherichia coli (UPEC) biofilm formation and protection against host innate defenses. Here we sequenced the genome of the curli-producing UPEC pyelonephritis strain MS7163 and showed it belongs to the highly virulent O45:K1:H7 neonatal meningitis-associated clone. MS7163 produced curli at human physiological temperature, and this correlated with biofilm growth, resistance of sessile cells to the human cationic peptide cathelicidin, and enhanced colonization of the mouse bladder. We devised a forward genetic screen using CR staining as a proxy for curli production and identified 41 genes that were required for optimal CR binding, of which 19 genes were essential for curli synthesis. Ten of these genes were novel or poorly characterized with respect to curli synthesis and included genes involved in purine de novo biosynthesis, a regulator that controls the Rcs phosphorelay system, and a novel repressor of curli production (referred to as rcpA). The involvement of these genes in curli production was confirmed by the construction of defined mutants and their complementation. The mutants did not express the curli major subunit CsgA and failed to produce curli based on CR binding. Mutation of purF (the first gene in the purine biosynthesis pathway) and rcpA also led to attenuated colonization of the mouse bladder. Overall, this work has provided new insight into the regulation of curli and the role of these amyloid fibers in UPEC biofilm formation and pathogenesis.

Evidence for up and down regulation of 450 genes by rpoB12 (rif) mutation and their implications in complexity of transcription modulation in Escherichia coli

Analyses of mutations in rpoB subunit of Escherichia coli that lead to resistance to rifampicin have been invaluable in providing insight into events during transcription continue to be discovered. Earlier we reported that rpoB12 suppresses over-expression of cps genes in Δlon mutant of E. coli, by interfering with the transcription of rcsA. Here we report Microarray based Transcriptome profile of Δlon and Δlon rpoB12 strains. The data analyses clearly reveal that rpoB12 mutation results in the differential expression of ∼450 genes. The transcription profiles of some of the genes namely, rcsA, gadE, csgD, bolA, ypdI, dnaJ, clpP, csrA and hdeA are significantly altered, particularly the genes implicated in virulence. Some of the phenotypic traits namely, biofilm formation, motility, curli synthesis and ability to withstand acidic stress in a lon+rpoB12 strain were assessed. The results clearly indicate that rpoB12 up-regulates biofilm formation and curli synthesis while it makes the cells sensitive for growth in acidic medium and inhibits motility almost completely. Furthermore, rpoB12 modulates the expression profile of a significant number of genes involved in stress responses, genes encoding small RNAs. Thus, this study reveals the versatile role of the rpoB12 mutation, especially its impact on the regulation of genes related to virulence and highlights its medical importance.

Nutrient Composition Promotes Switching between Pellicle and Bottom Biofilm in Salmonella.

Salmonella is one of the most frequently reported causes of foodborne illness worldwide. Non-typhoidal serovars cause gastroenteritis in humans. Salmonella can grow on surfaces forming biofilms, contributing to its persistence since biofilms are difficult to eradicate due to the high resistance to antimicrobials and disinfectants. It has been described that there are two crucial biofilm promoting factors in Salmonella: curli and cellulose. The expression of both factors is coordinately regulated by the transcriptional regulator CsgD. Most biofilm studies of Salmonella have been performed by growing bacteria in low osmolarity rich medium and low temperature (25°C). In such conditions, the biofilm is formed at the air–liquid interface (pellicle biofilm). Remarkably, when Salmonella grow in minimal medium, biofilm formation switches from the air–liquid interface to the solid–liquid interface (bottom biofilm). In this report, the switching between pellicle and bottom biofilm has been characterized. Our data indicate that curli, but not cellulose, is crucial for the formation of both kinds of biofilms. In minimal medium, conditions promoting formation of bottom biofilm, a high transcriptional expression of csgD and consequently of the genes involved in the synthesis of curli and cellulose was detected. The nutritional status of the cells seems to be pivotal for the spatial distribution of the biofilms formed. When bacteria is growing in minimal medium the addition of amino acids downregulates the expression of csgB and causes the switch between bottom and pellicle biofilm. The crosstalk between general metabolism and biofilm formation is also highlighted by the fact that the metabolic sensor cAMP modulates the type of biofilm generated by Salmonella. Moreover, cAMP regulates transcriptional expression of csgD and stimulates pellicle biofilm formation, suggesting that the physiological conditions define the type of biofilm formed by Salmonella. The consequences of the switching between pellicle and bottom biofilm during either infection or survival in natural environments remain undercover.

Curli synthesis and biofilm formation in enteric bacteria are controlled by a dynamic small RNA module made up of a pseudoknot assisted by an RNA chaperone

RydC pseudoknot aided by Hfq is a dynamic regulatory module. We report that RydC reduces expression of curli-specific gene D transcription factor required for adhesion and biofilm production in enterobacteria. During curli formation, csgD messenger RNA (mRNA) synthesis increases when endogenous levels of RydC are lacking. In Escherichia coli and Salmonella enterica, stimulation of RydC expression also reduces biofilm formation by impairing curli synthesis. Inducing RydC early on in growth lowers CsgA, -B and -D protein and mRNA levels. RydC’s 5′-domain interacts with csgD mRNA translation initiation signals to prevent initiation. Translation inhibition occurs by an antisense mechanism, blocking the translation initiation signals through pairing, and that mechanism is facilitated by Hfq. Although Hfq represses csgD mRNA translation without a small RNA (sRNA), it forms a ternary complex with RydC and facilitates pseudoknot unfolding to interact with the csgD mRNA translation initiation signals. RydC action implies Hfq-assisted unfolding and mRNA rearrangements, but once the pseudoknot is disrupted, Hfq is unnecessary for regulation. RydC is the sixth sRNA that negatively controls CsgD synthesis. Hfq induces structural changes in the mRNA domains targeted by these six sRNAs. What we describe is an ingenious process whereby pseudoknot opening is orchestrated by a chaperone to allow RNA control of gene expression.

Spontaneous non‐rdar mutations increase fitness of Salmonella in plants

Proliferation of human enteric pathogens within alternate hosts, like plants, leads to temporal changes in gene expression and also selects for the phenotypic variants of the enterics that are presumed to be more fit within plants. Human enteric pathogens recovered from produce-borne outbreaks exhibit peculiar phenotypes, for example many of them do not display the rdar (red dry and rough) phenotype. The non-rdar phenotype results from mutations in cellulose and/or curli synthesis or regulation. How often these mutants arise, and whether they are more fit within plants is not entirely clear. We addressed this hypothesis by sequentially passaging the type strain of Salmonella enterica sv. Typhimurium ATCC14028 through tomatoes. Two spontaneous mutants defective in their ability to form red dry and rough colonies were further characterized. Even though attachment of the mutants to tomato surfaces was modestly reduced, they were 5- to 50-fold more competitive than the wild-type inside tomato fruits. Because the mutants were outcompeted by the wild-type on common laboratory media, and not in tomatoes, the lack of the rdar phenotype is probably beneficial within tomatoes. Recombinase-based in vivo expression tests indicate that the agfB and yihT genes were regulated differently in the mutants, although the corresponding mutations cannot fully account for the increased competitive fitness of the mutants. One of the variants has a mutated rpoS, which also reduced the expression of a SPI-5 effector encoded by sopB. A survey of the Salmonella strains recovered from produce outbreaks revealed that some were similarly non-rdar, likely containing rpoS mutations. This report indicates that the 'perfect storm' scenario, typically used to model outbreaks of produce-borne gastroenteritis, needs to account for the ability of the pathogen to rapidly evolve and adapt to the crop production environments.

Small regulatory RNAs control the multi‐cellular adhesive lifestyle of Escherichia coli

Small regulatory RNA molecules have recently been recognized as important regulatory elements of developmental processes in both eukaryotes and bacteria. We here describe a striking example in Escherichia coli that can switch between a single-cell motile lifestyle and a multi-cellular, sessile and adhesive state that enables biofilm formation on surfaces. For this, the bacterium needs to reprogramme its gene expression, and in many E. coli and Salmonella strains the lifestyle shift relies on control cascades that inhibit flagellar expression and activate the synthesis of curli, extracellular adhesive fibres important for co-aggregation of cells and adhesion to biotic and abiotic surfaces. By combining bioinformatics, genetic and biochemical analysis we identified three small RNAs that act by an antisense mechanism to downregulate translation of CsgD, the master regulator of curli synthesis. Our demonstration that basal expression of each of the three RNA species is sufficient to downregulate CsgD synthesis and prevent curli formation indicates that all play a prominent role in the curli regulatory network. Our findings provide the first clue as to how the Rcs signalling pathway negatively regulates curli synthesis and increase the number of small regulatory RNAs that act directly on the csgD mRNA to five.

Engineering a novel c-di-GMP-binding protein for biofilm dispersal.

Bacteria prefer to grow attached to themselves or an interface, and it is important for an array of applications to make biofilms disperse. Here we report simultaneously the discovery and protein engineering of BdcA (formerly YjgI) for biofilm dispersal using the universal signal 3,5-cyclic diguanylic acid (c-di-GMP). The bdcA deletion reduced biofilm dispersal, and production of BdcA increased biofilm dispersal to wild-type level. Since BdcA increases motility and extracellular DNA production while decreasing exopolysaccharide, cell length and aggregation, we reasoned that BdcA decreases the concentration of c-di-GMP, the intracellular messenger that controls cell motility through flagellar rotation and biofilm formation through synthesis of curli and cellulose. Consistently, c-di-GMP levels increase upon deleting bdcA, and purified BdcA binds c-di-GMP but does not act as a phosphodiesterase. Additionally, BdcR (formerly YjgJ) is a negative regulator of bdcA. To increase biofilm dispersal, we used protein engineering to evolve BdcA for greater c-di-GMP binding and found that the single amino acid change E50Q causes nearly complete removal of biofilms via dispersal without affecting initial biofilm formation.

Two antisense RNAs target the transcriptional regulator CsgD to inhibit curli synthesis

Escherichia coli produces proteinaceous surface structures called curli that are involved in adhesion and biofilm formation. CsgD is the transcriptional activator of curli genes. We show here that csgD expression is, in part, controlled post-transcriptionally by two redundant small RNAs (sRNAs), OmrA and OmrB. Their overexpression results in curli deficiency, in accordance with the inhibition of chromosomally encoded, FLAG-tagged CsgD. Downregulation of csgD occurs by a direct antisense interaction within the csgD 5'-UTR, far upstream of the ribosome-binding site (RBS). OmrA/B downregulate plasmid-borne csgD-gfp fusions in vivo, and inhibit CsgD translation in vitro. The RNA chaperone Hfq is required for normal csgD mRNA and OmrA/B levels in the cell, and enhances sRNA-dependent inhibition of csgD translation in vitro. Translational inhibition involves two phylogenetically conserved secondary structure modules that are supported by chemical and enzymatic probing. The 5'-most element is necessary and sufficient for regulation, the one downstream comprises the RBS and affects translational efficiency. OmrA/B are two antisense RNAs that regulate a transcription factor to alter a morphotype and group behaviour.

Reconfiguring the quorum-sensing regulator SdiA of Escherichia coli to control biofilm formation via indole and N-acylhomoserine lactones.

SdiA is a homolog of quorum-sensing regulators that detects N-acylhomoserine lactone (AHL) signals from other bacteria. Escherichia coli uses SdiA to reduce its biofilm formation in the presence of both AHLs and its own signal indole. Here we reconfigured SdiA (240 amino acids) to control biofilm formation using protein engineering. Four SdiA variants were obtained with altered biofilm formation, including truncation variants SdiA1E11 (F7L, F59L, Y70C, M94K, and K153X) and SdiA14C3 (W9R, P49T, N87T, frameshift at N96, and L123X), which reduced biofilm formation by 5- to 20-fold compared to wild-type SdiA in the presence of endogenous indole. Whole-transcriptome profiling revealed that wild-type SdiA reduced biofilm formation by repressing genes related to indole synthesis and curli synthesis compared to when no SdiA was expressed, while variant SdiA1E11 induced genes related to indole synthesis in comparison to wild-type SdiA. These results suggested altered indole metabolism, and corroborating the DNA microarray results in regard to indole synthesis, variant SdiA1E11 produced ninefold more indole, which led to reduced swimming motility and cell density. Also, wild-type SdiA decreased curli production and tnaA transcription, while SdiA1E11 increased tnaA transcription (tnaA encodes tryptophanase, which forms indole) compared to wild-type SdiA. Hence, wild-type SdiA decreased biofilm formation by reducing curli production and motility, and SdiA1E11 reduced biofilm formation via indole. Furthermore, an AHL-sensitive variant (SdiA2D10, having four mutations at E31G, Y42F, R116H, and L165Q) increased biofilm formation sevenfold in the presence of N-octanoyl-DL-homoserine lactone and N-(3-oxododecatanoyl)-L-homoserine lactone. Therefore, SdiA can be evolved to increase or decrease biofilm formation, and biofilm formation may be controlled by altering sensors rather than signals.