Flagellar motility is mutagenic.

Flagella are highly complex rotary molecular machines that enable bacteria to not only migrate to optimal environments but also to promote range expansion, competitiveness, virulence, and antibiotic survival. Flagellar motility is an energy-demanding process, where the sum of its production (biosynthesis) and operation (rotation) costs has been estimated to total ~10% of the entire energy budget of an Escherichia coli cell. The acquisition of such a costly adaptation process is expected to secure short-term benefits by increasing competitiveness and survival, as well as long-term evolutionary fitness gains. While the role of flagellar motility in bacterial survival has been widely reported, its direct influence on the rate of evolution remains unclear. We show here that both production and operation costs contribute to elevated mutation rates. Our findings suggest that flagellar movement may be an important player in tuning the rate of bacterial evolution.

Deciphering the impact of exogenous fatty acids on Listeria monocytogenes at low temperature by transcriptome analysis.

Listeria monocytogenes is a ubiquitous and psychrotrophic foodborne pathogen commonly found in raw materials, ready-to-eat products, and food environments. We previously demonstrated that L. monocytogenes can grow faster at low temperature when unsaturated fatty acids (UFA) are present in its environment. This could question the maintenance of food safety for refrigerated foods, especially those reformulated with a higher ratio of UFA versus saturated fatty acids (SFA) to fit with nutritional recommendations. In this study, we used transcriptomics to understand the impact of UFA on the behavior of L. monocytogenes at low temperature. We first demonstrated that fabK, a key gene in SFA synthesis, is up-regulated in the presence of UFA but not SFA at low temperature. L. monocytogenes can thus regulate the synthesis of SFA in its membrane according to the type of FA available in its environment. Interestingly, we also observed up-regulation of genes involved in chemotaxis and flagellar assembly (especially cheY and flaA) in the presence of UFA but not SFA at low temperature. TEM observations confirmed that L. monocytogenes acquired a remarkable phenotype with numerous and long-looped flagella only in the presence of UFA at 5°C but not at 37°C. As flagella are well known to be involved in biofilm formation, this new finding raises questions about the structure and persistence of biofilms settled in refrigerated environments using unsaturated lipid-rich products.

Tyzzerella nexilis strains enriched in mobile genetic elements are involved in progressive multiple sclerosis.

Multiple sclerosis (MS) is an autoimmune-demyelinating disease with an inflammatory pathology formed by self-reactive lymphocytes with activated glial cells. Progressive MS, characterized by resistance to medications, significantly differs from the non-progressive form in gut microbiome profiles. After confirming an increased abundance of "Tyzzerella nexilis" in various cohorts of progressive MS, we identified a distinct cluster of T. nexilis strains enriched in progressive MS based on long-read metagenomics. The distinct T. nexilis cluster is characterized by a large number of mobile genetic elements (MGEs) and a lack of defense systems against MGEs. Microbial genes for sulfate reduction and flagella formation with pathogenic implications are specific to this cluster. Moreover, these flagellar genes are encoded on MGEs. Mono-colonization with MGE-enriched T. nexilis made germ-free mice more susceptible to experimental autoimmune encephalomyelitis. These results indicate that the progression of MS may be promoted by MGE-enriched T. nexilis with potentially pathogenic properties.

Deciphering the influence of NaCl on social behaviour of Bacillus subtilis.

Various environmental signals, such as temperature, pH, nutrient levels, salt content and the presence of other microorganisms, can influence biofilm's development and dynamics. However, the innate mechanisms that govern at the molecular and cellular levels remain elusive. Here, we report the impact of physiologically relevant concentrations of NaCl on biofilm formation and the associated differences in an undomesticated natural isolate of Bacillus subtilis. NaCl exposure and its uptake by bacterial cells induced substantial changes in the architecture of pellicle biofilm and an upsurge in the expansion of biofilm colonies on agar surfaces. We have observed the upregulation of genes involved in motility and the downregulation of genes involved in the biosynthesis of extracellular matrix components through the transcription factor sigD, suggesting the possible underlying mechanisms. To further support these observations, we have used ΔsigD and ΔsrfAC null mutants, which showed compromised NaCl-induced effects. Our results indicate that NaCl induces a lifestyle shift in B. subtilis from a sessile biofilm state to an independent unicellular motile state. Overall, we present evidence that NaCl can reprogramme gene expression and alter cellular morphology and the state of cells to adapt to motility, which facilitates the expansion of bacterial colonies.

Multicellularity and increasing Reynolds number impact on the evolutionary shift in flash-induced ciliary response in Volvocales.

BACKGROUND: Volvocales in green algae have evolved by multicellularity of Chlamydomonas-like unicellular ancestor. Those with various cell numbers exist, such as unicellular Chlamydomonas, four-celled Tetrabaena, and Volvox species with different cell numbers (~1,000, ~5,000, and ~10,000). Each cell of these organisms shares two cilia and an eyespot, which are used for swimming and photosensing. They are all freshwater microalgae but inhabit different fluid environments: unicellular species live in low Reynolds-number (Re) environments where viscous forces dominate, whereas multicellular species live in relatively higher Re where inertial forces become non-negligible. Despite significant changes in the physical environment, during the evolution of multicellularity, they maintained photobehaviors (i.e., photoshock and phototactic responses), which allows them to survive under changing light conditions. RESULTS: In this study, we utilized high-speed imaging to observe flash-induced changes in the ciliary beating manner of 27 Volvocales strains. We classified flash-induced ciliary responses in Volvocales into four patterns: "1: temporal waveform conversion", "2: no obvious response", "3: pause in ciliary beating", and "4: temporal changes in ciliary beating directions". We found that which species exhibit which pattern depends on Re, which is associated with the individual size of each species rather than phylogenetic relationships. CONCLUSIONS: These results suggest that only organisms that acquired different patterns of ciliary responses survived the evolutionary transition to multicellularity with a greater number of cells while maintaining photobehaviors. This study highlights the significance of the Re as a selection pressure in evolution and offers insights for designing propulsion systems in biomimetic micromachines.

A homozygous ARMC3 splicing variant causes asthenozoospermia and flagellar disorganization in a consanguineous family.

Male infertility due to asthenozoospermia is quite frequent, but its etiology is poorly understood. We recruited two infertile brothers, born to first-cousin parents from Pakistan, displaying idiopathic asthenozoospermia with mild stuttering disorder but no ciliary-related symptoms. Whole-exome sequencing identified a splicing variant (c.916+1G>A) in ARMC3, recessively co-segregating with asthenozoospermia in the family. The ARMC3 protein is evolutionarily highly conserved and is mostly expressed in the brain and testicular tissue of human. The ARMC3 splicing mutation leads to the exclusion of exon 8, resulting in a predicted truncated protein (p.Glu245_Asp305delfs*16). Quantitative real-time PCR revealed a significant decrease at mRNA level for ARMC3 and Western blot analysis did not detect ARMC3 protein in the patient's sperm. Individuals homozygous for the ARMC3 splicing variant displayed reduced sperm motility with frequent morphological abnormalities of sperm flagella. Transmission electron microscopy of the affected individual IV: 2 revealed vacuolation in sperm mitochondria at the midpiece and disrupted flagellar ultrastructure in the principal and end piece. Altogether, our results indicate that this novel homozygous ARMC3 splicing mutation destabilizes sperm flagella and leads to asthenozoospermia in our patients, providing a novel marker for genetic counseling and diagnosis of male infertility.

Investigating the interaction of zno nanoparticles with flagellum and fimbriae in multi-drug resistant uropathogenic bacteria encoding fli and fim genes.

Due to the increasing occurrence of drug resistant urinary tract infections (UTI) among children, there is a need to investigate alternative effective treatment protocols such as nanoparticles. Flagella and fimbriae are primary factors contributing the virulence of urinary tract infecting bacteria. The aim of this study was to assess the antibacterial effects of zinc oxide nanoparticles which have been synthesized using both chemical and green methods on multi-drug resistant (MDR) uropathogenic bacteria encoding fli and fim genes and investigating their binding ability to bacterial appendage proteins. A total of 30 urine culture samples were collected from children under 2 years old diagnosed with urinary tract infection. The isolates underwent antibiotic suseptibility assessment and the isolates demonstrating MDR were subjected to molecular amplification of fimG (fimbrial) and fliD and fliT (flagellal) genes. The confirmation of cellular appendages was achieved through silver nitrate staining. The antibacterial efficacy of the synthetized nanoparticles was assessed using the micro and macrodilution methods. The successful binding of nanoparticles to bacterial appendage proteins was confirmed through mobility shift and membrane filter assays. The dimensions of chemically synthesized ZnO nanoparticles and green nanoparticles were measured at 30 nm and 85 nm, respectively, with the exhibition of hexagonal geometries. The nanoparticles synthesized through chemical and green methods exhibited minimum inhibitory concentrations (MIC) of 0.0062-0.025 g/L and 0.3 g/L, respectively. The ability of ZnO nanoparticles to bind bacterial appendage proteins and to combat MDR uropathogenic bacteria are promising for new treatment protocols against UTI in children in future.

VirB11, a traffic ATPase, mediated flagella assembly and type IV pilus morphogenesis to control the motility and virulence of Xanthomonas albilineans.

Xanthomonas albilineans (Xal) is a gram-negative bacterial pathogen responsible for developing sugarcane leaf scald disease, which engenders significant economic losses within the sugarcane industry. In the current study, homologous recombination exchange was carried out to induce mutations within the virB/D4-like type IV secretion system (T4SS) genes of Xal. The results revealed that the virB11-deletion mutant (ΔvirB11) exhibited a loss in swimming and twitching motility. Application of transmission electron microscopy analysis further demonstrated that the ΔvirB11 failed to develop flagella formation and type IV pilus morphology and exhibited reduced swarming behaviour and virulence. However, these alterations had no discernible impact on bacterial growth. Comparative transcriptome analysis between the wild-type Xal JG43 and the deletion-mutant ΔvirB11 revealed 123 differentially expressed genes (DEGs), of which 28 and 10 DEGs were notably associated with flagellar assembly and chemotaxis, respectively. In light of these findings, we postulate that virB11 plays an indispensable role in regulating the processes related to motility and chemotaxis in Xal.

Salmonella Detection in Food Using a HEK-hTLR5 Reporter Cell-Based Sensor.

The development of a rapid, sensitive, specific method for detecting foodborne pathogens is paramount for supplying safe food to enhance public health safety. Despite the significant improvement in pathogen detection methods, key issues are still associated with rapid methods, such as distinguishing living cells from dead, the pathogenic potential or health risk of the analyte at the time of consumption, the detection limit, and the sample-to-result. Mammalian cell-based assays analyze pathogens' interaction with host cells and are responsive only to live pathogens or active toxins. In this study, a human embryonic kidney (HEK293) cell line expressing Toll-Like Receptor 5 (TLR-5) and chromogenic reporter system (HEK dual hTLR5) was used for the detection of viable Salmonella in a 96-well tissue culture plate. This cell line responds to low concentrations of TLR5 agonist flagellin. Stimulation of TLR5 ligand activates nuclear factor-kB (NF-κB)-linked alkaline phosphatase (AP-1) signaling cascade inducing the production of secreted embryonic alkaline phosphatase (SEAP). With the addition of a ρ-nitrophenyl phosphate as a substrate, a colored end product representing a positive signal is quantified. The assay's specificity was validated with the top 20 Salmonella enterica serovars and 19 non-Salmonella spp. The performance of the assay was also validated with spiked food samples. The total detection time (sample-to-result), including shortened pre-enrichment (4 h) and selective enrichment (4 h) steps with artificially inoculated outbreak-implicated food samples (chicken, peanut kernel, peanut butter, black pepper, mayonnaise, and peach), was 15 h when inoculated at 1-100 CFU/25 g sample. These results show the potential of HEK-DualTM hTLR5 cell-based functional biosensors for the rapid screening of Salmonella.

Flagellar point mutation causes social aggregation in laboratory-adapted Bacillus subtilis under conditions that promote swimming.

Motility allows microbes to explore and maximize success in their environment; however, many laboratory bacterial strains have a reduced or altered capacity for motility. Swimming motility in Bacillus subtilis depends on peritrichous flagella and is carried out individually as cells move by biased random walks toward attractants. Previously, we adapted Bacillus subtilis strain 3610 to the laboratory for 300 generations in lysogeny broth (LB) batch culture and isolated lab-adapted strains. Strain SH2 is motility-defective and in broth culture forms large, frequently spherical aggregates of cells. A single point mutation in the flagellin gene hag that causes amino acid 259 to switch from A to T is necessary and sufficient to cause these social cell aggregates, and aggregation occurs between flagellated cells bearing this point mutation regardless of the strain background. Cells associate when bearing this mutation, but flagellar rotation is needed to pull associating cells into spherical aggregates. Using electron microscopy, we are able to show that the SH2 flagellar filament has limited polymorphism when compared to other flagellar structures. This limited polymorphism hinders the flagellum's ability to function as a motility apparatus but appears to alter its function to that of cell aggregation/adhesion. We speculate that the genotype-specific aggregation of cells producing HagA259T flagella could have increased representation in a batch-culture experiment by allowing similar cells to go through a transfer together and also that this mutation could serve as an early step to evolve sociality in the natural world.IMPORTANCEThe first life forms on this planet were prokaryotic, and the earliest environments were aquatic, and from these relatively simple starting conditions, complex communities of microbes and ultimately multicellular organisms were able to evolve. Usually, motile cells in aqueous environments swim as individuals but become social by giving up motility and secreting extracellular substances to become a biofilm. Here, we identify a single point mutation in the flagellum that is sufficient to allow cells containing this mutation to specifically form large, suspended groups of cells. The specific change in the flagellar filament protein subunits causes a unique change in the flagellar structure. This could represent a distinct way for closely related cells to associate as an early precursor to sociality.

Lipopolysaccharide Core Truncation in Invasive Escherichia coli O157:H7 ATCC 43895 Impairs Flagella and Curli Biosynthesis and Reduces Cell Invasion Ability.

Escherichia coli O157:H7 (E. coli O157) is known for causing severe foodborne illnesses such as hemorrhagic colitis and hemolytic uremic syndrome. Although E. coli O157 is typically regarded as an extracellular pathogen and a weak biofilm producer, some E. coli O157 strains, including a clinical strain ATCC 43895, exhibit a notable ability to invade bovine crypt cells and other epithelial cells, as well as to form robust biofilm. This invasive strain persists in the bovine host significantly longer than non-invasive strains. Various surface-associated factors, including lipopolysaccharides (LPS), flagella, and other adhesins, likely contribute to this enhanced invasiveness and biofilm formation. In this study, we constructed a series of LPS-core deletion mutations (waaI, waaG, waaF, and waaC) in E. coli O157 ATCC 43895, resulting in stepwise truncations of the LPS. This approach enabled us to investigate the effects on the biosynthesis of key surface factors, such as flagella and curli, and the ability of this invasive strain to invade host cells. We confirmed the LPS structure and found that all LPS-core mutants failed to form biofilms, highlighting the crucial role of core oligosaccharides in biofilm formation. Additionally, the LPS inner-core mutants ΔwaaF and ΔwaaC lost the ability to produce flagella and curli. Furthermore, these inner-core mutants exhibited a dramatic reduction in adherence to and invasion of epithelial cells (MAC-T), showing an approximately 100-fold decrease in cell invasion compared with the outer-core mutants (waaI and waaG) and the wild type. These findings underscore the critical role of LPS-core truncation in impairing flagella and curli biosynthesis, thereby reducing the invasion capability of E. coli O157 ATCC 43895.

Conditional expression of flagellar motility, curli fimbriae, and biofilms in Shiga toxin- producing Escherichia albertii.

Escherichia albertii is an emerging foodborne pathogen. We previously reported that some avian Shiga toxin-producing E. albertii strains exhibited higher or comparable cytotoxicity in Vero-d2EGFP cells with several enterohemorrhagic E. coli (EHEC) outbreak strains. To better understand the environmental persistence of this pathogen, comparative genomics and phenotypic assays were applied to assess adhesion capability, motility, and biofilm formation in E. albertii. Among the 108 adherence-related genes, those involved in biogenesis of curli fimbriae, hemorrhagic E. coli pilus, type 1 fimbriae, and Sfm fimbriae were conserved in E. albertii. All 20 E. albertii strains carried a complete set of primary flagellar genes that were organized into four gene clusters, while five strains possessed genes related to the secondary flagella, also known as lateral flagella. Compared to EHEC strain EDL933, the eight chemotaxis genes located within the primary flagellar gene clusters were deleted in E. albertii. Additional deletion of motility genes flhABCD and motBC was identified in several E. albertii strains. Swimming motility was detected in three strains when grown in LB medium, however, when grown in 5% TSB or in the pond water-supplemented with 10% pigeon droppings, an additional four strains became motile. Although all E. albertii strains carried curli genes, curli fimbriae were detected only in four, eight, and nine strains following 24, 48, and 120 h incubation, respectively. Type 1 fimbriae were undetectable in any of the strains grown at 37°C or 28°C. Strong biofilms were detected in strains that produced curli fimbriae and in a chicken isolate that was curli fimbriae negative but carried genes encoding adhesive fimbriae K88, a signature of enterotoxigenic E. coli strains causing neonatal diarrhea in piglets. In all phenotypic traits examined, no correlation was revealed between the strains isolated from different sources, or between the strains with and without Shiga toxin genes. The phenotypic variations could not be explained solely by the genetic diversity or the difference in adherence genes repertoire, implying complex regulation in expression of various adhesins. Strains that exhibited a high level of cytotoxicity and were also proficient in biofilm production, may have potential to emerge into high-risk pathogens.

X-ray diffraction recording from a small amount of fibrous protein materials oriented by a micro shear-flow cell.

This paper describes a method for recording X-ray diffraction patterns from a small amount of fibrous protein materials while being oriented by using a micro shear-flow cell. This cell consists of two concentrically arranged glass tubes. The inner tube is stationary, while the outer one rotates at a high speed. The gap between the two tubes is about 100 µm, into which the suspension of fibrous protein materials is injected. By using synchrotron-radiation X-ray microbeams (diameter, 10 µm), clear diffraction images from oriented protein materials can be recorded. The required volume of the sample is only about 10 µl. This method can also be combined with the laser-flash photolysis of caged compounds. Examples of application of this method to the flagella of a green alga Chlamydomonas, and sperm of a tunicate Ciona are presented.

Molecular mechanisms and environmental adaptations of flagellar loss and biofilm growth of Rhodanobacter under environmental stress.

Biofilms aid bacterial adhesion to surfaces via direct and indirect mechanisms, and formation of biofilms is considered as an important strategy for adaptation and survival in suboptimal environmental conditions. However, the molecular underpinnings of biofilm formation in subsurface sediment/groundwater ecosystems where microorganisms often experience fluctuations in nutrient input, pH, and nitrate or metal concentrations are underexplored. We examined biofilm formation under different nutrient, pH, metal, and nitrate regimens of 16 Rhodanobacter strains isolated from subsurface groundwater wells spanning diverse levels of pH (3.5 to 5) and nitrates (13.7 to 146 mM). Eight Rhodanobacter strains demonstrated significant biofilm growth under low pH, suggesting adaptations for survival and growth at low pH. Biofilms were intensified under aluminum stress, particularly in strains possessing fewer genetic traits associated with biofilm formation, findings warranting further investigation. Through random barcode transposon-site sequencing (RB-TnSeq), proteomics, use of specific mutants, and transmission electron microscopy analysis, we discovered flagellar loss under aluminum stress, indicating a potential relationship between motility, metal tolerance, and biofilm growth. Comparative genomic analyses revealed the absence of flagella and chemotaxis genes and the presence of a putative type VI secretion system in the highly biofilm-forming strain FW021-MT20. In this study we identified genetic determinants associated with biofilm growth under metal stress in a predominant environmental genus, Rhodanobacter, and identified traits aiding survival and adaptation to contaminated subsurface environments.

Flagella and Cell Body Staining of Bacteria with Fluorescent Dyes.

Bacteria can propel themselves by rotating a flagellum or a flagellar bundle. To image this thin structure in motile bacteria, the flagella can be vitally stained with fluorophores. This chapter describes a flagellar staining protocol with the additional possibility of visualizing the cell body. It offers the opportunity to track conformational changes of flagella and simultaneously track the positions of the cell bodies. The additional use of a filter increases the number of motile cells and improves the signal-to-noise ratio of images. The flagellar staining requires a prior introduction of a surface-exposed cysteine, which is not covered in this chapter.

Enhancement of astaxanthin accumulation via energy reassignment by removing the flagella of Haematococcus pluvialis.

Astaxanthin biosynthesis in Haematococcus pluvialis is driven by energy. However, the effect of the flagella-mediated energy-consuming movement process on astaxanthin accumulation has not been well studied. In this study, the profiles of astaxanthin and NADPH contents in combination with the photosynthetic parameters with or without flagella enabled by pH shock were characterized. The results demonstrated that there was no significant alteration in cell morphology, with the exception of the loss of flagella observed in the pH shock treatment group. In contrast, the astaxanthin content in the flagella removal groups was 62.9%, 62.8% and 91.1% higher than that of the control at 4, 8 and 12 h, respectively. Simultaneously, the increased Y(II) and decreased Y(NO) suggest that cells lacking the flagellar movement process may allocate more energy towards astaxanthin biosynthesis. This finding was verified by NADPH analysis, which revealed higher levels in flagella removal cells. These results provide preliminary insights into the underlying mechanism of astaxanthin accumulation enabled by energy reassignment in movement-lacking cells.

Flagellar evolution and flagella-independent motility in Actinobacteria.

Actinobacterial species are mostly thought to be nonmotile. Recent studies have revealed the degenerate evolution of flagella in this phylum and different flagellar rod compositions from the classical model. Moreover, flagella-independent motility by various means has been reported in Streptomyces spp. and Mycobacterium spp., but the underlying mechanisms remain elusive.

Flagellar protein FliL: A many-splendored thing.

FliL is a bacterial flagellar protein demonstrated to associate with, and regulate ion flow through, the stator complex in a diverse array of bacterial species. FliL is also implicated in additional functions such as stabilizing the flagellar rod, modulating rotor bias, sensing the surface, and regulating gene expression. How can one protein do so many things? Its location is paramount to understanding its numerous functions. This review will look at the evidence, attempt to resolve some conflicting findings, and offer new thoughts on FliL.

Homozygous ACTL9 mutations cause irregular mitochondrial sheath arrangement and abnormal flagellum assembly in spermatozoa and male infertility.

PURPOSE: To identify novel variants in ACTL9 and new phenotypes responsible for male infertility. METHODS: Genomic DNA was extracted from peripheral blood samples for whole-exome sequencing (WES). Computer-assisted sperm analysis (CASA) was used to test the motility of spermatozoa. The ultrastructure of flagella and the mitochondrial sheath were assessed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Immunostaining was used to validate the localization and expression of ACTL9 and ACTL7A. An Actl9-mutated mouse model was used to validate the phenotypes by CASA and TEM. RESULTS: We identified novel homozygous variants in ACTL9 in two independent Chinese families. Spermatozoa with ACTL9 mutations showed decreased CASA parameters and a higher proportion of spermatozoa with abnormal morphology, exhibiting coiled flagella and a thickened midpiece. The spermatozoa were characterized by chaotic or irregular '9+2' structures and irregular mitochondrial sheath arrangements in the flagellum. Actl9 knock-in mice also showed abnormal CASA parameters and irregular '9+2' structures in flagella. CONCLUSIONS: Our study expands the mutation spectrum and phenotypic spectrum of ACTL9.

Flagellar Motility is Mutagenic.

Flagella are highly complex rotary molecular machines that enable bacteria to not only migrate to optimal environments but to also promote range expansion, competitiveness, virulence, and antibiotic survival. Flagellar motility is an energy-demanding process, where the sum of its production (biosynthesis) and operation (rotation) costs has been estimated to total ~10% of the entire energy budget of an E. coli cell. The acquisition of such a costly adaptation process is expected to secure short-term benefits by increasing competitiveness and survival, as well as long-term evolutionary fitness gains. While the role of flagellar motility in bacterial survival has been widely reported, its direct influence on the rate of evolution remains unclear. We show here that both production and operation costs contribute to elevated mutation frequencies. Our findings suggest that flagellar movement may be an important player in tuning the rate of bacterial evolution.