Micro robot mimicking microorganisms with two flagellar structures

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.

Structure of Straight flagella from a mutant Salmonella

Motility mediated by the flagella of Helicobacter pylori has been shown to be required for normal colonization and is thought to be important for the bacteria to move toward the gastric mucus in niches adjacent to the epithelium. Barnard et al. showed that CsrA appears to be necessary for full motility and the ability to infect mice, but its mechanism of regulation is still unclear. Motility and cell adhesion ability were determined in wild-type, csrA mutant, and revertant J99 strains. The bacterial shape and flagellar structure were evaluated by transmission electron microscopy. The expression of two major flagellins, flaA/flaB, and the alternative sigma factor rpoN (σ(54)) were determined by real-time quantitative RT-PCR and Western blot. The csrA mutant showed loss of motility and lower adhesion ability compared with the wild-type and revertant J99 strains. The csrA mutant was not flagellated. Transcription of flaA and flaB mRNA decreased to only 40% and 16%, respectively, in the csrA mutant compared with the wild-type J99 (p = .006 and <.0001, respectively), and Western blot analysis showed dramatically reduced FlaA/FlaB proteins in a csrA mutant. The disruption of csrA also decreased expression of rpoN to 48% in the csrA mutant, but the degradation rate of rpoN mRNA was not changed. These results suggest that CsrA regulates H. pylori J99 flagella formation and thereby affects bacterial motility.

THERE is a considerable variation in the morphology of the flagella in the lower plant groups and in the flagellates. The flagellar structures have been used as systematic characters since the pioneering work of Petersen1 and Vlk2. The following five types of flagella are recognized: (1) One-sided flimmer flagella which have one row of hair-like appendages (called flimmer hairs or mastigonemata or, somewhat misleadingly, cilia). The flimmer hairs are about 3µ long, and the row extends along the entire length of the flagellum on its one side. (2) Two-sided flimmer flagella which have two rows of flimmer hairs along the entire length of the flagellum. (3) Whip flimmer flagella which have a demarcated thin end region devoid of flimmer hairs and a main region which possesses two rows of flimmer hairs. The flimmer hairs stand perpendicular to the flagellar axis. (4) Whip flagella which are devoid of flimmer hairs but have a demarcated thin end region. These flagella are also called lash flagella. (5) Flagella which lack flimmer hairs and also lack a demarcated end region visible in the light microscope.

In the study, a comprehensive investigation on potential bacterial pathogens affecting largemouth bass (Micropterus salmoides) was performed. Monthly surveys were conducted from April to October 2024. Diseased largemouth bass exhibited diverse clinical symptoms, such as rot of gill and fin, ulcers on body surface, and petechial hemorrhages in liver. Following isolation and identification, a total of 21 potential bacterial pathogens (numbered strain 1 to 21, respectively) were identified. The genus Aeromonas had the highest proportion (67.14%), among which the frequency of Aeromonas veronii was 24.60%. TEM analysis revealed that the bacterial strains exhibited three predominant shapes (rod-shaped, spherical, and curved) with length ranging from 0.5 to 3 μm. Flagellar structures were observed in strains 1–4, 6–8, 11–17, and 19–21, with variations in number and growth sites. Three isolates (strains 9, 10, 18) demonstrated Gram-positive characteristic, and strains 5, 11, and 18 have capsule structures. Strains 5, 9, 10, and 18 were non-motile, and strains 1–4, 6, 7, 9–11, 16–18, and 21 exhibited β-hemolysis. Physiological and biochemical characteristics of the 21 bacterial isolates were comprehensively analyzed. Antibiotic sensitivity testing revealed that florfenicol and enrofloxacin exhibited excellent antibacterial effects. These data will enrich the potential bacterial diseases information and promote the healthy development of the largemouth bass industry.

The Gram-negative bacterial species Pectobacterium actinidiae causes summer canker in kiwifruit plants. However, little is known about its virulence factors and mechanisms of genetic adaptation. We aimed to identify the key determinants that control the virulence of P. actinidiae in kiwifruit by genomic and functional analyses. Analysis of four P. actinidiae isolates indicated low genetic variability with an average of 98.7% genome-level sequence similarity and 82% shared protein-coding gene content. Phylogenetic analysis, based on both bulk single nucleotide polymorphisms (SNPs) and single-copy genes, revealed that P. actinidiae strains cluster into a single clade, which is closely related to the clades of P. odoriferum (species with a completely different host range). Through comparison between these two clades of strains, 746 unique core orthologs/genes were clustered in the clades of P. actinidiae, especially key virulence determinants involved in the biosynthesis of secretion systems (type III, IV, and VI), iron, flagellar structure, and the quorum-sensing system. Our results provide insights into the pathogenomics underlying the genetic diversification and evolution of pathogenicity in P. actinidiae species.

C. reinhardtii sheds its flagella in response to acidification. Previously, we showed correlations between pH shock, deflagellation, and inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] production, but 100% of cells deflagellated by 5 s, which was the earliest that Ins(1,4,5)P3 accumulation could be accurately measured by techniques available to us at that time (Quarmby, L. M., Y. G. Yueh, J. L. Cheshire, L. R. Keller, W. J. Snell, and R. C. Crain. J. Cell Biol. 1992. 116:737-744). To learn about the causal relationship between Ins(1,4,5)P3 accumulation and deflagellation, we extended these studies to early times using a continuous-flow rapid-quench device. Within 1 s of acidification to pH 4.3-4.5, 100% of cells deflagellated. A transient peak of Ins(1,4,5)P3 was observed 250-350 ms after pH shock, preceding deflagellation. Preincubation with 10 microM neomycin, which prevents hydrolysis of phosphatidylinositol 4,5-bisphosphate, inhibited both the transient production of Ins(1,4,5)P3 and the subsequent deflagellation. The nonspecific Ca2+ channel blockers La3+ and Cd2+ prevented flagellar excision induced by mastoparan without inhibiting rapid Ins(1,4,5)P3 production. Likewise, the Ins(1,4,5)P3-gated channel inhibitors ruthenium red and heparin blocked deflagellation in response to mastoparan. These studies were extended to mutants defective in flagellar excision. Fa-1, a mutant defective in flagellar structure, produced Ins(1,4,5)P3 but failed to deflagellate. These results support a model in which acid pH activates a putative cellular receptor leading to G-protein dependent activation of phospholipase C and accumulation of Ins(1,4,5)P3. These events are upstream of Ins(1,4,5)P3-dependent Ca2+ entry from the medium, and of deflagellation.

Emergent flagella of Euglenozoa consist of two prominent structural elements: the axoneme built by microtubules with motor proteins to enable the movement of the flagellum and a highly organized protein structure of unknown function, called the paraxonemal rod (PAR), which consists of two major proteins paralleling the axoneme of euglenid and kinetoplastid emergent flagella. These flagellar structures are considered apomorphic characters of Euglenozoa. We examined the evolution of the genes par1 and par2 encoding the two major proteins, where we could show that these proteins are encoded by two very similar genes found in kinetoplastids and euglenids. The branching pattern indicated a gene duplication before the diversification into euglenids and kinetoplastids. In the clades of the genes, subtrees of euglenid and kinetoplastid monophyla arose. Both genes showed strong genetic diversity with biased GC content at taxon rather than at gene level. We also examined phylogenies inferred from PAR genes that are well in agreement with established small subunit rDNA analyses. Both showed further separation of the euglenid subtree into primary osmotrophs and a phototrophic clade, including secondarily derived osmotrophs.

A number of extracellular helical protein polymers are crucial for supporting bacterial motility. The bacterial flagellum is a polymeric appendage used to support cellular motility. Historically, structural studies of flagellar and other filaments were limited to those present as or locked into straightened states. Here, we present a robust workflow that produces biologically relevant high-resolution cryo-electron microscopy (cryo-EM) structures of bacterial flagellar filaments. We highlight how a simple purification method, centered around several centrifugation steps, exploits the process of filament ejection in Caulobacter crescentus and results in isolated filaments amenable to transmission electron microscopy (TEM) studies. The quality of the sample is validated by SDS-PAGE and negative stain TEM analysis before a sample is vitrified for cryogenic electron microscopy (cryo-EM) data collection. We provide a detailed protocol for reconstructing either straight or curved flagellar filaments by cryo-EM helical reconstruction methods, followed by an overview of model building and validation. In our hands, this workflow resulted in several flagellar structures below 3 Å resolution, with one data set reaching a global resolution of 2.1 Å. The application of this workflow supports structure-function studies to better understand the molecular interactions that regulate filament architecture in biologically relevant states. Future work will not only examine interactions that regulate bacterial flagellar and other filament organization but also provide a foundation for developing new helical biopolymers for biotech applications.Key features• Rapid high-quality purification of bacterial flagella via simple bacterial culturing, centrifugation, and resuspension methods.• High-throughput cryo-EM data collection of filamentous objects.• Use of cryoSPARC implementations of helical reconstruction algorithms to generate high-resolution 3D structures of bacterial flagella or other helical polymers.

Escherichia coli mutants with defects in 29 flagellar genes identified so far were examined by electron microscopy for possession of incomplete flagellar structures in membrane-associated fractions. The results are discussed in consideration of the known transcriptional interaction of flagellar genes. Hook-basal body structures were detected in flaD, flaS, flaT, flbC, and hag mutants. The flaE mutant had a polyhook-basal body structure. An intact basal body appeared in flaK mutants. Putative precursors of the basal body were detected in mutants with defects in flaM, flaU, flaV, and flaY. No structures homologous to the flagellar basal body or its parts were detected in mutants with defects in flaA, flaB, flaC, flaG, flaH, flaI, flaL, flaN, flaO, flaP, flaQ, flaR, flaW, flaX, flbA, flbB, and flbD. One flaZ mutant had an incomplete flagellar basal body structure and another formed no significant structure, suggesting that flaZ is responsible for both basal body assembly and the transcription of the hag gene.

Molecular Evaluation of Impacted Reproductive Physiology in Fathead Minnow Testes Provides Mechanistic Insights into Insensitive Munitions Toxicology

BackgroundPrimary Ciliary Dyskinesia (PCD) is a genetically heterogeneous ciliopathy caused by ultrastructural defects in ciliary or flagellar structure and is characterized by a number of clinical symptoms including recurrent respiratory infections progressing to permanent lung damage and infertility.Case presentationHere we describe our search to delineate the molecular basis in two affected sisters with clinically diagnosed PCD from a consanguineous Saudi Arabian family, in which all known genes have been excluded. A homozygosity mapping-based approach was utilized that ultimately identified one single affected-shared region of homozygosity using 10 additional unaffected family members. A plausible candidate gene was directly sequenced and analyzed for mutations. A novel homozygous missense aberration (p.Lys1154Gln) was identified in both sisters in the DNAH1 gene that segregated completely with the disease phenotype. Further confirmation of this interesting variant was provided by exome-wide analysis in the proband.ConclusionMolecular variation in DNAH1 may play a role in PCD and its potential contribution should be considered in patients where all known genes are excluded.Electronic supplementary materialThe online version of this article (doi:10.1186/s12881-015-0162-5) contains supplementary material, which is available to authorized users.

The phylogenetic relation and taxomomic sta tus between genusChloromonas and genusChlamydomonas are uncertain in phycological field, since that the microstructure of them are much similar and bo th are unice llar groups with two equal-lengthed flagellas, the difference is just in that, theChlamydomonas species have more than one pyrenoid on the chloroplast whi le theChloromonas species have none ever since being reported. Therefore, as to the unicellar flagellated groups, the character of having a pyrenoid or not is of great significance in taxonomy. Whereas, many scholars have doubted the s tability of this character of not having pyrenoid and that is why the argument is still in pendency for long years. The recent research forChloromonas species are mostly carried out on micros copic levels, but not in the fields of ultrastructure, life history and molecula r phylogenetics. In this article we have compared the ultrastructure and microst ructure of a newly reported alga ofChloromonas sinica by H. J. Hu et C. T. Yin (2002) to that ofChlamydomonas species, finding that they are in highly similarity except for thatChloromonas sinica does have no pyrenoid in its whole life history, which starts from the period of spores. The truth is proved by the ultrastructure evidence. What’s more, we have also study the Chloromonas sinica by its flagellar structure, character of cell wall and chloroplast fi gures, knowing that the pattern of fibrovascular structure in the flagella trans ition region ofChloromonas sinica is much the same with that ofChlamydom onas species, and it is also the same for the young cell wall character in Chloromonas sinica, being compared to that of sporangium wall in theChlamyd o monas species. And there are some special differed characters betweenChloro monas sinica according to otherChloromonas species in the observation: 1) it has no colour and leads a heterotrophic life while cultured without lights s upply. Only under illumination can the chloroplast develop normally and the cells turn green with bigger irregular discoid chloroplasts, while the colorless cells are having smaller round ones in contrast, which still can be seen clearly in the microscopy. Besides these, the thylakoids are very rear and short in the co lourless cells. While in mature cells of normal condition the thylakoids are full of the whole chloroplast, which also possess 1 or more starch grains. 2) the c olourless cell wall is thicker than normal, being of 3 layers; the green ones’ c ell wall is very thin and have less layers or even just have one layer. However, this difference in cell wall being connected to its nourish pattern or not need s to go deep into in the future. Though molecular phylogenetics research of genusChloromonas and genusChl amydomonas shows that they are polyphylogentic, plenty studies are needed to do to confirm if Chloromonas is a subgenus ofChlamydomonas, or a genus of Chlamydomonas. And now before getting enough evidence, it is more reasonab le to take theChloromonas as a seperate genus from the genus Chlamydomonas. &amp;nbsp;

This chapter presents selected examples of how genetic approaches have been used to identify Salmonella virulence factors. The genetic approaches are based on five distinct concepts and assumptions about what might characterize Salmonella virulence genes. The identification of Salmonella-specific genes is based on the potential relationship between a pathogen-specific gene and the pathogenic phenotype. The biochemical and molecular functions of the spv gene products have remained elusive, but current speculations suggest that they may be involved in bacterial modulation of host immune cells. In summary, the comparison of Salmonella strains with nonpathogenic strains or species has been a very useful approach to the identification of virulence genes. In many cases, the subsequent identification and mutagenesis of bacterial genes involved in putative Salmonella virulence phenotypes have led to the identification of virulence genes. An alternative and more general approach has been to study in vitro systems in which Salmonella species are able to survive interaction with primary or cultured macrophages. Examination of certain noninvasive mutants in animal infection models provides strong support for the importance of bacterial entry into intestinal cells. Bacterial binding and utilization of the enterochelin-iron complex is facilitated by Chr-TonB, an outer membrane receptor. Bacterial motility and chemotaxis require 50 different genes which encode chemical sensors, physical motors, directional switches, and components of the flagellar structure, as well as specific factors which export and assemble the flagellar structure. In fact, it has been speculated that Salmonella motility and chemotaxis may be important for virulence.

Flagellar axonemes isolated from sea urchin sperm were digested with trypsin for various time periods. The course of digestion was monitored turbidimetrically and was found to take two different courses depending on the presence or absence of ATP in the digestion mixture. It was found that ATP induced active disintegration of the axonemes after slight digestion. Samples of the digested axonemes were examined with the electron microscope to determine the effects of trypsin digestion on the substructures of the axonemes. The rate at which trypsin sensitized the axonemes to ATP paralleled the rate at which it damaged the radial spokes and the nexin links, while the dynein arms were removed much more slowly. The results suggest that inactive dynein arms form cross bridges between the adjacent doublet tubules in digested axonemes, and that when activated by the addition of ATP, they induce an active shearing force between adjacent doublets. The radial spokes and the nexin links are not directly involved in the production of mechanical force, but they may participate in regulating the sliding between tubules to produce a propagated bending wave.