Multiple Polar Flagella Research Articles

Control of the flagellation pattern in Helicobacter pylori by FlhF and FlhG.

FlhF and FlhG control the location and number of flagella, respectively, in many polar-flagellated bacteria. The roles of FlhF and FlhG are not well characterized in bacteria that have multiple polar flagella, such as Helicobacter pylori. Deleting flhG in H. pylori shifted the flagellation pattern where most cells had approximately four flagella to a wider and more even distribution in flagellar number. As reported in other bacteria, deleting flhF in H. pylori resulted in reduced motility, hypoflagellation, and the improper localization of flagella to nonpolar sites. Motile variants of H. pylori ∆flhF mutants that had a higher proportion of flagella localizing correctly to the cell pole were isolated, but we were unable to identify the genetic determinants responsible for the increased localization of flagella to the cell pole. One motile variant though produced more flagella than the ΔflhF parental strain, which apparently resulted from a missense mutation in fliF (encodes the MS ring protein), which changed Asn-255 to aspartate. Recombinant FliFN255D, but not recombinant wild-type FliF, formed ordered ring-like assemblies in vitro that were ~50 nm wide and displayed the MS ring architecture. We infer from these findings that the FliFN225D variant forms the MS ring more effectively in vivo in the absence of FlhF than wild-type FliF. IMPORTANCE Helicobacter pylori colonizes the human stomach where it can cause a variety of diseases, including peptic ulcer disease and gastric cancer. H. pylori uses flagella for motility, which is required for host colonization. FlhG and FlhF control the flagellation patterns in many bacteria. We found that in H. pylori, FlhG ensures that cells have approximately equal number of flagella and FlhF is needed for flagellum assembly and localization. FlhF is proposed to facilitate the assembly of FliF into the MS ring, which is one of the earliest structures formed in flagellum assembly. We identified a FliF variant that assembles the MS ring in the absence of FlhF, which supports the proposed role of FlhF in facilitating MS ring assembly.

Taxonomic Description and Complete Genome Sequencing of Pseudomonas silvicola sp. nov. Isolated from Cunninghamia laceolata

The Pseudomonas strain T1-3-2T isolated from the cone of Cunninghamia laceolata exhibited growth-promoting and antifungal activity. Strain T1-3-2T was characterized by a polyphasic taxonomy and complete genome sequencing analysis to explore its taxonomic position and biocontrol potentials fully. The results revealed that strain T1-3-2T shares a high degree of similarity with Pseudomonas eucalypticola and is distinct from any known Pseudomonas species. The G + C content was 61.65%, and the difference was greater than 1 compared to “P. eucalypticola”. Additionally, values of the average nucleotide identity blast (ANIb), average nucleotide identity MUMmer (ANIm), and DNA-DNA hybridization (DDH) between T1-3-2T and its closest known related species, “P. eucalypticola”, were below the thresholds necessary for species delineation. Furthermore, the T1-3-2T strain exhibited the distinctions with the multiple polar flagella and the specific quinone system with MK8 compared with that of “P. eucalypticola”. Collectively, these findings affirm the designation of strain T1-3-2T as a new Pseudomonas species, proposed to be named Pseudomonas silvicola, with T1-3-2T as the type strain. Genomic analyses revealed strain T1-3-2T contains three circular DNA contigs, including a 7,613,303 bp chromosome and two plasmids (952,764 bp and 84,880 bp). Bioinformatics analyses further offered potential insight into the molecular mechanisms whereby this strain can promote plant growth and control disease, revealing encoded genes related to antibiotic and secondary metabolite production, the uptake and biosynthesis of siderophores, and pyoverdine biosynthesis. These genomic data offer a valuable foundation for future efforts to apply the T1-3-2T strain in research contexts.

Pseudomonas fildesensis sp. nov., a psychrotolerant bacterium isolated from Antarctic soil of King George Island, South Shetland Islands.

The strain KG01T was isolated from a soil sample from King George Island, Antarctica. Cells of KG01T are rod-shaped and motile by means of multiple polar flagella. The absence of arginine dihydrolase activity could be a key feature to readily distinguish KG01T from its closest phylogenetic relative species. The main fatty acids of the strain include summed feature 3 (C16 : 1 ω7c and/or C15 : 0 iso 2-OH), C16 : 0 and C18 : 1 ω7c. Phylogenetic analysis based on the 16S rRNA gene sequence and on a multilocus sequence analysis (MLSA) using housekeeping genes (16S rRNA, rpoB, rpoD, gyrB) were carried out. These analyses allowed us to include the strain within the Pseudomonas fluorescens group, presenting the highest similarity of multilocus sequence with Pseudomonas veronii LMG 17761T (96.67 %). The genome of KG01T was sequenced and in silico compared with genomes of the most closely related species of the P. fluorescens group. The average nucleotide identity (ANIb) and average amino acid identity (AAI) values of the species phylogenetically closest to KG01T were less than 95-96 %, threshold currently accepted to define strain as belonging to a bacterial species, the highest scores being those to Pseudomonas veronii LMG 17761T (87.98 %) and Pseudomonas marginalis ICMP 3553T (91.90 %). Therefore, the phenotypic and genotypic analyses results, allow us to propose that KG01T represents a member of a novel species of the genus Pseudomonas, for which the name Pseudomonas fildesensis is proposed, and KG01T (=CECT 9084T;=DSM 102036T) is established as the type strain .

Unusual extracellular appendages deployed by the model strain Pseudomonas fluorescens C7R12.

Pseudomonas fluorescens is considered to be a typical plant-associated saprophytic bacterium with no pathogenic potential. Indeed, some P. fluorescens strains are well-known rhizobacteria that promote plant growth by direct stimulation, by preventing the deleterious effects of pathogens, or both. Pseudomonas fluorescens C7R12 is a rhizosphere-competent strain that is effective as a biocontrol agent and promotes plant growth and arbuscular mycorrhization. This strain has been studied in detail, but no visual evidence has ever been obtained for extracellular structures potentially involved in its remarkable fitness and biocontrol performances. On transmission electron microscopy of negatively stained C7R12 cells, we observed the following appendages: multiple polar flagella, an inducible putative type three secretion system typical of phytopathogenic Pseudomonas syringae strains and densely bundled fimbria-like appendages forming a broad fractal-like dendritic network around single cells and microcolonies. The deployment of one or other of these elements on the bacterial surface depends on the composition and affinity for the water of the microenvironment. The existence, within this single strain, of machineries known to be involved in motility, chemotaxis, hypersensitive response, cellular adhesion and biofilm formation, may partly explain the strong interactions of strain C7R12 with plants and associated microflora in addition to the type three secretion system previously shown to be implied in mycorrhizae promotion.

First Report of Bacterial Leaf Spot of Cucumber Caused by Pseudomonas cichorii in China

Cucumber (Cucumis sativus L.) is a globally important vegetable. From September to May every year, more than 600 groups from 29 provinces of China conduct experiments in crop breeding and seed reproduction in Hainan Province. A new cucumber bacterial leaf spot disease was discovered in 2015, during a survey in a cucumber breeding facility in Sanya, Hainan province. The disease incidence reached 90% in severely infected fields since 2015. Initial symptoms on cucumber leaves are water soaking and light green lesions. Leaves are thinner. Lesion margins gradually turn yellow, and the lesion centers turn brown. The range of irregular lesions is 1.5 to 2.0 cm long and 1 to 1.5 cm wide. Severely affected leaves show chlorotic lesions, which coalesce to produce broad necrotic areas. More than 120 bacterial isolates with consistent appearance were isolated from symptomatic cucumber leaf samples using nutrient agar (NA), and three isolates were selected for further characterization. Physiological and morphological characteristics of the isolated bacteria were determined using standard microbiological techniques (Schaad et al. 2001). Colonies are round, pale yellow, smooth, with undulate margins. Bacterial cells were gram negative, with multiple polar flagella, positive for catalase and oxidase, produced fluorescent blue color under ultraviolet light on King’s medium B agar, and metabolized glucose and fructose. Negative results were produced for growth at 41°C, gelatin hydrolysis, fructosan production, arginine dihydrolase presence, Tween 80 hydrolysis, use of trehalose, and sorbitol. Based on these morphological and biochemical characteristics, the pathogen was identified as Pseudomonas cichorii Stapp (Bergey et al. 1994). The bacterial identity was further confirmed by amplifying the 16S rRNA gene using primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′) and the rpoB gene using primers LAPS/LAPS27 (Ait Tayeb et al. 2005). The sequences were deposited in GenBank (accession nos. MF979525.1 [16S rRNA] and MF979524.1 [rpoB]) and exhibited a 99% identity with P. cichorii (accession nos. KU923374.1 and JQ994485.1). A pathogenicity assay was carried out using potted, greenhouse-grown cucumber plants. The inoculum was grown in NA for 24 h at 28°C and then was suspended in distilled water. Leaves (three replications, cv. Yansi) were sanitized in 0.1% mercuric chloride for 30 s, washed and rinsed with sterile distilled water, and then sprayed with a bacterial suspension (5.0 × 10⁸ CFU/ml). Distilled water was used as negative control. Inoculated and control plants were incubated at 90 to 100% relative humidity at 25°C. Water-soaked, thin, and light-green lesions appeared after 24 h, and enlarged lesions with yellow halos were observed after 48 h. The lesion margins gradually turned yellow, and the lesion centers turned brown after 5 days. These symptoms were not observed on control plants. The pathogenicity test was repeated three times. The same bacteria were consistently reisolated. Thus, the Koch’s postulates were fulfilled. To our knowledge, this is the first isolation of cucumber leaf spot caused by P. cichorii in China.

Structural Characterization of the Fla2 Flagellum of Rhodobacter sphaeroides.

Rhodobacter sphaeroides is a free-living alphaproteobacterium that contains two clusters of functional flagellar genes in its genome: one acquired by horizontal gene transfer (fla1) and one that is endogenous (fla2). We have shown that the Fla2 system is normally quiescent and under certain conditions produces polar flagella, while the Fla1 system is always active and produces a single flagellum at a nonpolar position. In this work we purified and characterized the structure and analyzed the composition of the Fla2 flagellum. The number of polar filaments per cell is 4.6 on average. By comparison with the Fla1 flagellum, the prominent features of the ultra structure of the Fla2 HBB are the absence of an H ring, thick and long hooks, and a smoother zone at the hook-filament junction. The Fla2 helical filaments have a pitch of 2.64 μm and a diameter of 1.4 μm, which are smaller than those of the Fla1 filaments. Fla2 filaments undergo polymorphic transitions in vitro and showed two polymorphs: curly (right-handed) and coiled. However, in vivo in free-swimming cells, we observed only a bundle of filaments, which should probably be left-handed. Together, our results indicate that Fla2 cell produces multiple right-handed polar flagella, which are not conventional but exceptional. R. sphaeroides possesses two functional sets of flagellar genes. The fla1 genes are normally expressed in the laboratory and were acquired by horizontal transfer. The fla2 genes are endogenous and are expressed in a Fla1(-) mutant grown phototrophically and in the absence of organic acids. The Fla1 system produces a single lateral or subpolar flagellum, and the Fla2 system produces multiple polar flagella. The two kinds of flagella are never expressed simultaneously, and both are used for swimming in liquid media. The two sets of genes are certainly ready for responding to specific environmental conditions. The characterization of the Fla2 system will help us to understand its role in the physiology of this microorganism.

Quorum Sensing Controls Flagellar Morphogenesis in Burkholderia glumae

Burkholderia glumae is a motile plant pathogenic bacterium that has multiple polar flagella and one LuxR/LuxI-type quorum sensing (QS) system, TofR/TofI. A QS-dependent transcriptional regulator, QsmR, activates flagellar master regulator flhDC genes. FlhDC subsequently activates flagellar gene expression in B. glumae at 37°C. Here, we confirm that the interplay between QS and temperature is critical for normal polar flagellar morphogenesis in B. glumae. In the wild-type bacterium, flagellar gene expression and flagellar number were greater at 28°C compared to 37°C. The QS-dependent flhC gene was significantly expressed at 28°C in two QS-defective (tofI::Ω and qsmR::Ω) mutants. Thus, flagella were present in both tofI::Ω and qsmR::Ω mutants at 28°C, but were absent at 37°C. Most tofI::Ω and qsmR::Ω mutant cells possessed polar or nonpolar flagella at 28°C. Nonpolarly flagellated cells processing flagella around cell surface of both tofI::Ω and qsmR::Ω mutants exhibited tumbling and spinning movements. The flhF gene encoding GTPase involved in regulating the correct placement of flagella in other bacteria was expressed in QS mutants in a FlhDC-dependent manner at 28°C. However, FlhF was mislocalized in QS mutants, and was associated with nonpolar flagellar formation in QS mutants at 28°C. These results indicate that QS-independent expression of flagellar genes at 28°C allows flagellar biogenesis, but is not sufficient for normal polar flagellar morphogenesis in B. glumae. Our findings demonstrate that QS functions together with temperature to control flagellar morphogenesis in B. glumae.

English

This study was done at Christian Medical College and Hospital, Ludhiana for the period of 5 years from January 2006 to December 2010 in the Department of Microbiology. A total of 306 endoscopic gastric biopsy specimens were collected from the suspected cases of the peptic ulcer and were subjected to rapid urease test for Helicobacter pylori. This test has high sensitivity and cent percent specificity. The prevalence was determined to be 09.48% and male: female ratio was determined to be 68.97:31.03. INTRODUCTION: Helicobacter pylori is a common cause of peptic ulcer. It is gram negative, microaerophilic and motile organism with multiple polar flagella. It associated with 90% of duodenal ulcer and 80% of gastric ulcer¹.H.pylori is also associated with gastric adenocarcinoma. H. pylori infection is mainly acquired through the faeco oral route. Water has been shown to be a source of H. pylori infection by Kleinet al².A study from Bombay reported that H. pylori is present in the dental plaques of 98% patients with dyspepsia³.

Conversion of mono-polar to peritrichous flagellation in Vibrio alginolyticus

Precise regulation of the number and positioning of flagella are critical in order for the mono-polar-flagellated bacterium Vibrio alginolyticus to swim efficiently. It has been shown that, in V. alginolyticus cells, the putative GTPase FlhF determines the polar location and production of flagella, while the putative ATPase FlhG interacts with FlhF, preventing it from localizing at the pole, and thus negatively regulating the flagellar number. In fact, no ΔflhF cells have flagella, while a very small fraction of ΔflhFG cells possess peritrichous flagella. In this study, the mutants that suppress inhibition of the swarming ability of ΔflhFG cells were isolated. The mutation induced an increase in the flagellar number and, furthermore, most Vibrio cells appeared to have peritrichous flagella. The sequence of the flagella related genes was successfully determined, however, the location of the suppressor mutation could not been found. When the flhF gene was introduced into the suppressor mutant, multiple polar flagella were generated in addition to peritrichous flagella. On the other hand, introduction of the flhG gene resulted in the loss of most flagella. These results suggest that the role of FlhF is bypassed through a suppressor mutation which is not related to the flagellar genes.

Pseudomonas arsenicoxydans sp nov., an arsenite-oxidizing strain isolated from the Atacama desert

A Gram-negative, arsenite-oxidizing bacterial strain, designated VC-1, was isolated from sediment samples from the Camarones Valley in the Atacama Desert, Chile. Strain VC-1 was strictly aerobic, oxidase and catalase positive, rod shaped, of about 5.5 μm in length and 0.5–1.0 μm in diameter. It was motile by means of multiple polar flagella. The phylogenetic reconstruction of the 16S rRNA gene sequence, an MLSA study by concatenating six genes, and DDH studies indicated that the strain differed genotypically from its closest relatives and was therefore recognized as a new species within the genus Pseudomonas. Phenotypic analysis combining metabolic tests, fatty acid profiles and MALDI-TOF profiles of total cell extracts supported the classification of the new species for which we propose the designation Pseudomonas arsenicoxydans sp. nov. The type strain is accessible under the culture collection numbers CCUG 58201 T and CECT 7543 T.

Sulfurihydrogenibium rodmanii sp. nov., a sulfur-oxidizing chemolithoautotroph from the Uzon Caldera, Kamchatka Peninsula, Russia, and emended description of the genus Sulfurihydrogenibium

Four thermophilic, sulfur-oxidizing, chemolithoautotrophic strains with >99 % 16S rRNA gene sequence similarity were isolated from terrestrial hot springs in the Geyser Valley and the Uzon Caldera, Kamchatka, Russia. One strain, designated UZ3-5T, was characterized fully. Cells of UZ3-5T were Gram-negative, motile, slightly oval rods (about 0.7 microm wide and 1.0 microm long) with multiple polar flagella. All four strains were obligately microaerophilic chemolithoautotrophs and could use elemental sulfur or thiosulfate as electron donors and oxygen (1-14 %, v/v) as the electron acceptor. Strain UZ3-5T grew at temperatures between 55 and 80 degrees C (optimally at 75 degrees C; 1.1 h doubling time), at pH 5.0-7.2 (optimally at pH 6.0-6.3) and at 0-0.9 % NaCl (optimally in the absence of NaCl). The G+C content of the genomic DNA of strain UZ3-5T was 35 mol%. Phylogenetic analysis revealed that strain UZ3-5T was a member of the genus Sulfurihydrogenibium, its closest relative in culture being Sulfurihydrogenibium azorense Az-Fu1T (98.3 % 16S rRNA gene sequence similarity). On the basis of its physiological and molecular characteristics, strain UZ3-5T represents a novel species of the genus Sulfurihydrogenibium, for which the name Sulfurihydrogenibium rodmanii sp. nov. is proposed. The type strain is UZ3-5T (=OCM 900T =ATCC BAA-1536T =DSM 19533T).

Chemotactic Control of the Two Flagellar Systems of Rhodobacter sphaeroides Is Mediated by Different Sets of CheY and FliM Proteins

Rhodobacter sphaeroides expresses two different flagellar systems, a subpolar flagellum (fla1) and multiple polar flagella (fla2). These structures are encoded by different sets of flagellar genes. The chemotactic control of the subpolar flagellum (fla1) is mediated by three of the six different CheY proteins (CheY6, CheY4, or CheY3). We show evidence that CheY1, CheY2, and CheY5 control the chemotactic behavior mediated by fla2 flagella and that RSP6099 encodes the fla2 FliM protein.

Regulation of Polar Flagellar Number by the flhF and flhG Genes in Vibrio alginolyticus

The number and location of bacterial flagella vary with the species. The Vibrio alginolyticus cell has a single polar flagellum, which is driven by sodium ions. We selected mutants on the basis of reduced swarming ability on soft agar plates. Among them, we found two mutants with multiple polar flagella, and named them KK148 and NMB155. In Pseudomonas species, it is known that FlhF and FleN, which are FtsY and MinD homologs, respectively, are involved in regulation of flagellar placement and number, respectively. We cloned homologous genes of V. alginolyticus, flhF and flhG. KK148 cells had a nonsense mutation in flhG; cells expressing transgenic flhG recovered the swarming ability and had a reduced number of polar flagella. NMB155 cells did not have a mutation in either flhF or flhG. In wild-type cells, expression of flhF increased the number of polar flagella; in contrast, expression of flhG reduced both the number of polar flagella and the swarming ability. These results suggest that FlhG negatively regulates the number of polar flagella in V. alginolyticus. KK148 cells expressing both flhF and flhG exhibited fewer polar flagella and better swarming ability than KK148 cells expressing flhG alone, suggesting that FlhG acts with FlhF.

Palaeococcus helgesonii sp. nov., a facultatively anaerobic, hyperthermophilic archaeon from a geothermal well on Vulcano Island, Italy.

A novel, hyperthermophilic archaeon was isolated from a shallow geothermal well that taps marine waters on the Island of Vulcano in the southern Tyrrhenian Sea, Italy. The cells were irregular cocci, 0.6-1.5 microm in diameter, with multiple polar flagella. Growth was observed at temperatures from 45 to 85 degrees C (optimum at approximately 80 degrees C), pH 5-8 (optimum at 6.5), and 0.5-6.0% NaCl (optimum at approximately 2.8%). The minimum doubling time was 50 min. The isolate was obligately chemoheterotrophic, utilizing complex organic compounds including yeast or beef extract, peptone, tryptone, or casein for best growth. The presence of elemental sulfur enhanced growth. The isolate grew anaerobically as well as microaerobically. The G+C content of the genomic DNA was 42.5 mol%. The 16S rRNA sequence indicated that the new isolate was a member of the Thermococcales within the euryarchaeota, representing the second species in the genus Palaeococcus. Its physiology and phylogeny differed in several key characteristics from those of Palaeococcus ferrophilus, justifying the establishment of a new species; the name Palaeococcus helgesonii sp. nov. is proposed, type strain PI1 (DSM 15127).

Palaeococcus ferrophilus gen. nov., sp. nov., a barophilic, hyperthermophilic archaeon from a deep-sea hydrothermal vent chimney.

A novel barophilic, hyperthermophilic archaeon was isolated from a deep-sea hydrothermal vent chimney at the Myojin Knoll in the Ogasawara-Bonin Arc, Japan. The cells were found to be irregular cocci and motile with multiple polar flagella. Growth was observed between 60 and 88 degrees C (opt. 83 degrees C; 30 min doubling time), pH 4.0 and 8.0 (opt. pH 6.0), 20 and 73 g sea salts l-1 (opt. 47 g l-1) and 0.1 and 60 MPa (opt. 30 MPa). The isolate was a strictly anaerobic chemoorganotroph capable of utilizing proteinaceous substrates such as yeast extract, peptone, tryptone and casein in the presence of elemental sulfur or ferrous iron. The G + C content of the genomic DNA was 53.5 mol%. Phylogenetic analysis based on 16S rDNA sequences indicated that the isolate was a member of an ancient lineage of the Thermococcales that diverged prior to the formation of the two genera Thermococcus and Pyrococcus. On the basis of the physiological and molecular properties of the new isolate, the name Palaeococcus ferrophilus gen. nov., sp. nov. is proposed. The type strain is strain DMJT (= JCM 10417) [corrected].

The relationship between Helicobacter pylori motility, morphology and phase of growth: implications for gastric colonization and pathology.

To explore the relationship between Helicobacter pylori motility, morphology and phase of growth, bacteria were isolated from antral biopsies of patients with duodenal ulcer or non-ulcer dyspepsia, and grown in liquid medium in batch and continuous culture systems. Motilities and morphologies of H. pylori in different phases of growth were examined with a Hobson BackTracker and by transmission electron microscopy. Morphologies of bacteria grown in vitro were also compared with those of bacteria in antral biopsies from patients with non-autoimmune gastritis. H. pylori had poor motility in lag phase, became highly motile in mid-exponential phase and lost motility in the decline phase of growth. Motilities of bacteria in the same phase of growth from patients with duodenal ulcer or non-ulcer dyspepsia were not significantly different. In the mid/late-exponential phase of growth bacteria had helical morphologies and multiple polar flagella, typical of H. pylori in the gastric mucus layer. In the decline phase of growth bacteria shed flagella, and had precoccoidal or coccoidal morphologies. These findings support the view that helical and coccoidal H. pylori are in different phases of growth with different roles in gastric colonization, indicate that bacterial motility per se is unlikely to be a determinant of H. pylori pathology, and suggest that H. pylori in the antral mucus layer is in a state of continuous (exponential phase) growth.

Cleavage of dimethylsulfoniopropionate and reduction of acrylate by Desulfovibrio acrylicus sp. nov.

From anoxic intertidal sediment, a dimethylsulfoniopropionate (DMSP)-cleaving anaerobe (strain W218) was isolated that reduced the acrylate formed to propionate. The bacterium was vibrio- to rod-shaped and motile by means of multiple polar flagella. It reduced sulfate, thiosulfate, and acrylate, and used lactate, fumarate, succinate, malate, pyruvate, ethanol, propanol, glycerol, glycine, serine, alanine, cysteine, hydrogen, and formate as electron donors. Sulfate and acrylate were reduced simultaneously; growth with sulfate was faster than with acrylate. Extracts of cells grown in the presence of DMSP contained high DMSP lyase activities (9.8 U/mg protein). The DNA mol% G+C was 45.1. On the basis of its characteristics and the 16S rRNA gene sequence, strain W218 was assigned to a new Desulfovibrio species for which the name Desulfovibrio acrylicus is proposed. A variety of other sulfate-reducing bacteria (eight of them originating from a marine or saline environment and five from other environments) did not reduce acrylate.