ABSTRACT
Purpose: To evaluate the utility of model-based deep learning reconstruction in prostate diffusion-weighted imaging (DWI). Methods: This retrospective study evaluated two prostate diffusion-weighted imaging (DWI) methods: deep learning reconstruction (DL-DWI) and traditional parallel imaging (PI-DWI). We examined 32 patients with radiologically diagnosed and histologically confirmed prostate cancer (PCa) lesions ≥10â¯mm. Image quality was evaluated both qualitatively (for overall quality, prostate conspicuity, and lesion conspicuity) and quantitatively, using the signal-to-noise ratio (SNR), contrast-to-noise ratio (CNR), and apparent diffusion coefficient (ADC) for prostate tissue. Results: In the qualitative evaluation, DL-DWI scored significantly higher than PI-DWI for all three parameters (p<0.0001). In the quantitative analysis, DL-DWI showed significantly higher SNR and CNR values compared to PI-DWI (p<0.0001). Both the prostate tissue and the lesions exhibited significantly higher ADC values in DL-DWI compared to PI-DWI (p<0.0001, p=0.0014, respectively). Conclusion: Model-based DL reconstruction enhanced both qualitative and quantitative aspects of image quality in prostate DWI. However, this study did not include comparisons with other DL-based methods, which is a limitation that warrants future research.
ABSTRACT
The motility of Vibrio species plays a pivotal role in their survival and adaptation to diverse environments and is intricately associated with pathogenicity in both humans and aquatic animals. Numerous mutant strains of Vibrio alginolyticus have been generated using UV or EMS mutagenesis to probe flagellar motility using molecular genetic approaches. Identifying these mutations promises to yield valuable insights into motility at the protein structural physiology level. In this study, we determined the complete genomic structure of 4 reference specimens of laboratory V. alginolyticus strains: a precursor strain, V. alginolyticus 138-2, two strains showing defects in the lateral flagellum (VIO5 and YM4), and one strain showing defects in the polar flagellum (YM19). Subsequently, we meticulously ascertained the specific mutation sites within the 18 motility-deficient strains related to the polar flagellum (they fall into three categories: flagellar-deficient, multi-flagellar, and chemotaxis-deficient strains) by whole genome sequencing and mapping to the complete genome of parental strains VIO5 or YM4. The mutant strains had an average of 20.6 (±12.7) mutations, most of which were randomly distributed throughout the genome. However, at least two or more different mutations in six flagellar-related genes were detected in 18 mutants specifically selected as chemotaxis-deficient mutants. Genomic analysis using a large number of mutant strains is a very effective tool to comprehensively identify genes associated with specific phenotypes using forward genetics.
Subject(s)
Chemotaxis , Vibrio alginolyticus , Animals , Humans , Vibrio alginolyticus/genetics , Mutation , MutagenesisABSTRACT
PURPOSE: We present a novel algorithm for the automated detection of cerebral microbleeds (CMBs) on 2D gradient-recalled echo T2* weighted images (T2*WIs). This approach combines a morphology filter bank with a convolutional neural network (CNN) to improve the efficiency of CMB detection. A technical evaluation was performed to ascertain the algorithm's accuracy. METHODS: In this retrospective study, 60 patients with CMBs on T2*WIs were included. The gold standard was set by three neuroradiologists based on the Microbleed Anatomic Rating Scale guidelines. Images with CMBs were extracted from the training dataset comprising 30 cases using a morphology filter bank, and false positives (FPs) were removed based on the threshold of size and signal intensity. The extracted images were used to train the CNN (Vgg16). To determine the effectiveness of the morphology filter bank, the outcomes of the following two methods for detecting CMBs from the 30-case test dataset were compared: (a) employing the morphology filter bank and additional FP removal and (b) comprehensive detection without filters. The trained CNN processed both sets of initial CMB candidates, and the final CMB candidates were compared with the gold standard. The sensitivity and FPs per patient of both methods were compared. RESULTS: After CNN processing, the morphology-filter-bank-based method had a 95.0% sensitivity with 4.37 FPs per patient. In contrast, the comprehensive method had a 97.5% sensitivity with 25.87 FPs per patient. CONCLUSION: Through effective CMB candidate refinement with a morphology filter bank and FP removal with a CNN, we achieved a high CMB detection rate and low FP count. Combining a CNN and morphology filter bank may facilitate the accurate automated detection of CMBs on T2*WIs.
ABSTRACT
PURPOSE: Prolonged scanning of time-resolved 3D phase-contrast MRI (4D flow MRI) limits its routine use in clinical practice. An echo-planar imaging (EPI)-based sequence and compressed sensing can reduce the scan duration. We aimed to determine the impact of EPI for 4D flow MRI on the scan duration, image quality, and quantitative flow metrics. METHODS: This was a prospective study of 15 healthy volunteers (all male, mean age 33 ± 5 years). Conventional sensitivity encoding (SENSE), EPI with SENSE (EPI), and compressed SENSE (CS) (reduction factors: 6 and 12, respectively) were scanned.Scan duration, qualitative indexes of image quality, and quantitative flow parameters of net flow volume, maximum flow velocity, wall shear stress (WSS), and energy loss (EL) in the ascending aorta were assessed. Two-dimensional phase-contrast cine MRI (2D-PC) was considered the gold standard of net flow volume and maximum flow velocity. RESULTS: Compared to SENSE, EPI and CS12 shortened scan durations by 71% and 73% (EPI, 4 min 39 sec; CS6, 7 min 29 sec; CS12, 4 min 14 sec; and SENSE, 15 min 51 sec). Visual image quality was significantly better for EPI than for SENSE and CS (P < 0.001). The net flow volumes obtained with SENSE, EPI, and CS12 and those obtained with 2D-PC were correlated well (r = 0.950, 0.871, and 0.850, respectively). However, the maximum velocity obtained with EPI was significantly underestimated (P < 0.010). The average WSS was significantly higher with EPI than with SENSE, CS6, and CS12 (P < 0.001, P = 0.040, and P = 0.012, respectively). The EL was significantly lower with EPI than with CS6 and CS12 (P = 0.002 and P = 0.007, respectively). CONCLUSION: EPI reduced the scan duration, improved visual image quality, and was associated with more accurate net flow volume than CS. However, the flow velocity, WSS, and EL values obtained with EPI and other sequences may not be directly comparable.
ABSTRACT
We investigated the ability of echo-planar imaging with L1-regularized iterative sensitivity encoding-based diffusion-weighted imaging (DWI) to improve the image quality and reduce the scanning time in prostate magnetic resonance imaging. We retrospectively analyzed 109 cases of prostate magnetic resonance imaging. We compared variables in the quantitative and qualitative assessments among 3 imaging groups: conventional parallel imaging-based DWI (PI-DWI) with an acquisition time of 3 minutes 15 seconds; echo-planar imaging with L1-regularized iterative sensitivity encoding-based DWI (L1-DWI) with a normal acquisition time (L1-DWINEX12) of 3 minutes 15 seconds; and L1-DWI with a half acquisition time (L1-DWINEX6) of 1 minute 45 seconds. As a quantitative assessment, the signal-to-noise ratio (SNR) of DWI (SNR-DWI), the contrast-to-noise ratio (CNR) of DWI (CNR-DWI), and the CNR of apparent diffusion coefficient were measured. As a qualitative assessment, the image quality and visual detectability of prostate carcinoma were evaluated. In the quantitative analysis, L1-DWINEX12 showed significantly higher SNR-DWI than PI-DWI (P = .0058) and L1-DWINEX6 (P < .0001). In the qualitative analysis, the image quality score for L1-DWINEX12 was significantly higher than those of PI-DWI and L1-DWINEX6. A non-inferiority assessment demonstrated that L1-DWINEX6 was non-inferior to PI-DWI in terms of both quantitative CNR-DWI and qualitative grading of image quality with a <20% inferior margin. L1-DWI successfully demonstrated a reduced scanning time while maintaining good image quality.
Subject(s)
Echo-Planar Imaging , Prostate , Male , Humans , Echo-Planar Imaging/methods , Prostate/diagnostic imaging , Retrospective Studies , Magnetic Resonance Imaging , Diffusion Magnetic Resonance Imaging/methods , Signal-To-Noise Ratio , Reproducibility of ResultsABSTRACT
Symptomatic vitamin C deficiency, scurvy, is a relatively rare disease in developed countries, but it has been reported in patients with autism spectrum disorder or developmental delay who tend to have selective diets. Patients with scurvy often demonstrate musculoskeletal manifestations with unknown pathophysiology. Herein, we report a case of scurvy in an 11-year-old boy who presented with iron-deficiency anaemia, systemic osteomyelitis, myositis predominantly in the lower extremities, and right ventricular volume overload with mild pulmonary hypertension and was diagnosed with scurvy. He had a mild developmental disorder and a selective diet, which resulted in severe vitamin C deficiency. He received intravenous and oral vitamin C supplementation, which relieved his arthralgia and muscle pain in a week. Following 4 months of vitamin C supplementation, he demonstrated no abnormal manifestations on laboratory or imaging examination and recovered without sequelae. Inflammatory cytokine and chemokine evaluations demonstrated elevated levels of interleukin (IL)-6, IL-17A, and IL-23, which are associated with T-helper (Th) 17 cell activation. This study is the first to suggest the association between the inflammation seen in scurvy, rheumatic manifestations in the patient, and Th17 cell activation. Further analysis of the association between the inflammation and vitamin C supplementation may contribute to new insights for the comprehension and treatment of other inflammatory diseases, such as rheumatic diseases.
Subject(s)
Arthritis, Rheumatoid , Ascorbic Acid Deficiency , Autism Spectrum Disorder , Scurvy , Male , Humans , Child , Scurvy/complications , Scurvy/diagnosis , Interleukin-6 , Autism Spectrum Disorder/complications , Interleukin-23 , Interleukin-17 , Ascorbic Acid/therapeutic use , Ascorbic Acid Deficiency/complications , Inflammation/complications , Arthritis, Rheumatoid/complicationsABSTRACT
The marine bacterium Vibrio alginolyticus has a single polar flagellum, the number of which is regulated positively by FlhF and negatively by FlhG. FlhF is intrinsically localized at the cell pole, whereas FlhG is localized there through putative interactions with the polar landmark protein HubP. Here we focused on the role of HubP in the regulation of flagellar number in V. alginolyticus Deletion of hubP increased the flagellar number and completely disrupted the polar localization of FlhG. It was thought that the flagellar number is determined primarily by the absolute amount of FlhF localized at the cell pole. Here we found that deletion of hubP increased the flagellar number although it did not increase the polar amount of FlhF. We also found that FlhG overproduction did not reduce the polar localization of FlhF. These results show that the absolute amount of FlhF is not always the determinant of flagellar number. We speculate that cytoplasmic FlhG works as a quantitative regulator, controlling the amount of FlhF localized at the pole, and HubP-anchored polar FlhG works as a qualitative regulator, directly inhibiting the activity of polar FlhF. This regulation by FlhF, FlhG, and HubP might contribute to achieving optimal flagellar biogenesis at the cell pole in V. alginolyticus IMPORTANCE: For regulation of the flagellar number in marine Vibrio, two proteins, FlhF and FlhG, work as positive and negative regulators, respectively. In this study, we found that the polar landmark protein HubP is involved in the regulation of flagellar biogenesis. Deletion of hubP increased the number of flagella without increasing the amount of pole-localizing FlhF, indicating that the number of flagella is not determined solely by the absolute amount of pole-localizing FlhF, which is inconsistent with the previous model. We propose that cytoplasmic FlhG and HubP-anchored polar FlhG negatively regulate flagellar formation through two independent schemes.
Subject(s)
Bacterial Proteins/physiology , Flagella/physiology , Monomeric GTP-Binding Proteins/physiology , Vibrio alginolyticus/genetics , Bacterial Proteins/genetics , Gene Deletion , Gene Expression Regulation, Bacterial , Monomeric GTP-Binding Proteins/genetics , Vibrio alginolyticus/physiologyABSTRACT
For construction of the bacterial flagellum, flagellar proteins are exported via its specific export apparatus from the cytoplasm to the distal end of the growing flagellar structure. The flagellar export apparatus consists of a transmembrane (TM) export gate complex and a cytoplasmic ATPase complex consisting of FliH, FliI, and FliJ. FlhA is a TM export gate protein and plays important roles in energy coupling of protein translocation. However, the energy coupling mechanism remains unknown. Here, we performed a cross-complementation assay to measure robustness of the energy transduction system of the export apparatus against genetic perturbations. Vibrio FlhA restored motility of a Salmonella ΔflhA mutant but not that of a ΔfliH-fliI flhB(P28T) ΔflhA mutant. The flgM mutations significantly increased flagellar gene expression levels, allowing Vibrio FlhA to exert its export activity in the ΔfliH-fliI flhB(P28T) ΔflhA mutant. Pull-down assays revealed that the binding affinities of Vibrio FlhA for FliJ and the FlgN-FlgK chaperone-substrate complex were much lower than those of Salmonella FlhA. These suggest that Vibrio FlhA requires the support of FliH and FliI to efficiently and properly interact with FliJ and the FlgN-FlgK complex. We propose that FliH and FliI ensure robust and efficient energy coupling of protein export during flagellar assembly.
Subject(s)
Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Flagella/metabolism , Proton-Translocating ATPases/metabolism , Salmonella/metabolism , Sigma Factor/genetics , Vibrio/genetics , Base Sequence , Genome, Bacterial/genetics , Protein Transport , Proton-Translocating ATPases/genetics , Salmonella/genetics , Sequence Analysis, DNA , Sigma Factor/metabolismABSTRACT
A 74-year-old woman with advanced gastric cancer was admitted to our hospital. A central venous (CV) port catheter was implanted into the right subclavian vein for preoperative chemotherapy and parenteral nutritional management. On the 35th day after implantation, she complained of diarrhea, fever and dyspnea. The chest radiograph showed a right-sided massive pleural effusion. As the patient progressively fell into severe respiratory distress, endotracheal intubation was performed for management of respiration by mechanical ventilation. Initially, given the patient's symptoms, she was diagnosed with septic shock. Therefore, after placement of a CV catheter through the right femoral vein, in consideration of the possibility of a port infection, she was treated with thoracentesis and infusion of antibiotics. The patient gradually recovered, and again received parenteral nutrition through the CV port catheter. After the infusion was administered, she complained of dyspnea. A CT scan of the chest revealed a right pleural effusion and displacement of the tip of the CV port catheter out of the wall of the superior vena cava. We diagnosed delayed vascular injury (DVI), and the CV port catheter was removed. She soon recovered with conservative treatment. We speculated that the initial respiratory symptoms such as the pleural effusion were caused by DVI. DVI should therefore be recognized as a complication related to implanted CV port catheters.
Subject(s)
Bacillaceae Infections/microbiology , Bacillus cereus , Catheters, Indwelling/adverse effects , Central Venous Catheters/adverse effects , Stomach Neoplasms , Vascular System Injuries/microbiology , Aged , Female , Humans , Stomach Neoplasms/drug therapy , Stomach Neoplasms/surgeryABSTRACT
The membrane motor proteins, PomA (polar flagellar motility protein A) and PomB (polar flagellar motility protein B), of Vibrio alginolyticus form a stator complex that converts energy from the ion flow to mechanical work in bacterial flagellar motors. The cytoplasmic domain of PomA is believed to interact with the rotor protein FliG to make a torque. In this study, to investigate the function of the cytoplasmic domain of PomA, we constructed a series of fragments that flank the cytoplasmic loop of PomA between the second and third transmembrane (TM) domains (A-loop) and the C-terminal region, and expressed them in Escherichia coli together with PomA and PotB (a chimeric protein of PomB and MotB). We observed a dominant-negative effect of one PomA fragment on motility. We confirmed that these PomA fragments localized both in the membrane fraction and in the cytoplasmic fraction, and induced bacterial growth delay. Effect of additional point and deletion mutations into this fragment implies that the C-terminal region and TM domains used as a linker play a significant part in these observations. From these results, we conclude that the PomA fragments retain the structure important for functions. We expect that further constructions will provide a variety of experimental approaches to characterize the interaction between PomA and FliG.
Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Flagella/metabolism , Recombinant Fusion Proteins/metabolism , Sodium Channels/chemistry , Sodium Channels/metabolism , Sodium/metabolism , Vibrio alginolyticus/metabolism , Conserved Sequence , Cytoplasm/metabolism , Escherichia coli/growth & development , Escherichia coli/metabolism , Movement , Mutant Proteins/chemistry , Mutant Proteins/metabolism , Mutation/genetics , Protein Binding , Protein Structure, Secondary , Protein Structure, Tertiary , Sequence DeletionABSTRACT
Bacterial flagellar motors exploit the electrochemical potential gradient of a coupling ion as energy source and are composed of stator and rotor proteins. Vibrio alginolyticus has a Na(+)-driven motor and its stator is composed of PomA and PomB. Recently, we isolated increased motility strains (sp1-sp4) from the PomA-N194D/PomB-D24N mutant whose motility was quite weak. To detect the responsible mutation, we have used a next-generation sequencer and determined the entire genome sequences of the sp1 and sp2 strains. Candidate mutations were identified in the gene encoding the a-subunit of F1Fo-ATPase (uncB). To confirm this, we constructed a deletion strain, which gave the increased motility phenotype. The amount of membrane-bound ATPase was reduced in the sp2 and ΔuncB mutants. From these results, we conclude that a mutation in the uncB gene causes the increased motility phenotype in V. alginolyticus. They confer faster motility in low concentrations of sodium than in the parental strain and this phenotype is suppressed in the presence of KCN. Those results may suggest that the proton gradient generated by the respiratory chain is increased by the uncB mutation, consequently the sodium motive force is increased and causes the increased motility phenotype.
Subject(s)
Bacterial Proton-Translocating ATPases/metabolism , Flagella/metabolism , Mutation, Missense , Vibrio alginolyticus/enzymology , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Bacterial Proton-Translocating ATPases/genetics , Flagella/genetics , Sodium Channels/genetics , Sodium Channels/metabolism , Vibrio alginolyticus/geneticsABSTRACT
The marine bacterium Vibrio alginolyticus has a single polar flagellum. Formation of that flagellum is regulated positively and negatively by FlhF and by FlhG, respectively. The ΔflhF mutant makes no flagellum, whereas the ΔflhFG double-deletion mutant usually lacks a flagellum. However, the ΔflhFG mutant occasionally reverts to become motile by forming peritrichous flagella. We have isolated a suppressor pseudorevertant from the ΔflhFG strain (ΔflhFG-sup). The suppressor strain forms peritrichous flagella in the majority of cells. We identified candidate suppressor mutations by comparing the genome sequence of the parental strain, VIO5, with the genome sequences of the suppressor strains. Two mutations were mapped to a gene, named sflA (suppressor of ΔflhFG), at the VEA003730 locus of the Vibrio sp. strain EX25 genome. This gene is specific for Vibrio species and is predicted to encode a transmembrane protein with a DnaJ domain. When the wild-type gene was introduced into the suppressor strain, motility was impaired. Introducing a mutant version of the sflA gene into the ΔflhFG strain conferred the suppressor phenotype. Thus, we conclude that loss of the sflA gene is responsible for the suppressor phenotype and that the wild-type SflA protein plays a role in preventing polar-type flagella from forming on the lateral cell wall.
Subject(s)
Bacterial Proteins/metabolism , Flagella/metabolism , Gene Expression Regulation, Bacterial/physiology , Vibrio alginolyticus/metabolism , Amino Acid Sequence , Bacterial Proteins/genetics , Conserved Sequence , Flagella/genetics , Genes, Suppressor , Genome, Bacterial , Molecular Sequence Data , Multigene Family/genetics , Mutation , Phenotype , Phylogeny , Species Specificity , Vibrio alginolyticus/cytology , Vibrio alginolyticus/geneticsABSTRACT
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.
Subject(s)
Cell Polarity , Flagella/chemistry , Vibrio alginolyticus/physiology , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Flagella/genetics , Flagella/physiology , Flagellin/genetics , Flagellin/metabolism , Gene Expression Regulation, Bacterial , Mutation , Vibrio alginolyticus/chemistry , Vibrio alginolyticus/geneticsABSTRACT
PURPOSE: For lung cancer patients with poor pulmonary function because of emphysema or fibrosis, it is important to predict the amplitude of internal tumor motion to minimize the irradiation of the functioning lung tissue before undergoing stereotactic body radiotherapy. METHODS AND MATERIALS: Two board-certified diagnostic radiologists independently assessed the degree of pulmonary emphysema and fibrosis on computed tomography scans in 71 patients with peripheral lung tumors before real-time tumor-tracking radiotherapy. The relationships between the computed tomography findings of the lung parenchyma and the motion of the fiducial marker near the lung tumor were investigated. Of the 71 patients, 30 had normal pulmonary function, and 29 had obstructive pulmonary dysfunction (forced expiratory volume in 1 s/forced vital capacity ratio of <70%), 6 patients had constrictive dysfunction (percentage of vital capacity <80%), and 16 had mixed dysfunction. RESULTS: The upper region was associated with smaller tumor motion, as expected (p = .0004), and the presence of fibrosis (p = .088) and pleural tumor contact (p = .086) were weakly associated with tumor motion. The presence of fibrotic changes in the lung tissue was associated with smaller tumor motion in the upper region (p <.05) but not in the lower region. The findings of emphysema and pulmonary function tests were not associated with tumor motion. CONCLUSION: Tumors in the upper lung region with fibrotic changes have smaller motion than those in the upper region of the lungs without fibrotic changes. The tumor motion in the lower lung region was not significantly different between patients with and without lung fibrosis. Emphysema was not associated with the amplitude of tumor motion.
Subject(s)
Fiducial Markers , Lung Neoplasms/diagnostic imaging , Movement , Pulmonary Emphysema/diagnostic imaging , Pulmonary Fibrosis/diagnostic imaging , Radiosurgery , Adult , Aged , Aged, 80 and over , Area Under Curve , Female , Forced Expiratory Volume/physiology , Humans , Lung/radiation effects , Lung Diseases, Obstructive/diagnostic imaging , Lung Diseases, Obstructive/physiopathology , Lung Neoplasms/physiopathology , Lung Neoplasms/surgery , Male , Middle Aged , Pulmonary Emphysema/pathology , Pulmonary Emphysema/physiopathology , Pulmonary Fibrosis/pathology , Pulmonary Fibrosis/physiopathology , Radiation Injuries/prevention & control , Radiosurgery/adverse effects , Statistics, Nonparametric , Tomography, X-Ray Computed/methods , Vital Capacity/physiologyABSTRACT
The polar flagellum of Vibrio alginolyticus is driven by sodium ion flux via a stator complex, composed of PomA and PomB, across the cell membrane. The interaction between PomA and the rotor component FliG is believed to generate torque required for flagellar rotation. Previous research reported that a GFP-fused FliG retained function in the Vibrio flagellar motor. In this study, we found that N-terminal or C-terminal fusion of GFP has different effects on both torque generation and the switching frequency of the direction of flagellar motor rotation. We could detect the GFP-fused FliG in the basal-body (rotor) fraction although its association with the basal body was less stable than that of intact FliG. Furthermore, the fusion of GFP to the C-terminus of FliG, which is believed to be directly involved in torque generation, resulted in very slow motility and prohibited the directional change of motor rotation. On the other hand, the fusion of GFP to the N-terminus of FliG conferred almost the same swimming speed as intact FliG. These results are consistent with the premise that the C-terminal domain of FliG is directly involved in torque generation and the GFP fusions are useful to analyze the functions of various domains of FliG.
ABSTRACT
Precise regulation of the number and placement of flagella is critical for the mono-flagellated bacterium Vibrio alginolyticus to swim efficiently. We previously proposed a model in which the putative GTPase FlhF determines the polar location and generation of the flagellum, the putative ATPase FlhG interacts with FlhF to prevent FlhF from localizing to the pole, and thus FlhG negatively regulates the flagellar number in V. alginolyticus cells. To investigate the role of the GTP-binding motif of FlhF, we generated a series of alanine-replacement mutations at the positions that are highly conserved among homologous proteins. The results indicate that there is a correlation between the polar localization and the ability to produce flagella in the mutants. We investigated whether the mutations in the GTP-binding motif affected the ability to interact with FlhG. In contrast to our prediction, no significant difference was detected in the interaction with FlhG between the wild-type and mutant FlhFs. We showed that the GTP-binding motif of FlhF is important for polar localization of the flagellum but not for the interaction with FlhG.
Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Cell Polarity , Flagella/metabolism , Guanosine Triphosphate/metabolism , Monomeric GTP-Binding Proteins/chemistry , Monomeric GTP-Binding Proteins/metabolism , Vibrio alginolyticus/cytology , Vibrio alginolyticus/metabolism , Alanine/metabolism , Amino Acid Motifs , Amino Acid Sequence , DNA Mutational Analysis , Green Fluorescent Proteins/metabolism , Immunoprecipitation , Molecular Sequence Data , Movement , Mutant Proteins/chemistry , Mutant Proteins/metabolism , Mutation/genetics , Plasmids/genetics , Protein Binding , Protein Transport , Recombinant Fusion Proteins/metabolismABSTRACT
Vibrio alginolyticus has two types of flagella, polar (Pof) and lateral (Laf). From a Laf-defective mutant (Pof+Laf-), polar-flagellar-length mutants which have short Pof and long Pof were isolated. The mean lengths of the helical axis in wild-type, short and long Pof were 5.5.0.9 µm, 2.5.0.6 µm and 11.2.3.6 µm, respectively. The swimming speeds of the short- and long-Pof mutants were slower than that of the wild-type strain. The relationship between swimming speed and flagellar length in a population of mutant cells was examined. In the short-Pof mutant, the decrease of swimming speed seemed to be derived from the decrease in flagellar length. In the long-Pof mutant, there was almost no correlation between swimming speed and flagellar length, and the slow swimming was explained by the helical shape of the flagella, whose pitch and radius were 1.4 µm and 0.062 µm, respectively, whereas those of the wild-type flagella were 1.5 µm and 0.16 µm. The relative amounts of the various molecular components of the long Pof were different from those of the wild-type or the short Pof. This seems to be the reason for the difference in flagellar shape and length, though the mutation may be pleiotropic and affect flagellar function or regulation.