Minor Pilins of the Type IV Pilus System Participate in the Negative Regulation of Swarming Motility

Pseudomonas aeruginosa exhibits distinct surface-associated behaviors, including biofilm formation, flagellum-mediated swarming motility, and type IV pilus-driven twitching. Here, we report a role for the minor pilins, PilW and PilX, components of the type IV pilus assembly machinery, in the repression of swarming motility. Mutating either the pilW or pilX gene alleviates the inhibition of swarming motility observed for strains with elevated levels of the intracellular signaling molecule cyclic di-GMP (c-di-GMP) due to loss of BifA, a c-di-GMP-degrading phosphodiesterase. Blocking PilD peptidase-mediated processing of PilW and PilX renders the unprocessed proteins defective for pilus assembly but still functional in c-di-GMP-mediated swarming repression, indicating our ability to separate these functions. Strains with mutations in pilW or pilX also fail to exhibit the increase in c-di-GMP levels observed when wild-type (WT) or bifA mutant cells are grown on a surface. We also provide data showing that c-di-GMP levels are increased upon PilY1 overexpression in surface-grown cells and that this c-di-GMP increase does not occur in the absence of the SadC diguanylate cyclase. Increased levels of endogenous PilY1, PilX, and PilA are observed when cells are grown on a surface compared to liquid growth, linking surface growth and enhanced signaling via SadC. Our data support a model wherein PilW, PilX, and PilY1, in addition to their role(s) in type IV pilus biogenesis, function to repress swarming via modulation of intracellular c-di-GMP levels. By doing so, these pilus assembly proteins contribute to P. aeruginosa\u27s ability to coordinately regulate biofilm formation with its two surface motility systems

A Three-Component Regulatory System Regulates Biofilm Maturation and Type III Secretion in Pseudomonas aeruginosa

Biofilms are structured communities found associated with a wide range of surfaces. Here we report the identification of a three-component regulatory system required for biofilm maturation by Pseudomonas aeruginosa strain PA14. A transposon mutation that altered biofilm formation in a 96-well dish assay originally defined this locus, which is comprised of genes for a putative sensor histidine kinase and two response regulators and has been designated sadARS. Nonpolar mutations in any of the sadARS genes result in biofilms with an altered mature structure but do not confer defects in growth or early biofilm formation, swimming, or twitching motility. After 2 days of growth under flowing conditions, biofilms formed by the mutants are indistinguishable from those formed by the wild-type (WT) strain. However, by 5 days, mutant biofilms appear to be more homogeneous than the WT in that they fail to form large and distinct macrocolonies and show a drastic reduction in water channels. We propose that the sadARS three-component system is required for later events in biofilm formation on an abiotic surface. Semiquantitative reverse transcription-PCR analysis showed that there is no detectable change in expression of the sadARSgenes when cells are grown in a planktonic culture versus a biofilm, indicating that this locus is not itself induced during or in response to biofilm formation. DNA microarray studies were used to identify downstream targets of the SadARS system. Among the genes regulated by the SadARS system are those required for type III secretion. Mutations in type III secretion genes result in strains with enhanced biofilm formation. We propose a possible mechanism for the role that the SadARS system plays in biofilm formation

Використання тренажерів із віртуальною та доповненою реальністю в системі професійної підготовки зварників: минуле, сучасне та майбутнє

The article discusses the theory and methods of simulation training, its significance in the context of training specialists for areas where the lack of primary qualification is critical. The most widespread hardware and software solutions for the organization welders' simulation training that use VR- and AR- technologies have been analyzed. A review of the technological infrastructure and software tools for the virtual teaching-and-production laboratory of electric welding has been made on the example of the achievements of Fronius, MIMBUS, Seabery. The features of creating a virtual simulation of the welding process using modern equipment based on studies of the behavioral reactions of the welder have been shown. It is found the simulators allow not only training, but also one can build neuro-fuzzy logic and design automated and robotized welding systems. The functioning peculiarities of welding's simulators with AR have been revealed. It is shown they make it possible to ensure the forming basic qualities of a future specialist, such as concentration, accuracy and agility. The psychological and technical aspects of the coaching programs for the training and retraining of qualified welders have been illustrated. The conclusions about the significant advantages of VR- and AR-technologies in comparison with traditional ones have been made. Possible directions of the development of simulation training for welders have been revealed. Among them the AR-technologies have been presented as such that gaining wide popularity as allow to realize the idea of mass training in basic professional skills.У статті розглянуто теорію та методи симуляційного навчання, його значення в контексті підготовки фахівців для галузей, де недостатня початкова кваліфікація є критичною. Проаналізовано найпоширеніші апаратні та програмні рішення для навчання симуляцій організації зварників, які використовують VR- та AR-технології. Огляд технологічної інфраструктури та програмних засобів для віртуальної навчально-виробничої лабораторії електрозварювання було зроблено на прикладі досягнень Fronius, MIMBUS, Seabery. Показано особливості створення віртуального моделювання зварювального процесу з використанням сучасного обладнання на основі досліджень поведінкових реакцій зварника. Встановлено, що тренажери дозволяють не тільки навчатись, але й можна будувати нейро-нечітку логіку та проектувати автоматизовані та роботизовані системи зварювання. Виявлено особливості функціонування зварювальних тренажерів з АР. Показано, що вони дозволяють забезпечити формуючі основні якості майбутнього фахівця, такі як концентрація, акуратність та спритність. Проілюстровано психологічні та технічні аспекти навчальних програм для підготовки та перепідготовки кваліфікованих зварювальників. Зроблено висновки про суттєві переваги VR- та AR-технологій порівняно з традиційними. Розкрито можливі напрями розвитку симуляційної підготовки зварників. Серед них AR-технології були представлені як такі, що набувають широкої популярності, оскільки дозволяють реалізувати ідею масового навчання базовим професійним навичкам

Aminoglycoside Resistance of Pseudomonas Aeruginosa Biofilms Modulated by Extracellular Polysaccharide

Pseudomonas aeruginosa is an opportunistic pathogen that produces sessile communities known as biofilms that are highly resistant to antibiotic treatment. Limited information is available on the exact role of various components of the matrix in biofilm-associated antibiotic resistance. Here we show that the presence of extracellular polysaccharide reduced the extent of biofilm-associated antibiotic resistance for one class of antibiotics. Minimal bactericidal concentration (MBC) for planktonic and biofilm cells of P. aeruginosa PA14 was measured using a 96 well microtiter plate assay. The MBC of biofilm-grown ΔpelA mutant, which does not produce the Pel polysaccharide, was 4-fold higher for tobramycin and gentamicin, and unchanged for ΔbifA mutant, which overproduces Pel, when compared to the wild type. Biofilms of pelA mutants in two clinical isolates of P. aeruginosa showed 4- and 8-fold higher MBC for tobramycin as compared to wild type. There was no difference in the biofilm resistance of any of these strains when tested with fluoroquinolones. This work forms a basis for future studies revealing the mechanisms of biofilm-associated antibiotic resistance to aminoglycoside antibiotics by P. aeruginosa

Aminoglycoside Resistance of Pseudomonas Aeruginosa Biofilms Modulated by Extracellular Polysaccharide

Pseudomonas aeruginosa is an opportunistic pathogen that produces sessile communities known as biofilms that are highly resistant to antibiotic treatment. Limited information is available on the exact role of various components of the matrix in biofilm-associated antibiotic resistance. Here we show that the presence of extracellular polysaccharide reduced the extent of biofilm-associated antibiotic resistance for one class of antibiotics. Minimal bactericidal concentration (MBC) for planktonic and biofilm cells of P. aeruginosa PA14 was measured using a 96 well microtiter plate assay. The MBC of biofilm-grown ΔpelA mutant, which does not produce the Pel polysaccharide, was 4-fold higher for tobramycin and gentamicin, and unchanged for ΔbifA mutant, which overproduces Pel, when compared to the wild type. Biofilms of pelA mutants in two clinical isolates of P. aeruginosa showed 4- and 8-fold higher MBC for tobramycin as compared to wild type. There was no difference in the biofilm resistance of any of these strains when tested with fluoroquinolones. This work forms a basis for future studies revealing the mechanisms of biofilm-associated antibiotic resistance to aminoglycoside antibiotics by P. aeruginosa

Cyclic Di-GMP-Mediated Repression of Swarming Motility by Pseudomonas aeruginosa PA14 Requires the MotAB Stator

The second messenger cyclic diguanylate (c-di-GMP) plays a critical role in the regulation of motility. In Pseudomonas aeruginosa PA14, c-di-GMP inversely controls biofilm formation and surface swarming motility, with high levels of this dinucleotide signal stimulating biofilm formation and repressing swarming. P. aeruginosa encodes two stator complexes, MotAB and MotCD, that participate in the function of its single polar flagellum. Here we show that the repression of swarming motility requires a functional MotAB stator complex. Mutating the motAB genes restores swarming motility to a strain with artificially elevated levels of c-di-GMP as well as stimulates swarming in the wild-type strain, while overexpression of MotA from a plasmid represses swarming motility. Using point mutations in MotA and the FliG rotor protein of the motor supports the conclusion that MotA-FliG interactions are critical for c-di-GMP-mediated swarming inhibition. Finally, we show that high c-di-GMP levels affect the localization of a green fluorescent protein (GFP)-MotD fusion, indicating a mechanism whereby this second messenger has an impact on MotCD function. We propose that when c-di-GMP level is high, the MotAB stator can displace MotCD from the motor, thereby affecting motor function. Our data suggest a newly identified means of c-di-GMP-mediated control of surface motility, perhaps conserved among Pseudomonas, Xanthomonas, and other organisms that encode two stator systems