Effect of arsenite on swimming motility delays surface colonization in Herminiimonas arsenicoxydans

Herminiimonas arsenicoxydans is a Gram-negative bacterium able to detoxify arsenic-contaminated environments by oxidizing arsenite [As(III)] to arsenate [As(V)] and by scavenging arsenic ions in an extracellular matrix. Its motility and colonization behaviour have been previously suggested to be influenced by arsenite. Using time-course confocal laser scanning microscopy, we investigated its biofilm development in the absence and presence of arsenite. Arsenite was shown to delay biofilm initiation in the wild-type strain; this was partly explained by its toxicity, which caused an increased growth lag time. However, this delayed adhesion step in the presence of arsenite was not observed in either a swimming motility defective fliL mutant or an arsenite oxidase defective aoxB mutant; both strains displayed the wild-type surface properties and growth capacities. We propose that during the biofilm formation process arsenite acts on swimming motility as a result of the arsenite oxidase activity, preventing the switch between planktonic and sessile lifestyles. Our study therefore highlights the existence, under arsenite exposure, of a competition between swimming motility, resulting from arsenite oxidation, and biofilm initiation.

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Cell-to-Cell Signaling in Xylella fastidiosa Suppresses Movement and Xylem Vessel Colonization in Grape

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-21-10-1309 ◽

2008 ◽

Vol 21 (10) ◽

pp. 1309-1315 ◽

Cited By ~ 71

Author(s):

Subhadeep Chatterjee ◽

Karyn L. Newman ◽

Steven E. Lindow

Keyword(s):

Cell Signaling ◽

Type Strain ◽

Laser Scanning ◽

Wild Type Strain ◽

Xylella Fastidiosa ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Deficient Mutant ◽

Wild Type ◽

Wide Range

Cell-to-cell signaling mediated by a fatty acid diffusible signaling factor (DSF) is central to the regulation of the virulence of Xylella fastidiosa. DSF production by X. fastidiosa is dependent on rpfF and, although required for insect colonization, appears to reduce its virulence to grape. To understand what aspects of colonization of grape are controlled by DSF in X. fastidiosa and, thus, those factors that contribute to virulence, we assessed the colonization of grape by a green fluorescent protein–marked rpfF-deficient mutant. The rpfF-deficient mutant was detected at a greater distance from the point of inoculation than the wild-type strain at a given sampling time, and also attained a population size that was up to 100-fold larger than that of the wild-type strain at a given distance from the point of inoculation. Confocal laser-scanning microscopy revealed that approximately 10-fold more vessels in petioles of symptomatic leaves harbored at least some cells of either the wild type or rpfF mutant when compared with asymptomatic leaves and, thus, that disease symptoms were associated with the extent of vessel colonization. Importantly, the rpfF mutant colonized approximately threefold more vessels than the wild-type strain. Although a wide range of colony sizes were observed in vessels colonized by both the wild type and rpfF mutant, the proportion of colonized vessels harboring large numbers of cells was significantly higher in plants inoculated with the rpfF mutant than with the wild-type strain. These studies indicated that the hypervirulence phenotype of the rpfF mutant is due to both a more extensive spread of the pathogen to xylem vessels and unrestrained multiplication within vessels leading to blockage. These results suggest that movement and multiplication of X. fastidiosa in plants are linked, perhaps because cell wall degradation products are a major source of nutrients. Thus, DSF-mediated cell-to-cell signaling, which restricts movement and colonization of X. fastidiosa, may be an adaptation to endophytic growth of the pathogen that prevents the excessive growth of cells in vessels.

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Role of the luxS Gene in Initial Biofilm Formation by Streptococcus mutans

Journal of Molecular Microbiology and Biotechnology ◽

10.1159/000371816 ◽

2015 ◽

Vol 25 (1) ◽

pp. 60-68 ◽

Cited By ~ 24

Author(s):

Zhiyan He ◽

Jingping Liang ◽

Zisheng Tang ◽

Rui Ma ◽

Huasong Peng ◽

...

Keyword(s):

Streptococcus Mutans ◽

Mutant Strain ◽

Biofilm Formation ◽

Type Strain ◽

Laser Scanning ◽

Wild Type Strain ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Wild Type ◽

Initial Biofilm

Quorum sensing (QS) is a process by which bacteria communicate with each other by secreting chemical signals called autoinducers (AIs). Among Gram-negative and Gram-positive bacteria, AI-2 synthesized by the LuxS enzyme is widespread. The aim of this study was to evaluate the effect of QS luxS gene on initial biofilm formation by Streptococcus mutans. The bacterial cell surface properties, including cell hydrophobicity (bacterial adherence to hydrocarbons) and aggregation, which are important for initial adherence during biofilm development, were investigated. The biofilm adhesion assay was evaluated by the MTT method. The structures of the 5-hour biofilms were observed by using confocal laser scanning microscopy, and QS-related gene expressions were investigated by real-time PCR. The luxS mutant strain exhibited higher biofilm adherence and aggregation, but lower hydrophobicity than the wild-type strain. The confocal laser scanning microscopy images revealed that the wild-type strain tended to form smaller aggregates with uniform distribution, whereas the luxS mutant strain aggregated into distinct clusters easily discernible in the generated biofilm. Most of the genes examined were downregulated in the biofilms formed by the luxS mutant strain, except the gtfB gene. QS luxS gene can affect the initial biofilm formation by S. mutans.

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Contribution of alginate and levan production to biofilm formation by Pseudomonas syringae

Microbiology ◽

10.1099/mic.0.28875-0 ◽

2006 ◽

Vol 152 (10) ◽

pp. 2909-2918 ◽

Cited By ~ 94

Author(s):

Heike Laue ◽

Alexander Schenk ◽

Hongqiao Li ◽

Lotte Lambertsen ◽

Thomas R. Neu ◽

...

Keyword(s):

Spatial Distribution ◽

Pseudomonas Syringae ◽

Biofilm Formation ◽

Laser Scanning ◽

Time Course ◽

Complex Structure ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Bacterial Cells ◽

Biofilm Development

Exopolysaccharides (EPSs) play important roles in the attachment of bacterial cells to a surface and/or in building and maintaining the three-dimensional, complex structure of bacterial biofilms. To elucidate the spatial distribution and function of the EPSs levan and alginate during biofilm formation, biofilms of Pseudomonas syringae strains with different EPS patterns were compared. The mucoid strain PG4180.muc, which produces levan and alginate, and its levan- and/or alginate-deficient derivatives all formed biofilms in the wells of microtitre plates and in flow chambers. Confocal laser scanning microscopy with fluorescently labelled lectins was applied to investigate the spatial distribution of levan and an additional as yet unknown EPS in flow-chamber biofilms. Concanavalin A (ConA) bound specifically to levan and accumulated in cell-depleted voids in the centres of microcolonies and in blebs. No binding of ConA was observed in biofilms of the levan-deficient mutants or in wild-type biofilms grown in the absence of sucrose as confirmed by an enzyme-linked lectin-sorbent assay using peroxidase-linked ConA. Time-course studies revealed that expression of the levan-forming enzyme, levansucrase, occurred mainly during early exponential growth of both planktonic and sessile cells. Thus, accumulation of levan in biofilm voids hints to a function as a nutrient storage source for later stages of biofilm development. The presence of a third EPS besides levan and alginate was indicated by binding of the lectin from Naja mossambica to a fibrous structure in biofilms of all P. syringae derivatives. Production of the as yet uncharacterized additional EPS might be more important for biofilm formation than the syntheses of levan and alginate.

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Statistical Analysis of Pseudomonas aeruginosa Biofilm Development: Impact of Mutations in Genes Involved in Twitching Motility, Cell-to-Cell Signaling, and Stationary-Phase Sigma Factor Expression

Applied and Environmental Microbiology ◽

10.1128/aem.68.4.2008-2017.2002 ◽

2002 ◽

Vol 68 (4) ◽

pp. 2008-2017 ◽

Cited By ~ 197

Author(s):

Arne Heydorn ◽

Bjarne Ersbøll ◽

Junichi Kato ◽

Morten Hentzer ◽

Matthew R. Parsek ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Laser Scanning ◽

Fluorescent Protein ◽

Current Model ◽

Flow Chamber ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Wild Type ◽

Time Points ◽

Biofilm Development

ABSTRACT Four strains of Pseudomonas aeruginosa (wild type, ΔpilHIJK mutant, lasI mutant, and rpoS mutant) were genetically tagged with the green fluorescent protein, and the development of flow chamber-grown biofilms by each of them was investigated by confocal laser scanning microscopy. The structural developments of the biofilms were quantified by the computer program COMSTAT (A. Heydorn, A. T. Nielsen, M. Hentzer, C. Sternberg, M. Givskov, B. K. Ersbøll, and S. Molin, Microbiology 146:2395-2407, 2000). Two structural key variables, average thickness and roughness, formed the basis for an analysis of variance model comprising the four P. aeruginosa strains, five time points (55, 98, 146, 242, and 314 h), and three independent rounds of biofilm experiments. The results showed that the wild type, the ΔpilHIJK mutant, and the rpoS mutant display conspicuously different types of temporal biofilm development, whereas the lasI mutant was indistinguishable from the wild type at all time points. The wild type and the lasI mutant formed uniform, densely packed biofilms. The rpoS mutant formed densely packed biofilms that were significantly thicker than those of the wild type, whereas the ΔpilHIJK mutant formed distinct microcolonies that were regularly spaced and almost uniform in size. The results are discussed in relation to the current model of P. aeruginosa biofilm development.

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The PTS Components in Klebsiella pneumoniae Affect Bacterial Capsular Polysaccharide Production and Macrophage Phagocytosis Resistance

Microorganisms ◽

10.3390/microorganisms9020335 ◽

2021 ◽

Vol 9 (2) ◽

pp. 335

Author(s):

Novaria Sari Dewi Panjaitan ◽

Yu-Tze Horng ◽

Chih-Ching Chien ◽

Hung-Chi Yang ◽

Ren-In You ◽

...

Keyword(s):

Survival Rate ◽

Klebsiella Pneumoniae ◽

Laser Scanning ◽

Bacterial Resistance ◽

Capsular Polysaccharide ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Intracellular Bacteria ◽

Wild Type ◽

Macrophage Phagocytosis

Capsular polysaccharide (CPS) is a crucial virulence factor for Klebsiella pneumoniae infection. We demonstrated an association of CPS production with two phosphoenolpyruvate:carbohydrate phosphotransferase systems (PTSs). Deficiency of crr, encoding enzyme IIA of PTS, in K. pneumoniae enhanced the transcriptional activities of galF, wzi and gnd, which are in the cps gene cluster, leading to high CPS production. A crr mutant exhibited a higher survival rate in 1% hydrogen peroxide than the wild-type. The crr mutant showed less sensitivity to engulfment by macrophage (RAW 264.7) than the wild-type by observing the intracellular bacteria using confocal laser scanning microscopy (CLSM) and by calculating the colony-forming units (CFU) of intracellular bacteria. After long-term incubation, the survival rate of the intracellular crr mutant was higher than that of the wild-type. Deficiency of crr enhanced the transcriptional activities of etcABC which encodes another putative enzyme II complex of a PTS. Deletion of etcABC in the crr mutant reduced CPS production and the transcriptional activities of galF compared to those of the crr mutant. These results indicated that one PTS component, Crr, represses CPS production by repressing another PTS component, EtcABC, in K. pneumoniae. In addition, PTS plays a role in bacterial resistance to macrophage phagocytosis.

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Importance of Initial Interfacial Steps during Chalcopyrite Bioleaching by a Thermoacidophilic Archaeon

Microorganisms ◽

10.3390/microorganisms8071009 ◽

2020 ◽

Vol 8 (7) ◽

pp. 1009

Author(s):

Camila Safar ◽

Camila Castro ◽

Edgardo Donati

Keyword(s):

High Temperatures ◽

Laser Scanning ◽

Laser Scanning Microscopy ◽

Copper Recovery ◽

Confocal Laser ◽

Mineral Surface ◽

Thermophilic Microorganisms ◽

Valuable Metals ◽

Refractory Mineral ◽

Biofilm Development

Studies of thermophilic microorganisms have shown that they have a considerable biotechnological potential due to their optimum growth and metabolism at high temperatures. Thermophilic archaea have unique characteristics with important biotechnological applications; many of these species could be used in bioleaching processes to recover valuable metals from mineral ores. Particularly, bioleaching at high temperatures using thermoacidophilic microorganisms can greatly improve metal solubilization from refractory mineral species such as chalcopyrite (CuFeS2), one of the most abundant and widespread copper-bearing minerals. Interfacial processes such as early cell adhesion, biofilm development, and the formation of passive layers on the mineral surface play important roles in the initial steps of bioleaching processes. The present work focused on the investigation of different bioleaching conditions using the thermoacidophilic archaeon Acidianus copahuensis DSM 29038 to elucidate which steps are pivotal during the chalcopyrite bioleaching. Fluorescent in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM) were used to visualize the microorganism–mineral interaction. Results showed that up to 85% of copper recovery from chalcopyrite could be achieved using A. copahuensis. Improvements in these yields are intimately related to an early contact between cells and the mineral surface. On the other hand, surface coverage by inactivated cells as well as precipitates significantly reduced copper recoveries.

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Bacterial lipopolysaccharides variably modulate in vitro biofilm formation of Candida species

Journal of Medical Microbiology ◽

10.1099/jmm.0.021832-0 ◽

2010 ◽

Vol 59 (10) ◽

pp. 1225-1234 ◽

Cited By ~ 51

Author(s):

H. M. H. N. Bandara ◽

O. L. T. Lam ◽

R. M. Watt ◽

L. J. Jin ◽

L. P. Samaranayake

Keyword(s):

Biofilm Formation ◽

Laser Scanning ◽

Significant Inhibition ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Dependent Manner ◽

Bacterial Lipopolysaccharides ◽

Species Specific ◽

Biofilm Development

The objective of this study was to evaluate the effect of the bacterial endotoxin LPS on Candida biofilm formation in vitro. The effect of the LPS of Pseudomonas aeruginosa, Klebsiella pneumoniae, Serratia marcescens and Salmonella typhimurium on six different species of Candida, comprising Candida albicans ATCC 90028, Candida glabrata ATCC 90030, Candida krusei ATCC 6258, Candida tropicalis ATCC 13803, Candida parapsilosis ATCC 22019 and Candida dubliniensis MYA 646, was studied using a standard biofilm assay. The metabolic activity of in vitro Candida biofilms treated with LPS at 90 min, 24 h and 48 h was quantified by XTT reduction assay. Viable biofilm-forming cells were qualitatively analysed using confocal laser scanning microscopy (CLSM), while scanning electron microscopy (SEM) was employed to visualize the biofilm structure. Initially, adhesion of C. albicans was significantly stimulated by Pseudomonas and Klebsiella LPS. A significant inhibition of Candida adhesion was noted for the following combinations: C. glabrata with Pseudomonas LPS, C. tropicalis with Serratia LPS, and C. glabrata, C. parapsilosis or C. dubliniensis with Salmonella LPS (P<0.05). After 24 h of incubation, a significant stimulation of initial colonization was noted for the following combinations: C. albicans/C. glabrata with Klebsiella LPS, C. glabrata/C. tropicalis/C. krusei with Salmonella LPS. In contrast, a significant inhibition of biofilm formation was observed in C. glabrata/C. dubliniensis/C. krusei with Pseudomonas LPS, C. krusei with Serratia LPS, C. dubliniensis with Klebsiella LPS and C. parapsilosis/C. dubliniensis /C. krusei with Salmonella LPS (P<0.05). On further incubation for 48 h, a significant enhancement of biofilm maturation was noted for the following combinations: C. glabrata/C. tropicalis with Serratia LPS, C. dubliniensis with Klebsiella LPS and C. glabrata with Salmonella LPS, and a significant retardation was noted for C. parapsilosis/C. dubliniensis/C. krusei with Pseudomonas LPS, C. tropicalis with Serratia LPS, C. glabrata/C. parapsilosis/C. dubliniensis with Klebsiella LPS and C. dubliniensis with Salmonella LPS (P<0.05). These findings were confirmed by SEM and CLSM analyses. In general, the inhibition of the biofilm development of LPS-treated Candida spp. was accompanied by a scanty architecture with a reduced numbers of cells compared with the profuse and densely colonized control biofilms. These data are indicative that bacterial LPSs modulate in vitro Candida biofilm formation in a species-specific and time-dependent manner. The clinical and the biological relevance of these findings have yet to be explored.

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The fate of medial edge epithelial cells during palatal fusion in vitro: an analysis by DiI labelling and confocal microscopy

Development ◽

10.1242/dev.114.2.379 ◽

1992 ◽

Vol 114 (2) ◽

pp. 379-388 ◽

Cited By ~ 5

Author(s):

M.J. Carette ◽

M.W. Ferguson

Keyword(s):

Laser Scanning ◽

Time Course ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Palatal Fusion ◽

Morphological Criteria ◽

Medial Edge ◽

Mesenchymal Transformation

Fusion of bilateral shelves, to form the definitive mammalian secondary palate, is critically dependent on removal of the medial edge cells that constitute the midline epithelial seam. Conflicting views suggest that programmed apoptotic death or epithelial-mesenchymal transformation of these cells is predominantly involved. Due in part to the potentially ambiguous interpretation of static images and the notable absence of fate mapping studies, the process by which this is achieved has, however, remained mechanistically equivocal. Using an in vitro mouse model, we have selectively labelled palatal epithelia with DiI and examined the fate of medial edge epithelial (MEE) cells during palatal fusion by localisation using a combination of conventional histology and confocal laser scanning microscopy (CLSM). In dynamic studies using CLSM, we have made repetitive observations of the same palatal cultures in time-course investigations. Our results concurred with the established morphological criteria of seam degeneration; however, they provided no evidence of MEE cell death or transformation. Instead we report that MEE cells migrate nasally and orally out of the seam and are recruited into, and constitute, epithelial triangles on both the oral and nasal aspects of the palate. Subsequently these cells become incorporated into the oral and nasal epithelia on the surface of the palate. We hypothesize an alternative method of seam degeneration in vivo which largely conserves the MEE population by recruiting it into the nasal and oral epithelia.

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Tyrosine phosphorylation following alterations in arteriolar intraluminal pressure and wall tension

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.2001.281.3.h1047 ◽

2001 ◽

Vol 281 (3) ◽

pp. H1047-H1056 ◽

Cited By ~ 15

Author(s):

Timothy V. Murphy ◽

Brian E. Spurrell ◽

Michael A. Hill

Keyword(s):

Tyrosine Phosphorylation ◽

Fluorescence Intensity ◽

Laser Scanning ◽

Time Course ◽

Transmural Pressure ◽

Laser Scanning Microscopy ◽

Intraluminal Pressure ◽

Confocal Laser ◽

Wall Tension ◽

Myogenic Activity

Arterioles respond to increased transmural pressure with myogenic constriction. The present study investigated the role of tyrosine phosphorylation in myogenic activity. Cannulated segments of a rat cremaster arteriole were fixed under pressure, followed by incubation with fluorescein isothiocyanate (FITC)-conjugated anti-phosphotyrosine. Smooth muscle cell fluorescence intensity was measured with the use of confocal laser-scanning microscopy. Anti-phosphotyrosine fluorescence intensity in muscle cells of arterioles maintained at 100 mmHg was reduced by the tyrosine kinase inhibitor tyrphostin A47 (30 μM) and increased by the tyrosine phosphatase inhibitor pervanadate (100 μM). In time-course experiments, anti-phosphotyrosine fluorescence increased slowly (over 5 min) after an acute increase in intraluminal pressure, and was dissociated from myogenic contraction (within 1 min). In contrast, angiotensin II (0.1 μM) caused rapid constriction and increased tyrosine phosphorylation. Anti-phosphotyrosine fluorescence was also pressure dependent (10–100 mmHg). Abolition of myogenic activity, either through removal of extracellular Ca2+, or exposure to verapamil (5 μM) or forskolin (0.1 μM) caused a further increase in anti-phosphotyrosine fluorescence. We conclude that transmural pressure and/or wall tension in arterioles causes increased tyrosine phosphorylation; however, this is not involved in the acute phase of myogenic constriction but may be involved in later responses, such as sustained myogenic tone or mechanisms possibly related to growth.

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Microbial Composition and Structure of Aerobic Granular Sewage Biofilms

Applied and Environmental Microbiology ◽

10.1128/aem.01002-07 ◽

2007 ◽

Vol 73 (19) ◽

pp. 6233-6240 ◽

Cited By ~ 109

Author(s):

S. D. Weber ◽

W. Ludwig ◽

K.-H. Schleifer ◽

J. Fried

Keyword(s):

Activated Sludge ◽

Laser Scanning ◽

Extracellular Polymeric Substances ◽

Phase 1 ◽

Simultaneous Detection ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Microbial Composition ◽

Granule Formation ◽

Biofilm Development

ABSTRACT Aerobic activated sludge granules are dense, spherical biofilms which can strongly improve purification efficiency and sludge settling in wastewater treatment processes. In this study, the structure and development of different granule types were analyzed. Biofilm samples originated from lab-scale sequencing batch reactors which were operated with malthouse, brewery, and artificial wastewater. Scanning electron microscopy, light microscopy, and confocal laser scanning microscopy together with fluorescence in situ hybridization (FISH) allowed insights into the structure of these biofilms. Microscopic observation revealed that granules consist of bacteria, extracellular polymeric substances (EPS), protozoa and, in some cases, fungi. The biofilm development, starting from an activated sludge floc up to a mature granule, follows three phases. During phase 1, stalked ciliated protozoa of the subclass Peritrichia, e.g., Epistylis spp., settle on activated sludge flocs and build tree-like colonies. The stalks are subsequently colonized by bacteria. During phase 2, the ciliates become completely overgrown by bacteria and die. Thereby, the cellular remnants of ciliates act like a backbone for granule formation. During phase 3, smooth, compact granules are formed which serve as a new substratum for unstalked ciliate swarmers settling on granule surfaces. These mature granules comprise a dense core zone containing bacterial cells and EPS and a loosely structured fringe zone consisting of either ciliates and bacteria or fungi and bacteria. Since granules can grow to a size of up to several millimeters in diameter, we developed and applied a modified FISH protocol for the study of cryosectioned biofilms. This protocol allows the simultaneous detection of bacteria, ciliates, and fungi in and on granules.

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