Advanced understanding of prokaryotic biofilm formation using a cost-effective and versatile multi-panel adhesion (mPAD) mount

AbstractMost microorganisms exist in biofilms, which comprise aggregates of cells surrounded by an extracellular matrix that provides protection from external stresses. Based on the conditions under which they form, biofilm structures vary in significant ways. For instance, biofilms that develop when microbes are incubated under static conditions differ from those formed when microbes encounter the shear forces of a flowing liquid. Moreover, biofilms develop dynamically over time. Here, we describe a cost-effective, 3D-printed coverslip holder that facilitates surface adhesion assays under a broad range of standing and shaking culture conditions. This multi-panel adhesion (mPAD) mount further allows cultures to be sampled at multiple time points, ensuring consistency and comparability between samples and enabling analyses of the dynamics of biofilm formation. As a proof of principle, using the mPAD mount for shaking, oxic cultures, we confirm previous flow chamber experiments showing that Pseudomonas aeruginosa wild type and a phenazine deletion mutant (Δphz) form similar biofilms. Extending this analysis to anoxic conditions, we reveal that microcolony and biofilm formation can only be observed under shaking conditions and are decreased in the Δphz mutant compared to wild-type cultures, indicating that phenazines are crucial for the formation of biofilms if oxygen as an electron acceptor is not available. Furthermore, while the model archaeon Haloferax volcanii does not require archaella for attachment to surfaces under static conditions, we demonstrate that H. volcanii mutants that lack archaella are negatively affected in their early stages of biofilm formation under shaking conditions.ImportanceDue to the versatility of the mPAD mount, we anticipate that it will aid the analysis of biofilm formation in a broad range of bacteria and archaea. Thereby, it contributes to answering critical biological questions about the regulatory and structural components of biofilm formation and understanding this process in a wide array of environmental, biotechnological, and medical contexts.

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Advanced understanding of prokaryotic biofilm formation using a cost-effective and versatile multi-panel adhesion (mPAD) mount

Applied and Environmental Microbiology ◽

10.1128/aem.02283-21 ◽

2022 ◽

Author(s):

Stefan Schulze ◽

Heather Schiller ◽

Jordan Solomonic ◽

Orkan Telhan ◽

Kyle Costa ◽

...

Keyword(s):

Biofilm Formation ◽

Cost Effective ◽

Flow Chamber ◽

Culture Conditions ◽

Multiple Time ◽

Wild Type ◽

Surface Attachment ◽

Flowing Liquid ◽

Multiple Time Points ◽

Static Conditions

Most microorganisms exist in biofilms, which comprise aggregates of cells surrounded by an extracellular matrix that provides protection from external stresses. Based on the conditions under which they form, biofilm structures vary in significant ways. For instance, biofilms that develop when microbes are incubated under static conditions differ from those formed when microbes encounter the shear forces of a flowing liquid. Moreover, biofilms develop dynamically over time. Here, we describe a cost-effective, 3D-printed coverslip holder that facilitates surface adhesion assays under a broad range of standing and shaking culture conditions. This multi-panel adhesion (mPAD) mount further allows cultures to be sampled at multiple time points, ensuring consistency and comparability between samples and enabling analyses of the dynamics of biofilm formation. As a proof of principle, using the mPAD mount for shaking, oxic cultures, we confirm previous flow chamber experiments showing that Pseudomonas aeruginosa wild type and a phenazine deletion mutant (Δ phz ) form biofilms with similar structure but reduced density in the mutant strain. Extending this analysis to anoxic conditions, we reveal that microcolony and biofilm formation can only be observed under shaking conditions and are decreased in the Δ phz mutant compared to wild-type cultures, indicating that phenazines are crucial for the formation of biofilms if oxygen as an electron acceptor is unavailable. Furthermore, while the model archaeon Haloferax volcanii does not require archaella for surface attachment under static conditions, we demonstrate that H. volcanii mutants that lack archaella are impaired in early stages of biofilm formation under shaking conditions. Importance: Due to the versatility of the mPAD mount, we anticipate that it will aid the analysis of biofilm formation in a broad range of bacteria and archaea. Thereby, it contributes to answering critical biological questions about the regulatory and structural components of biofilm formation and understanding this process in a wide array of environmental, biotechnological, and medical contexts.

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Bacterial Adhesion to Nanomodified Surfaces: Dynamic Flow Effects onS. aureusandP. aeruginosa

MRS Proceedings ◽

10.1557/opl.2013.241 ◽

2013 ◽

Vol 1498 ◽

pp. 79-84

Author(s):

Mary C. Machado ◽

Keiko M. Tarquinio ◽

Thomas J. Webster

Keyword(s):

Bacterial Adhesion ◽

Biofilm Formation ◽

Cost Effective ◽

Clinical Problem ◽

Flow Chamber ◽

High Morbidity ◽

Pressure Gradients ◽

Dynamic Flow ◽

Endotracheal Tubes ◽

Flow Effects

AbstractVentilator associated pneumonia (VAP) is a serious and costly clinical problem. Specifically, receiving mechanical ventilation over 24 hours increases the risk of VAP and is associated with high morbidity, mortality and medical costs. Cost effective endotracheal tubes (ETTs) that are resistant to bacterial infection would help to prevent this problem. The objective of this study was to determine differences in bacterial growth on nanomodified and unmodified ETTs under dynamic airway conditions. A bench top model based upon the general design of Hartmann et al. (1999) was constructed to test of the effectiveness of nanomodified ETTs under the airflow conditions present in the airway. Twenty-four hour studies performed in a dynamic flow chamber showed a marked difference in the biofilm formation on different areas of unmodified tubes. Areas where tubes were curved, such as at the entrance to the mouth and the connection between the oropharynx and the larynx, seemed to collect the largest amount of biofilm.The biofilm formation on ETTs in the airflow system after 24 hours showed a large difference depending upon where tubes were oriented within the apparatus. This illustrates the importance of dynamic flow on biofilm formation in pediatric ETTs. It is of particular interest that increased biofilm density on both unmodified and nanomodified tubes appeared to occur at curves in the tube where changes in flow pattern occurred. This emphasizes the need for more accurate models of airflow within pediatric ETTs, suggesting that not only does flow affect pressure gradients along the tube, but in fact, determines the composition of the film itself. More testing is needed to determine the effects of biofilm formation on the efficiency of ETT under airflow, however this study provides significant evidence for nanomodification alone (without the use of antibiotics) to decrease bacteria function.

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Time-resolved mapping of genetic interactions to model rewiring of signaling pathways

10.1101/384800 ◽

2018 ◽

Author(s):

Florian Heigwer ◽

Christian Scheeder ◽

Thilo Miersch ◽

Barbara Schmitt ◽

Claudia Blass ◽

...

Keyword(s):

Signaling Pathways ◽

Genetic Interactions ◽

Ras Signaling ◽

Multiple Time ◽

Time Resolved ◽

External Stresses ◽

Cellular Pathways ◽

Context Dependent ◽

Multiple Time Points ◽

Over Time

AbstractContext-dependent changes in genetic vulnerabilities are important to understand the wiring of cellular pathways and variations in different environmental conditions. However, methodological frameworks to investigate the plasticity of genetic networks over time or in response to external stresses are lacking. To analyze the plasticity of genetic interactions, we performed an arrayed combinatorial RNAi screen in Drosophila cells at multiple time points and after pharmacological inhibition of Ras signaling activity. Using an image-based morphology assay to capture a broad range of phenotypes, we assessed the effect of 12768 pairwise RNAi perturbations in six different conditions. We found that genetic interactions form in different trajectories and developed an algorithm, termed MODIFI, to analyze how genetic interactions rewire over time. Using this framework, we identified more statistically significant interactions compared to endpoints assays and further observed several examples of context-dependent crosstalk between signaling pathways such as an interaction between Ras and Rel which is dependent on MEK activity.

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Reduced Resistance to Air Flow from Nanomodified Endotracheal Tubes

MRS Proceedings ◽

10.1557/opl.2011.778 ◽

2011 ◽

Vol 1316 ◽

Author(s):

Mary C. Machado ◽

Keiko M. Tarquinio ◽

Thomas J. Webster

Keyword(s):

Biofilm Formation ◽

Cost Effective ◽

Clinical Problem ◽

Flow Chamber ◽

High Morbidity ◽

Ventilator Associated Pneumonia ◽

Pressure Gradients ◽

Dynamic Flow ◽

Endotracheal Tubes ◽

Significant Evidence

AbstractVentilator associated pneumonia (VAP) is a serious and costly clinical problem. Specifically, receiving mechanical ventilation over 24 hours increases the risk of VAP and is associated with high morbidity, mortality and medical costs. Cost effective endotracheal tubes (ETTs) that are resistant to bacterial infection would help to prevent this problem. The objective of this study was to determine differences in bacterial growth on nanomodified and unmodified ETTs under dynamic airway conditions, a bench top model based upon the general design of Hartmann et al. (1999) was constructed to test of the effectiveness of nanomodified ETTs under the airflow conditions present in the airway. Twenty-four hour studies performed in a dynamic flow chamber showed a marked difference in the biofilm formation on different areas of unmodified tubes. Areas where tubes were curved, such as at the entrance to the mouth and the connection between the oropharynx and the larynx, seemed to collect the largest amount of biofilm. On the nanomodified tubes biofilm formation was markedly different occurring on smaller pieces.The biofilm formation on ETTs in the airflow system after 24 hours showed a large difference depending upon where tubes were oriented within the apparatus. This illustrates the importance of dynamic flow on biofilm formation in pediatric ETTs. It is of particular interest that increased biofilm density on both unmodified and nanomodified tubes appeared to occur at curves in the tube where changes in flow pattern occured. This emphasizes the need for more accurate models of airflow within pediatric ETTs, suggesting that not only does flow affect pressure gradients along the tube, but in fact, determines the composition of the film itself. More testing is needed to determine the effects of biofilm formation on the efficiency of ETT under airflow, however this study provides significant evidence for nanomodification alone (without the use of antibiotics) to decrease bacteria function.

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Motility and Chemotaxis in Agrobacterium tumefaciens Surface Attachment and Biofilm Formation

Journal of Bacteriology ◽

10.1128/jb.00566-07 ◽

2007 ◽

Vol 189 (22) ◽

pp. 8005-8014 ◽

Cited By ~ 112

Author(s):

Peter M. Merritt ◽

Thomas Danhorn ◽

Clay Fuqua

Keyword(s):

Agrobacterium Tumefaciens ◽

Biofilm Formation ◽

Bacterial Species ◽

Wild Type ◽

Swarming Motility ◽

Surface Attachment ◽

Directed Motility ◽

Suppressor Mutants ◽

Static Conditions ◽

Specific Deletion

ABSTRACT Bacterial motility mechanisms, including swimming, swarming, and twitching, are known to have important roles in biofilm formation, including colonization and the subsequent expansion into mature structured surface communities. Directed motility requires chemotaxis functions that are conserved among many bacterial species. The biofilm-forming plant pathogen Agrobacterium tumefaciens drives swimming motility by utilizing a small group of flagella localized to a single pole or the subpolar region of the cell. There is no evidence for twitching or swarming motility in A. tumefaciens. Site-specific deletion mutations that resulted in either aflagellate, flagellated but nonmotile, or flagellated but nonchemotactic A. tumefaciens derivatives were examined for biofilm formation under static and flowing conditions. Nonmotile mutants were significantly deficient in biofilm formation under static conditions. Under flowing conditions, however, the aflagellate mutant rapidly formed aberrantly dense, tall biofilms. In contrast, a nonmotile mutant with unpowered flagella was clearly debilitated for biofilm formation relative to the wild type. A nontumbling chemotaxis mutant was only weakly affected with regard to biofilm formation under nonflowing conditions but was notably compromised in flow, generating less adherent biomass than the wild type, with a more dispersed cellular arrangement. Extragenic suppressor mutants of the chemotaxis-impaired, straight-swimming phenotype were readily isolated from motility agar plates. These mutants regained tumbling at a frequency similar to that of the wild type. Despite this phenotype, biofilm formation by the suppressor mutants in static cultures was significantly deficient. Under flowing conditions, a representative suppressor mutant manifested a phenotype similar to yet distinct from that of its nonchemotactic parent.

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Glycoproteomics of Haloferax volcanii reveals an extensive glycoproteome and concurrence of different N-glycosylation pathways

10.1101/2021.01.21.427637 ◽

2021 ◽

Author(s):

Stefan Schulze ◽

Friedhelm Pfeiffer ◽

Benjamin A. Garcia ◽

Mechthild Pohlschroder

Keyword(s):

Biofilm Formation ◽

Cell Biology ◽

Culture Conditions ◽

Haloferax Volcanii ◽

Spectrometric Analysis ◽

Wild Type ◽

Cellular Processes ◽

Phenotypic Characterisation ◽

Mutant Strains ◽

Glycosylation Pathway

AbstractGlycosylation is one of the most complex post-translational protein modifications. Its importance has been established not only for eukaryotes but also for a variety of prokaryotic cellular processes, such as biofilm formation, motility and mating. However, comprehensive glycoproteomic analyses are largely missing in prokaryotes. Here we extend the phenotypic characterisation of N-glycosylation pathway mutants in Haloferax volcanii and provide a detailed glycoproteome for this model archaeon through the mass spectrometric analysis of intact glycopeptides. Using in-depth glycoproteomic datasets generated for the wild-type and mutant strains as well as a reanalysis of datasets within the Archaeal Proteome Project, we identify the largest archaeal glycoproteome described so far. We further show that different N-glycosylation pathways can modify the same glycosites under the same culture conditions. The extent and complexity of the Hfx. volcanii N-glycoproteome revealed here provides new insights into the roles of N-glycosylation in archaeal cell biology.

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Culture Conditions of Roseobacter Strain 27-4 Affect Its Attachment and Biofilm Formation as Quantified by Real-Time PCR

Applied and Environmental Microbiology ◽

10.1128/aem.72.4.3011-3015.2006 ◽

2006 ◽

Vol 72 (4) ◽

pp. 3011-3015 ◽

Cited By ~ 28

Author(s):

Jesper Bartholin Bruhn ◽

Janus Anders Juul Haagensen ◽

Dorthe Bagge-Ravn ◽

Lone Gram

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Biofilm Formation ◽

Three Dimensional ◽

Growth Conditions ◽

Culture Conditions ◽

Probiotic Bacterium ◽

Antibacterial Compound ◽

Pcr Method ◽

Static Conditions

ABSTRACT The fish probiotic bacterium Roseobacter strain 27-4 grows only as rosettes and produces its antibacterial compound under static growth conditions. It forms three-dimensional biofilms when precultured under static conditions. We quantified attachment of Roseobacter strain 27-4 using a direct real-time PCR method and demonstrated that the bacteria attached more efficiently to surfaces during static growth than under aerated conditions.

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Comprehensive glycoproteomics shines new light on the complexity and extent of glycosylation in archaea

PLoS Biology ◽

10.1371/journal.pbio.3001277 ◽

2021 ◽

Vol 19 (6) ◽

pp. e3001277

Author(s):

Stefan Schulze ◽

Friedhelm Pfeiffer ◽

Benjamin A. Garcia ◽

Mechthild Pohlschroder

Keyword(s):

Biofilm Formation ◽

Cell Biology ◽

Culture Conditions ◽

Phenotypic Characterization ◽

Spectrometric Analysis ◽

Wild Type ◽

Cellular Processes ◽

Mutant Strains ◽

Glycosylation Pathway

Glycosylation is one of the most complex posttranslational protein modifications. Its importance has been established not only for eukaryotes but also for a variety of prokaryotic cellular processes, such as biofilm formation, motility, and mating. However, comprehensive glycoproteomic analyses are largely missing in prokaryotes. Here, we extend the phenotypic characterization of N-glycosylation pathway mutants in Haloferax volcanii and provide a detailed glycoproteome for this model archaeon through the mass spectrometric analysis of intact glycopeptides. Using in-depth glycoproteomic datasets generated for the wild-type (WT) and mutant strains as well as a reanalysis of datasets within the Archaeal Proteome Project (ArcPP), we identify the largest archaeal glycoproteome described so far. We further show that different N-glycosylation pathways can modify the same glycosites under the same culture conditions. The extent and complexity of the Hfx. volcanii N-glycoproteome revealed here provide new insights into the roles of N-glycosylation in archaeal cell biology.

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Production of Antibacterial Compounds and Biofilm Formation by Roseobacter Species Are Influenced by Culture Conditions

Applied and Environmental Microbiology ◽

10.1128/aem.02238-06 ◽

2006 ◽

Vol 73 (2) ◽

pp. 442-450 ◽

Cited By ~ 100

Author(s):

Jesper Bartholin Bruhn ◽

Lone Gram ◽

Robert Belas

Keyword(s):

Biofilm Formation ◽

Selective Advantage ◽

Growth Conditions ◽

Culture Conditions ◽

Rosette Formation ◽

Antibacterial Compounds ◽

Antibacterial Compound ◽

Marine Algal ◽

Static Conditions ◽

Air Liquid Interface

ABSTRACT Bacterial communities associated with marine algae are often dominated by members of the Roseobacter clade, and in the present study, we describe Roseobacter phenotypes that may provide this group of bacteria with selective advantages when colonizing this niche. Nine of 14 members of the Roseobacter clade, of which half were isolated from cultures of the dinoflagellate Pfiesteria piscicida, produced antibacterial compounds. Many non-Roseobacter marine bacteria were inhibited by sterile filtered supernatants of Silicibacter sp. TM1040 and Phaeobacter (formerly Roseobacter) strain 27-4, which had the highest production of antibacterial compound. In contrast, Roseobacter strains were susceptible only when exposed to concentrated compound. The production of antibacterial compound was influenced by the growth conditions, as production was most pronounced when bacteria were grown in liquid medium under static conditions. Under these conditions, Silicibacter sp. TM1040 cells attached to one another, forming rosettes, as has previously been reported for Phaeobacter 27-4. A spontaneous Phaeobacter 27-4 mutant unable to form rosettes was also defective in biofilm formation and the production of antibacterial compound, indicating a possible link between these phenotypes. Rosette formation was observed in 8 of 14 Roseobacter clade strains examined and was very pronounced under static growth in 5 of these strains. Attachment to surfaces and biofilm formation at the air-liquid interface by these five strains was greatly facilitated by growth conditions that favored rosette formation, and rosette-forming strains were 13 to 30 times more efficient in attaching to glass compared to strains under conditions where rosette formation was not pronounced. We hypothesize that the ability to produce antibacterial compounds that principally inhibit non-Roseobacter species, combined with an enhancement in biofilm formation, may give members of the Roseobacter clade a selective advantage and help to explain the dominance of members of this clade in association with marine algal microbiota.

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Isolation and characterization of a motility-defective mutant of Euglena gracilis

PeerJ ◽

10.7717/peerj.10002 ◽

2020 ◽

Vol 8 ◽

pp. e10002

Author(s):

Shuki Muramatsu ◽

Kohei Atsuji ◽

Koji Yamada ◽

Kazunari Ozasa ◽

Hideyuki Suzuki ◽

...

Keyword(s):

Mutant Strain ◽

Euglena Gracilis ◽

Model Organism ◽

Cost Effective ◽

Industrial Applications ◽

Culture Conditions ◽

Wild Type ◽

Mass Cultivation ◽

Isolation And Characterization ◽

Defective Mutant

Euglena gracilis is a green photosynthetic microalga that swims using its flagellum. This species has been used as a model organism for over half a century to study its metabolism and the mechanisms of its behavior. The development of mass-cultivation technology has led to E. gracilis application as a feedstock in various products such as foods. Therefore, breeding of E. gracilis has been attempted to improve the productivity of this feedstock for potential industrial applications. For this purpose, a characteristic that preserves the microalgal energy e.g., reduces motility, should be added to the cultivars. The objective of this study was to verify our hypothesis that E. gracilis locomotion-defective mutants are suitable for industrial applications because they save the energy required for locomotion. To test this hypothesis, we screened for E. gracilis mutants from Fe-ion-irradiated cell suspensions and established a mutant strain, ${\mathrm{M}}_{3}^{-}$ZFeL, which shows defects in flagellum formation and locomotion. The mutant strain exhibits a growth rate comparable to that of the wild type when cultured under autotrophic conditions, but had a slightly slower growth under heterotrophic conditions. It also stores 1.6 times the amount of paramylon, a crystal of β-1,3-glucan, under autotrophic culture conditions, and shows a faster sedimentation compared with that of the wild type, because of the deficiency in mobility and probably the high amount of paramylon accumulation. Such characteristics make E. gracilis mutant cells suitable for cost-effective mass cultivation and harvesting.

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