scholarly journals BigR, a Transcriptional Repressor from Plant-Associated Bacteria, Regulates an Operon Implicated in Biofilm Growth

2007 ◽  
Vol 189 (17) ◽  
pp. 6185-6194 ◽  
Author(s):  
Rosicler L. Barbosa ◽  
Celso E. Benedetti
Keyword(s):  
Biofilm Formation ◽  
Molecular Mechanisms ◽  
Constitutive Expression ◽  
Vascular Occlusion ◽  
Bacterial Biofilm ◽  
Plant Interactions ◽  
Biofilm Growth ◽  
Associated Bacteria ◽  
Tobacco Roots ◽  
Biofilm Development

ABSTRACT Xylella fastidiosa is a plant pathogen that colonizes the xylem vessels, causing vascular occlusion due to bacterial biofilm growth. However, little is known about the molecular mechanisms driving biofilm formation in Xylella-plant interactions. Here we show that BigR (for “biofilm growth-associated repressor”) is a novel helix-turn-helix repressor that controls the transcription of an operon implicated in biofilm growth. This operon, which encodes BigR, membrane proteins, and an unusual beta-lactamase-like hydrolase (BLH), is restricted to a few plant-associated bacteria, and thus, we sought to understand its regulation and function in X. fastidiosa and Agrobacterium tumefaciens. BigR binds to a palindromic AT-rich element (the BigR box) in the Xylella and Agrobacterium blh promoters and strongly represses the transcription of the operon in these cells. The BigR box overlaps with two alternative −10 regions identified in the blh promoters, and mutations in this box significantly affected transcription, indicating that BigR competes with the RNA polymerase for the same promoter site. Although BigR is similar to members of the ArsR/SmtB family of regulators, our data suggest that, in contrast to the initial prediction, it does not act as a metal sensor. Increased activity of the BigR operon was observed in both Xylella and Agrobacterium biofilms. In addition, an A. tumefaciens bigR mutant showed constitutive expression of operon genes and increased biofilm formation on glass surfaces and tobacco roots, indicating that the operon may play a role in cell adherence or biofilm development.

scholarly journals Metabolic Remodeling during Biofilm Development ofBacillus subtilis

mBio ◽  
2019 ◽  
Vol 10 (3) ◽  
Author(s):  
Tippapha Pisithkul ◽  
Jeremy W. Schroeder ◽  
Edna A. Trujillo ◽  
Ponlkrit Yeesin ◽  
David M. Stevenson ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Regulatory Networks ◽  
Developmental Process ◽  
Central Carbon Metabolism ◽  
Bacterial Biofilm ◽  
Biofilm Growth ◽  
Time Resolved ◽  
Content Type ◽  
Metabolic Remodeling ◽  
Biofilm Development

ABSTRACTBiofilms are structured communities of tightly associated cells that constitute the predominant state of bacterial growth in natural and human-made environments. Although the core genetic circuitry that controls biofilm formation in model bacteria such asBacillus subtilishas been well characterized, little is known about the role that metabolism plays in this complex developmental process. Here, we performed a time-resolved analysis of the metabolic changes associated with pellicle biofilm formation and development inB. subtilisby combining metabolomic, transcriptomic, and proteomic analyses. We report surprisingly widespread and dynamic remodeling of metabolism affecting central carbon metabolism, primary biosynthetic pathways, fermentation pathways, and secondary metabolism. Most of these metabolic alterations were hitherto unrecognized as biofilm associated. For example, we observed increased activity of the tricarboxylic acid (TCA) cycle during early biofilm growth, a shift from fatty acid biosynthesis to fatty acid degradation, reorganization of iron metabolism and transport, and a switch from acetate to acetoin fermentation. Close agreement between metabolomic, transcriptomic, and proteomic measurements indicated that remodeling of metabolism during biofilm development was largely controlled at the transcriptional level. Our results also provide insights into the transcription factors and regulatory networks involved in this complex metabolic remodeling. Following upon these results, we demonstrated that acetoin production via acetolactate synthase is essential for robust biofilm growth and has the dual role of conserving redox balance and maintaining extracellular pH. This report represents a comprehensive systems-level investigation of the metabolic remodeling occurring duringB. subtilisbiofilm development that will serve as a useful road map for future studies on biofilm physiology.IMPORTANCEBacterial biofilms are ubiquitous in natural environments and play an important role in many clinical, industrial, and ecological settings. Although much is known about the transcriptional regulatory networks that control biofilm formation in model bacteria such asBacillus subtilis, very little is known about the role of metabolism in this complex developmental process. To address this important knowledge gap, we performed a time-resolved analysis of the metabolic changes associated with bacterial biofilm development inB. subtilisby combining metabolomic, transcriptomic, and proteomic analyses. Here, we report a widespread and dynamic remodeling of metabolism affecting central carbon metabolism, primary biosynthetic pathways, fermentation pathways, and secondary metabolism. This report serves as a unique hypothesis-generating resource for future studies on bacterial biofilm physiology. Outside the biofilm research area, this work should also prove relevant to any investigators interested in microbial physiology and metabolism.

Bifunctional Composites for Biofilms Modulation on Cervical Restorations

2021 ◽  
pp. 002203452110181
Author(s):  
A.A. Balhaddad ◽  
I.M. Garcia ◽  
L. Mokeem ◽  
M.S. Ibrahim ◽  
F.M. Collares ◽  
...  
Keyword(s):  
Free Energy ◽  
Surface Free Energy ◽  
Contact Angles ◽  
Bacterial Biofilm ◽  
Dental Composite ◽  
Log P ◽  
Rrna Gene ◽  
Cervical Lesions ◽  
Biofilm Growth ◽  
Biofilm Development

Cervical composites treating root carious and noncarious cervical lesions usually extend subgingivally. The subgingival margins of composites present poor plaque control, enhanced biofilm accumulation, and cause gingival irritation. A potential material to restore such lesions should combine agents that interfere with bacterial biofilm development and respond to acidic conditions. Here, we explore the use of new bioresponsive bifunctional dental composites against mature microcosm biofilms derived from subgingival plaque samples. The designed formulations contain 2 bioactive agents: dimethylaminohexadecyl methacrylate (DMAHDM) at 3 to 5 wt.% and 20 wt.% nanosized amorphous calcium phosphate (NACP) in a base resin. Composites with no DMAHDM and NACP were used as controls. The newly formulated 5% DMAHDM–20% NACP composite was analyzed by micro-Raman spectroscopy and transmission electron microscopy. The wettability and surface-free energy were also assessed. The inhibitory effect on the in vitro biofilm growth and the 16S rRNA gene sequencing of survival bacterial colonies derived from the composites were analyzed. Whole-biofilm metabolic activity, polysaccharide production, and live/dead images of the biofilm grown over the composites complement the microbiological assays. Overall, the designed formulations had higher contact angles with water and lower surface-free energy compared to the commercial control. The DMAHDM-NACP composites significantly inhibited the growth of total microorganisms, Porphyromonas gingivalis, Prevotella intermedia/nigrescens, Aggregatibacter actinomycetemcomitans, and Fusobacterium nucleatum by 3 to 5-log ( P < 0.001). For the colony isolates from control composites, the composition was typically dominated by the genera Veillonella, Fusobacterium, Streptococcus, Eikenella, and Leptotrichia, while Fusobacterium and Veillonella dominated the 5% DMAHDM–20% NACP composites. The DMAHDM-NACP composites contributed to over 80% of reduction in metabolic and polysaccharide activity. The suppression effect on plaque biofilms suggested that DMAHDM-NACP composites might be used as a bioactive material for cervical restorations. These results may propose an exciting path to prevent biofilm growth and improve dental composite restorations’ life span.

scholarly journals Improvement of XTT assay performance for studies involving Candida albicans biofilms

2008 ◽  
Vol 19 (4) ◽  
pp. 364-369 ◽  
Author(s):  
Wander José da Silva ◽  
Jayampath Seneviratne ◽  
Nipuna Parahitiyawa ◽  
Edvaldo Antonio Ribeiro Rosa ◽  
Lakshman Perera Samaranayake ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Biofilm Formation ◽  
Cell Activity ◽  
Growth Yield ◽  
Xtt Assay ◽  
Biofilm Growth ◽  
Assay Performance ◽  
Reduction Assay ◽  
Cell Layers ◽  
Biofilm Development

2, 3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide (XTT) reduction assay has been used to study Candida biofilm formation. However, considering that the XTT reduction assay is dependent on cell activity, its use for evaluating mature biofilms may lead to inaccuracies since biofilm bottom cell layers tend to be relatively quiescent at later stages of biofilm formation. The aim of this study was to improve XTT reduction assay by adding glucose supplements to the standard XTT formulation. Candida albicans ATCC 90028 was used to form 24-, 48- and 72-h biofilms. The oxidative activity at 90, 180 and 270 min of incubation was evaluated. The control consisted of standard XTT formulation without glucose supplements, and was modified by the addition of 50, 100 and 200 mM of glucose. The XTT assay with 200 mM glucose showed more accurate and consistent readings correlating with biofilm development at 24, 48 and 72 h. Biofilm growth yield after 180 min incubation, when evaluated with the 200 mM glucose supplemented XTT, produced the most consistent readings on repetitive testing. It may be concluded that glucose supplementation of XTT could minimize variation and produce more accurate data for the XTT assay.

scholarly journals The Histidine Kinase BinK Is a Negative Regulator of Biofilm Formation and Squid Colonization

2016 ◽  
Vol 198 (19) ◽  
pp. 2596-2607 ◽  
Author(s):  
John F. Brooks ◽  
Mark J. Mandel
Keyword(s):  
Histidine Kinase ◽  
Biofilm Formation ◽  
Bacterial Biofilm ◽  
Negative Regulator ◽  
Epithelial Tissue ◽  
Temperature Modulation ◽  
Content Type ◽  
Animal Hosts ◽  
Biofilm Development

ABSTRACTBacterial colonization of animal epithelial tissue is a dynamic process that relies on precise molecular communication. Colonization ofEuprymna scolopesbobtail squid byVibrio fischeribacteria requires bacterial aggregation in host mucus as the symbiont transitions from a planktonic lifestyle in seawater to a biofilm-associated state in the host. We have identified a gene,binK(biofilm inhibitor kinase; VF_A0360), which encodes an orphan hybrid histidine kinase that negatively regulates theV. fischerisymbiotic biofilm (Syp)in vivoandin vitro. We identifiedbinKmutants as exhibiting a colonization advantage in a global genetic screen, a phenotype that we confirmed in controlled competition experiments. Bacterial biofilm aggregates in the host are larger in strains lacking BinK, whereas overexpression of BinK suppresses biofilm formation and squid colonization. Signaling through BinK is required for temperature modulation of biofilm formation at 28°C. Furthermore, we present evidence that BinK acts upstream of SypG, the σ54-dependent transcriptional regulator of thesypbiofilm locus. The BinK effects are dependent on intact signaling in the RscS-Syp biofilm pathway. Therefore, we propose that BinK antagonizes the signal from RscS and serves as an integral component inV. fischeribiofilm regulation.IMPORTANCEBacterial lifestyle transitions underlie the colonization of animal hosts from environmental reservoirs. Formation of matrix-enclosed, surface-associated aggregates (biofilms) is common in beneficial and pathogenic associations, but investigating the genetic basis of biofilm development in live animal hosts remains a significant challenge. Using the bobtail squid light organ as a model, we analyzed putative colonization factors and identified a histidine kinase that negatively regulates biofilm formation at the host interface. This work reveals a novelin vivobiofilm regulator that influences the transition of bacteria from their planktonic state in seawater to tight aggregates of cells in the host. The study enriches our understanding of biofilm regulation and beneficial colonization by an animal's microbiome.

scholarly journals Extracellular DNA and Type IV Pilus Expression Regulate the Structure and Kinetics of Biofilm Formation by Nontypeable Haemophilus influenzae

mBio ◽  
2017 ◽  
Vol 8 (6) ◽  
Author(s):  
Jayajit Das ◽  
Elaine Mokrzan ◽  
Vinal Lakhani ◽  
Lucia Rosas ◽  
Joseph A. Jurcisek ◽  
...  
Keyword(s):  
Middle Ear ◽  
In Silico ◽  
Biofilm Formation ◽  
Rational Design ◽  
Bacterial Biofilm ◽  
Confocal Imaging ◽  
Extracellular Dna ◽  
Fractal Structures ◽  
Biofilm Development

ABSTRACT Biofilms formed in the middle ear by nontypeable Haemophilus influenzae (NTHI) are central to the chronicity, recurrence, and refractive nature of otitis media (OM). However, mechanisms that underlie the emergence of specific NTHI biofilm structures are unclear. We combined computational analysis tools and in silico modeling rooted in statistical physics with confocal imaging of NTHI biofilms formed in vitro during static culture in order to identify mechanisms that give rise to distinguishing morphological features. Our analysis of confocal images of biofilms formed by NTHI strain 86-028NP using pair correlations of local bacterial densities within sequential planes parallel to the substrate showed the presence of fractal structures of short length scales (≤10 μm). The in silico modeling revealed that extracellular DNA (eDNA) and type IV pilus (Tfp) expression played important roles in giving rise to the fractal structures and allowed us to predict a substantial reduction of these structures for an isogenic mutant (ΔcomE) that was significantly compromised in its ability to release eDNA into the biofilm matrix and had impaired Tfp function. This prediction was confirmed by analysis of confocal images of in vitro ΔcomE strain biofilms. The fractal structures potentially generate niches for NTHI survival in the hostile middle ear microenvironment by dramatically increasing the contact area of the biofilm with the surrounding environment, facilitating nutrient exchange, and by generating spatial positive feedback to quorum signaling. IMPORTANCE NTHI is a major bacterial pathogen for OM, which is a common ear infection in children worldwide. Chronic OM is associated with bacterial biofilm formation in the middle ear; therefore, knowledge of the mechanisms that underlie NTHI biofilm formation is important for the development of therapeutic strategies for NTHI-associated OM. Our combined approach using confocal imaging of NTHI biofilms formed in vitro and mathematical tools for analysis of pairwise density correlations and agent-based modeling revealed that eDNA and Tfp expression were important factors in the development of fractal structures in NTHI biofilms. These structures may help NTHI survive in hostile environments, such as the middle ear. Our in silico model can be used in combination with laboratory or animal modeling studies to further define the mechanisms that underlie NTHI biofilm development during OM and thereby guide the rational design of, and optimize time and cost for, benchwork and preclinical studies. IMPORTANCE NTHI is a major bacterial pathogen for OM, which is a common ear infection in children worldwide. Chronic OM is associated with bacterial biofilm formation in the middle ear; therefore, knowledge of the mechanisms that underlie NTHI biofilm formation is important for the development of therapeutic strategies for NTHI-associated OM. Our combined approach using confocal imaging of NTHI biofilms formed in vitro and mathematical tools for analysis of pairwise density correlations and agent-based modeling revealed that eDNA and Tfp expression were important factors in the development of fractal structures in NTHI biofilms. These structures may help NTHI survive in hostile environments, such as the middle ear. Our in silico model can be used in combination with laboratory or animal modeling studies to further define the mechanisms that underlie NTHI biofilm development during OM and thereby guide the rational design of, and optimize time and cost for, benchwork and preclinical studies.

Biofilm formation by a biotechnologically important tropical marine yeast isolate, Yarrowia lipolytica NCIM 3589

2008 ◽  
Vol 58 (6) ◽  
pp. 1221-1229 ◽  
Author(s):  
D. H. Dusane ◽  
Y. V. Nancharaiah ◽  
V. P. Venugopalan ◽  
A. R. Kumar ◽  
S. S. Zinjarde
Keyword(s):  
Yarrowia Lipolytica ◽  
Biofilm Formation ◽  
Edible Oils ◽  
Carbon Sources ◽  
Three Dimensional ◽  
Ecological Niches ◽  
Lag Phase ◽  
Biofilm Growth ◽  
Water Media ◽  
Biofilm Development

Biofilm formation by Yarrowia lipolytica, a biotechnologically important fungus in microtitre plates, on glass slide surfaces and in flow cell was investigated. In microtitre plates, there was a short lag phase of adhesion followed by a period of rapid biofilm growth. The fungus formed extensive biofilms on glass slides, whereas in flow-cells a multicellular, three-dimensional microcolony structure was observed. The isolate formed biofilms in seawater and in fresh water media at neutral pH when grown in microtitre plates. The carbon sources differentially affected formation of biofilms in microtitre plates. Lactic acid, erythritol, glycerol, glucose and edible oils supported the formation of biofilms, while alkanes resulted in sub-optimal biofilm development. A variation in the morphology of the fungus was observed with different carbon sources. The results point to the possible existence of highly structured biofilms in varied ecological niches from where the yeast is isolated.

scholarly journals Architectural transitions in Vibrio cholerae biofilms at single-cell resolution

2016 ◽  
Vol 113 (14) ◽  
pp. E2066-E2072 ◽  
Author(s):  
Knut Drescher ◽  
Jörn Dunkel ◽  
Carey D. Nadell ◽  
Sven van Teeffelen ◽  
Ivan Grnja ◽  
...  
Keyword(s):  
Single Cell ◽  
Vibrio Cholerae ◽  
Biofilm Formation ◽  
Bacterial Species ◽  
Bacterial Biomass ◽  
Biofilm Growth ◽  
Glass Substrates ◽  
Critical Transitions ◽  
Cell Shapes ◽  
Biofilm Development

Many bacterial species colonize surfaces and form dense 3D structures, known as biofilms, which are highly tolerant to antibiotics and constitute one of the major forms of bacterial biomass on Earth. Bacterial biofilms display remarkable changes during their development from initial attachment to maturity, yet the cellular architecture that gives rise to collective biofilm morphology during growth is largely unknown. Here, we use high-resolution optical microscopy to image all individual cells in Vibrio cholerae biofilms at different stages of development, including colonies that range in size from 2 to 4,500 cells. From these data, we extracted the precise 3D cellular arrangements, cell shapes, sizes, and global morphological features during biofilm growth on submerged glass substrates under flow. We discovered several critical transitions of the internal and external biofilm architectures that separate the major phases of V. cholerae biofilm growth. Optical imaging of biofilms with single-cell resolution provides a new window into biofilm formation that will prove invaluable to understanding the mechanics underlying biofilm development.

scholarly journals purL gene expression affects biofilm formation and symbiotic persistence of Photorhabdus temperata in the nematode Heterorhabditis bacteriophora

Microbiology ◽  
2011 ◽  
Vol 157 (9) ◽  
pp. 2595-2603 ◽  
Author(s):  
Ruisheng An ◽  
Parwinder S. Grewal
Keyword(s):  
Gene Expression ◽  
Metabolic Pathway ◽  
Biofilm Formation ◽  
Heterorhabditis Bacteriophora ◽  
Bacterial Biofilm ◽  
Infective Juvenile ◽  
Plant Interactions ◽  
Symbiotic Interaction ◽  
The Impact ◽  
Photorhabdus Temperata

Extensive studies of the well-known legume and rhizobium symbiosis model system suggest that the purine metabolic pathway plays a key role in microbe–plant interactions, although the exact mechanism is unknown. Here, we report the impact of a key purine metabolic gene, purL, on the symbiotic interaction between the bacterium Photorhabdus temperata and its nematode partner Heterorhabditis bacteriophora. Real-time PCR assays showed that the purL gene was upregulated in P. temperata in the nematode infective juvenile compared with artificial media. Mutation of the purL gene by in-frame deletion dramatically decreased the capacity of the bacterium to persist in infective juveniles and its ability to form biofilm in vitro. It was further demonstrated that purL gene expression was positively related to bacterial biofilm formation and the symbiotic persistence of the bacterium in nematode infective juveniles. A ΔpurL mutant lost the ability to support infective juvenile formation in the media which weakly supported biofilm formation, suggesting that a critical level of biofilm formation is required by the bacteria to support infective juvenile formation and thus establish their partnership. In addition, the defects in both biofilm formation and symbiotic ability due to the disruption of the purL gene could be partially restored by the addition of 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), an intermediate of the purine biosynthesis pathway. Overall, these data indicate that the purine metabolic pathway is important in microbe–animal symbioses, and that it may influence symbiotic interactions at the level of biofilm formation.

scholarly journals Phenotypic Characterization of Streptococcus pneumoniae Biofilm Development

2006 ◽  
Vol 188 (7) ◽  
pp. 2325-2335 ◽  
Author(s):  
Magee Allegrucci ◽  
F. Z. Hu ◽  
K. Shen ◽  
J. Hayes ◽  
Garth D. Ehrlich ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Biofilm Formation ◽  
Protein Identification ◽  
Development Process ◽  
Developmental Stages ◽  
Developmental Process ◽  
Phenotypic Characterization ◽  
Biofilm Growth ◽  
Cell Counts ◽  
Biofilm Development

ABSTRACT Streptococcus pneumoniae is among the most common pathogens associated with chronic otitis media with effusion, which has been hypothesized to be a biofilm disease. S. pneumoniae has been shown to form biofilms, however, little is known about the developmental process, the architecture, and the changes that occur upon biofilm development. In the current study we made use of a continuous-culture biofilm system to characterize biofilm development of 14 different S. pneumoniae strains representing at least 10 unique serotypes. The biofilm development process was found to occur in three distinct stages, including initial attachment, cluster formation, and biofilm maturation. While all 14 pneumococcal strains displayed similar developmental stages, the mature biofilm architecture differed significantly among the serotypes tested. Overall, three biofilm architectural groups were detected based on biomass, biofilm thickness, and cluster size. The biofilm viable cell counts and total protein concentration increased steadily over the course of biofilm development, reaching ∼8 × 108 cells and ∼15 mg of protein per biofilm after 9 days of biofilm growth. Proteomic analysis confirmed the presence of distinct biofilm developmental stages by the detection of multiple phenotypes over the course of biofilm development. The biofilm development process was found to correlate not only with differential production of proteins but also with a dramatic increase in the number of detectable proteins, indicating that biofilm formation by S. pneumoniae may be a far more complex process than previously anticipated. Protein identification revealed that proteins involved in virulence, adhesion, and resistance were more abundant under biofilm growth conditions. A possible role of the identified proteins in biofilm formation is discussed.

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