scholarly journals Functional Genomics of Enterococcus faecalis: Multiple Novel Genetic Determinants for Biofilm Formation in the Core Genome

2009 ◽  
Vol 191 (8) ◽  
pp. 2806-2814 ◽  
Author(s):  
Katie S. Ballering ◽  
Christopher J. Kristich ◽  
Suzanne M. Grindle ◽  
Ana Oromendia ◽  
David T. Beattie ◽  
...  
Keyword(s):  
Enterococcus Faecalis ◽  
Biofilm Formation ◽  
Core Genome ◽  
Genetic Locus ◽  
Regulatory Protein ◽  
Genetic Determinants ◽  
Genetic Loci ◽  
Putative Promoter Region ◽  
Biofilm Growth ◽  
The Core

ABSTRACT The ability of Enterococcus faecalis to form robust biofilms on host tissues and on abiotic surfaces such as catheters likely plays a major role in the pathogenesis of opportunistic antibiotic-resistant E. faecalis infections and in the transfer of antibiotic resistance genes. We have carried out a comprehensive analysis of genetic determinants of biofilm formation in the core genome of E. faecalis. Here we describe 68 genetic loci predicted to be involved in biofilm formation that were identified by recombinase in vivo expression technology (RIVET); most of these genes have not been studied previously. Differential expression of a number of these determinants during biofilm growth was confirmed by quantitative reverse transcription-PCR, and genetic complementation studies verified a role in biofilm formation for several candidate genes. Of particular interest was genetic locus EF1809, predicted to encode a regulatory protein of the GntR family. We isolated 14 independent nonsibling clones containing the putative promoter region for this gene in the RIVET screen; EF1809 also showed the largest increase in expression during biofilm growth of any of the genes tested. Since an in-frame deletion of EF1809 resulted in a severe biofilm defect that could be complemented by the cloned wild-type gene, we have designated EF1809 ebrA (enterococcal biofilm regulator). Most of the novel genetic loci identified in our studies are highly conserved in gram-positive bacterial pathogens and may thus constitute a pool of uncharacterized genes involved in biofilm formation that may be useful targets for drug discovery.

scholarly journals Development and Use of an Efficient System for Random mariner Transposon Mutagenesis To Identify Novel Genetic Determinants of Biofilm Formation in the Core Enterococcus faecalis Genome

2008 ◽  
Vol 74 (11) ◽  
pp. 3377-3386 ◽  
Author(s):  
Christopher J. Kristich ◽  
Vy T. Nguyen ◽  
Thinh Le ◽  
Aaron M. T. Barnes ◽  
Suzanne Grindle ◽  
...  
Keyword(s):  
Transposable Element ◽  
Enterococcus Faecalis ◽  
Biofilm Formation ◽  
Transposon Mutagenesis ◽  
Genetic Determinants ◽  
Direct Role ◽  
Efficient System ◽  
The Core ◽  
New Genes ◽  
Functional Genomic Analysis

ABSTRACT Enterococcus faecalis is a gram-positive commensal bacterium of the gastrointestinal tract and an important opportunistic pathogen. Despite the increasing clinical significance of the enterococci, most of the genetic analysis of these organisms has focused on mobile genetic elements, and existing tools for manipulation and analysis of the core E. faecalis chromosome are limited. We are interested in a comprehensive analysis of the genetic determinants for biofilm formation encoded within the core E. faecalis genome. To identify such determinants, we developed a substantially improved system for transposon mutagenesis in E. faecalis based on a mini-mariner transposable element. Mutagenesis of wild-type E. faecalis with this element yielded predominantly mutants carrying a single copy of the transposable element, and insertions were distributed around the entire chromosome in an apparently random fashion. We constructed a library of E. faecalis transposon insertion mutants and screened this library to identify mutants exhibiting a defect in biofilm formation. Biofilm-defective mutants were found to carry transposon insertions both in genes that were previously known to play a role in biofilm formation and in new genes lacking any known function; for several genes identified in the screen, complementation analysis confirmed a direct role in biofilm formation. These results provide significant new information about the genetics of enterococcal biofilm formation and demonstrate the general utility of our transposon system for functional genomic analysis of E. faecalis.

scholarly journals Comparative biofilm assays using Enterococcus faecalis OG1RF identify new determinants of biofilm formation

2021 ◽  
Author(s):  
Julia L. E. Willett ◽  
Jennifer L. Dale ◽  
Lucy M. Kwiatkowski ◽  
Jennifer L. Powers ◽  
Michelle L. Korir ◽  
...  
Keyword(s):  
Enterococcus Faecalis ◽  
Growth Medium ◽  
Biofilm Formation ◽  
Time Course ◽  
Opportunistic Pathogen ◽  
Genetic Determinants ◽  
Experimental Conditions ◽  
Biofilm Reactors ◽  
Biofilm Architecture ◽  
Biofilm Development

AbstractEnterococcus faecalis is a common commensal organism and a prolific nosocomial pathogen that causes biofilm-associated infections. Numerous E. faecalis OG1RF genes required for biofilm formation have been identified, but few studies have compared genetic determinants of biofilm formation and biofilm morphology across multiple conditions. Here, we cultured transposon (Tn) libraries in CDC biofilm reactors in two different media and used Tn sequencing (TnSeq) to identify core and accessory biofilm determinants, including many genes that are poorly characterized or annotated as hypothetical. Multiple secondary assays (96-well plates, submerged Aclar, and MultiRep biofilm reactors) were used to validate phenotypes of new biofilm determinants. We quantified biofilm cells and used fluorescence microscopy to visualize biofilms formed by 6 Tn mutants identified using TnSeq and found that disrupting these genes (OG1RF_10350, prsA, tig, OG1RF_10576, OG1RF_11288, and OG1RF_11456) leads to significant time- and medium-dependent changes in biofilm architecture. Structural predictions revealed potential roles in cell wall homeostasis for OG1RF_10350 and OG1RF_11288 and signaling for OG1RF_11456. Additionally, we identified growth medium-specific hallmarks of OG1RF biofilm morphology. This study demonstrates how E. faecalis biofilm architecture is modulated by growth medium and experimental conditions, and identifies multiple new genetic determinants of biofilm formation.ImportanceE. faecalis is an opportunistic pathogen and a leading cause of hospital-acquired infections, in part due to its ability to form biofilms. A complete understanding of the genes required for E. faecalis biofilm formation as well as specific features of biofilm morphology related to nutrient availability and growth conditions is crucial for understanding how E. faecalis biofilm-associated infections develop and resist treatment in patients. We employed a comprehensive approach to analysis of biofilm determinants by combining TnSeq primary screens with secondary phenotypic validation using diverse biofilm assays. This enabled identification of numerous core (important under many conditions) and accessory (important under specific conditions) biofilm determinants in E. faecalis OG1RF. We found multiple genes whose disruption results in drastic changes to OG1RF biofilm morphology. These results expand our understanding of the genetic requirements for biofilm formation in E. faecalis that affect the time course of biofilm development as well as the response to specific nutritional conditions.

scholarly journals Revisiting Bap Multidomain Protein: More Than Sticking Bacteria Together

2020 ◽  
Vol 11 ◽  
Author(s):  
Jaione Valle ◽  
Xianyang Fang ◽  
Iñigo Lasa
Keyword(s):  
Biofilm Formation ◽  
Core Genome ◽  
Current Knowledge ◽  
Surface Proteins ◽  
Structure And Properties ◽  
Diverse Range ◽  
The Core ◽  
Self Assembling ◽  
Abiotic Surfaces ◽  
Range Of Functions

One of the major components of the staphylococcal biofilm is surface proteins that assemble as scaffold components of the biofilm matrix. Among the different surface proteins able to contribute to biofilm formation, this review is dedicated to the Biofilm Associated Protein (Bap). Bap is part of the accessory genome of Staphylococcus aureus but orthologs of Bap in other staphylococcal species belong to the core genome. When present, Bap promotes adhesion to abiotic surfaces and induces strong intercellular adhesion by self-assembling into amyloid like aggregates in response to the levels of calcium and the pH in the environment. During infection, Bap enhances the adhesion to epithelial cells where it binds directly to the host receptor Gp96 and inhibits the entry of the bacteria into the cells. To perform such diverse range of functions, Bap comprises several domains, and some of them include several motifs associated to distinct functions. Based on the knowledge accumulated with the Bap protein of S. aureus, this review aims to summarize the current knowledge of the structure and properties of each domain of Bap and their contribution to Bap functionality.

scholarly journals ClpP participates in stress tolerance, biofilm formation, antimicrobial tolerance, and virulence of Enterococcus faecalis

2020 ◽  
Author(s):  
Jinxin Zheng ◽  
Yang Wu ◽  
Zhiwei Lin ◽  
Guangfu Wang ◽  
Sibo Jiang ◽  
...  
Keyword(s):  
Ribosomal Protein ◽  
Stress Tolerance ◽  
Enterococcus Faecalis ◽  
Mutant Strain ◽  
Biofilm Formation ◽  
Protein Quality ◽  
Galleria Mellonella ◽  
Regulatory Protein ◽  
Proteomics Data ◽  
Acetyl Esterase

Abstract Background ClpP is important for bacterial growth and plays an indispensable role in cellular protein quality control systems by refolding or degrading damaged proteins, but the physiological significance of ClpP in Enterococcus faecalis remains obscure. A clpP deletion mutant (△ clpP ) was constructed using the E. faecalis OG1RF strain to clarify the effect of ClpP on E. faecalis. The global abundance of proteins was determined by a mass spectrometer with tandem mass tag labeling. Results The Δ clpP mutant strain showed impaired growth at 20°C or 45°C at 5% NaCl or 2 mM H 2 O 2 . The number of surviving Δ clpP mutants decreased after exposure to the high concentration (50× minimal inhibitory concentration) of linezolid or minocycline for 96 h. The Δ clpP mutant strain also demonstrated decreased biofilm formation but increased virulence in a Galleria mellonella model. The mass spectrometry proteomics data indicated that the abundances of 135 proteins changed (111 increased, 24 decreased) in the Δ clpP mutant strain. Among those, the abundances of stress response or virulence relating proteins: FsrA response regulator, gelatinase GelE, regulatory protein Spx ( spxA ), heat-inducible transcription repressor HrcA, transcriptional regulator CtsR, ATPase/chaperone ClpC, acetyl esterase/lipase, and chaperonin GroEL increased in the Δ clpP mutant strain; however, the abundances of ribosomal protein L4/L1 family protein ( rplD ), ribosomal protein L7/L12 ( rplL2 ), 50S ribosomal protein L13 ( rplM ), L18 ( rplR ), L20 ( rplT ), 30S ribosomal protein S14 ( rpsN2 ) and S18 ( rpsR ) all decreased. The abundances of biofilm formation-related adapter protein MecA increased, while the abundances of dihydroorotase ( pyrC ), orotate phosphoribosyltransferase ( pyrE ), and orotidine-5'-phosphate decarboxylase ( pyrF ) all decreased in the Δ clpP mutant strain. Conclusion The present study demonstrates that ClpP participates in stress tolerance, biofilm formation, antimicrobial tolerance, and virulence of E. faecalis.

scholarly journals ClpP participates in stress tolerance, biofilm formation, antimicrobial tolerance, and virulence of Enterococcus faecalis

2020 ◽  
Author(s):  
Jinxin Zheng ◽  
Yang Wu ◽  
Zhiwei Lin ◽  
Guangfu Wang ◽  
Sibo Jiang ◽  
...  
Keyword(s):  
Ribosomal Protein ◽  
Stress Tolerance ◽  
Enterococcus Faecalis ◽  
Mutant Strain ◽  
Biofilm Formation ◽  
Protein Quality ◽  
Galleria Mellonella ◽  
Regulatory Protein ◽  
Proteomics Data ◽  
Acetyl Esterase

Abstract Background ClpP is important for bacterial growth and plays an indispensable role in cellular protein quality control systems by refolding or degrading damaged proteins, but the physiological significance of ClpP in Enterococcus faecalis remains obscure. A clpP deletion mutant (△ clpP ) was constructed using the E. faecalis OG1RF strain to clarify the effect of ClpP on E. faecalis. The global abundance of proteins was determined by a mass spectrometer with tandem mass tag labeling.Results The Δ clpP mutant strain showed impaired growth at 20°C or 45°C at 5% NaCl or 2 mM H 2 O 2 . The number of surviving Δ clpP mutants decreased after exposure to the high concentration (50× minimal inhibitory concentration) of linezolid or minocycline for 96 h. The Δ clpP mutant strain also demonstrated decreased biofilm formation but increased virulence in a Galleria mellonella model. The mass spectrometry proteomics data indicated that the abundances of 135 proteins changed (111 increased, 24 decreased) in the Δ clpP mutant strain. Among those, the abundances of stress response or virulence relating proteins: FsrA response regulator, gelatinase GelE, regulatory protein Spx ( spxA ), heat-inducible transcription repressor HrcA, transcriptional regulator CtsR, ATPase/chaperone ClpC, acetyl esterase/lipase, and chaperonin GroEL increased in the Δ clpP mutant strain; however, the abundances of ribosomal protein L4/L1 family protein ( rplD ), ribosomal protein L7/L12 ( rplL2 ), 50S ribosomal protein L13 ( rplM ), L18 ( rplR ), L20 ( rplT ), 30S ribosomal protein S14 ( rpsN2 ) and S18 ( rpsR ) all decreased. The abundances of biofilm formation-related adapter protein MecA increased, while the abundances of dihydroorotase ( pyrC ), orotate phosphoribosyltransferase ( pyrE ), and orotidine-5'-phosphate decarboxylase ( pyrF ) all decreased in the Δ clpP mutant strain.Conclusion The present study demonstrates that ClpP participates in stress tolerance, biofilm formation, antimicrobial tolerance, and virulence of E. faecalis.

scholarly journals The Response of Acinetobacter baumannii to Hydrogen Sulfide Reveals Two Independent Persulfide-Sensing Systems and a Connection to Biofilm Regulation

mBio ◽  
2020 ◽  
Vol 11 (3) ◽  
Author(s):  
Brenna J. C. Walsh ◽  
Jiefei Wang ◽  
Katherine A. Edmonds ◽  
Lauren D. Palmer ◽  
Yixiang Zhang ◽  
...  
Keyword(s):  
Hydrogen Sulfide ◽  
Acinetobacter Baumannii ◽  
Biofilm Formation ◽  
Sulfide Oxidation ◽  
Regulatory Protein ◽  
Transcriptional Regulators ◽  
Human Pathogen ◽  
Secondary System ◽  
Biofilm Growth ◽  
Content Type

ABSTRACT Acinetobacter baumannii is an opportunistic nosocomial pathogen that is the causative agent of several serious infections in humans, including pneumonia, sepsis, and wound and burn infections. A. baumannii is also capable of forming proteinaceous biofilms on both abiotic and epithelial cell surfaces. Here, we investigate the response of A. baumannii toward sodium sulfide (Na2S), known to be associated with some biofilms at oxic/anoxic interfaces. The addition of exogenous inorganic sulfide reveals that A. baumannii encodes two persulfide-sensing transcriptional regulators, a primary σ54-dependent transcriptional activator (FisR), and a secondary system controlled by the persulfide-sensing biofilm growth-associated repressor (BigR), which is only induced by sulfide in a fisR deletion strain. FisR activates an operon encoding a sulfide oxidation/detoxification system similar to that characterized previously in Staphylococcus aureus, while BigR regulates a secondary persulfide dioxygenase (PDO2) as part of yeeE-yedE-pdo2 sulfur detoxification operon, found previously in Serratia spp. Global S-sulfuration (persulfidation) mapping of the soluble proteome reveals 513 persulfidation targets well beyond FisR-regulated genes and includes five transcriptional regulators, most notably the master biofilm regulator BfmR and a poorly characterized catabolite regulatory protein (Crp). Both BfmR and Crp are well known to impact biofilm formation in A. baumannii and other organisms, respectively, suggesting that persulfidation of these regulators may control their activities. The implications of these findings on bacterial sulfide homeostasis, persulfide signaling, and biofilm formation are discussed. IMPORTANCE Although hydrogen sulfide (H2S) has long been known as a respiratory poison, recent reports in numerous bacterial pathogens reveal that H2S and more downstream oxidized forms of sulfur collectedly termed reactive sulfur species (RSS) function as antioxidants to combat host efforts to clear the infection. Here, we present a comprehensive analysis of the transcriptional and proteomic response of A. baumannii to exogenous sulfide as a model for how this important human pathogen manages sulfide/RSS homeostasis. We show that A. baumannii is unique in that it encodes two independent persulfide sensing and detoxification pathways that govern the speciation of bioactive sulfur in cells. The secondary persulfide sensor, BigR, impacts the expression of biofilm-associated genes; in addition, we identify two other transcriptional regulators known or projected to regulate biofilm formation, BfmR and Crp, as highly persulfidated in sulfide-exposed cells. These findings significantly strengthen the connection between sulfide homeostasis and biofilm formation in an important human pathogen.

scholarly journals Physiological significance of ClpP in Enterococcus faecalis and its regulating proteins were identified by Tandem Mass Tag Mass Spectrometry

2019 ◽  
Author(s):  
Jinxin Zheng ◽  
Yang Wu ◽  
Zhiwei Lin ◽  
Guangfu Wang ◽  
Sibo Jiang ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Ribosomal Protein ◽  
Enterococcus Faecalis ◽  
Mutant Strain ◽  
Biofilm Formation ◽  
Protein Quality ◽  
Regulatory Protein ◽  
Physiological Significance ◽  
Tandem Mass ◽  
Acetyl Esterase

Abstract Background ClpP is important for bacterial growth and plays an indispensable role in cellular protein quality control systems by refolding or degrading damaged proteins, but the physiological significance of ClpP in Enterococcus faecalis is still obscure. Thus a clpP deletion mutant (△clpP) was constructed in E. faecalis OG1RF strain to elucidate a more comprehensive picture of the effect of ClpP on E. faecalis. The global abundance of proteins was determined by a mass spectrometer with Tandem Mass Tags labeling. Results The ΔclpP mutant strain showed impaired growth at 20°C or 45°C, at 5% NaCl or 2 mM H2O2. The surviving bacteria of the ΔclpP mutant strain reduced after exposure to the high concentration (50 x MIC) of linezolid or minocycline for 96 h. The ΔclpP mutant strain also demonstrated decreased biofilm formation but increased virulence in a Galleria mellonella model. The mass spectrometry proteomics data indicated that the abundances of 135 proteins changed (111 proteins increased, 24 proteins decreased) in the ΔclpP mutant strain. Among those differential abundance proteins, the abundances of stress response or virulence relating proteins: FsrA response regulator, gelatinase GelE, regulatory protein Spx (spxA), heat-inducible transcription repressor HrcA, transcriptional regulator CtsR, ATPase/chaperone ClpC, acetyl esterase/lipase, and chaperonin GroEL increased in the ΔclpP mutant strain; however, the abundances of ribosomal protein L4/L1 family protein (rplD), ribosomal protein L7/L12 (rplL2), 50S ribosomal protein L13 (rplM), L18 (rplR), L20 (rplT), 30S ribosomal protein S14 (rpsN2) and S18 (rpsR) all reduced. The abundances of biofilm formation related adapter protein MecA increased, while the abundances of dihydroorotase (pyrC), orotate phosphoribosyltransferase (pyrE) and orotidine-5'-phosphate decarboxylase (pyrF) all decreased in the ΔclpP mutant strain. Conclusion The present study demonstrates that ClpP participates in stress tolerance, biofilm formation, antimicrobials tolerance, and virulence of E. faecalis.

scholarly journals CD25890, a conserved protein that modulates sporulation initiation in Clostridioides difficile

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Diogo Martins ◽  
Michael A. DiCandia ◽  
Aristides L. Mendes ◽  
Daniela Wetzel ◽  
Shonna M. McBride ◽  
...  
Keyword(s):  
Life Cycle ◽  
Biofilm Formation ◽  
Regulatory Protein ◽  
Well Being ◽  
Human Intestine ◽  
Healthy Humans ◽  
Clostridioides Difficile ◽  
Nutritional Conditions ◽  
Sporulation Initiation

AbstractBacteria that reside in the gastrointestinal tract of healthy humans are essential for our health, sustenance and well-being. About 50–60% of those bacteria have the ability to produce resilient spores that are important for the life cycle in the gut and for host-to-host transmission. A genomic signature for sporulation in the human intestine was recently described, which spans both commensals and pathogens such as Clostridioides difficile and contains several genes of unknown function. We report on the characterization of a signature gene, CD25890, which, as we show is involved in the control of sporulation initiation in C. difficile under certain nutritional conditions. Spo0A is the main regulatory protein controlling entry into sporulation and we show that an in-frame deletion of CD25890 results in increased expression of spo0A per cell and increased sporulation. The effect of CD25890 on spo0A is likely indirect and mediated through repression of the sinRR´ operon. Deletion of the CD25890 gene, however, does not alter the expression of the genes coding for the cytotoxins or the genes involved in biofilm formation. Our results suggest that CD25890 acts to modulate sporulation in response to the nutrients present in the environment.

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