scholarly journals Exploring the transcriptome of luxI- and ΔainS mutants and the impact of N-3-oxo-hexanoyl-L- and N-3-hydroxy-decanoyl-L-homoserine lactones on biofilm formation in Aliivibrio salmonicida

2018 ◽  
Author(s):  
Miriam Khider ◽  
Hilde Hansen ◽  
Jostein A. Johansen ◽  
Erik Hjerde ◽  
Nils Peder Willassen
Keyword(s):  
Biofilm Formation ◽  
Transcriptome Profiling ◽  
Homoserine Lactone ◽  
Colony Morphology ◽  
Phenotypic Traits ◽  
Dependent Manner ◽  
Rna Seq ◽  
Homoserine Lactones ◽  
A Cell ◽  
The Impact

Background. The marine bacterium A. salmonicida uses the quorum sensing (QS) systems, AinS/R and LuxI/R to produce eight acyl-homoserine lactones (AHLs) in a cell density dependent manner. Biofilm formation is one of the QS regulated phenotypes, which requires the expression of exopolysaccharides (EPS).We previously demonstrated that inactivation of LitR, the master regulator of QS in A. salmonicida resulted in biofilm formation, which was, similar to the biofilm formed by the AHL deficient mutant ΔainSluxI-.In this work, we have identified genes regulated by AinS and LuxI using RNA sequensing (RNA-Seq), and studied their role in biofilm formation, colony morphology and motility. We have also studied the effect of two AHLs on the biofilm formation. Results.The transcriptome profiling of ΔainS and luxI- mutants allowed us to identify essential genes regulated by QS in A. salmonicida. Relative to the wild-type, the ΔainS and luxI- mutants revealed 40 and 500 differentially expressed genes (DEGs), respectively. The functional analysis demonstrated that the most pronounced DEGs were involved in bacterial motility and chemotaxis, exopolysaccharide production, and surface structures related to adhesion. Inactivation of luxI but not ainS genes resulted in wrinkled colony morphology. While inactivation of both genes (ΔainSluxI-) resulted in strains able to form wrinkled colonies and mushroom structured biofilm. Moreover, when the ΔainSluxI- mutant was supplemented with N-3-oxo-hexanoyl-L- homoserine lactone (3OC6-HSL) and N-3-hydroxy-decanoyl-L-homoserine lactone(3OHC10-HSL), the biofilm did not develop. We also show that LuxI is needed for motility and repression for EPS production, where repression of EPS is likely operated through the RpoQ-sigma factor. Conclusion.These findings imply that LuxI and AinS synthases have a critical contribution to the QS-dependent regulation on gene expression and the phenotypic traits related to it.

scholarly journals Exploring the transcriptome of luxI- and ΔainS mutants and the impact of N-3-oxo-hexanoyl-L- and N-3-hydroxy-decanoyl-L-homoserine lactones on biofilm formation in Aliivibrio salmonicida

2018 ◽  
Author(s):  
Miriam Khider ◽  
Hilde Hansen ◽  
Jostein A. Johansen ◽  
Erik Hjerde ◽  
Nils Peder Willassen
Keyword(s):  
Biofilm Formation ◽  
Transcriptome Profiling ◽  
Homoserine Lactone ◽  
Colony Morphology ◽  
Phenotypic Traits ◽  
Dependent Manner ◽  
Rna Seq ◽  
Homoserine Lactones ◽  
A Cell ◽  
The Impact

Background. The marine bacterium A. salmonicida uses the quorum sensing (QS) systems, AinS/R and LuxI/R to produce eight acyl-homoserine lactones (AHLs) in a cell density dependent manner. Biofilm formation is one of the QS regulated phenotypes, which requires the expression of exopolysaccharides (EPS).We previously demonstrated that inactivation of LitR, the master regulator of QS in A. salmonicida resulted in biofilm formation, which was, similar to the biofilm formed by the AHL deficient mutant ΔainSluxI-.In this work, we have identified genes regulated by AinS and LuxI using RNA sequensing (RNA-Seq), and studied their role in biofilm formation, colony morphology and motility. We have also studied the effect of two AHLs on the biofilm formation. Results.The transcriptome profiling of ΔainS and luxI- mutants allowed us to identify essential genes regulated by QS in A. salmonicida. Relative to the wild-type, the ΔainS and luxI- mutants revealed 40 and 500 differentially expressed genes (DEGs), respectively. The functional analysis demonstrated that the most pronounced DEGs were involved in bacterial motility and chemotaxis, exopolysaccharide production, and surface structures related to adhesion. Inactivation of luxI but not ainS genes resulted in wrinkled colony morphology. While inactivation of both genes (ΔainSluxI-) resulted in strains able to form wrinkled colonies and mushroom structured biofilm. Moreover, when the ΔainSluxI- mutant was supplemented with N-3-oxo-hexanoyl-L- homoserine lactone (3OC6-HSL) and N-3-hydroxy-decanoyl-L-homoserine lactone(3OHC10-HSL), the biofilm did not develop. We also show that LuxI is needed for motility and repression for EPS production, where repression of EPS is likely operated through the RpoQ-sigma factor. Conclusion.These findings imply that LuxI and AinS synthases have a critical contribution to the QS-dependent regulation on gene expression and the phenotypic traits related to it.

scholarly journals Exploring the transcriptome ofluxI−andΔainSmutants and the impact of N-3-oxo-hexanoyl-L- and N-3-hydroxy-decanoyl-L-homoserine lactones on biofilm formation inAliivibrio salmonicida

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e6845
Author(s):  
Miriam Khider ◽  
Hilde Hansen ◽  
Erik Hjerde ◽  
Jostein A. Johansen ◽  
Nils Peder Willassen
Keyword(s):  
Biofilm Formation ◽  
Expression Patterns ◽  
Critical Role ◽  
Transcriptome Profiling ◽  
Global Gene Expression ◽  
Gene Clusters ◽  
Homoserine Lactone ◽  
Dependent Manner ◽  
Homoserine Lactones ◽  
The Impact

BackgroundBacterial communication through quorum sensing (QS) systems has been reported to be important in coordinating several traits such as biofilm formation. InAliivibrio salmonicidatwo QS systems the LuxI/R and AinS/R, have been shown to be responsible for the production of eight acyl-homoserine lactones (AHLs) in a cell density dependent manner. We have previously demonstrated that inactivation of LitR, the master regulator of the QS system resulted in biofilm formation, similar to the biofilm formed by the AHL deficient mutantΔainSluxI−. In this study, we aimed to investigate the global gene expression patterns ofluxIandainSautoinducer synthases mutants using transcriptomic profiling. In addition, we examined the influence of the different AHLs on biofilm formation.ResultsThe transcriptome profiling ofΔainSandluxI−mutants allowed us to identify genes and gene clusters regulated by QS inA. salmonicida. Relative to the wild type, theΔainSandluxI−mutants revealed 29 and 500 differentially expressed genes (DEGs), respectively. The functional analysis demonstrated that the most pronounced DEGs were involved in bacterial motility and chemotaxis, exopolysaccharide production, and surface structures related to adhesion. Inactivation ofluxI, but notainSgenes resulted in wrinkled colony morphology. While inactivation of both genes (ΔainSluxI−) resulted in strains able to form wrinkled colonies and mushroom structured biofilm. Moreover, when theΔainSluxI−mutant was supplemented with N-3-oxo-hexanoyl-L-homoserine lactone (3OC6-HSL) or N-3-hydroxy-decanoyl-L-homoserine lactone (3OHC10-HSL), the biofilm did not develop. We also show that LuxI is needed for motility and for repression of EPS production, where repression of EPS is likely operated through the RpoQ-sigma factor.ConclusionThese findings imply that the LuxI and AinS autoinducer synthases play a critical role in the regulation of biofilm formation, EPS production, and motility.

scholarly journals Engineering acyl-homoserine lactone-interfering enzymes toward bacterial control

2020 ◽  
Vol 295 (37) ◽  
pp. 12993-13007 ◽  
Author(s):  
Raphaël Billot ◽  
Laure Plener ◽  
Pauline Jacquet ◽  
Mikael Elias ◽  
Eric Chabrière ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Homoserine Lactone ◽  
Communication Process ◽  
Dependent Manner ◽  
Biotechnological Potential ◽  
Bacterial Communication ◽  
Homoserine Lactones ◽  
A Cell ◽  
Eukaryotic Organisms ◽  
Bacterial Control

Enzymes able to degrade or modify acyl-homoserine lactones (AHLs) have drawn considerable interest for their ability to interfere with the bacterial communication process referred to as quorum sensing. Many proteobacteria use AHL to coordinate virulence and biofilm formation in a cell density–dependent manner; thus, AHL-interfering enzymes constitute new promising antimicrobial candidates. Among these, lactonases and acylases have been particularly studied. These enzymes have been isolated from various bacterial, archaeal, or eukaryotic organisms and have been evaluated for their ability to control several pathogens. Engineering studies on these enzymes were carried out and successfully modulated their capacity to interact with specific AHL, increase their catalytic activity and stability, or enhance their biotechnological potential. In this review, special attention is paid to the screening, engineering, and applications of AHL-modifying enzymes. Prospects and future opportunities are also discussed with a view to developing potent candidates for bacterial control.

scholarly journals Analysis of Quorum-Sensing-Dependent Control of Rhizosphere-Expressed (rhi) Genes in Rhizobium leguminosarum bv. viciae

1999 ◽  
Vol 181 (12) ◽  
pp. 3816-3823 ◽  
Author(s):  
Belen Rodelas ◽  
James K. Lithgow ◽  
Florence Wisniewski-Dye ◽  
Andrea Hardman ◽  
Adam Wilkinson ◽  
...  
Keyword(s):  
Rhizobium Leguminosarum ◽  
Culture Supernatant ◽  
Homoserine Lactone ◽  
Dependent Manner ◽  
Chemical Analyses ◽  
Wild Type ◽  
Homoserine Lactones ◽  
Acyl Homoserine Lactones ◽  
A Cell ◽  
Culture Supernatants

ABSTRACT The rhi genes of Rhizobium leguminosarumbiovar viciae are expressed in the rhizosphere and play a role in the interaction with legumes, such as the pea. Previously (K. M. Gray, J. P. Pearson, J. A. Downie, B. E. A. Boboye, and E. P. Greenberg, J. Bacteriol. 178:372–376, 1996) therhiABC operon had been shown to be regulated by RhiR and to be induced by addedN-(3-hydroxy-7-cis-tetradecenoyl)-l-homoserine lactone (3OH,C14:1-HSL). Mutagenesis of a cosmid carrying the rhiABC and rhiR gene region identified a gene (rhiI) that affects the level of rhiAexpression. Mutation of rhiI slightly increased the number of nodules formed on the pea. The rhiI gene is (likerhiA) regulated by rhiR in a cell density-dependent manner. RhiI is similar to LuxI and other proteins involved in the synthesis of N-acyl-homoserine lactones (AHLs). Chemical analyses of spent culture supernatants demonstrated that RhiI produces N-(hexanoyl)-l-homoserine lactone (C6-HSL) andN-(octanoyl)-l-homoserine lactone (C8-HSL). Both of these AHLs induced rhiA-lacZand rhiI-lacZ expression on plasmids introduced into anAgrobacterium strain that produces no AHLs, showing thatrhiI is positively regulated by autoinduction. However, in this system no induction of rhiA or rhiI with 3OH,C14:1-HSL was observed. Analysis of the spent culture supernatant of the wild-type R. leguminosarum bv. viciae revealed that at least seven different AHLs are made. Mutation ofrhiI decreased the amounts of C6-HSL and C8-HSL but did not block their formation, and in this background the rhiI mutation did not significantly affect the expression levels of the rhiI gene orrhiABC genes or the accumulation of RhiA protein. These observations suggest that there are additional loci involved in AHL production in R. leguminosarum bv. viciae and that they affect rhiI and rhiABC expression. We postulate that the previously observed induction of rhiA by 3OH,C14:1-HSL may be due to an indirect effect caused by induction of other AHL production loci.

scholarly journals 3′ MACE RNA-sequencing allows for transcriptome profiling in human tissue samples after long-term storage

2020 ◽  
Vol 100 (10) ◽  
pp. 1345-1355 ◽  
Author(s):  
Stefaniya Boneva ◽  
Anja Schlecht ◽  
Daniel Böhringer ◽  
Hans Mittelviefhaus ◽  
Thomas Reinhard ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Storage Time ◽  
Transcriptome Profiling ◽  
Rna Seq ◽  
Fresh Tissue ◽  
Long Term Storage ◽  
Ffpe Samples ◽  
The Impact ◽  
Term Storage

Abstract This study aims to compare the potential of standard RNA-sequencing (RNA-Seq) and 3′ massive analysis of c-DNA ends (MACE) RNA-sequencing for the analysis of fresh tissue and describes transcriptome profiling of formalin-fixed paraffin-embedded (FFPE) archival human samples by MACE. To compare MACE to standard RNA-Seq on fresh tissue, four healthy conjunctiva from four subjects were collected during vitreoretinal surgery, halved and immediately transferred to RNA lysis buffer without prior fixation and then processed for either standard RNA-Seq or MACE RNA-Seq analysis. To assess the impact of FFPE preparation on MACE, a third part was fixed in formalin and processed for paraffin embedding, and its transcriptional profile was compared with the unfixed specimens analyzed by MACE. To investigate the impact of FFPE storage time on MACE results, 24 FFPE-treated conjunctival samples from 24 patients were analyzed as well. Nineteen thousand six hundred fifty-nine transcribed genes were detected by both MACE and standard RNA-Seq on fresh tissue, while 3251 and 2213 transcripts were identified explicitly by MACE or RNA-Seq, respectively. Standard RNA-Seq tended to yield longer detected transcripts more often than MACE technology despite normalization, indicating that the MACE technology is less susceptible to a length bias. FFPE processing revealed negligible effects on MACE sequencing results. Several quality-control measurements showed that long-term storage in paraffin did not decrease the diversity of MACE libraries. We noted a nonlinear relation between storage time and the number of raw reads with an accelerated decrease within the first 1000 days in paraffin, while the numbers remained relatively stable in older samples. Interestingly, the number of transcribed genes detected was independent on FFPE storage time. RNA of sufficient quality and quantity can be extracted from FFPE samples to obtain comprehensive transcriptome profiling using MACE technology. We thus present MACE as a novel opportunity for utilizing FFPE samples stored in histological archives.

scholarly journals Nitrite-Oxidizing Bacterium Nitrobacter winogradskyi ProducesN-Acyl-Homoserine Lactone Autoinducers

2015 ◽  
Vol 81 (17) ◽  
pp. 5917-5926 ◽  
Author(s):  
Brett L. Mellbye ◽  
Peter J. Bottomley ◽  
Luis A. Sayavedra-Soto
Keyword(s):  
Mass Spectrometry ◽  
Chemostat Culture ◽  
Rhodopseudomonas Palustris ◽  
Homoserine Lactone ◽  
Amino Acid Sequences ◽  
Acyl Homoserine Lactone ◽  
Dependent Manner ◽  
Content Type ◽  
Genes Encoding ◽  
A Cell

ABSTRACTNitrobacter winogradskyiis a chemolithotrophic bacterium that plays a role in the nitrogen cycle by oxidizing nitrite to nitrate. Here, we demonstrate a functionalN-acyl-homoserine lactone (acyl-HSL) synthase in this bacterium. TheN. winogradskyigenome contains genes encoding a putative acyl-HSL autoinducer synthase (nwi0626,nwiI) and a putative acyl-HSL autoinducer receptor (nwi0627,nwiR) with amino acid sequences 38 to 78% identical to those inRhodopseudomonas palustrisand otherRhizobiales. Expression ofnwiIandnwiRcorrelated with acyl-HSL production during culture.N. winogradskyiproduces two distinct acyl-HSLs,N-decanoyl-l-homoserine lactone (C10-HSL) and a monounsaturated acyl-HSL (C10:1-HSL), in a cell-density- and growth phase-dependent manner, during batch and chemostat culture. The acyl-HSLs were detected by bioassay and identified by ultraperformance liquid chromatography with information-dependent acquisition mass spectrometry (UPLC-IDA-MS). The C=C bond in C10:1-HSL was confirmed by conversion into bromohydrin and detection by UPLC-IDA-MS.

scholarly journals Codon bias determines sorting of ion channel protein

2020 ◽  
Author(s):  
Marina Kithil ◽  
Anja Jeannine Engel ◽  
Markus Langhans ◽  
Oliver Rauh ◽  
Matea Cartolano ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Secretory Pathway ◽  
Model Systems ◽  
Dependent Manner ◽  
Intracellular Proteins ◽  
Polypeptide Folding ◽  
A Cell ◽  
Identical Amino Acid Sequence ◽  
Cycle Dependent ◽  
The Impact

AbstractThe choice of codons can influence local translation kinetics during protein synthesis. The question of whether the modulation of polypeptide folding and binding to chaperons influences sorting of nascent membrane proteins remains unclear. Here, we use two similar K+ channels as model systems to examine the impact of codon choice on protein sorting. By monitoring transient expression of GFP tagged proteins in mammalian cells we find that targeting of one channel to the secretory pathway is insensitive to codon optimization. In contrast, sorting of the second channel to the mitochondria is very sensitive to codon choice. The protein with an identical amino acid sequence is sorted in a codon and cell cycle dependent manner either to mitochondria or the secretory pathway. The data establish that a gene with either rare or frequent codons serves together with a cell-state depending decoding mechanism as a secondary code for sorting intracellular proteins.

scholarly journals Fibrinogen-InducedStreptococcus mutansBiofilm Formation and Adherence to Endothelial Cells

2013 ◽  
Vol 2013 ◽  
pp. 1-8 ◽  
Author(s):  
Telma Blanca Lombardo Bedran ◽  
Jabrane Azelmat ◽  
Denise Palomari Spolidorio ◽  
Daniel Grenier
Keyword(s):  
Endothelial Cells ◽  
Infective Endocarditis ◽  
Biofilm Formation ◽  
Bacterial Species ◽  
Bactericidal Effect ◽  
Dependent Manner ◽  
Bacterial Cells ◽  
Human Fibrinogen ◽  
Polystyrene Support ◽  
The Impact

Streptococcus mutans, the predominant bacterial species associated with dental caries, can enter the bloodstream and cause infective endocarditis. The aim of this study was to investigateS. mutansbiofilm formation and adherence to endothelial cells induced by human fibrinogen. The putative mechanism by which biofilm formation is induced as well as the impact of fibrinogen onS. mutansresistance to penicillin was also evaluated. Bovine plasma dose dependently induced biofilm formation byS. mutans. Of the various plasma proteins tested, only fibrinogen promoted the formation of biofilm in a dose-dependent manner. Scanning electron microscopy observations revealed the presence of complex aggregates of bacterial cells firmly attached to the polystyrene support.S. mutansin biofilms induced by the presence of fibrinogen was markedly resistant to the bactericidal effect of penicillin. Fibrinogen also significantly increased the adherence ofS. mutansto endothelial cells. NeitherS. mutanscells nor culture supernatants converted fibrinogen into fibrin. However, fibrinogen is specifically bound to the cell surface ofS. mutansand may act as a bridging molecule to mediate biofilm formation. In conclusion, our study identified a new mechanism promotingS. mutansbiofilm formation and adherence to endothelial cells which may contribute to infective endocarditis.

scholarly journals Two Dissimilar N-Acyl-Homoserine Lactone Acylases of Pseudomonas syringae Influence Colony and Biofilm Morphology

2008 ◽  
Vol 75 (1) ◽  
pp. 45-53 ◽  
Author(s):  
Ryan W. Shepherd ◽  
Steven E. Lindow
Keyword(s):  
Pseudomonas Syringae ◽  
Biofilm Formation ◽  
Homoserine Lactone ◽  
Acyl Homoserine Lactone ◽  
Associated Bacteria ◽  
Homoserine Lactones ◽  
Amide Bonds ◽  
Acyl Homoserine Lactones ◽  
Complex Habitat ◽  
Long Chain

ABSTRACT Plant aerial surfaces comprise a complex habitat for microorganisms, and many plant-associated bacteria, such as the pathogen Pseudomonas syringae, exhibit density-dependent survival on leaves by utilizing quorum sensing (QS). QS is often mediated by diffusible signals called N-acyl-homoserine lactones (AHLs), and P. syringae utilizes N-3-oxo-hexanoyl-dl-homoserine lactone (3OC6HSL) to control traits influencing epiphytic fitness and virulence. The P. syringae pathovar syringae B728a genome sequence revealed two putative AHL acylases, termed HacA (Psyr_1971) and HacB (Psyr_4858), which are N-terminal nucleophile hydrolases that inactivate AHLs by cleaving their amide bonds. HacA is a secreted AHL acylase that degrades only long-chain (C ≥ 8) AHLs, while HacB is not secreted and degrades all tested AHLs. Targeted disruptions of hacA, hacB, and hacA and hacB together do not alter endogenous 3OC6HSL levels under the tested conditions. Surprisingly, targeted disruptions of hacA alone and hacA and hacB together confer complementable phenotypes that are very similar to autoaggregative phenotypes seen in other species. While AHL acylases might enable P. syringae B728a to degrade signals of competing species and block expression of their QS-dependent traits, these enzymes also play fundamental roles in biofilm formation.

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