Structural Features of the Pseudomonas fluorescens Biofilm Adhesin LapA Required for LapG-Dependent Cleavage, Biofilm Formation, and Cell Surface Localization

Boyd, Chelsea D.; Smith, T. Jarrod; El-Kirat-Chatel, Sofiane; Newell, Peter D.; Dufrêne, Yves F.; O’Toole, George A.

doi:10.1128/jb.01629-14

Cited by 95 publications

(137 citation statements)

References 67 publications

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“…While there is no obvious primary sequence motif in the extended peptide that could explain the increased level of proteolysis, we speculate whether secondary structures, not present in CdrA randomP but predicted to be in the faster-processed substrates, could be responsible for more efficient protease-substrate interactions. This notion is in agreement with prior suggestions that LapG recognizes helical structures upstream of its cleavage sites (18). In terms of key residues, Asp 2097 , Pro 2098 , and L 2101 (but not L 2100 ) of CdrA are critical for cleavage by LapG (Fig.…”

Section: Resultssupporting

confidence: 81%

Cyclic Di-GMP-Regulated Periplasmic Proteolysis of a Pseudomonas aeruginosa Type Vb Secretion System Substrate

et al. 2016

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We previously identified a second-messenger-regulated signaling system in the environmental bacterium Pseudomonas fluorescens which controls biofilm formation in response to levels of environmental inorganic phosphate. This system contains the transmembrane cyclic di-GMP (c-di-GMP) receptor LapD and the periplasmic protease LapG. LapD regulates LapG and controls the ability of this protease to process a large cell surface adhesin protein, LapA. While LapDG orthologs can be identified in diverse bacteria, predictions of LapG substrates are sparse. Notably, the opportunistic pathogen Pseudomonas aeruginosa harbors LapDG orthologs, but neither the substrate of LapG nor any associated secretion machinery has been identified to date. Here, we identified P. aeruginosa CdrA, a protein known to mediate cell-cell aggregation and biofilm maturation, as a substrate of LapG. We also demonstrated LapDG to be a minimal system sufficient to control CdrA localization in response to changes in the intracellular concentration of c-di-GMP. Our work establishes this biofilm signaling node as a regulator of a type Vb secretion system substrate in a clinically important pathogen. IMPORTANCEHere, the biological relevance of a conserved yet orphan signaling system in the opportunistic pathogen Pseudomonas aeruginosa is revealed. In particular, we identified the adhesin CdrA, the cargo of a two-partner secretion system, as a substrate of a periplasmic protease whose activity is controlled by intracellular c-di-GMP levels and a corresponding transmembrane receptor via an inside-out signaling mechanism. The data indicate a posttranslational control mechanism of CdrA via c-di-GMP, in addition to its established transcriptional regulation via the same second messenger. Bacteria in nature exist as free-swimming motile organisms or as sessile communities adhered to solid surfaces that are enveloped in a self-produced matrix of adhesive proteins, polysaccharides, and nucleic acids (1). These biofilms protect bacterial communities from their surrounding environment, and as a result, infections caused by biofilm-forming pathogens are often tolerant to traditional antibiotic therapies and the immune system. Understanding the molecular mechanisms governing the regulation of bacterial biofilm formation is thus paramount to finding new avenues for treating such chronic infections.Biofilm formation is regulated via a bacterial second messenger, cyclic di-GMP (c-di-GMP), enzymes for its biosynthesis and degradation, and binding proteins that monitor levels of this cyclic dinucleotide. High levels of c-di-GMP are often associated with a switch to a sessile lifestyle through the binding of the second messenger to receptor proteins (2). Our previous studies identified a central c-di-GMP-specific receptor, LapD, and its associated signaling system in the environmental bacterium and model system for biofilm formation Pseudomonas fluorescens (3-6) (Fig. 1A). In particular, we found that the inner membrane-localized LapD receptor is autoinhibited at...

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Section: Resultssupporting

confidence: 81%

Cyclic Di-GMP-Regulated Periplasmic Proteolysis of a Pseudomonas aeruginosa Type Vb Secretion System Substrate

et al. 2016

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“…So, AFM has recently and rapidly evolved into a tool for quantifying the adhesion force between living cells, cells and surface, and even single molecules (Baro & Reifenberger, 2012;Beaussart et al, 2014). First used by Bremer et al to visualize biofilms (Bremer et al, 1992), AFM has been mainly applied to gain valuable insights in biofilm structure and mechanisms underlying adhesion, as well as single-and multi-strain biofilm formation (Figure 12) (Boyd et al, 2014;Cabral et al, 2014;Lim et al, 2011;Ovchinnikova et al, 2013;Potthoff et al, 2015). It has also proven useful for quantitative biofilm analysis, especially to confirm findings obtained by quantitative (viable count, CV staining) or other imaging (light microscopy, SEM) techniques (Ansari et al, 2013;Chatterjee et al, 2014;Li et al, 2015;Salunke et al, 2014).…”

Section: ;mentioning

confidence: 99%

Critical review on biofilm methods

Azeredo

Azevedo

Briandet

et al. 2016

Critical Reviews in Microbiology

784

679

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Biofilms are widespread in nature and constitute an important strategy implemented by microorganisms to survive in sometimes harsh environmental conditions. They can be beneficial or have a negative impact particularly when formed in industrial settings or on medical devices. As such, research into the formation and elimination of biofilms is important for many disciplines. Several new methodologies have been recently developed for, or adapted to, biofilm studies that have contributed to deeper knowledge on biofilm physiology, structure and composition. In this review, traditional and cutting-edge methods to study biofilm biomass, viability, structure, composition and physiology are addressed. Moreover, as there is a lack of consensus among the diversity of techniques used to grow and study biofilms. This review intends to remedy this, by giving a critical perspective, highlighting the advantages and limitations of several methods. Accordingly, this review aims at helping scientists in finding the most appropriate and up-to-date methods to study their biofilms. ARTICLE HISTORY

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“…Therefore, we assessed the impact of elevated Fis levels on the abundance of the LapA protein also by monitoring the fluorescence of a LapA-GFP fusion protein in 18 h grown cells. According to previous studies, the LapA C-terminus contains a signal sequence for a type I transport system and the transporter LapB is needed to transport LapA out of the cytoplasm (Boyd et al, 2014;Hinsa et al, 2003). To localize LapA-GFP in the cytoplasm, we deleted the lapB and lapC genes and fused the GFP protein to the LapA C-terminal end.…”

Section: Overexpression Of Fis Increases the Amount Ofmentioning

confidence: 99%

Fis overexpression enhances Pseudomonas putida biofilm formation by regulating the ratio of LapA and LapF

et al. 2014

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Bacteria form biofilm as a response to a number of environmental signals that are mediated by global transcription regulators and alarmones. Here we report the involvement of the global transcription regulator Fis in Pseudomonas putida biofilm formation through regulation of lapA and lapF genes. The major component of P. putida biofilm is proteinaceous and two large adhesive proteins, LapA and LapF, are known to play a key role in its formation. We have previously shown that Fis overexpression enhances P. putida biofilm formation. In this study, we used mini-Tn5 transposon mutagenesis to select potential Fis-regulated genes involved in biofilm formation. A total of 90 % of the studied transposon mutants carried insertions in the lap genes. Since our experiments showed that Fis-enhanced biofilm is mostly proteinaceous, the amounts of LapA and LapF from P. putida cells lysates were quantified using SDS-PAGE. Fis overexpression increases the quantity of LapA 1.6 times and decreases the amount of LapF at least 4 times compared to the wild-type cells. The increased LapA expression caused by Fis overexpression was confirmed by FACS analysis measuring the amount of LapA-GFP fusion protein. Our results suggest that the profusion of LapA in the Fis-overexpressed cells causes enhanced biofilm formation in mature stages of P. putida biofilm and LapF has a minor role in P. putida biofilm formation.

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Structural Features of the Pseudomonas fluorescens Biofilm Adhesin LapA Required for LapG-Dependent Cleavage, Biofilm Formation, and Cell Surface Localization

Cited by 95 publications

References 67 publications

Cyclic Di-GMP-Regulated Periplasmic Proteolysis of a Pseudomonas aeruginosa Type Vb Secretion System Substrate

Cyclic Di-GMP-Regulated Periplasmic Proteolysis of a Pseudomonas aeruginosa Type Vb Secretion System Substrate

Critical review on biofilm methods

Fis overexpression enhances Pseudomonas putida biofilm formation by regulating the ratio of LapA and LapF

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