SummaryThe opportunistic pathogen Pseudomonas aeruginosa has two acyl-homoserine lactone (acyl-HSL) signalling systems, LasR-I and RhlR-I. LasI catalyses the synthesis of N-3-oxododecanoyl homoserine lactone (3OC12) and LasR is a transcription factor that requires 3OC12 as a ligand. RhlI catalyses the synthesis of N -butanoyl homoserine lactone (C4) and RhlR is a transcription factor that responds to C4. LasR and RhlR control the transcription of hundreds of P. aeruginosa genes. There is a third P. aeruginosa LasR-RhlR homologue encoded by qscR for which there is no cognate acyl-HSL synthase gene. To test the hypothesis that QscR functions by direct control of specific promoters in an acyl-HSL-dependent manner we purified QscR and characterized QscR activity in vitro . We also studied QscR activity in recombinant Escherichia coli . QscR binds to promoters that have elements similar in sequence to those found in LasRor RhlR-dependent promoters but QscR does not bind to the LasR-or RhlR-specific promoters we examined. QscR binding to DNA requires 3OC12, but QscR exhibits a relaxed acyl-HSL specificity compared with the 3OC12-cognate signal receptor LasR. Our results support the hypothesis that there is a specific QscRdependent regulon. We show that QscR controls genes in this regulon directly and that regulation is dependent on an acyl-HSL produced by LasI. Because of its relaxed signal specificity QscR may also respond to acyl-HSLs made by other bacteria in mixed bacterial communities.
The opportunistic pathogen Pseudomonas aeruginosa possesses two complete acyl-homoserine lactone (acyl-HSL) signaling systems. One system consists of LasI and LasR, which generate a 3-oxododecanoyl-homoserine lactone signal and respond to that signal, respectively. The other system is RhlI and RhlR, which generate butanoyl-homoserine lactone and respond to butanoyl-homoserine lactone, respectively. These quorum-sensing systems control hundreds of genes. There is also an orphan LasR-RhlR homolog, QscR, for which there is no cognate acyl-HSL synthetic enzyme. We previously reported that a qscR mutant is hypervirulent and showed that QscR transiently represses a few quorum-sensing-controlled genes. To better understand the role of QscR in P. aeruginosa gene regulation and to better understand the relationship between QscR, LasR, and RhlR control of gene expression, we used transcription profiling to identify a QscR-dependent regulon. Our analysis revealed that QscR activates some genes and represses others. Some of the repressed genes are not regulated by the LasR-I or RhlR-I systems, while others are. The LasI-generated 3-oxododecanoyl-homoserine lactone serves as a signal molecule for QscR. Thus, QscR appears to be an integral component of the P. aeruginosa quorum-sensing circuitry. QscR uses the LasI-generated acyl-homoserine lactone signal and controls a specific regulon that overlaps with the already overlapping LasR-and RhlR-dependent regulons.
The aim of this study was to analyze the cleaning efficiency of polysaccharidases and proteolytic enzymes against biofilms of bacterial species found in food industry processing lines and to study enzyme effects on the composition of extracellular polymeric substances (EPS) and biofilm removal in a Clean-in-Place (CIP) procedure. The screening of 7 proteases and polysaccharidases for removal of biofilms of 16 bacterial species was first evaluated using a microtiter plate assay. The alkaline pH buffer removed more biofilm biomass as well as affecting a larger range of bacterial species. The two serine proteases and alpha-amylase were the most efficient enzymes. Proteolytic enzymes promoted biofilm removal of a larger range of bacterial species than polysaccharidases. Using three isolates derived from two bacterial species widely found in food processing lines (Pseudomonas fluorescens and the Bacillus cereus group), biofilms were developed on stainless steel slides and enzymatic solutions were used to remove the biofilms using CIP procedure. Serine proteases were more efficient in removing cells of Bacillus biofilms than polysaccharidases. However, polysaccharidases were more efficient in removing P. fluorescens biofilms than serine proteases. Solubilization of enzymes with a buffer containing surfactants, and dispersing and chelating agents enhanced the efficiency of polysaccharidases and proteases respectively in removing biofilms of Bacillus and P. fluorescens. A combination of enzymes targeting several components of EPS, surfactants, dispersing and chelating agents would be an efficient alternative to chemical cleaning agents.
Pseudomonas aeruginosa biofilms can develop mushroom-like structures with stalks and caps consisting of discrete subpopulations of cells. Self-produced rhamnolipid surfactants have been shown to be important in development of the mushroom-like structures. The quorum-sensing-controlled rhlAB operon is required for rhamnolipid synthesis. We have introduced an rhlA-gfp fusion into a neutral site in the P. aeruginosa genome to study rhlAB promoter activity in rhamnolipid-producing biofilms. Expression of the rhlA-gfp fusion in biofilms requires the quorum-sensing signal butanoyl-homoserine lactone, but other factors are also required for expression. Early in biofilm development rhlA-gfp expression is low, even in the presence of added butanoylhomoserine lactone. Expression of the fusion becomes apparent after microcolonies with a depth of >20 m have formed and, as shown by differential labeling with rfp or fluorescent dyes, rhlA-gfp is preferentially expressed in the stalks rather than the caps of mature mushrooms. The rhlA-gfp expression pattern is not greatly influenced by addition of butanoyl-homoserine lactone to the biofilm growth medium. We propose that rhamnolipid synthesis occurs in biofilms after stalks have formed but prior to capping in the mushroom-like structures. The differential expression of rhlAB may play a role in the development of normal biofilm architecture.Pseudomonas aeruginosa is an opportunistic pathogen that can cause acute infections or chronic biofilm infections (3,5,6,16,35). In part because it is an emerging pathogen (33), the biology of P. aeruginosa biofilms has received increasing attention recently.At least under specific laboratory conditions, P. aeruginosa biofilm development involves a number of discrete steps. First, individual cells attach to a surface. This is followed by the formation of microcolonies on the surface. Finally, the microcolonies mature into mushroom-like structures in which the cells are embedded in a self-produced extracellular polymeric matrix (6,20). We have begun to learn about genes involved in the development of normal biofilm structures. The development of mushroom-like structures is dependent upon the available carbon and energy source (19). A recent report showed that the mushroom-like structures consist of stalks on which caps form. Cap production requires a form of surface movement called twitching motility (18). Apparently, cells in the stalk do not move and a subpopulation of cells use twitching motility to migrate up the stalk and form a cap. Quorum sensing influences the ability of P. aeruginosa to develop stalks, so that quorum-sensing mutants form a thin, rather uniform layer on surfaces (8). Furthermore, P. aeruginosa produces extracellular rhamnolipid surfactants, and the synthesis of these rhamnolipids is controlled by quorum sensing (25). Rhamnolipid synthesis mutants form abnormal unstructured biofilms and may show enhanced dispersal from surfaces (7). The involvement of rhamnolipids in biofilm formation in part could explain why quorum se...
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