Glycosyl hydrolase from Pseudomonas fluorescens inhibits the biofilm formation of Pseudomonads.

Biofilms are complex microbial communities embedded in extracellular matrix. Pathogens within the biofilm become more resistant to the antibiotics than planktonic counterparts. Novel strategies are required to encounter biofilms. Exopolysaccharides are one of the major components of biofilm matrix and play a vital role in biofilm architecture. In previous studies, a glycosyl hydrolase, PslGPA, from Pseudomonas aeruginosa was found to be able to inhibit biofilm formation by disintegrating exopolysaccharide in biofilms. Here, we investigate the potential spectrum of PslG homologous protein with anti-biofilm activity. One glycosyl hydrolase from Pseudomonas fluorescens, PslGPF, exhibits anti-biofilm activities and the key catalytic residues of PslGPF are conserved with those of PslGPA. PslGPF at concentrations as low as 50 nM efficiently inhibits the biofilm formation of P. aeruginosa and disassemble its preformed biofilm. Furthermore, PslGPF exhibits anti-biofilm activity on a series of Pseudomonads, including P. fluorescens, Pseudomonas stutzeri and Pseudomonas syringae pv. phaseolicola. PslGPF stays active under various temperatures. Our findings suggest that P. fluorescens glycosyl hydrolase PslGPF has potential to be a broad spectrum inhibitor on biofilm formation of a wide range of Pseudomonads.

A Library of Promoter-gfp Fusion Reporters for Studying Systematic Expression Pattern of Cyclic-di-GMP Metabolism-Related Genes in Pseudomonas aeruginosa.

The opportunistic pathogen Pseudomonas aeruginosa is an environmental microorganism and is a model organism for biofilm research. Cyclic dimeric GMP (c-di-GMP) is a bacterial second messenger that plays critical roles in biofilm formation. P. aeruginosa contains approximately 40 genes that encode enzymes that participate in the metabolism of c-di-GMP (biosynthesis or degradation), yet it lacks tools that aid investigation of the systematic expression pattern of those genes. In this study, we constructed a promoter-gfp fusion reporter library that consists of 41 reporter plasmids. Each plasmid contains a promoter of corresponding c-di-GMP metabolism-related (CMR) genes from P. aeruginosa reference strain PAO1; thus, each promoter-gfp fusion reporter can be used to detect the promoter activity as well as the transcription of corresponding gene. The promoter activity was tested in P. aeruginosa and Escherichia coli. Among the 41 genes, the promoters of 26 genes showed activity in both P. aeruginosa and E. coli. The library was applied to determine the influence of different temperatures, growth media, and subinhibitory concentrations of antibiotics on the transcriptional profile of the 41 CMR genes in P. aeruginosa. The results showed that different growth conditions did affect the transcription of different genes, while the promoter activity of a few genes was kept at the same level under several different growth conditions. In summary, we provide a promoter-gfp fusion reporter library for systematic monitoring or study of the regulation of CMR genes in P. aeruginosa. In addition, the functional promoters can also be used as a biobrick for synthetic biology studies. IMPORTANCE The opportunistic pathogen P. aeruginosa can cause acute and chronic infections in humans, and it is one of the main pathogens in nosocomial infections. Biofilm formation is one of the most important causes for P. aeruginosa persistence in hosts and evasion of immune and antibiotic attacks. c-di-GMP is a critical second messenger to control biofilm formation. In P. aeruginosa reference strain PAO1, 41 genes are predicted to participate in the making and breaking of this dinucleotide. A major missing piece of information in this field is the systematic expression profile of those genes in response to changing environment. Toward this goal, we constructed a promoter-gfp transcriptional fusion reporter library that consists of 41 reporter plasmids, each of which contains a promoter of corresponding c-di-GMP metabolism-related genes in P. aeruginosa. This library provides a helpful tool to understand the complex regulation network related to c-di-GMP and to discover potential therapeutic targets.

Dual functions: A coumarin-chalcone conjugate inhibits cyclic-di-GMP and quorum-sensing signaling to reduce biofilm formation and virulence of pathogens.

Antibiotic resistance or tolerance of pathogens is one of the most serious global public health threats. Bacteria in biofilms show extreme tolerance to almost all antibiotic classes. Thus, use of antibiofilm drugs without bacterial-killing effects is one of the strategies to combat antibiotic tolerance. In this study, we discovered a coumarin-chalcone conjugate C9, which can inhibit the biofilm formation of three common pathogens that cause nosocomial infections, namely, Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli, with the best antibiofilm activity against P. aeruginosa. Further investigations indicate that C9 decreases the synthesis of the key biofilm matrix exopolysaccharide Psl and bacterial second messenger cyclic-di-GMP. Meanwhile, C9 can interfere with the regulation of the quorum sensing (QS) system to reduce the virulence of P. aeruginosa. C9 treatment enhances the sensitivity of biofilm to several antibiotics and reduces the survival rate of P. aeruginosa under starvation or oxidative stress conditions, indicating its excellent potential for use as an antibiofilm-forming and anti-QS drug.

Cell division factor ZapE regulates Pseudomonas aeruginosa biofilm formation by impacting the pqs quorum sensing system.

Pseudomonas aeruginosa is one of the leading nosocomial pathogens that causes both severe acute and chronic infections. The strong capacity of P. aeruginosa to form biofilms can dramatically increase its antibiotic resistance and lead to treatment failure. The biofilm resident bacterial cells display distinct gene expression profiles and phenotypes compared to their free-living counterparts. Elucidating the genetic determinants of biofilm formation is crucial for the development of antibiofilm drugs. In this study, a high-throughput transposon-insertion site sequencing (Tn-seq) approach was employed to identify novel P. aeruginosa biofilm genetic determinants. When analyzing the novel biofilm regulatory genes, we found that the cell division factor ZapE (PA4438) controls the P. aeruginosa pqs quorum sensing system. The ∆zapE mutant lost fitness against the wild-type PAO1 strain in biofilms and its production of 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS) had been reduced. Further biochemical analysis showed that ZapE interacts with PqsH, which encodes the synthase that converts 2-heptyl-4-quinolone (HHQ) to PQS. In addition, site-directed mutagenesis of the ATPase active site of ZapE (K72A) abolished the positive regulation of ZapE on PQS signaling. As ZapE is highly conserved among the Pseudomonas group, our study suggests that it is a potential drug target for the control of Pseudomonas infections.

Regulation of Biofilm Exopolysaccharide Biosynthesis and Degradation in Pseudomonas aeruginosa.

Microbial communities enmeshed in a matrix of macromolecules, termed as biofilms, are the natural setting of bacteria. Exopolysaccharide is a critical matrix component of biofilms. Here, we focus on biofilm matrix exopolysaccharides in Pseudomonas aeruginosa. This opportunistic pathogen can adapt to a wide range of environments and can form biofilms or aggregates in a variety of surfaces or environments, such as the lungs of people with cystic fibrosis, catheters, wounds, and contact lenses. The ability to synthesize multiple exopolysaccharides is one of the advantages that facilitate bacterial survival in different environments. P. aeruginosa can produce several exopolysaccharides, including alginate, Psl, Pel, and lipopolysaccharide. In this review, we highlight the roles of each exopolysaccharide in P. aeruginosa biofilm development and how bacteria coordinate the biosynthesis of multiple exopolysaccharides and bacterial motility. In addition, we present advances in antibiofilm strategies targeting matrix exopolysaccharides, with a focus on glycoside hydrolases.

Inactivation of mixed Escherichia coli O157:H7 biofilms on lettuce by bacteriophage in combination with slightly acidic hypochlorous water (SAHW) and mild heat treatment.

Escherichia coli O157:H7 is one of the most important foodborne pathogens that can persist in leafy green vegetables and subsequently produce biofilms. Biofilm formation is an ongoing concern in the food industry as biofilms are relatively resistant to a variety of antimicrobial treatments. In the present study, we evaluated the combined effects of phage FP43 and mild-heated slightly acidic hypochlorous water (SAHW) in reducing established biofilms on lettuce. Prior to the sequential treatments involving phage-SAHW and SAHW-phage for long-term storage, equal inoculum densities of E. coli O157:H7 and E. coli O91:H- were added on iceberg lettuce surfaces and the lettuce samples were stored at 10 °C for 48 h to allow biofilm formation. The sequential treatment with phage FP43 and SAHW significantly decreased the number of adhered cells, especially the combination of phage FP43 at 25 °C for 2 h and mild-heated SAHW, which considerably eliminated E. coli viable biofilm cells to undetectable levels (>3 log CFU/piece). However, the biofilms were not completely removed, as evidenced via SEM observation. Additionally, sequential treatment with SAHW and phage caused continuous reductions in viable counts, decreasing the viability of E. coli O157:H7 and total E. coli to the lower limit of detection after incubation for 5 d. Meanwhile, bacterial regrowth was observed after treatment with SAHW alone. These results indicated that the combination of phage and SAHW could be considered as a promising strategy to control the formation of E. coli O157:H7 biofilms on lettuce.

Mutations in surface-sensing receptor WspA lock the Wsp signal transduction system into a constitutively active state.

Pseudomonas aeruginosa rugose small-colony variants (RSCVs) are frequently isolated from chronic infections, yet, they are rarely reported in environmental isolates. Here, during the comparative genomic analysis of two P. aeruginosa strains isolated from crude oil, we discovered a spontaneous in-frame deletion, wspAΔ280-307 , which led to hyper-biofilm and RSCV phenotypes. WspA is a homologue of methyl-accepting chemotaxis proteins (MCPs) that senses surfaces to regulate biofilm formation by stimulating cyclic-di-guanosine monophosphate (c-di-GMP) synthesis through the Wsp system. However, the methylation sites of WspA have never been identified. In this study, we identified E280 and E294 of WspA as methylation sites. The wspAΔ280-307 mutation enabled the Wsp system to lock into a constitutively active state that is independent of regulation by methylation. The result is an enhanced production of c-di-GMP. Sequence alignment revealed three conserved repeat sequences within the amino acid residues 280-313 (aa280-313) region of WspA homologues, suggesting that a spontaneous deletion within this DNA encoding region was likely a result of intragenic recombination and that similar mutations might occur in several related bacterial genera. Our results provide a plausible explanation for the selection of RSCVs and a mechanism to confer a competitive advantage for P. aeruginosa in a crude-oil environment.

Rugose small colony variant and its hyper-biofilm in Pseudomonas aeruginosa: Adaption, evolution, and biotechnological potential.

One of the hallmarks of the environmental bacterium Pseudomonas aeruginosa is its excellent ecological flexibility, which can thrive in diverse ecological niches. In different ecosystems, P. aeruginosa may use different strategies to survive, such as forming biofilms in crude oil environment, converting to mucoid phenotype in the cystic fibrosis (CF) lung, or becoming persisters when treated with antibiotics. Rugose small colony variants (RSCVs) are the adaptive mutants of P. aeruginosa, which can be frequently isolated from chronic infections. During the past years, there has been a renewed interest in using P. aeruginosa as a model organism to investigate the RSCVs formation, persistence and pathogenesis, as RSCVs represent a hyper-biofilm formation, high adaptability, high-tolerance sub-population in biofilms. This review will briefly summarize recent advances regarding the phenotypic, genetic and host interaction associated with RSCVs, with an emphasis on P. aeruginosa. Meanwhile, some non-pathogenic bacteria such as Pseudomonas fluorescence, Pseudomonas putida and Bacillus subtilis will be also included. Remarkable emphasis is given on intrinsic functions of such hyper-biofilm formation characteristic as well as its potential applications in several biocatalytic transformations including wastewater treatment, microbial fermentation, and plastic degradation. Hopefully, this review will attract the interest of researchers in various fields and shape future research focused not only on evolutionary biology but also on biotechnological applications related to RSCVs.

Enhanced mechanism of extracellular electron transfer between semiconducting minerals anatase and Pseudomonas aeruginosa PAO1 in euphotic zone.

Focusing the marine euphotic zone, which is the pivotal region for interaction of solar light-mineral-microorganism and the elements cycle, we have conducted the research on the mechanism of semiconducting minerals promoting extracellular electron transfer with microorganisms in depth. Therein, anatase which is one of the most representative semiconducting minerals in marine euphotic zone was selected. The mineralogical characterization of anatase was identified by ESEM, AFM, EDS, Raman, XRD, and its semiconducting characteristics was determined by UV-Vis and Mott-Schottky plots. Determined by the electrochemical measurement of I-t curves, the photocurrent density of anatase was more prominent than dark current density. Pseudomonas aeruginosa PAO1 was widely distributed in the euphotic zone, and its mutants of operons deficient in biosynthesis pyocyanin (Δphz1Δphz2) and pili deficient (ΔpilA) were employed in this study. I-t curves indicated that both direct and indirect extracellular electron transfer processes occurred between anatase and PAO1. The indirect electron transfer depending on pyocyanin secreted by PAO1 was the main electron transfer mode. This work demonstrated the light-driven extracellular electron transfer and further revealed the photo-catalyzed mechanisms between anatase and PAO1 in marine euphotic zone.

Effect of Polyhexamethylene Biguanide in Combination with Undecylenamidopropyl Betaine or PslG on Biofilm Clearance.

Hospital-acquired infection is a great challenge for clinical treatment due to pathogens' biofilm formation and their antibiotic resistance. Here, we investigate the effect of antiseptic agent polyhexamethylene biguanide (PHMB) and undecylenamidopropyl betaine (UB) against biofilms of four pathogens that are often found in hospitals, including Gram-negative bacteria Pseudomonas aeruginosa and Escherichia coli, Gram-positive bacteria Staphylococcus aureus, and pathogenic fungus, Candida albicans. We show that 0.02% PHMB, which is 10-fold lower than the concentration of commercial products, has a strong inhibitory effect on the growth, initial attachment, and biofilm formation of all tested pathogens. PHMB can also disrupt the preformed biofilms of these pathogens. In contrast, 0.1% UB exhibits a mild inhibitory effect on biofilm formation of the four pathogens. This concentration inhibits the growth of S. aureus and C. albicans yet has no growth effect on P. aeruginosa or E. coli. UB only slightly enhances the anti-biofilm efficacy of PHMB on P. aeruginosa biofilms. However, pretreatment with PslG, a glycosyl hydrolase that can efficiently inhibit and disrupt P. aeruginosa biofilm, highly enhances the clearance effect of PHMB on P. aeruginosa biofilms. Meanwhile, PslG can also disassemble the preformed biofilms of the other three pathogens within 30 min to a similar extent as UB treatment for 24 h.

A molecular mechanism for how sigma factor AlgT and transcriptional regulator AmrZ inhibit twitching motility in Pseudomonas aeruginosa.

Pseudomonas aeruginosa isolates from cystic fibrosis patients are often mucoid (due to the overexpression of exopolysaccharide alginate) yet lost motility. It remains unclear about how P. aeruginosa coordinately regulates alginate production and the type IV pili-driven twitching motility. Here we showed that sigma 22 factor (AlgT/U), an activator of alginate biosynthesis, repressed twitching motility by inhibiting the expression of pilin (PilA) through the intermediate transcriptional regulator AmrZ, which directly bound to the promoter region of pilA in both mucoid strain FRD1 and non-mucoid strain PAO1. Four conserved AmrZ-binding sites were found in pilA promoters among 10 P. aeruginosa strains although their entire pilA promoters had low identity. AmrZ has been reported to be essential for twitching in PAO1. We found that AmrZ was also required for twitching in mucoid FRD1, yet a high level of AmrZ inhibited twitching motility. This result was consistent with the phenomenon that twitching is frequently repressed in mucoid strains, in which the expression of AmrZ was highly activated by AlgT. Additionally, AlgT also inhibited the transcription of pilMNOP operon, which is involved in efficient pilus assembly. Our data elucidated a mechanism for how AlgT and AmrZ coordinately controlled twitching motility in P. aeruginosa.

Integrated Comparative Genomic Analysis and Phenotypic Profiling of Pseudomonas aeruginosa Isolates From Crude Oil.

Pseudomonas aeruginosa is an environmental microorganism that can thrive in diverse ecological niches including plants, animals, water, soil, and crude oil. It also one of the microorganism widely used in tertiary recovery of crude oil and bioremediation. However, the genomic information regarding the mechanisms of survival and adapation of this bacterium in crude oil is still limited. In this study, three Pseudomonads strains (named as IMP66, IMP67, and IMP68) isolated from crude oil were taken for whole-genome sequencing by using a hybridized PacBio and Illumina approach. The phylogeny analysis showed that the three strains were all P. aeruginosa species and clustered in clade 1, the group with PAO1 as a representitive. Subsequent comparative genomic analysis revealed a high degree of individual genomic plasticity, with a probable alkane degradation genomic island, one type I-F CRISPR-Cas system and several prophages integrated into their genomes. Nine genes encoding alkane hydroxylases (AHs) homologs were found in each strain, which might enable these strains to degrade alkane in crude oil. P. aeruginosa can produce rhamnolipids (RLs) biosurfactant to emulsify oil, which enables their survival in crude oil enviroments. Our previous report showed that IMP67 and IMP68 were high RLs producers, while IMP66 produced little RLs. Genomic analysis suggested that their RLs yield was not likely due to differences at genetic level. We then further analyzed the quorum sensing (QS) signal molecules that regulate RLs synthesis. IMP67 and IMP68 produced more N-acyl-homoserine lactones (AHLs) signal molecules than that of PAO1 and IMP66, which could explain their high RLs yield. This study provides evidence for adaptation of P. aeruginosa in crude oil and proposes the potential application of IMP67 and IMP68 in microbial-enhanced oil recovery and bioremediation.

Evaluation and characterization of the predicted diguanylate cyclase-encoding genes in Pseudomonas aeruginosa.

Opportunistic pathogen Pseudomonas aeruginosa can cause acute and chronic infections in humans. It is notorious for its resistance to antibiotics due to the formation of biofilms. Cyclic-di-GMP is a bacterial second messenger that plays important roles during biofilm development. There are 40 genes in P. aeruginosa predicted to participate in c-di-GMP biosynthesis or degradation. It is time-consuming for the functional characterization of these genes. Here, we cloned 16 genes from P. aeruginosa PAO1 that are predicted to encode diguanylate cyclases (DGCs, responsible for c-di-GMP biosynthesis) and constructed their corresponding in-frame deletion mutants. We evaluated the methods to measure the intracellular c-di-GMP concentration by using deletion mutants and PAO1 strains containing a plasmid expressing one of the 16 genes, respectively. Functional outputs of all PAO1-derived stains were also detected and evaluated, including biofilm formation, production of exopolysaccharide, swimming and swarming motilities. Our data showed that measuring the c-di-GMP level only characterized a few DGC by using either pCdrA::gfp as a reporter or LC/MS/MS. Functional output results indicated that overexpression of a DGC gave more pronounced phenotypes than the corresponding deletion mutant and suggested that the swimming motility assay could be a quick way to briefly estimate a predicted DGC for further studies. The overall evaluation suggested 15 out of 16 predicted DGCs were functional DGCs, wherein six were characterized to encode DGCs previously. Altogether, we have provided not only a cloning library of 16 DGC-encoding genes and their corresponding in-frame deletion mutants but also paved ways to briefly characterize a predicted DGC.

Chinese medicinal herb extract inhibits PQS-mediated quorum sensing system in Pseudomonas aeruginosa.

ETHNOPHARMACOLOGICAL RELEVANCE: Chinese medicinal herbs have long been recognized as important resources that can be used for the struggle against diseases and a significant component of health care system for thousands of years. AIM OF THE STUDY: In order to understand their roles in the treatment against bacterial infections, we examined the underlying mechanisms of one of the medicinal herb extracts (MHE) (Artemisiae argyi Folium, the root bark of Cortex dictamni and the root of Solanum melongena) on the human opportunistic pathogen Pseudomonas aeruginosa. MATERIALS AND METHODS: We combined phenotypic assays, transcriptional analysis and chemical investigations to identify the mechanisms underlying MHE inhibition. The standard sample was prepared and transcriptional reporters for quorum sensing systems were constructed. Electrophoretic mobility shift assays were used to clarify the mechanism. GC-MS and molecular docking were used to identify the chemicals in MHE and potential binding agents. RESULTS: We found that co-culturing of MHE with bacterial cells did not change the growth rate but substantially attenuate the production of virulence factors such as phenazine pyocyanin, siderophore pyoverdine and biofilm formation. Transcriptional responses of three major quorum sensing (QS) systems of P. aeruginosa to MHE showed that Pseudomonas quinolone signaling (PQS) system was completely repressed, rhlR/rhlI QS system was moderately inhibited, while lasR/lasI QS system was only slightly affected, suggesting that MHE might selectively target the PQS system to inhibit bacterial virulence. Furthermore, electrophoretic mobility shift assays (EMSA) showed that MHE inhibited the binding of MvfR the corresponding pqsA promoter region, suggesting that MHE serves as a competitive agent to quench the QS functionality in P. aeruginosa. CONCLUSION: We prove that MHE functions as an effective countermeasure against bacterial infections.

Diguanylate Cyclases and Phosphodiesterases Required for Basal-Level c-di-GMP in Pseudomonas aeruginosa as Revealed by Systematic Phylogenetic and Transcriptomic Analyses.

Cyclic diguanosine monophosphate (c-di-GMP) is an important second messenger involved in bacterial switching from motile to sessile lifestyles. In the opportunistic pathogen Pseudomonas aeruginosa, at least 40 genes are predicted to encode proteins for the making and breaking of this signal molecule. However, there is still paucity of information concerning the systemic expression pattern of these genes and the functions of uncharacterized genes. In this study, we analyzed the phylogenetic distribution of genes from P. aeruginosa that were predicted to have a GGDEF domain and found five genes (PA5487, PA0285, PA0290, PA4367, and PA5017) with highly conserved distribution across 52 public complete pseudomonad genomes. PA5487 was further characterized as a typical diguanylate cyclase (DGC) and was named dgcH A systemic analysis of the gene expression data revealed that the expression of dgcH is highly invariable and that dgcH probably functions as a conserved gene to maintain the basal level of c-di-GMP, as reinforced by gene expression analyses. The other four conserved genes also had an expression pattern similar to that of dgcH The functional analysis suggested that PA0290 encoded a DGC, while the others functioned as phosphodiesterases (PDEs). Our data revealed that there are five DGC and PDE genes that maintain the basal level of c-di-GMP in P. aeruginosaIMPORTANCEPseudomonas aeruginosa is an opportunistic pathogen that can cause infections in animals, humans, and plants. The formation of biofilms by P. aeruginosa is the central mode of action to persist in hosts and evade immune and antibiotic attacks. Cyclic-di-GMP (c-di-GMP) is an important second messenger involved in the regulation of biofilm formation. In P. aeruginosa PAO1 strain, there are around 40 genes that encode enzymes for making and breaking this dinucleotide. A major missing piece of information in this field is the phylogeny and expression profile of those genes. Here, we took a systemic approach to investigate this mystery. We found that among 40 c-di-GMP metabolizing genes, 5 have well-conserved phylogenetic distribution and invariable expression profiles, suggesting that there are enzymes required for the basal level of c-di-GMP in P. aeruginosa This study thus provides putative therapeutic targets against P. aeruginosa infections.

Crystal structure of the glycoside hydrolase PssZ from Listeria monocytogenes.

Biofilms are microbial communities that are embedded in the extracellular matrix. The exopolysaccharide (EPS) is a key component of the biofilm matrix that maintains the structure of the biofilm and protects the bacteria from antimicrobials. Microbial glycoside hydrolases have been exploited to disrupt biofilms by breaking down EPSs. PssZ has recently been identified as a glycoside hydrolase that can disperse aggregates of Listeria monocytogenes. In this study, the crystal structure of PssZ has been determined at 1.6 Šresolution. PssZ belongs to glycoside hydrolase family 8 and adopts a classical (α/α)6-barrel fold. This architecture forms a deep groove which may serve as the substrate-binding pocket. The conserved catalytic residues (Glu72, Trp110, Asn119, Phe167, Tyr183 and Asp232) are localized at the centre of the groove. This crystal structure will help to improve the understanding of the hydrolytic mechanism of PssZ and its application as a biofilm disrupter.

The advance of assembly of exopolysaccharide Psl biosynthesis machinery in Pseudomonas aeruginosa.

Biofilms are microbial communities embedded in extracellular matrix. Exopolysaccharide Psl (ePsl) is a key biofilm matrix component that initiates attachment, maintains biofilms architecture, and protects bacteria within biofilms of Pseudomonas aeruginosa, an opportunistic pathogen. There are at least 12 Psl proteins involved in the biosynthesis of this exopolysaccharide. However, it remains unclear about the function of each Psl protein and how these proteins work together during the biosynthesis of ePsl. PslG has been characterized as a degrader of ePsl in extracellular or periplasm and PslD is predicted to be a transporter. In this study, we found that PslG and its glycoside hydrolytic activity were also involved in the biosynthesis of ePsl. PslG localized mainly in the inner membrane and some in the periplasm. The inner membrane association of PslG was critical for the biosynthesis of ePsl. The expression of PslA, PslD, and PslE helped PslG remain in the inner membrane. The bacterial two-hybrid results suggested that PslE could interacted with either PslA, PslD, or PslG. The strongest interaction was found between PslE and PslD. Consistently, PslD was disabled to localize on the outer membrane in the ΔpslE strain, suggesting that the PslE-PslD interaction affected the localization of PslD. Our results shed light on the assembly of ePsl biosynthesis machinery and suggested that the membrane-associated PslG was a part of ePsl biosynthesis proteins complex.

Development of an effective fluorescence probe for discovery of aminopeptidase inhibitors to suppress biofilm formation.

The human pathogen Pseudomonas aeruginosa can easily form biofilms. The extracellular matrix produced by the bacterial cells acts as a physical barrier to hinder the antibiotics treatment. It is necessary to destroy the biofilm in order to improve the efficacy of antibiotics. However, it has been a significant challenge to develop effective small molecules targeting the components of biofilm matrix. In this study, we report the development of a new effective fluorescence probe that could be used in the high throughput screening to identify novel small molecule inhibitors targeting the most abundant component in the biofilm formation: P. aeruginosa aminopeptidase (PaAP). Through screening of an in-house chemical library, a commercially available drug, balsalazide, has been identified as a novel PaAP inhibitor, which exhibited remarkable anti-biofilm effect. Our study indicated that the newly developed fluorescence probe is applicable in exploring new aminopeptidase inhibitors, and it also warrants further investigation of balsalazide as a new anti-biofilm agent to treat P. aeruginosa infection in combination with known antibiotics.

Biological synthesis of high-conductive pili in aerobic bacterium Pseudomonas aeruginosa.

Bioelectrical nanowires as ecomaterials have great potential on environmental applications. A wide range of bacteria can express type IV pili (T4P), which are long protein fibers assembled from PilA. The T4P of Geobacter sulfurreducens are well known as "microbial nanowires," yet T4P of Pseudomonas aeruginosa (PaT4P) was believed to be poorly conductive. P. aeruginosa is an aerobic and electrochemically active bacterium. Its T4P have been known to be responsible for surface attachment, twitching motility and biofilm formation. Here, we show that PaT4P can be highly conductive while assembled by a truncated P. aeruginosa PilA (PaPilA) containing only N-terminus 61 amino acids. Furthermore, increasing the number of aromatic amino acids in the PaPilA1-61 significantly enhances the conductivity of pili and the bioelectricity output of P. aeruginosa in microbial fuel cell system, suggesting a potential application of PaT4P as a conductive nanomaterial. The N-terminal region of PilA from diverse eubacteria is highly conserved, implying a general way to synthesize highly conductive microbial nanowires and to increase the bioelectricity output of microbial fuel cell.

Overshadow Effect of Psl on Bacterial Response to Physiochemically Distinct Surfaces Through Motility-Based Characterization.

Biofilms of Pseudomonas aeruginosa are ubiquitously found on surfaces of many medical devices, which are the major cause of hospital-acquired infections. A large amount of work has been focused on bacterial attachment on surfaces. However, how bacterial cells evolve on surfaces after their attachment is the key to get better understanding and further control of biofilm formation. In this work, by employing both single-cell- and collective-motility of cells, we characterized the bacterial surface movement on physiochemically distinct surfaces. The measurement of cell surface motility showed consistent results that gold and especially platinum surfaces displayed a stronger capability in microcolony formation than polyvinyl chloride and polycarbonate surfaces. More interestingly, we found that overproduction of Psl led to a narrower variance in cell surface motility among tested surfaces, indicating an overshadow effect of Psl for bacteria by screening the influence of physicochemical properties of solid surfaces. Our results provide insights into how Pseudomonas aeruginosa cells adapt their motion to physiochemically distinct surfaces, and thus would be beneficial for developing new anti-biofouling techniques in biomedical engineering.