RESUMO
The molecular basis of second messenger signaling relies on an array of proteins that synthesize, degrade or bind the molecule to produce coherent functional outputs. Cyclic di-GMP (c-di-GMP) has emerged as a eubacterial nucleotide second messenger regulating a plethora of key behaviors, like the transition from planktonic cells to biofilm communities. The striking multiplicity of c-di-GMP control modules and regulated cellular functions raised the question of signaling specificity. Are c-di-GMP signaling routes exclusively dependent on a central hub or can they be locally administrated? In this study, we show an example of how c-di-GMP signaling gains output specificity in Pseudomonas aeruginosa. We observed the occurrence in P. aeruginosa of a c-di-GMP synthase gene, hsbD, in the proximity of the hptB and flagellar genes cluster. We show that the HptB pathway controls biofilm formation and motility by involving both HsbD and the anti-anti-sigma factor HsbA. The rewiring of c-di-GMP signaling into the HptB cascade relies on the original interaction between HsbD and HsbA and on the control of HsbD dynamic localization at the cell poles.
Assuntos
Movimento Celular/genética , Proteínas de Escherichia coli/genética , Fósforo-Oxigênio Liases/genética , Pseudomonas aeruginosa/genética , Biofilmes/crescimento & desenvolvimento , Ciclo Celular/genética , Divisão Celular/genética , GMP Cíclico/genética , Proteínas de Escherichia coli/metabolismo , Flagelos/genética , Regulação Bacteriana da Expressão Gênica , Fósforo-Oxigênio Liases/metabolismo , Fosforilação , Pseudomonas aeruginosa/patogenicidadeRESUMO
[This corrects the article DOI: 10.1371/journal.pgen.1006354.].
RESUMO
UNLABELLED: Nucleotide signaling molecules are important intracellular messengers that regulate a wide range of biological functions. The human pathogen Staphylococcus aureus produces the signaling nucleotide cyclic di-AMP (c-di-AMP). This molecule is common among Gram-positive bacteria and in many organisms is essential for survival under standard laboratory growth conditions. In this study, we investigated the interaction of c-di-AMP with the S. aureus KdpD protein. The sensor kinase KdpD forms a two-component signaling system with the response regulator KdpE and regulates the expression of the kdpDE genes and the kdpFABC operon coding for the Kdp potassium transporter components. Here we show that the S. aureus KdpD protein binds c-di-AMP specifically and with an affinity in the micromolar range through its universal stress protein (USP) domain. This domain is located within the N-terminal cytoplasmic region of KdpD, and amino acids of a conserved SXS-X20-FTAXY motif are important for this binding. We further show that KdpD2, a second KdpD protein found in some S. aureus strains, also binds c-di-AMP, and our bioinformatics analysis indicates that a subclass of KdpD proteins in c-di-AMP-producing bacteria has evolved to bind this signaling nucleotide. Finally, we show that c-di-AMP binding to KdpD inhibits the upregulation of the kdpFABC operon under salt stress, thus indicating that c-di-AMP is a negative regulator of potassium uptake in S. aureus. IMPORTANCE: Staphylococcus aureus is an important human pathogen and a major cause of food poisoning in Western countries. A common method for food preservation is the use of salt to drive dehydration. This study sheds light on the regulation of potassium uptake in Staphylococcus aureus, an important aspect of this bacterium's ability to tolerate high levels of salt. We show that the signaling nucleotide c-di-AMP binds to a regulatory component of the Kdp potassium uptake system and that this binding has an inhibitory effect on the expression of the kdp genes encoding a potassium transporter. c-di-AMP binds to the USP domain of KdpD, thus providing for the first time evidence for the ability of such a domain to bind a cyclic dinucleotide.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Fosfatos de Dinucleosídeos/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Potássio/metabolismo , Proteínas Quinases/metabolismo , Staphylococcus aureus/enzimologia , Proteínas de Bactérias/genética , Proteínas de Transporte/genética , Fosfatos de Dinucleosídeos/genética , Regulação para Baixo , Filogenia , Ligação Proteica , Proteínas Quinases/genética , Estrutura Terciária de Proteína , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismoRESUMO
The second messenger cyclic di-GMP (c-di-GMP) controls the transition between different lifestyles in bacterial pathogens. Here, we report the identification of DgcP (diguanylate cyclase conserved in Pseudomonads), whose activity in the olive tree pathogen Pseudomonas savastanoi pv. savastanoi is dependent on the integrity of its GGDEF domain. Furthermore, deletion of the dgcP gene revealed that DgcP negatively regulates motility and positively controls biofilm formation in both the olive tree pathogen P. savastanoi pv. savastanoi and the human opportunistic pathogen Pseudomonas aeruginosa. Overexpression of the dgcP gene in P. aeruginosaâ PAK led to increased exopolysaccharide production and upregulation of the type VI secretion system; in turn, it repressed the type III secretion system, which is a hallmark of chronic infections and persistence for P. aeruginosa. Deletion of the dgcP gene in P. savastanoi pv. savastanoi NCPPB 3335 and P. aeruginosaâ PAK reduced their virulence in olive plants and in a mouse acute lung injury model respectively. Our results show that diguanylate cyclase DgcP is a conserved Pseudomonas protein with a role in virulence, and confirm the existence of common c-di-GMP signalling pathways that are capable of regulating plant and human Pseudomonas spp. infections.
Assuntos
Lesão Pulmonar Aguda/microbiologia , Proteínas de Escherichia coli/genética , Fósforo-Oxigênio Liases/genética , Doenças das Plantas/microbiologia , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidade , Animais , Biofilmes/crescimento & desenvolvimento , GMP Cíclico/análogos & derivados , GMP Cíclico/metabolismo , Modelos Animais de Doenças , Humanos , Camundongos , Olea/microbiologia , Estrutura Terciária de Proteína , Deleção de Sequência , Transdução de Sinais/genética , Sistemas de Secreção Tipo III/genética , Sistemas de Secreção Tipo III/metabolismo , Sistemas de Secreção Tipo VI/genética , Sistemas de Secreção Tipo VI/metabolismo , Virulência/genéticaRESUMO
Pseudomonas aeruginosa is a Gram-negative opportunistic human pathogen and a threat for immunocompromised and cystic fibrosis patients. It is responsible for acute and chronic infections and can switch between these lifestyles upon taking an informed decision involving complex regulatory networks. The RetS/LadS/Gac/Rsm network and the cyclic-di-GMP (c-di-GMP) signaling pathways are both central to this phenomenon redirecting the P. aeruginosa population toward a biofilm mode of growth, which is associated with chronic infections. While these two pathways were traditionally studied independently from each other, we recently showed that cellular levels of c-di-GMP are increased in the hyperbiofilm retS mutant. Here, we have formally established the link between the two networks by showing that the SadC diguanylate cyclase is central to the Gac/Rsm-associated phenotypes, notably, biofilm formation. Importantly, SadC is involved in the signaling that converges onto the RsmA translational repressor either via RetS/LadS or via HptB/HsbR. Although the level of expression of the sadC gene does not seem to be impacted by the regulatory cascade, the production of the SadC protein is tightly repressed by RsmA. This adds to the growing complexity of the signaling network associated with c-di-GMP in P. aeruginosa. While this organism possesses more than 40 c-di-GMP-related enzymes, it remains unclear how signaling specificity is maintained within the c-di-GMP network. The finding that SadC but no other diguanylate cyclase is related to the formation of biofilm governed by the Gac/Rsm pathway further contributes to understanding of this insulation mechanism.
Assuntos
Biofilmes/crescimento & desenvolvimento , GMP Cíclico/análogos & derivados , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Fósforo-Oxigênio Liases/metabolismo , Pseudomonas aeruginosa/fisiologia , Proteínas Repressoras/metabolismo , GMP Cíclico/metabolismo , Proteínas de Escherichia coli/genética , Fósforo-Oxigênio Liases/genética , Pseudomonas aeruginosa/genética , Transdução de SinaisRESUMO
We present a case of a male neonate who experienced a 13.5% weight loss at 96 hours of life, despite receiving adequate calorie intake and exhibiting no feeding difficulties. The pregnancy was uneventful, and maternal serological investigation was normal. A routine ultrasound at 34 weeks of gestational age revealed late oligohydramnios. The neonate was delivered at 35 weeks of gestational age by forceps, weighing 2600 g. Physical examination disclosed bilateral cryptorchidism. Laboratory studies unveiled acute kidney injury (AKI) with hyperkalaemia. Renal ultrasound revealed bilateral hydronephrosis and renal dysplasia with pyelocalyceal dilatation. Despite early recognition and treatment, the newborn developed chronic kidney disease (CKD). AKI is an important and under-recognised cause of significant neonatal weight loss.This case underscores the significance of considering AKI as a potential and under-recognised cause of neonatal weight loss. It emphasises the importance of maintaining a high clinical suspicion for early AKI diagnosis to mitigate the risk of progression to CKD.
Assuntos
Injúria Renal Aguda , Hidronefrose , Insuficiência Renal Crônica , Recém-Nascido , Gravidez , Feminino , Humanos , Masculino , Injúria Renal Aguda/diagnóstico , Injúria Renal Aguda/etiologia , Insuficiência Renal Crônica/complicações , Idade Gestacional , Hidronefrose/complicações , Unidades de Terapia Intensiva Neonatal , Fatores de RiscoRESUMO
Introduction Wheezing is common in preschool-aged children, affecting about half of all children within their first six years of life. Children who have recurrent wheezing experience disease-related morbidity, including increased emergency visits and hospitalizations. Early-life lower respiratory tract viral infections are linked to recurrent wheezing and eventual asthma onset. Identifying high-risk children is crucial, with the frequency and severity of wheezing episodes being good predictors of long-term outcomes. Aim To identify predictors of severe exacerbations in children with recurrent wheezing. Methods We conducted a retrospective cohort study involving 168 pediatric patients with recurrent wheezing followed up at our outpatient clinic. The outcome of interest was the occurrence of a severe exacerbation, defined as any exacerbation requiring hospitalization and the need for supplemental oxygenation or ventilatory support. Results The median age of the first wheezing exacerbation was five months, with a predominance of the male gender. Approximately two-thirds of the patients had a family history of atopy. Comorbid allergic rhinitis and atopic dermatitis were present in 15.4% and 16.7% of patients, respectively. Twenty percent of patients had a severe wheezing exacerbation as the first form of presentation, and 30% presented at least one severe exacerbation from the first presentation to the last follow-up. Patients with severe exacerbations were younger at the first episode (median age 4 months, IQR 2-7, versus 7 months, IQR 4-12, p=0.027) and more frequently had a family history of atopy (71.7% versus 55.6%, p=0.050). In this cohort, patients who initially presented with a severe episode are at increased risk of incident severe exacerbations during follow-up, HR 2.24 (95%CI 1.01-4.95). Conclusions We know that the severity of exacerbations in children with recurrent wheezing correlates with the long-term outcomes of the disease. Therefore, preventing severe exacerbations can positively impact the prognosis of these patients. In this analysis, we found independent predictors of severe exacerbations to be the first clinical episode before the age of three months and a family history of atopy. We also found that patients whose initial presentation was severe have a higher risk of new severe exacerbations. Therefore, these subgroups of patients should be closely monitored by pediatricians.
RESUMO
INTRODUCTION: Bronchopulmonary dysplasia (BPD) is the most common complication associated with extreme prematurity. Although several criteria defining severity were developed over time, there are a few studies describing the differences in BPD phenotype and neonatal morbidities and complications between severity groups. We aimed to describe these differences in BPD patients of a neonatal intensive care unit (NICU). METHODS: We conducted an observational retrospective cohort study through a medical record review over a five-year period. Participants were newborns admitted to an NICU who were diagnosed with BPD. We performed a descriptive statistical analysis of gestational complications and the use of antenatal corticosteroid therapy, birth-related data, and complications throughout the NICU stay, as well as the respiratory support used. We also compared different severity groups across these variables. The patients were divided into severe and non-severe BPD using the severity criteria of the 2001 NICHD/NHLBI/ORD consensus workshop. RESULTS: A total of 101 newborns with BPD participated in the study and 73 had data on BPD severity. The median gestational age was 27 weeks, ranging from 23 to 32 weeks. Of these 73 newborns, 36 had mild BPD (49.3%), 10 had moderate BPD (13.7%), and 27 had severe BPD (37.0%). When comparing severe and non-severe BPD, we found that extreme prematurity, extremely low birth weight, and small size for gestational age were more frequent in the severe BPD group (p-value=0.012, p-value<0.001, and p-value=0.012, respectively). Infants with severe BPD had a longer duration of invasive ventilation than those with mild or moderate BPD (p-value<0.001). Late sepsis, necrotizing enterocolitis, severe brain injury, and retinopathy of prematurity were more frequent in severe BPD (p-value=0.017, p-value=0.045, p-value=0.033, p-value=0.003, respectively). DISCUSSION: Previously published evidence describing causal links between BPD development and comorbidities exists but data on their impact on BPD severity are scarce. In our study, severe BPD seemed to be associated with a higher frequency of comorbidities and complications. Further studies are needed to ascertain the impact of each morbidity on the severity of BPD and if measures to prevent them could lead to potentially milder BPD disease.
RESUMO
Salt poisoning is a rare cause of severe hypernatremia in children resulting from the ingestion of toxic amounts of sodium chloride, either from accidental or intentional administration of salted solutions. We present the case of a newborn admitted to a pediatric emergency department for lethargy and reduced oral intake; his laboratory evaluation showed severe hypernatremia ([Na+] of 174 mmol/L). The infant developed convulsive status epilepticus during treatment. Neuroimaging showed a tetraventricular hemorrhage, a large right-sided parenchymal hemorrhage with midline shift, and several left hemorrhagic foci. Etiologic evaluation for hypernatremia did not reveal a renal or extrarenal source of water loss nor an intercurrent illness to explain the reduced oral intake. A careful review of how the parents prepared the infant formula revealed an error in dosing the ratio of powder/water, resulting in hyperosmolar infant formula. The infant was diagnosed with salt poisoning as the major cause of hypernatremia. After careful correction of hypernatremia and the use of antiseizure medication, the patient improved and was discharged. The parents were given a careful review of instructions for infant formula preparation. Due to its rarity, a high index of suspicion is mandatory for a correct diagnosis of salt poisoning. Timely and adequate treatment is needed due to the high risk of intracerebral bleeding, seizures, and irreversible neurologic injury. Children, particularly newborns and infants, depend upon adults to ingest water and, thus, have more difficulty in maintaining electrolyte balance. Therefore, it is of utmost importance that parents are educated about childcare, particularly on the importance of careful infant formula preparation.
RESUMO
Science outreach is key to closing the gap between science and society. However, it often fails to reach those who feel excluded from science or are dismissive of it. By sharing our experience at Native Scientist, we demonstrate how outreach activities can help improve equity, diversity, and inclusion (EDI).
Assuntos
Diversidade Cultural , Equidade de Gênero , Ciência , Criança , Feminino , Humanos , MasculinoRESUMO
Acute bacterial infections are associated with motility and cytotoxicity via the type III secretion system (T3SS), while chronic infections are linked to biofilm formation and reduced virulence. In Pseudomonas aeruginosa, the transition between motility and sessility involves regulatory networks including the RetS/GacS sensors, as well as the second messenger c-di-GMP. The RetS/GacS signalling cascade converges on small RNAs, RsmY and RsmZ, which control a range of functions via RsmA. A retS mutation induces biofilm formation, and high levels of c-di-GMP produce a similar response. In this study, we connect RetS and c-di-GMP pathways by showing that the retS mutant displays high levels of c-di-GMP. Furthermore, a retS mutation leads to repression of the T3SS, but also upregulates the type VI secretion system (T6SS), which is associated with chronic infections. Strikingly, production of the T3SS and T6SS can be switched by artificially modulating c-di-GMP levels. We show that the diguanylate cyclase WspR is specifically involved in the T3SS/T6SS switch and that RsmY and RsmZ are required for the c-di-GMP-dependent response. These results provide a firm link between the RetS/GacS and the c-di-GMP pathways, which coordinate bacterial lifestyles, as well as secretion systems that determine the infection strategy of P. aeruginosa.
Assuntos
Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos , GMP Cíclico/análogos & derivados , Pseudomonas aeruginosa/metabolismo , Fatores de Virulência/metabolismo , Proteínas de Bactérias/genética , Biofilmes , GMP Cíclico/metabolismo , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Fósforo-Oxigênio Liases/metabolismo , Pseudomonas aeruginosa/genética , Transdução de Sinais , Fatores de Virulência/genéticaRESUMO
The serotype-specific glucosyltransferase, GtrV, is responsible for glucosylation of the O-antigen repeating unit of Shigella flexneri serotype 5a strains. GtrV is an integral inner membrane protein with two essential periplasmic loops: the large Loop 2 and the C-terminal Loop 10. In this study, the full length of the Loop 2 was shown to be necessary for GtrV function. Site-directed mutagenesis within this loop revealed that conserved aromatic and charged amino acids have a critical role in the formation of the active site. Sequential deletions of the C-terminal end indicated that this region may be essential for assembly of the protein in the cytoplasmic membrane. The highly conserved FWAED motif is thought to form the substrate-binding site and was found to be critical in GtrV and GtrX, a serotype-specific glucosyltransferase with homology to GtrV. The data presented constitutes a targeted analysis of the formation of the GtrV active site and highlights the essential role of the large periplasmic Loop 2 in its function.
Assuntos
Aminoácidos/metabolismo , Domínio Catalítico , Glucosiltransferases/química , Proteínas de Membrana/química , Shigella flexneri/enzimologia , Sequência de Aminoácidos , Glucosiltransferases/metabolismo , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Periplasma/metabolismo , Biossíntese de Proteínas , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/metabolismo , Deleção de Sequência , Homologia de Sequência de Aminoácidos , Relação Estrutura-AtividadeRESUMO
Divergent transcription of a regulatory gene and a cognate promoter under its control is a common theme in bacterial regulatory circuits. This genetic organization is found for the dmpR gene that encodes the substrate-responsive specific regulator of the sigma(54)-dependent Po promoter, which controls (methyl)phenol catabolism. Here we identify the Pr promoter of dmpR as a sigma(70)-dependent promoter that is regulated by a novel mechanism in which sigma(54)-RNA polymerase occupancy of the non-overlapping sigma(54)-Po promoter stimulates sigma(70)-Pr output. In addition, we show that DmpR stimulates its own production through Po activity both in vivo and in vitro. Hence, the demonstrated regulatory circuit reveals a novel role for sigma(54)-RNA polymerase, namely regulation of a sigma(70)-dependent promoter, and a new mechanism that places a single promoter under dual control of two alternative forms of RNA polymerase. We present a model in which guanosine tetra-phosphate plays a major role in the interplay between sigma(54)- and sigma(70)-dependent transcription to ensure metabolic integration to couple sigma(70)-Pr output to both low-energy conditions and the presence of substrate.
Assuntos
Proteínas de Bactérias/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Pseudomonas putida/genética , RNA Polimerase Sigma 54/metabolismo , Fator sigma/metabolismo , Transativadores/metabolismo , Transcrição Gênica , Proteínas de Bactérias/genética , RNA Polimerases Dirigidas por DNA/genética , Regulação Bacteriana da Expressão Gênica , Guanosina Tetrafosfato/metabolismo , Óperon , Regiões Promotoras Genéticas , Pseudomonas putida/metabolismo , RNA Polimerase Sigma 54/genética , RNA Bacteriano/genética , Fator sigma/genética , Ressonância de Plasmônio de Superfície , Transativadores/genéticaRESUMO
Pseudomonas aeruginosa is a Gram-negative bacterial pathogen associated with acute and chronic infections. The universal cyclic-di-GMP second messenger is instrumental in the switch from a motile lifestyle to resilient biofilm as in the cystic fibrosis lung. The SadC diguanylate cyclase is associated with this patho-adaptive transition. Here, we identify an unrecognized SadC partner, WarA, which we show is a methyltransferase in complex with a putative kinase, WarB. We established that WarA binds to cyclic-di-GMP, which potentiates its methyltransferase activity. Together, WarA and WarB have structural similarities with the bifunctional Escherichia coli lipopolysaccharide (LPS) O antigen regulator WbdD. Strikingly, WarA influences P. aeruginosa O antigen modal distribution and interacts with the LPS biogenesis machinery. LPS is known to modulate the immune response in the host, and by using a zebrafish infection model, we implicate WarA in the ability of P. aeruginosa to evade detection by the host.
Assuntos
GMP Cíclico/análogos & derivados , Evasão da Resposta Imune , Lipopolissacarídeos/metabolismo , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/patogenicidade , Animais , GMP Cíclico/metabolismo , Modelos Animais de Doenças , Metiltransferases/metabolismo , Ligação Proteica , Infecções por Pseudomonas/microbiologia , Infecções por Pseudomonas/patologia , Peixe-ZebraRESUMO
For the last decade, chemical control of bacterial virulence has received considerable attention. Ajoene, a sulfur-rich molecule from garlic has been shown to reduce expression of key quorum sensing regulated virulence factors in the opportunistic pathogen Pseudomonas aeruginosa. Here we show that the repressing effect of ajoene on quorum sensing occurs by inhibition of small regulatory RNAs (sRNA) in P. aeruginosa as well as in Staphylococcus aureus, another important human pathogen that employs quorum sensing to control virulence gene expression. Using various reporter constructs, we found that ajoene lowered expression of the sRNAs RsmY and RsmZ in P. aeruginosa and the small dual-function regulatory RNA, RNAIII in S. aureus, that controls expression of key virulence factors. We confirmed the modulation of RNAIII by RNA sequencing and found that the expression of many QS regulated genes encoding virulence factors such as hemolysins and proteases were lowered in the presence of ajoene in S. aureus. Importantly, our findings show that sRNAs across bacterial species potentially may qualify as targets of anti-virulence therapy and that ajoene could be a lead structure in search of broad-spectrum compounds transcending the Gram negative-positive borderline.
Assuntos
Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Percepção de Quorum/efeitos dos fármacos , Percepção de Quorum/genética , Pequeno RNA não Traduzido , Dissulfetos/farmacologia , Genes Bacterianos , Fenótipo , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/genética , Sulfóxidos , Transcriptoma , Fatores de Virulência/genéticaRESUMO
OprF is the major outer membrane porin in bacteria belonging to the Pseudomonas genus. In previous studies, we have shown that OprF is required for full virulence expression of the opportunistic pathogen Pseudomonas aeruginosa. Here, we describe molecular insights on the nature of this relationship and report that the absence of OprF leads to increased biofilm formation and production of the Pel exopolysaccharide. Accordingly, the level of c-di-GMP, a key second messenger in biofilm control, is elevated in an oprF mutant. By decreasing c-di-GMP levels in this mutant, both biofilm formation and pel gene expression phenotypes were restored to wild-type levels. We further investigated the impact on two small RNAs, which are associated with the biofilm lifestyle, and found that expression of rsmZ but not of rsmY was increased in the oprF mutant and this occurs in a c-di-GMP-dependent manner. Finally, the extracytoplasmic function (ECF) sigma factors AlgU and SigX displayed higher activity levels in the oprF mutant. Two genes of the SigX regulon involved in c-di-GMP metabolism, PA1181 and adcA (PA4843), were up-regulated in the oprF mutant, partly explaining the increased c-di-GMP level. We hypothesized that the absence of OprF leads to a cell envelope stress that activates SigX and results in a c-di-GMP elevated level due to higher expression of adcA and PA1181. The c-di-GMP level can in turn stimulate Pel synthesis via increased rsmZ sRNA levels and pel mRNA, thus affecting Pel-dependent phenotypes such as cell aggregation and biofilm formation. This work highlights the connection between OprF and c-di-GMP regulatory networks, likely via SigX (ECF), on the regulation of biofilm phenotypes.
RESUMO
Biofilm dispersal is the last and least understood stage of the biofilm life cycle. Several recent studies have characterized dispersal events in response to various cues and signals. Here we describe a range of methods useful for the investigation of dispersal in the biofilm model organism and opportunistic pathogen Pseudomonas aeruginosa.