ABSTRACT
Streptococcus sanguinis is a prevalent member of human microbiome capable of acting as a causative agent of oral and respiratory infections. S. sanguinis competitive success within the infection niche is dependent on acquisition of metal ions and vitamins. Among the systems that bacteria use for micronutrient uptake is the energy coupling factor (ECF) transporter system EcfAAT. Here we describe physiological changes arising from EcfAAT transporter disruption. We found that EcfAAT contributes to S. sanguinis antibiotic sensitivity as well as metal and membrane homeostasis. Specifically, our work found that disruption of EcfAAT results in increased polymyxin susceptibility. We performed assessment of cell-associated metal content and found depletion of iron, magnesium, and manganese. Furthermore, membrane composition analysis revealed significant enrichment in unsaturated fatty acid species resulting in increased membrane fluidity. Our results demonstrate how disruption of a single EcfAAT transporter can have broad consequences on bacterial cell homeostasis. ECF transporters are of interest within the context of infection biology in bacterial species other than streptococci, hence work described here will further the understanding of how micronutrient uptake systems contribute to bacterial pathogenesis.
ABSTRACT
Most microbial cells found in nature exist in matrix-covered, surface-attached communities known as biofilms. This mode of growth is initiated by the ability of the microbe to sense a surface on which to grow. The opportunistic pathogen Pseudomonas aeruginosa (Pa) PA14 utilizes a single polar flagellum and type 4 pili (T4P) to sense surfaces. For Pa, T4P-dependent "twitching" motility is characterized by effectively pulling the cell across a surface through a complex process of cooperative binding, pulling, and unbinding. T4P retraction is powered by hexameric ATPases. Pa cells that have engaged a surface increase production of the second messenger cyclic AMP (cAMP) over multiple generations via the Pil-Chp system. This rise in cAMP allows cells and their progeny to become better adapted for surface attachment and activates virulence pathways through the cAMP-binding transcription factor Vfr. While many studies have focused on mechanisms of T4P twitching and regulation of T4P production and function by the Pil-Chp system, the mechanism by which Pa senses and relays a surface-engagement signal to the cell is still an open question. Here we review the current state of the surface sensing literature for Pa, with a focus on T4P, and propose an integrated model of surface sensing whereby the retraction motor PilT senses and relays the signal to the Pil-Chp system via PilJ to drive cAMP production and adaptation to a surface lifestyle.
Subject(s)
Fimbriae, Bacterial , Pseudomonas aeruginosa , Pseudomonas aeruginosa/physiology , Pseudomonas aeruginosa/metabolism , Pseudomonas aeruginosa/genetics , Fimbriae, Bacterial/metabolism , Fimbriae, Bacterial/physiology , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Gene Expression Regulation, Bacterial , Cyclic AMP/metabolismABSTRACT
The Pel exopolysaccharide is one of the most mechanistically conserved and phylogenetically diverse bacterial biofilm matrix determinants. Pel is a major contributor to the structural integrity of Pseudomonas aeruginosa biofilms, and its biosynthesis is regulated by the binding of cyclic-3',5'-dimeric guanosine monophosphate (c-di-GMP) to the PelD receptor. c-di-GMP is synthesized from two molecules of guanosine triphosphate (GTP) by diguanylate cyclases with GGDEF domains and degraded by phosphodiesterases with EAL or HD-GYP domains. As the P. aeruginosa genome encodes 43 c-di-GMP metabolic enzymes, one way signaling specificity can be achieved is through direct interaction between specific enzyme-receptor pairs. Here, we show that the inner membrane hybrid GGDEF-EAL enzyme, BifA, directly interacts with PelD via its cytoplasmic HAMP, GGDEF, and EAL domains. Despite having no catalytic function, the degenerate active site motif of the BifA GGDEF domain (GGDQF) has retained the ability to bind GTP with micromolar affinity. Mutations that abolish GTP binding result in increased biofilm formation but stable global c-di-GMP levels. Our data suggest that BifA forms a dimer in solution and that GTP binding induces conformational changes in dimeric BifA that enhance the BifA-PelD interaction and stimulate its phosphodiesterase activity, thus reducing c-di-GMP levels and downregulating Pel biosynthesis. Structural comparisons between the dimeric AlphaFold2 model of BifA and the structures of other hybrid GGDEF-EAL proteins suggest that the regulation of BifA by GTP may occur through a novel mechanism.IMPORTANCEc-di-GMP is the most common cyclic dinucleotide used by bacteria to regulate phenotypes such as motility, biofilm formation, virulence factor production, cell cycle progression, and cell differentiation. While the identification and initial characterization of c-di-GMP metabolic enzymes are well established, our understanding of how these enzymes are regulated to provide signaling specificity remains understudied. Here we demonstrate that the inactive GGDEF domain of BifA binds GTP and regulates the adjacent phosphodiesterase EAL domain, ultimately downregulating Pel-dependent P. aeruginosa biofilm formation through an interaction with PelD. This discovery adds to the growing body of literature regarding how hybrid GGDEF-EAL enzymes are regulated and provides additional precedence for studying how direct interactions between c-di-GMP metabolic enzymes and effectors result in signaling specificity.
Subject(s)
Escherichia coli Proteins , Pseudomonas aeruginosa , Pseudomonas aeruginosa/genetics , Pseudomonas aeruginosa/metabolism , Bacterial Proteins/metabolism , Guanosine Triphosphate/metabolism , Escherichia coli Proteins/metabolism , Cyclic GMP/metabolism , Phosphoric Diester Hydrolases/metabolism , Biofilms , Gene Expression Regulation, BacterialABSTRACT
Biofilm formation begins when bacteria contacting a surface induce cellular changes to become better adapted for surface growth. One of the first changes to occur for Pseudomonas aeruginosa after surface contact is an increase in the nucleotide second messenger 3',5'-cyclic AMP (cAMP). It has been demonstrated that this increase in intracellular cAMP is dependent on functional type IV pili (T4P) relaying a signal to the Pil-Chp system, but the mechanism by which this signal is transduced remains poorly understood. Here, we investigate the role of the type IV pilus retraction motor PilT in sensing a surface and relaying that signal to cAMP production. We show that mutations in PilT, and in particular those impacting the ATPase activity of this motor protein, reduce surface-dependent cAMP production. We identify a novel interaction between PilT and PilJ, a member of the Pil-Chp system, and propose a new model whereby P. aeruginosa uses its PilT retraction motor to sense a surface and to relay that signal via PilJ to increased production of cAMP. We discuss these findings in light of current T4P-dependent surface sensing models for P. aeruginosa. IMPORTANCE T4P are cellular appendages that allow P. aeruginosa to sense a surface, leading to the production of cAMP. This second messenger not only activates virulence pathways but leads to further surface adaptation and irreversible attachment of cells. Here, we demonstrate the importance of the retraction motor PilT in surface sensing. We also present a new surface sensing model in P. aeruginosa whereby the T4P retraction motor PilT senses and transmits the surface signal, likely via its ATPase domain and interaction with PilJ, to mediate production of the second messenger cAMP.
Subject(s)
Bacterial Proteins , Pseudomonas aeruginosa , Bacterial Proteins/genetics , Pseudomonas aeruginosa/genetics , Fimbriae, Bacterial/metabolism , Second Messenger Systems , Cyclic AMP/metabolism , Adenosine Triphosphatases/metabolism , Fimbriae Proteins/geneticsABSTRACT
Biofilm formation begins when bacteria contacting a surface induce cellular changes to become better adapted for surface growth. One of the first changes to occur for Pseudomonas aeruginosa after surface contact is an increase in the nucleotide second messenger 3',5'-cyclic adenosine monophosphate (cAMP). It has been demonstrated that this increase in intracellular cAMP is dependent on functional Type IV pili (T4P) relaying a signal to the Pil-Chp system, but the mechanism by which this signal is transduced remains poorly understood. Here, we investigate the role of the Type IV pili retraction motor PilT in sensing a surface and relaying that signal to cAMP production. We show that mutations affecting the structure of PilT and in particular ATPase activity of this motor protein, reduce surface-dependent cAMP production. We identify a novel interaction between PilT and PilJ, a member of the Pil-Chp system, and propose a new model whereby P. aeruginosa uses its retraction motor to sense a surface and to relay that signal via PilJ to increased production of cAMP. We discuss these findings in light of current TFP-dependent surface sensing models for P. aeruginosa . Importance: T4P are cellular appendages that allow P. aeruginosa to sense a surface leading to the production of cAMP. This second messenger not only activates virulence pathways but leads to further surface adaptation and irreversible attachment of cells. Here, we demonstrate the importance of the retraction motor PilT in surface sensing. We also present a new surface sensing model in P. aeruginosa whereby the T4P retraction motor PilT senses and transmits the surface signal, likely via its ATPase domain and interaction with PilJ, to mediate production of the second messenger cAMP.
ABSTRACT
Biofilm formation represents a critical strategy whereby bacteria can tolerate otherwise damaging environmental stressors and antimicrobial insults. While the mechanisms bacteria use to establish a biofilm and disperse from these communities have been well-studied, we have only a limited understanding of the mechanisms required to maintain these multicellular communities. Indeed, until relatively recently, it was not clear that maintaining a mature biofilm could be considered an active, regulated process with dedicated machinery. Using Pseudomonas aeruginosa as a model system, we review evidence from recent studies that support the model that maintenance of these persistent, surface-attached communities is indeed an active process. Biofilm maintenance mechanisms include transcriptional regulation and second messenger signaling (including the production of extracellular polymeric substances). We also discuss energy-conserving pathways that play a key role in the maintenance of these communities. We hope to highlight the need for further investigation to uncover novel biofilm maintenance pathways and suggest the possibility that such pathways can serve as novel antibiofilm targets.
Subject(s)
Cyclic GMP , Gene Expression Regulation, Bacterial , Biofilms , Cyclic GMP/metabolism , Extracellular Polymeric Substance Matrix/metabolism , Pseudomonas aeruginosa/metabolismABSTRACT
While the early stages of biofilm formation have been well characterized, less is known about the requirements for Pseudomonas aeruginosa to maintain a mature biofilm. We utilized a P. aeruginosa-phage interaction to identify rmcA and morA, two genes which encode bis-(3',5')-cyclic dimeric GMP (c-di-GMP)-degrading phosphodiesterases (PDEs) and are important for the regulation of biofilm maintenance. Deletion of these genes initially results in an elevated biofilm phenotype characterized by increased production of c-di-GMP, Pel polysaccharide, and/or biofilm biomass. In contrast to the wild-type strain, these mutants were unable to maintain the biofilm when exposed to carbon-limited conditions. The susceptibility to nutrient limitation, as well as subsequent loss of biofilm viability of these mutants, was phenotypically reproduced with a stringent response mutant (ΔrelA ΔspoT), indicating that the ΔrmcA and ΔmorA mutants may be unable to appropriately respond to nutrient limitation. Genetic and biochemical data indicate that RmcA and MorA physically interact with the Pel biosynthesis machinery, supporting a model whereby unregulated Pel biosynthesis contributes to the death of the ΔrmcA and ΔmorA mutant strains in an established biofilm under nutrient limitation. These findings provide evidence that c-di-GMP-mediated regulation is required for mature biofilms of P. aeruginosa to effectively respond to changing availability of nutrients. Furthermore, the PDEs involved in biofilm maintenance are distinct from those required for establishing a biofilm, suggesting that a wide variety of c-di-GMP metabolizing enzymes in organisms such as P. aeruginosa allows for discrete control over the formation, maintenance or dispersion of biofilms.IMPORTANCE Recent advances in our understanding of c-di-GMP signaling have provided key insights into the regulation of biofilms. Despite an improved understanding of how biofilms initially form, the processes that facilitate the long-term maintenance of these multicellular communities remain opaque. We found that P. aeruginosa requires two phosphodiesterases, RmcA and MorA, to maintain a mature biofilm and that biofilms lacking these PDEs succumb to nutrient limitation and die. The biofilm maintenance deficiency observed in ΔrmcA and ΔmorA mutants was also found in the stringent response-defective ΔrelA ΔspoT strain, suggesting that a regulatory intersection between c-di-GMP signaling, extracellular polysaccharide biosynthesis, and the nutrient limitation response is important for biofilm persistence. We uncover components of an important regulatory system needed for P. aeruginosa biofilms to persist in nutrient-poor conditions and provide some of the first evidence that maintaining a mature biofilm is an active process.
Subject(s)
Biofilms , Cyclic GMP/analogs & derivatives , Phosphoric Diester Hydrolases/physiology , Pseudomonas aeruginosa/physiology , Cyclic GMP/metabolism , Signal Transduction/physiologyABSTRACT
The second messenger cyclic diguanylate (c-di-GMP) plays a critical role in the regulation of motility. In Pseudomonas aeruginosa PA14, c-di-GMP inversely controls biofilm formation and surface swarming motility, with high levels of this dinucleotide signal stimulating biofilm formation and repressing swarming. P. aeruginosa encodes two stator complexes, MotAB and MotCD, that participate in the function of its single polar flagellum. Here we show that the repression of swarming motility requires a functional MotAB stator complex. Mutating the motAB genes restores swarming motility to a strain with artificially elevated levels of c-di-GMP as well as stimulates swarming in the wild-type strain, while overexpression of MotA from a plasmid represses swarming motility. Using point mutations in MotA and the FliG rotor protein of the motor supports the conclusion that MotA-FliG interactions are critical for c-di-GMP-mediated swarming inhibition. Finally, we show that high c-di-GMP levels affect the localization of a green fluorescent protein (GFP)-MotD fusion, indicating a mechanism whereby this second messenger has an impact on MotCD function. We propose that when c-di-GMP level is high, the MotAB stator can displace MotCD from the motor, thereby affecting motor function. Our data suggest a newly identified means of c-di-GMP-mediated control of surface motility, perhaps conserved among Pseudomonas, Xanthomonas, and other organisms that encode two stator systems.
Subject(s)
Bacterial Proteins/metabolism , Cyclic GMP/analogs & derivatives , Gene Expression Regulation, Bacterial , Locomotion , Pseudomonas aeruginosa/physiology , Bacterial Proteins/genetics , Cyclic GMP/metabolism , Gene Deletion , Gene Expression , Point Mutation , Pseudomonas aeruginosa/metabolismABSTRACT
Pseudomonas aeruginosa exhibits distinct surface-associated behaviors, including biofilm formation, flagellum-mediated swarming motility, and type IV pilus-driven twitching. Here, we report a role for the minor pilins, PilW and PilX, components of the type IV pilus assembly machinery, in the repression of swarming motility. Mutating either the pilW or pilX gene alleviates the inhibition of swarming motility observed for strains with elevated levels of the intracellular signaling molecule cyclic di-GMP (c-di-GMP) due to loss of BifA, a c-di-GMP-degrading phosphodiesterase. Blocking PilD peptidase-mediated processing of PilW and PilX renders the unprocessed proteins defective for pilus assembly but still functional in c-di-GMP-mediated swarming repression, indicating our ability to separate these functions. Strains with mutations in pilW or pilX also fail to exhibit the increase in c-di-GMP levels observed when wild-type (WT) or bifA mutant cells are grown on a surface. We also provide data showing that c-di-GMP levels are increased upon PilY1 overexpression in surface-grown cells and that this c-di-GMP increase does not occur in the absence of the SadC diguanylate cyclase. Increased levels of endogenous PilY1, PilX, and PilA are observed when cells are grown on a surface compared to liquid growth, linking surface growth and enhanced signaling via SadC. Our data support a model wherein PilW, PilX, and PilY1, in addition to their role(s) in type IV pilus biogenesis, function to repress swarming via modulation of intracellular c-di-GMP levels. By doing so, these pilus assembly proteins contribute to P. aeruginosa's ability to coordinately regulate biofilm formation with its two surface motility systems.
Subject(s)
Fimbriae Proteins/metabolism , Gene Expression Regulation, Bacterial/physiology , Movement , Pseudomonas aeruginosa/physiology , Amino Acid Sequence , Cyclic GMP/analogs & derivatives , Cyclic GMP/genetics , Cyclic GMP/metabolism , Fimbriae Proteins/genetics , Fimbriae, Bacterial/physiology , Gene Deletion , Mutation , PhenotypeABSTRACT
UNLABELLED: Pulmonary damage caused by chronic colonization of the cystic fibrosis (CF) lung by microbial communities is the proximal cause of respiratory failure. While there has been an effort to document the microbiome of the CF lung in pediatric and adult patients, little is known regarding the developing microflora in infants. We examined the respiratory and intestinal microbiota development in infants with CF from birth to 21 months. Distinct genera dominated in the gut compared to those in the respiratory tract, yet some bacteria overlapped, demonstrating a core microbiota dominated by Veillonella and Streptococcus. Bacterial diversity increased significantly over time, with evidence of more rapidly acquired diversity in the respiratory tract. There was a high degree of concordance between the bacteria that were increasing or decreasing over time in both compartments; in particular, a significant proportion (14/16 genera) increasing in the gut were also increasing in the respiratory tract. For 7 genera, gut colonization presages their appearance in the respiratory tract. Clustering analysis of respiratory samples indicated profiles of bacteria associated with breast-feeding, and for gut samples, introduction of solid foods even after adjustment for the time at which the sample was collected. Furthermore, changes in diet also result in altered respiratory microflora, suggesting a link between nutrition and development of microbial communities in the respiratory tract. Our findings suggest that nutritional factors and gut colonization patterns are determinants of the microbial development of respiratory tract microbiota in infants with CF and present opportunities for early intervention in CF with altered dietary or probiotic strategies. IMPORTANCE: While efforts have been focused on assessing the microbiome of pediatric and adult cystic fibrosis (CF) patients to understand how chronic colonization by these microbes contributes to pulmonary damage, little is known regarding the earliest development of respiratory and gut microflora in infants with CF. Our findings suggest that colonization of the respiratory tract by microbes is presaged by colonization of the gut and demonstrated a role of nutrition in development of the respiratory microflora. Thus, targeted dietary or probiotic strategies may be an effective means to change the course of the colonization of the CF lung and thereby improve patient outcomes.
Subject(s)
Biota , Cystic Fibrosis/microbiology , Gastrointestinal Tract/microbiology , Metagenome , Respiratory System/microbiology , Age Factors , Bacteria/classification , Bacteria/genetics , Cluster Analysis , Humans , Infant , Infant, NewbornABSTRACT
Diverse microbial communities chronically colonize the lungs of cystic fibrosis patients. Pyrosequencing of amplicons for hypervariable regions in the 16S rRNA gene generated taxonomic profiles of bacterial communities for sputum genomic DNA samples from 22 patients during a state of clinical stability (outpatients) and 13 patients during acute exacerbation (inpatients). We employed quantitative PCR (qPCR) to confirm the detection of Pseudomonas aeruginosa and Streptococcus by the pyrosequencing data and human oral microbe identification microarray (HOMIM) analysis to determine the species of the streptococci identified by pyrosequencing. We show that outpatient sputum samples have significantly higher bacterial diversity than inpatients, but maintenance treatment with tobramycin did not impact overall diversity. Contrary to the current dogma in the field that Pseudomonas aeruginosa is the dominant organism in the majority of cystic fibrosis patients, Pseudomonas constituted the predominant genera in only half the patient samples analyzed and reported here. The increased fractional representation of Streptococcus in the outpatient cohort relative to the inpatient cohort was the strongest predictor of clinically stable lung disease. The most prevalent streptococci included species typically associated with the oral cavity (Streptococcus salivarius and Streptococcus parasanguis) and the Streptococcus milleri group species. These species of Streptococcus may play an important role in increasing the diversity of the cystic fibrosis lung environment and promoting patient stability.
Subject(s)
Cystic Fibrosis/microbiology , Pseudomonas aeruginosa/genetics , Sputum/microbiology , Streptococcus/classification , Streptococcus/genetics , Adult , Anti-Bacterial Agents/administration & dosage , Anti-Bacterial Agents/therapeutic use , Base Sequence , DNA, Bacterial/genetics , Female , Humans , Lung/microbiology , Male , Metagenome , Middle Aged , Pseudomonas aeruginosa/isolation & purification , RNA, Ribosomal, 16S/genetics , Sequence Analysis, DNA , Streptococcus/isolation & purification , Tobramycin/administration & dosage , Tobramycin/therapeutic use , Young AdultABSTRACT
Here, we report the characterization of 122 Pseudomonas aeruginosa clinical isolates from three distinct geographical locations: Dartmouth Hitchcock Medical Center in New Hampshire, USA, the Charles T. Campbell Eye Microbiology Lab at the University of Pittsburgh Medical Center, USA, and the Aravind Eye Hospital in Madurai, India. We identified and located clustered regularly interspaced short palindromic repeats (CRISPR) in 45/122 clinical isolates and sequenced these CRISPR, finding that Yersinia subtype CRISPR regions (33â%) were more prevalent than the Escherichia CRISPR region subtype (6â%) in these P. aeruginosa clinical isolates. Further, we observed 132 unique spacers from these 45 CRISPR that are 100â% identical to prophages or sequenced temperate bacteriophage capable of becoming prophages. Most intriguingly, all of these 132 viral spacers matched to temperate bacteriophage/prophages capable of inserting into the host chromosome, but not to extrachromosomally replicating lytic P. aeruginosa bacteriophage. We next assessed the ability of the more prevalent Yersinia subtype CRISPR regions to mediate resistance to bacteriophage infection or lysogeny by deleting the entire CRISPR region from sequenced strain UCBPP-PA14 and six clinical isolates. We found no change in CRISPR-mediated resistance to bacteriophage infection or lysogeny rate even for CRISPR with spacers 100â% identical to a region of the infecting bacteriophage. Lastly, to show these CRISPR and cas genes were expressed and functional, we demonstrated production of small CRISPR RNAs. This work provides both the first examination to our knowledge of CRISPR regions within clinical P. aeruginosa isolates and a collection of defined CRISPR-positive and -negative strains for further CRISPR and cas gene studies.
Subject(s)
DNA, Intergenic/genetics , Inverted Repeat Sequences , Pseudomonas aeruginosa/genetics , Bacteriophages/genetics , Conserved Sequence , DNA, Bacterial/genetics , Genes, Bacterial , India , Lysogeny , Molecular Sequence Annotation , New Hampshire , Pennsylvania , Prophages/genetics , Pseudomonas aeruginosa/isolation & purification , Pseudomonas aeruginosa/virology , Sequence Analysis, DNA , Sequence DeletionABSTRACT
INTRODUCTION: This cross-sectional study was conducted to assess the relationship between iron levels in the plasma and sputum of cystic fibrosis (CF) patients. METHODS: Demographic, clinical, and iron-related laboratory data were prospectively obtained from 25 patients with stable clinical features and 14 patients with worsened clinical features since their most recent evaluations. RESULTS: Compared to patients with stable clinical features, those who experienced clinical deterioration demonstrated significantly worse lung function and were more frequently malnourished and diabetic. Members of the latter group were also significantly more hypoferremic and had higher sputum iron content than patients with stable clinical features. No significant correlation was found between plasma and sputum iron levels when the groups were analyzed together and separately. CONCLUSIONS: Sputum iron content does not correlate with iron-related hematologic tests. Hypoferremia is common in CF and correlates with poor lung function and overall health.
Subject(s)
Cystic Fibrosis/physiopathology , Iron/blood , Lung/physiopathology , Adult , Anemia/etiology , Cross-Sectional Studies , Cystic Fibrosis/blood , Cystic Fibrosis/complications , Diabetes Mellitus/etiology , Female , Humans , Iron, Dietary/blood , Male , Middle Aged , Prospective Studies , Sputum/chemistry , Young AdultABSTRACT
Here, we report the characterization of 122 Pseudomonas aeruginosa clinical isolates from three distinct geographical locations: Dartmouth Hitchcock Medical Center in New Hampshire, USA, the Charles T. Campbell Eye Microbiology Lab at the University of Pittsburgh Medical Center, USA, and the Aravind Eye Hospital in Madurai, India. We identified and located clustered regularly interspaced short palindromic repeats (CRISPR) in 45/122 clinical isolates and sequenced these CRISPR, finding that Yersinia subtype CRISPR regions (33â%) were more prevalent than the Escherichia CRISPR region subtype (6â%) in these P. aeruginosa clinical isolates. Further, we observed 132 unique spacers from these 45 CRISPR that are 100â% identical to prophages or sequenced temperate bacteriophage capable of becoming prophages. Most intriguingly, all of these 132 viral spacers matched to temperate bacteriophage/prophages capable of inserting into the host chromosome, but not to extrachromosomally replicating lytic P. aeruginosa bacteriophage. We next assessed the ability of the more prevalent Yersinia subtype CRISPR regions to mediate resistance to bacteriophage infection or lysogeny by deleting the entire CRISPR region from sequenced strain UCBPP-PA14 and six clinical isolates. We found no change in CRISPR-mediated resistance to bacteriophage infection or lysogeny rate even for CRISPR with spacers 100â% identical to a region of the infecting bacteriophage. Lastly, to show these CRISPR and cas genes were expressed and functional, we demonstrated production of small CRISPR RNAs. This work provides both the first examination to our knowledge of CRISPR regions within clinical P. aeruginosa isolates and a collection of defined CRISPR-positive and -negative strains for further CRISPR and cas gene studies.
ABSTRACT
Bacterial biofilms are highly recalcitrant to antibiotic treatment, which holds serious consequences for therapy of infections that involve biofilms. The genetic mechanisms of this biofilm antibiotic resistance appear to fall into two general classes: innate resistance factors and induced resistance factors. Innate mechanisms are activated as part of the biofilm developmental pathway, the factors being integral parts of biofilm structure and physiology. Innate pathways include decreased diffusion of antibiotics through the biofilm matrix, decreased oxygen and nutrient availability accompanied by altered metabolic activity, formation of persisters, and other specific molecules not fitting into the above groups. Induced resistance factors include those resulting from induction by the antimicrobial agent itself. Biofilm antibiotic resistance is likely manifested as an intricate mixture of innate and induced mechanisms. Many researchers are currently trying to overcome this extreme biofilm antibiotic resistance by developing novel therapies aimed at disrupting biofilms and killing the constituent bacteria. These studies have led to the identification of several molecules that effectively disturb biofilm physiology, often by interrupting bacterial quorum sensing. In this manner, manipulation of innate and induced resistance pathways holds much promise for treatment of biofilm infections.
Subject(s)
Anti-Bacterial Agents/pharmacology , Bacterial Infections/microbiology , Biofilms/drug effects , Biofilms/growth & development , Bacterial Infections/drug therapy , Bacterial Physiological Phenomena , Drug Resistance, Bacterial , HumansABSTRACT
Pseudomonas aeruginosa is capable of twitching, swimming, and swarming motility. The latter form of translocation occurs on semisolid surfaces, requires functional flagella and biosurfactant production, and results in complex motility patterns. From the point of inoculation, bacteria migrate as defined groups, referred to as tendrils, moving in a coordinated manner capable of sensing and responding to other groups of cells. We were able to show that P. aeruginosa produces extracellular factors capable of modulating tendril movement, and genetic analysis revealed that modulation of these movements was dependent on rhamnolipid biosynthesis. An rhlB mutant (deficient in mono- and dirhamnolipid production) and an rhlC mutant (deficient in dirhamnolipid production) exhibited altered swarming patterns characterized by irregularly shaped tendrils. In addition, agar supplemented with rhamnolipid-containing spent supernatant inhibited wild-type (WT) swarming, whereas agar supplemented with spent supernatant from mutants that do not make rhamnolipids had no effect on WT P. aeruginosa swarming. Addition of purified rhamnolipids to swarming medium also inhibited swarming motility of the WT strain. We also show that a sadB mutant does not sense and/or respond to other groups of swarming cells and this mutant was capable of swarming on media supplemented with rhamnolipid-containing spent supernatant or purified rhamnolipids. The abilities to produce and respond to rhamnolipids in the context of group behavior are discussed.
Subject(s)
Glycolipids/physiology , Pseudomonas aeruginosa/chemistry , Pseudomonas aeruginosa/physiology , Bacterial Proteins/genetics , Bacterial Proteins/physiology , Gene Deletion , Genes, Bacterial , Glycolipids/biosynthesis , Glycolipids/genetics , Movement , Mutation , Pseudomonas aeruginosa/geneticsABSTRACT
Staphylococcus aureus is a common pathogen associated with nosocomial infections. It can persist in clinical settings and gain increased resistance to antimicrobial agents through biofilm formation. We have found that alpha-toxin, a secreted, multimeric, hemolytic toxin encoded by the hla gene, plays an integral role in biofilm formation. The hla mutant was unable to fully colonize plastic surfaces under both static and flow conditions. Based on microscopy studies, we propose that alpha-hemolysin is required for cell-to-cell interactions during biofilm formation.
Subject(s)
Bacterial Toxins/metabolism , Biofilms/growth & development , Hemolysin Proteins/metabolism , Staphylococcus aureus/physiology , Bacterial Toxins/genetics , Hemolysin Proteins/genetics , Microscopy, Phase-Contrast , Mutation , Phenotype , Polysaccharides, Bacterial/genetics , Polysaccharides, Bacterial/metabolism , Staphylococcus aureus/geneticsABSTRACT
Biofilms are communities of microorganisms attached to a surface. It has become clear that biofilm-grown cells express properties distinct from planktonic cells, one of which is an increased resistance to antimicrobial agents. Recent work has indicated that slow growth and/or induction of an rpoS-mediated stress response could contribute to biocide resistance. The physical and/or chemical structure of exopolysaccharides or other aspects of biofilm architecture could also confer resistance by exclusion of biocides from the bacterial community. Finally, biofilm-grown bacteria might develop a biofilm-specific biocide-resistant phenotype. Owing to the heterogeneous nature of the biofilm, it is likely that there are multiple resistance mechanisms at work within a single community. Recent research has begun to shed light on how and why surface-attached microbial communities develop resistance to antimicrobial agents.
Subject(s)
Anti-Bacterial Agents/pharmacology , Biofilms/drug effects , Drug Resistance, Microbial , Bacterial Proteins/metabolism , Biofilms/growth & development , Drug Resistance, Multiple , Klebsiella pneumoniae/drug effects , Klebsiella pneumoniae/growth & development , Klebsiella pneumoniae/metabolism , Phenotype , Polysaccharides, Bacterial/chemistry , Polysaccharides, Bacterial/metabolism , Pseudomonas aeruginosa/drug effects , Pseudomonas aeruginosa/growth & development , Pseudomonas aeruginosa/metabolism , Sigma Factor/metabolism , Staphylococcus epidermidis/drug effects , Staphylococcus epidermidis/growth & development , Staphylococcus epidermidis/metabolismABSTRACT
Biofilms are complex communities of microorganisms attached to surfaces or associated with interfaces. Despite the focus of modern microbiology research on pure culture, planktonic (free-swimming) bacteria, it is now widely recognized that most bacteria found in natural, clinical, and industrial settings persist in association with surfaces. Furthermore, these microbial communities are often composed of multiple species that interact with each other and their environment. The determination of biofilm architecture, particularly the spatial arrangement of microcolonies (clusters of cells) relative to one another, has profound implications for the function of these complex communities. Numerous new experimental approaches and methodologies have been developed in order to explore metabolic interactions, phylogenetic groupings, and competition among members of the biofilm. To complement this broad view of biofilm ecology, individual organisms have been studied using molecular genetics in order to identify the genes required for biofilm development and to dissect the regulatory pathways that control the plankton-to-biofilm transition. These molecular genetic studies have led to the emergence of the concept of biofilm formation as a novel system for the study of bacterial development. The recent explosion in the field of biofilm research has led to exciting progress in the development of new technologies for studying these communities, advanced our understanding of the ecological significance of surface-attached bacteria, and provided new insights into the molecular genetic basis of biofilm development.