Michael J. Franklin

Biosynthesis of the Pseudomonas aeruginosa Extracellular Polysaccharides, Alginate, Pel, and Psl

Pseudomonas aeruginosa thrives in many aqueous environments and is an opportunistic pathogen that can cause both acute and chronic infections. Environmental conditions and host defenses cause differing stresses on the bacteria, and to survive in vastly different environments, P. aeruginosa must be able to adapt to its surroundings. One strategy for bacterial adaptation is to self-encapsulate with matrix material, primarily composed of secreted extracellular polysaccharides. P. aeruginosa has the genetic capacity to produce at least three secreted polysaccharides; alginate, Psl, and Pel. These polysaccharides differ in chemical structure and in their biosynthetic mechanisms. Since alginate is often associated with chronic pulmonary infections, its biosynthetic pathway is the best characterized. However, alginate is only produced by a subset of P. aeruginosa strains. Most environmental and other clinical isolates secrete either Pel or Psl. Little information is available on the biosynthesis of these polysaccharides. Here, we review the literature on the alginate biosynthetic pathway, with emphasis on recent findings describing the structure of alginate biosynthetic proteins. This information combined with the characterization of the domain architecture of proteins encoded on the Psl and Pel operons allowed us to make predictive models for the biosynthesis of these two polysaccharides. The results indicate that alginate and Pel share certain features, including some biosynthetic proteins with structurally or functionally similar properties. In contrast, Psl biosynthesis resembles the EPS/CPS capsular biosynthesis pathway of Escherichia coli, where the Psl pentameric subunits are assembled in association with an isoprenoid lipid carrier. These models and the environmental cues that cause the cells to produce predominantly one polysaccharide over the others are subjects of current investigation.

Role of alginate O acetylation in resistance of mucoid Pseudomonas aeruginosa to opsonic phagocytosis

ABSTRACT Establishment and maintenance of chronic lung infections with mucoid Pseudomonas aeruginosa in patients with cystic fibrosis (CF) require that the bacteria avoid host defenses. Elaboration of the extracellular, O-acetylated mucoid exopolysaccharide, or alginate, is a major microbial factor in resistance to immune effectors. Here we show that O acetylation of alginate maximizes the resistance of mucoid P. aeruginosa to antibody-independent opsonic killing and is the molecular basis for the resistance of mucoid P. aeruginosa to normally nonopsonic but alginate-specific antibodies found in normal human sera and sera of infected CF patients. O acetylation of alginate appears to be critical for P. aeruginosa resistance to host immune effectors in CF patients.

Heterogeneity in Pseudomonas aeruginosa biofilms includes expression of ribosome hibernation factors in the antibiotic-tolerant subpopulation and hypoxia-induced stress response in the metabolically active population

Bacteria growing in biofilms are physiologically heterogeneous, due in part to their adaptation to local environmental conditions. Here, we characterized the local transcriptome responses of Pseudomonas aeruginosa growing in biofilms by using a microarray analysis of isolated biofilm subpopulations. The results demonstrated that cells at the top of the biofilms had high mRNA abundances for genes involved in general metabolic functions, while mRNA levels for these housekeeping genes were low in cells at the bottom of the biofilms. Selective green fluorescent protein (GFP) labeling showed that cells at the top of the biofilm were actively dividing. However, the dividing cells had high mRNA levels for genes regulated by the hypoxia-induced regulator Anr. Slow-growing cells deep in the biofilms had little expression of Anr-regulated genes and may have experienced long-term anoxia. Transcripts for ribosomal proteins were associated primarily with the metabolically active cell fraction, while ribosomal RNAs were abundant throughout the biofilms, indicating that ribosomes are stably maintained even in slowly growing cells. Consistent with these results was the identification of mRNAs for ribosome hibernation factors (the rmf and PA4463 genes) at the bottom of the biofilms. The dormant biofilm cells of a P. aeruginosa Δrmf strain had decreased membrane integrity, as shown by propidium iodide staining. Using selective GFP labeling and cell sorting, we show that the dividing cells are more susceptible to killing by tobramycin and ciprofloxacin. The results demonstrate that in thick P. aeruginosa biofilms, cells are physiologically distinct spatially, with cells deep in the biofilm in a viable but antibiotic-tolerant slow-growth state.

Photodynamic Therapy for Acinetobacter baumannii Burn Infections in Mice

ABSTRACT Multidrug-resistant Acinetobacter baumannii infections represent a growing problem, especially in traumatic wounds and burns suffered by military personnel injured in Middle Eastern conflicts. Effective treatment with traditional antibiotics can be extremely difficult, and new antimicrobial approaches are being investigated. One of these alternatives to antimicrobials could be the combination of nontoxic photosensitizers (PSs) and visible light, known as photodynamic therapy (PDT). We report on the establishment of a new mouse model of full-thickness thermal burns infected with a bioluminescent derivative of a clinical Iraqi isolate of A. baumannii and its PDT treatment by topical application of a PS produced by the covalent conjugation of chlorin(e6) to polyethylenimine, followed by illumination of the burn surface with red light. Application of 108A. baumannii cells to the surface of 10-s burns made on the dorsal surface of shaved female BALB/c mice led to chronic infections that lasted, on average, 22 days and that were characterized by a remarkably stable bacterial bioluminescence. PDT carried out on day 0 soon after application of the bacteria gave over 3 log units of loss of bacterial luminescence in a light exposure-dependent manner, while PDT carried out on day 1 and day 2 gave an approximately 1.7-log reduction. The application of PS dissolved in 10% or 20% dimethyl sulfoxide without light gave only a modest reduction in the bacterial luminescence from mouse burns. Some bacterial regrowth in the treated burn was observed but was generally modest. It was also found that PDT did not lead to the inhibition of wound healing. The data suggest that PDT may be an effective new treatment for multidrug-resistant localized A. baumannii infections.

The dual roles of AlgG in C-5-epimerization and secretion of alginate polymers in Pseudomonas aeruginosa

Pseudomonas aeruginosa strains causing chronic pulmonary infections in cystic fibrosis patients produce high levels of alginate, an exopolysaccharide that confers a mucoid phenotype. Alginate is a linear polymer of d ‐mannuronate (M) and variable amounts of its C‐5‐epimer, l ‐guluronate (G). AlgG is a periplasmic C‐5‐epimerase that converts poly d ‐mannuronate to the mixed M+G sequence of alginate. To understand better the role and mechanism of AlgG activity, a mutant was constructed in the mucoid strain FRD1 with a defined non‐polar deletion of algG . Instead of producing poly mannuronate, the algG deletion mutant secreted dialysable uronic acids, as does a mutant lacking the periplasmic protein AlgK. High levels of unsaturated ends and the nuclear magnetic resonance spectroscopy pattern revealed that the small, secreted uronic acids were the products of extensive polymer digestion by AlgL, a periplasmic alginate lyase co‐expressed with AlgG and AlgK. Thus, AlgG is bifunctional with (i) epimerase activity and (ii) a role in protecting alginate from degradation by AlgL during transport through the periplasm. AlgK appears to share the second role. AlgG and AlgK may be part of a periplasmic protein complex, or scaffold, that guides alginate polymers to the outer membrane secretin (AlgE). To characterize the epimerase activity of AlgG further, the algG4 allele of poly mannuronate‐producing FRD462 was shown to encode a protein lacking only the epimerase function. The sequence of algG4 has a Ser‐272 to Asn substitution in a serine–threonine‐rich and conserved region of AlgG, which revealed a critical residue for C‐5‐epimerase activity.

Contribution of Stress Responses to Antibiotic Tolerance in Pseudomonas aeruginosa Biofilms

ABSTRACT Enhanced tolerance of biofilm-associated bacteria to antibiotic treatments is likely due to a combination of factors, including changes in cell physiology as bacteria adapt to biofilm growth and the inherent physiological heterogeneity of biofilm bacteria. In this study, a transcriptomics approach was used to identify genes differentially expressed during biofilm growth of Pseudomonas aeruginosa. These genes were tested for statistically significant overlap, with independently compiled gene lists corresponding to stress responses and other putative antibiotic-protective mechanisms. Among the gene groups tested were those associated with biofilm response to tobramycin or ciprofloxacin, drug efflux pumps, acyl homoserine lactone quorum sensing, osmotic shock, heat shock, hypoxia stress, and stationary-phase growth. Regulons associated with Anr-mediated hypoxia stress, RpoS-regulated stationary-phase growth, and osmotic stress were significantly enriched in the set of genes induced in the biofilm. Mutant strains deficient in rpoS, relA and spoT, or anr were cultured in biofilms and challenged with ciprofloxacin and tobramycin. When challenged with ciprofloxacin, the mutant strain biofilms had 2.4- to 2.9-log reductions in viable cells compared to a 0.9-log reduction of the wild-type strain. Interestingly, none of the mutants exhibited a statistically significant alteration in tobramycin susceptibility compared to that with the wild-type biofilm. These results are consistent with a model in which multiple genes controlled by overlapping starvation or stress responses contribute to the protection of a P. aeruginosa biofilm from ciprofloxacin. A distinct and as yet undiscovered mechanism protects the biofilm bacteria from tobramycin.

New Technologies for Studying Biofilms

Bacteria have traditionally been studied as single-cell organisms. In laboratory settings, aerobic bacteria are usually cultured in aerated flasks, where the cells are considered essentially homogenous. However, in many natural environments, bacteria and other microorganisms grow in mixed communities, often associated with surfaces. Biofilms are comprised of surface-associated microorganisms, their extracellular matrix material, and environmental chemicals that have adsorbed to the bacteria or their matrix material. While this definition of a biofilm is fairly simple, biofilms are complex and dynamic. Our understanding of the activities of individual biofilm cells and whole biofilm systems has developed rapidly, due in part to advances in molecular, analytical, and imaging tools and the miniaturization of tools designed to characterize biofilms at the enzyme level, cellular level, and systems level.

Biofilms formed by the archaeon Haloferax volcanii exhibit cellular differentiation and social motility, and facilitate horizontal gene transfer

BackgroundArchaea share a similar microbial lifestyle with bacteria, and not surprisingly then, also exist within matrix-enclosed communities known as biofilms. Advances in biofilm biology have been made over decades for model bacterial species, and include characterizations of social behaviors and cellular differentiation during biofilm development. Like bacteria, archaea impact ecological and biogeochemical systems. However, the biology of archaeal biofilms is only now being explored. Here, we investigated the development, composition and dynamics of biofilms formed by the haloarchaeon Haloferax volcanii DS2.ResultsBiofilms were cultured in static liquid and visualized with fluorescent cell membrane dyes and by engineering cells to express green fluorescent protein (GFP). Analysis by confocal scanning laser microscopy showed that H. volcanii cells formed microcolonies within 24 h, which developed into larger clusters by 48 h and matured into flake-like towers often greater than 100 μm in height after 7 days. To visualize the extracellular matrix, biofilms formed by GFP-expressing cells were stained with concanavalin A, DAPI, Congo red and thioflavin T. Stains colocalized with larger cellular structures and indicated that the extracellular matrix may contain a combination of polysaccharides, extracellular DNA and amyloid protein. Following a switch to biofilm growth conditions, a sub-population of cells differentiated into chains of long rods sometimes exceeding 25 μm in length, compared to their planktonic disk-shaped morphology. Time-lapse photography of static liquid biofilms also revealed wave-like social motility. Finally, we quantified gene exchange between biofilm cells, and found that it was equivalent to the mating frequency of a classic filter-based experimental method.ConclusionsThe developmental processes, functional properties and dynamics of H. volcanii biofilms provide insight on how haloarchaeal species might persist, interact and exchange DNA in natural communities. H. volcanii demonstrates some biofilm phenotypes similar to bacterial biofilms, but also has interesting phenotypes that may be unique to this organism or to this class of organisms, including changes in cellular morphology and an unusual form of social motility. Because H. volcanii has one of the most advanced genetic systems for any archaeon, the phenotypes reported here may promote the study of genetic and developmental processes in archaeal biofilms.

The role of bacteria in pit propagation of carbon steel

Pit propagation in carbon steel exposed to a phosphate‐containing electrolyte required either stagnant conditions or microbial colonization of anodic regions. A scanning vibrating electrode (SVE) was used to resolve formation and inactivation of anodic and cathodic sites on carbon steel. In sterile, continuously aerated medium, pits initiated and repassivated, while in the absence of aeration, pits initiated and propagated. Pit propagation was also observed in continuously aerated medium inoculated with a heterotrophic bacterium, originally isolated from a corrosion tubercle formed on a steel pipe in a fresh water environment. Autoradiography of bacteria following uptake of 14C‐acetate into cellular material in combination with SVE analysis demonstrated that sites of anodic activity coincided with sites of bacterial activity. Prelabeled bacteria also preferentially attached to corrosion products over the anodic sites. Confocal laser scanning microscopy demonstrated that attraction to anodic sites did not depend on bacterial viability and was not specific for iron as a substratum. The results suggest that bacteria may preferentially attach to the corrosion products formed over corrosion pits. The biofilms over these anodic sites may create stagnant conditions within corrosion pits that result in pit propagation.

Genotypic and Phenotypic Variation in Pseudomonas aeruginosa Reveals Signatures of Secondary Infection and Mutator Activity in Certain Cystic Fibrosis Patients with Chronic Lung Infections

ABSTRACT Evolutionary adaptation of Pseudomonas aeruginosa to the cystic fibrosis lung is limited by genetic variation, which depends on rates of horizontal gene transfer and mutation supply. Because each may increase following secondary infection or mutator emergence, we sought to ascertain the incidence of secondary infection and genetic variability in populations containing or lacking mutators. Forty-nine strains collected over 3 years from 16 patients were phenotyped for antibiotic resistance and mutator status and were genotyped by repetitive-sequence PCR (rep-PCR), pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing (MLST). Though phenotypic and genetic polymorphisms were widespread and clustered more strongly within than between longitudinal series, their distribution revealed instances of secondary infection. Sequence data, however, indicated that interlineage recombination predated initial strain isolation. Mutator series were more likely to be multiply antibiotic resistant, but not necessarily more variable in their nucleotide sequences, than nonmutators. One mutator and one nonmutator series were sequenced at mismatch repair loci and analyzed for gene content using DNA microarrays. Both were wild type with respect to mutL, but mutators carried an 8-bp mutS deletion causing a frameshift mutation. Both series lacked 126 genes encoding pilins, siderophores, and virulence factors whose inactivation has been linked to adaptation during chronic infection. Mutators exhibited loss of severalfold more genes having functions related to mobile elements, motility, and attachment. A 105-kb, 86-gene deletion was observed in one nonmutator that resulted in loss of virulence factors related to pyoverdine synthesis and elements of the multidrug efflux regulon. Diminished DNA repair activity may facilitate but not be absolutely required for rapid evolutionary change.