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

Field application of a genetically engineered microorganism for polycyclic aromatic hydrocarbon bioremediation process monitoring and control

On October 30, 1996, the US Environmental Protection Agency (EPA) commenced the first test release of genetically engineered microorganisms (GEMs) for use in bioremediation. The specific objectives of the investigation were multifaceted and include (1) testing the hypothesis that a GEM can be successfully introduced and maintained in a bioremediation process, (2) testing the concept of using, at the field scale, reporter organisms for direct bioremediation process monitoring and control, and (3) acquiring data that can be used in risk assessment decision making and protocol development for future field release applications of GEMs. The genetically engineered strain under investigation is Pseudomonas fluorescens strain HK44 (King et al., 1990). The original P. fluorescens parent strain was isolated from polycyclic aromatic hydrocarbon (PAH) contaminated manufactured gas plant soil. Thus, this bacterium is able to biodegrade naphthalene (as well as other substituted naphthalenes and other PAHs) and is able to function as a living bioluminescent reporter for the presence of naphthalene contamination, its bioavailability, and the functional process of biodegradation. A unique component of this field investigation was the availability of an array of large subsurface soil lysimeters. This article describes the experience associated with the release of a genetically modified microorganism, the lysimeter facility and its associated instrumentation, as well as representative data collected during the first eighteen months of operation

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Infrared monitoring of the adhesion of Catenaria anguillulae zoospores to solid surfaces

Electron microscopic studies of nematodes infected with the chytridiomycetous fungusCatenaria anguillulae indicated that zoospores of the fungus adhered to the cuticle of nematodes by a layer of extracellular polymers. The chemical composition of the adhesive polymers and their interaction with a solid surface were examined with Fourier transform infrared spectroscopy, using an attenuated total reflectance cell. On-line monitoring of the adhesion of zoospores to a germanium crystal with this technique showed that the adhesive polymers consisted of a protein(s) containing amide I and II bands. The adsorption of these proteins, measured as the increase in the amide II band, had a rapid initial phase of ca. 20 minutes, followed by a slower increase during the course of incubation. Fluorescein isothiocyanate staining of the attached cells at the end of the experiment showed that the adhesion of the zoospores occurred before the formation of the cyst wall

Monitoring microbiol adhesion and biofilm formation by attenuated total reflection/Fourier transform infrared spectroscopy

A major problem in accurately defining bacterial adhesion mechanisms and processes occurring in biofilms on surfaces is the lack of techniques that nondestructively provide on-line information about the microorganisms, their extracellular polymers, and metabolites. The attenuated total reflectance (ATR) technique of Fourier transform infrared spectroscopy (FT-IR) is ideally suited to monitor molecular interactions at the solution/internal reflection element (IRE) interface, and we report its application to biofilm research. Two methodologies were utilized to obtain the ATF/FT-IR spectra of living Caulobacter crescentus cells attached to germanium crystals. Initially, spectra of attached bacteria in high purity water produced molecular details of the attachment process without spectral interferences from components of the medium. A growth medium, utilized in the second method, allowed direct examination of the infrared absorption bands associated with the actively growing microorganisms on the surface of the IRE in the spectral region of 2000 to 1200 cm-1. Using the amide II band as a marker for biofilm biomass, the detection limit was determined to be approximately 5 × 105 cells·cm-2. These results proved that the ATR-FT/IR methodologies can be utilized to provide chemical information from bacteria and bacterial products located within approximately 1 μm of the surface without spectral interferences due to components of the medium

Peptide ormosils as cellular substrates

Peptide-functionalized thin films exhibit significant potential for integration into implantable devices and cell-based technologies. A new type of neuroactive peptide-modified silica was developed using sol-gel reaction chemistry to produce thin films from four different peptide silane precursors. Peptide silanes containing binding sequences from laminin ( YIGSR and KDI), fibronectin ( RGD), and EGF repeats from laminin and tenascin ( NID) were produced using standard solid-state FMOC peptide synthesis conditions and the covalent attachment of 3\u27-( aminopropyl) trimethoxysilane ( APTMS), using carbonyldiimadazole ( CDI) as a linking molecule. Precursor formation was confirmed with MALDI- MS. Thin films were produced by dip-coating using the peptide precursors combined with hydrolyzed tetramethoxysilane. Atomic force microscopy indicated that the surface topography was not affected by low concentrations of peptide precursor ( 0.0025 mol%), but higher concentrations of peptide precursor ( 0.01 mol%) resulted in features that were 50 - 75 nm in height. The height features observed were consistent in size with previously determined topographical morphology supportive of neuronal cell lines. The surfaces were biologically active and modulated the phenotype of the embryonic carcinoma stem cell line, P19. Combinations of the peptide silanes resulted in altered cell types after retinoic acid treatment. More neurons were observed on RGD/YIGSR and RGD/YIGSR/NID surfaces compared to tetramethoxysilane ( TMOS) controls. More supporting cells were observed compared to collagen coated tissue culture plates. In addition, neurites were significantly longer on the peptide ormosils compared to controls. This work demonstrates a novel method for producing biologically active peptide ormosils using peptide-modified precursors

Role of Alginate and Its O Acetylation in Formation of Pseudomonas aeruginosa Microcolonies and Biofilms

Attenuated total reflection/Fourier transform-infrared spectrometry (ATR/FT-IR) and scanning confocal laser microscopy (SCLM) were used to study the role of alginate and alginate structure in the attachment and growth of Pseudomonas aeruginosa on surfaces. Developing biofilms of the mucoid (alginate-producing) cystic fibrosis pulmonary isolate FRD1, as well as mucoid and nonmucoid mutant strains, were monitored by ATR/FT-IR for 44 and 88 h as IR absorbance bands in the region of 2,000 to 1,000 cm(−1). All strains produced biofilms that absorbed IR radiation near 1,650 cm(−1) (amide I), 1,550 cm(−1) (amide II), 1,240 cm(−1) (P⩵O stretching, C—O—C stretching, and/or amide III vibrations), 1,100 to 1,000 cm(−1) (C—OH and P—O stretching) 1,450 cm(−1), and 1,400 cm(−1). The FRD1 biofilms produced spectra with an increase in relative absorbance at 1,060 cm(−1) (C—OH stretching of alginate) and 1,250 cm(−1) (C—O stretching of the O-acetyl group in alginate), as compared to biofilms of nonmucoid mutant strains. Dehydration of an 88-h FRD1 biofilm revealed other IR bands that were also found in the spectrum of purified FRD1 alginate. These results provide evidence that alginate was present within the FRD1 biofilms and at greater relative concentrations at depths exceeding 1 μm, the analysis range for the ATR/FT-IR technique. After 88 h, biofilms of the nonmucoid strains produced amide II absorbances that were six to eight times as intense as those of the mucoid FRD1 parent strain. However, the cell densities in biofilms were similar, suggesting that FRD1 formed biofilms with most cells at depths that exceeded the analysis range of the ATR/FT-IR technique. SCLM analysis confirmed this result, demonstrating that nonmucoid strains formed densely packed biofilms that were generally less than 6 μm in depth. In contrast, FRD1 produced microcolonies that were approximately 40 μm in depth. An algJ mutant strain that produced alginate lacking O-acetyl groups gave an amide II signal approximately fivefold weaker than that of FRD1 and produced small microcolonies. After 44 h, the algJ mutant switched to the nonmucoid phenotype and formed uniform biofilms, similar to biofilms produced by the nonmucoid strains. These results demonstrate that alginate, although not required for P. aeruginosa biofilm development, plays a role in the biofilm structure and may act as intercellular material, required for formation of thicker three-dimensional biofilms. The results also demonstrate the importance of alginate O acetylation in P. aeruginosa biofilm architecture

A Low-Molecular-Weight Alginate Oligosaccharide Disrupts Pseudomonal Microcolony Formation and Enhances Antibiotic Effectiveness

In chronic respiratory disease the formation of dense, 3-dimensional ‘micro colonies' by Pseudomonas aeruginosa within the airway plays an important role in contributing to resistance to treatment. An in vitro biofilm model of pseudomonal microcolony formation using artificial sputum (AS) medium was established to study the effects of low molecular weight alginate oligomers (OligoG CF-5/20) on pseudomonal growth, microcolony formation and the efficacy of colistin. The studies employed clinical cystic fibrosis (CF) isolates (n=3) and reference non-mucoid and mucoid multi-drug resistant (MDR) CF isolates (n=7). Bacterial growth, biofilm development and disruption were studied using cell-viability assays and image analysis using scanning electron- and confocal laser scanning microscopy. Pseudomonal growth in AS medium was associated with increased ATP production (p10 μm) microcolonies. In conventional growth medium, colistin retained an ability to inhibit growth of planktonic bacteria, although the MIC was increased (0.1 to 0.4 μg/ml) in AS medium versus. In contrast, in an established biofilm model in the AS medium, the efficacy of colistin was decreased. OligoG CF-5/20 (≥2%) treatment however, induced dose-dependent biofilm disruption (p0.2%; p<0.05) reductions in pseudomonal quorum sensing signaling. These findings reinforce the potential clinical significance of microcolony formation in the CF lung, and highlight a novel approach to treat MDR pseudomonal infections