Browsing by Author "Nivens, David E."

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Biosynthesis of the Pseudomonas aeruginosa extracellular polysaccharides, alginate, Pel, and Psl
(2011-08) Franklin, Michael J.; Nivens, David E.; Weadge, J. T.; Howell, P. L.
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.
Calcium-induced virulence factors associated with the extracellular matrix of mucoid Pseudomonas aeruginosa biofilms
(2005-06) Sarkisova, S. A.; Patrauchan, Marianna A.; Berglund, Deborah L.; Nivens, David E.; Franklin, Michael J.
Pseudomonas aeruginosa colonizes the pulmonary tissue of patientswith cystic fibrosis (CF), leading to biofilm-associated infections.The pulmonary fluid of CF patients usually contains elevated concentrations of cations and may contain the P. aeruginosa redox-active pigment pyocyanin, which is known to disrupt calcium homeostasis of host cells. Since divalent cations are important bridging ions for bacterial polysaccharides and since they may play regulatory roles in bacterial gene expression, we investigated the effect of calcium ions on the extracellular matrix constituents of P. aeruginosa biofilms. For mucoid strain P. aeruginosa FRD1,calcium addition (1.0 and 10 mM as CaCl2) resulted in biofilmsthat were at least 10-fold thicker than biofilms without added calcium. Scanning confocal laser microscopy showed increased spacing between cells for the thick biofilms, and Fourier transform infrared spectroscopy revealed that the material between cells is primarily alginate. An algD transcriptional reporter demonstrated that calcium addition caused an eightfold increase in alg gene expression in FRD1 biofilms. Calcium addition also resulted in increased amounts of three extracellular proteases (AprA, LasB, and PrpL). Immunoblots of the biofilm extracellular material established that AprA was harbored within the biofilm extracellular matrix. An aprA deletion mutation and a mutation in gene for a putative P. aeruginosa calmodulin-like protein did not significantly affect calcium-induced biofilm structure. Two-dimensional gel electrophoresis showed increased amounts of phenazine biosynthetic proteins in FRD1 biofilms and in calcium-amended planktonic cultures. Spectrochemical analyses showed that the calcium addition causes a three- to fivefold increase in pyocyanin production. These results demonstrate that calcium addition affects the structure and extracellular matrix composition of mucoid P. aeruginosa biofilms, through increased expression and stability of bacterial extracellular products. The calcium-induced extracellular matrix of mucoid P. aeruginosa consists primarily of the virulence factor alginate and also harbors extracellular proteases and perhaps pyocyanin, a biomolecule that may further disrupt cellular calcium levels.
Reversible Acceleration of the Corrosion of AISI 304 Stainless Steel Exposed to Seawater Induced by Growth and Secretions of the Marine Bacterium Vibrio natriegens
(1986-04) Nivens, David E.; Nichols, P. D.; Henson, J. M.; Geesey, Gill G.; White, D. C.
Role of alginate and alginate O-acetylation in the formation of Pseudomonas aeruginosa microcolonies and biofilms
(2001-02) Nivens, David E.; Ohman, Dennis E.; Williams, Jessica; Franklin, Michael J.
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. aeruginosabiofilm architecture.
Sampling and quantification of biofilms in food processing and other environments
(2009) Nivens, David E.; Co, B. M.; Franklin, Michael J.
In the food industry, assessment of food contact surfaces is necessary to determine whether equipment is properly cleaned and/or sanitized and whether living problematic microorganisms are present. Existing quantitative detection technologies are limited by the inability to directly detect living cells in sporadically dispersed biofilms on large surface areas. Thus, precise and accurate sampling strategies must be coupled with detection technology. This chapter discusses sampling methods and standard (e.g., plating and ATP-bioluminescence) and emerging (e.g., spectrometry, immunosensor, and nucleic acid-based) quantitative techniques to detect biofilms on food contact surfaces with a survey of function, analytical performance, and limitations.