Pseudomonas aeruginosa colonizes the pulmonary tissue of patients with 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 CaCl 2 ) resulted in biofilms that 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.

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Genomic and proteomic approaches were used to investigate phthalate and benzoate catabolism in Rhodococcus sp. strain RHA1, a polychlorinated biphenyl-degrading actinomycete. Sequence analyses identified genes involved in the catabolism of benzoate (ben) and phthalate (pad), the uptake of phthalate (pat), and two branches of the ␤-ketoadipate pathway (catRABC and pcaJIHGBLFR). The regulatory and structural ben genes are separated by genes encoding a cytochrome P450. The pad and pat genes are contained on a catabolic island that is duplicated on plasmids pRHL1 and pRHL2 and includes predicted terephthalate catabolic genes (tpa). Proteomic analyses demonstrated that the ␤-ketoadipate pathway is functionally convergent. Specifically, the pad and pat gene products were only detected in phthalate-grown cells. Similarly, the ben and cat gene products were only detected in benzoate-grown cells. However, pca-encoded enzymes were present under both growth conditions. Activity assays for key enzymes confirmed these results. Disruption of pcaL, which encodes a fusion enzyme, abolished growth on phthalate. In contrast, after a lag phase, growth of the mutant on benzoate was similar to that of the wild type. Proteomic analyses revealed 20 proteins in the mutant that were not detected in wild-type cells during growth on benzoate, including a CatD homolog that apparently compensated for loss of PcaL. Analysis of completed bacterial genomes indicates that the convergent ␤-ketoadipate pathway and some aspects of its genetic organization are characteristic of rhodococci and related actinomycetes. In contrast, the high redundancy of catabolic pathways and enzymes appears to be unique to RHA1 and may increase its potential to adapt to new carbon sources.

Aims: The aim of this work was to study the biodegradation of benzyldimethylalkylammonium chloride (BAC) by Aeromonas hydrophila sp. K, an organism isolated from polluted soil and capable of utilizing BAC as sole source of carbon and energy. Methods and Results: High performance liquid chromatography and gas chromatography–mass spectrometry (GC‐MS) analysis was used to study BAC degradation pathway. It was shown that during BAC biodegradation, formation of benzyldimethylamine, benzylmethylamine, benzylamine, benzaldehyde and benzoic acid occurred. Formation of benzyldimethylamine as the initial metabolite suggested that the cleavage of Calkyl‐N bond occurred as the first step of BAC catabolism. Liberation of benzylmethylamine and benzylamine likely resulted from subsequent demethylation reactions, followed by deamination with formation of benzaldehyde. Benzaldehyde was rapidly converted into benzoic acid, which was further degraded. Conclusions: Aer. hydrophila sp. K is able to degrade BAC. A degradation pathway for BAC and related compounds is proposed. Significance and Impact of Study: These findings are significant for understanding biodegradation pathways of benzyl‐containing quaternary ammonium compounds.

Pseudomonas aeruginosa is an opportunistic pathogen that forms biofilms on mucous plugs in the lungs of cystic fibrosis (CF) patients, resulting in chronic infections. Pulmonary P. aeruginosa isolates often display a mucoid (alginate-producing) phenotype, whereas non-mucoid strains are generally associated with acute infections. We characterized the cytosolic proteomes of biofilm-associated and planktonic forms of a CF pulmonary isolate, P. aeruginosa FRD1, and a non-mucoid strain, PAO1. Since Ca 2+ metabolism is altered in CF pulmonary fluids, we also analysed the effect of Ca 2+ on the proteome responses of these strains. Both strains altered the abundances of 40-60 % of their proteins in response to biofilm growth and/or [Ca 2+ ].Differentially expressed proteins clustered into 12 groups, based on their abundance profiles. From these clusters, 146 proteins were identified by using MALDI-TOF/TOF mass spectrometry. Similarities as well as strain-specific differences were observed. Both strains altered the production of proteins involved in iron acquisition, pyocyanin biosynthesis, quinolone signalling and nitrogen metabolism, proteases, and proteins involved in oxidative and general stress responses. Individual proteins from these classes were highly represented in the biofilm proteomes of both strains. Strain-specific differences concerned the proteins within these functional groups, particularly for enzymes involved in iron acquisition and polysaccharide metabolism, and proteases. The results demonstrate that a mucoid CF isolate of P. aeruginosa responds to biofilm-associated growth and [Ca 2+ ] in a fashion similar to strain PAO1, but that strain-specific differences may allow this CF isolate to successfully colonize the pulmonary environment.

Bacteria undergo a variety of physiological changes following a switch from planktonic growth to surface-associated biofilm growth. Here, it is shown that biofilm development of a marine isolate, Pseudoalteromonas sp. 1398, results in global changes in its cytosolic and extracellular proteomes. Calcium influences these proteome responses, and affects the amount of surface-associated biomass and extracellular matrix material produced by Pseudoalteromonas sp. 1398. Four extracellular proteins, characterized by N-terminal sequencing, showed increased abundances, while one protein, flagellin, showed reduced abundance at higher [Ca 2+ ].Immunoblotting and transmission-electron-microscopy analysis confirmed that higher [Ca 2+ ] and surface-associated growth results in the repression of flagella production. Two-dimensional gel electrophoresis (2DGE) studies combined with cluster analysis of global proteome responses demonstrated that Ca 2+ had a greater regulatory influence on Pseudoalteromonas sp. growing in biofilms than on planktonic cultures. Approximately 22 % of the total cytosolic proteins resolved by 2DGE had differing abundances in response to a switch from planktonic growth to surface-associated growth when the cells were cultivated in 1 mM Ca 2+ . At higher [Ca 2+ ] this number increased to 38 %. Fifteen cellular proteins that were differentially expressed in response to biofilm growth and/or Ca 2+ were analysed by N-terminal sequencing and/or MS/MS. These proteins were identified as factors involved in cellular metabolic functions, putative proteases and transport proteins, although there were several proteins that had not been previously characterized. These results indicate that Ca 2+ causes global changes in matrix material, as well as in cellular and extracellular protein profiles of Pseudoalteromonas sp. 1398. These changes are more pronounced when the bacterium grows in biofilms than when it grows in planktonic culture.

Pseudomonas aeruginosa is an opportunistic human pathogen that causes severe, life-threatening infections in patients with cystic fibrosis (CF), endocarditis, wounds, or artificial implants. During CF pulmonary infections, P. aeruginosa often encounters environments where the levels of calcium (Ca 2؉ ) are elevated. Previously, we showed that P. aeruginosa responds to externally added Ca 2؉ through enhanced biofilm formation, increased production of several secreted virulence factors, and by developing a transient increase in the intracellular Ca 2؉ level, followed by its removal to the basal submicromolar level. However, the molecular mechanisms responsible for regulating Ca . In addition, a mutation in carP had a pleotropic effect in a Ca 2؉ -dependent manner, altering swarming motility, pyocyanin production, and tobramycin sensitivity. Overall, the results indicate that the two-component system CarSR is responsible for sensing high levels of external Ca 2؉ and responding through its regulatory targets that modulate Ca 2؉ homeostasis, surface-associated motility, and the production of the virulence factor pyocyanin. P seudomonas aeruginosa, a natural inhabitant of soil and water, is able to infect a variety of hosts, including plants and humans. In humans, it causes severe acute and chronic infections by colonizing respiratory and urinary tracts and burned or wounded epithelia, cornea, and muscles (1-3). The versatility of P. aeruginosa pathogenicity is associated with diverse metabolic capabilities, multiple mechanisms of resistance, a large repertoire of virulence factors, and adaptability, due in part to the tightly coordinated regulation of gene expression. A large portion of the P. aeruginosa PAO1 genome, approximately 9.4%, encodes transcriptional regulators (4, 5), including two-component regulators: 89 response regulators, 55 sensor kinases, and 14 sensorresponse regulator hybrids (2). The regulatory targets for most of these regulatory systems are unknown. IMPORTANCE During infectious disease,Calcium plays an important signaling role in both eukaryotic and prokaryotic cells. In prokaryotes, Ca 2ϩ is an essential nutrient, since it is a necessary cofactor for many enzymes. However, Ca 2ϩ can be toxic to cells at high concentrations; therefore, bacteria maintain a low-submicromolar intracellular concentration of Ca 2ϩ (6). P. aeruginosa may encounter environments where

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