This study examined the role of the periplasmic oxidative defense proteins, copper, zinc superoxide dismutase (SodC), and thiol peroxidase (Tpx), from the Shiga toxin-producing Escherichia coli O157:H7 (STEC) in the formation of biofilms. Proteomic analyses have shown significantly higher expression levels of both periplasmic antioxidant systems (SodC and Tpx) in STEC cells grown under biofilm conditions than under planktonic conditions. An analysis of their growth phase-dependent gene expression indicated that a high level of the sodC expression occurred during the stationary phase and that the expression of the tpx gene was strongly induced only during the exponential growth phase. Exogenous hydrogen peroxide reduced the aerobic growth of the STEC sodC and tpx mutants by more than that of their parental strain. The two mutants also displayed significant reductions in their attachment to both biotic (HT-29 epithelial cell) and abiotic surfaces (polystyrene and polyvinyl chloride microplates) during static aerobic growth. However, the growth rates of both wild-type and mutants were similar under aerobic growth conditions. The formation of an STEC biofilm was only observed with the wild-type STEC cells in glass capillary tubes under continuous flow-culture conditions compared with the STEC sodC and tpx mutants. To the best of our knowledge, this is the first mutational study to show the contribution of sodC and tpx gene products to the formation of an E. coli O157:H7 biofilm. These results also suggest that these biofilms are physiologically heterogeneous and that oxidative stress defenses in both the exponential and stationary growth stages play important roles in the formation of STEC biofilms.

The role of periplasmic disulfide oxidoreductase DsbA in Shiga toxin-producing Escherichia coli O157:H7 (STEC) was investigated. Deletion of dsbA (DeltadsbA) significantly decreased cell motility and alkaline phosphatase activity in STEC. STEC DeltadsbA also showed greater sensitivity to menadione and under low pH conditions. Significant reductions in surface attachment to both biotic (HT-29 epithelial cells) and abiotic (polystyrene and polyvinyl chloride) surfaces were observed in STEC DeltadsbA. In addition, no biofilm formation was detected in STEC DeltadsbA compared to wild-type cells in glass capillary tubes under continuous flow-culture system conditions. In the nematode model Caenorhabditis elegans-killing assay, the deletion of dsbA in STEC resulted in attenuated virulence compared to wild-type cells. STEC DeltadsbA was also found to have a reduced ability to colonize the nematode gut. These results suggest that DsbA plays important roles in biofilm formation and virulence in STEC cells.

A method is developed for encapsulation of bacterial biocatalysts in silica gels formed by silica nanoparticles (SNP) and a silicon alkoxide crosslinker. Formulation of the gel was optimized by changing the SNP size, SNP to crosslinker ratio and crosslinker functionality. Hydrolysis and condensation reactions of silicon alkoxide were controlled by water to alkoxide ratio (r) and pH of the solution. FTIR analysis verified that a reactive and temporally stable silicon alkoxide crosslinker was obtained. As a case study, recombinant Escherichia coli (E. coli) cells expressing the atrazine dechlorinating enzyme AtzA were encapsulated. Synthesized catalytic biomaterials (silica gel encapsulated bacterial biocatalysts) were evaluated based on their gelation time, biocatalytic activity and mechanical strength. Diffusivity assays and SEM were used for characterization of the gel structure. We found that SNP to crosslinker ratio affected all the features of the gel, whereas crosslinker functionality primarily affected the gelation time and SNP size affected the mechanical strength and diffusivity. Based on systematic evaluation, we selected three gel formulations and subjected them to long-term activity measurements in a continuous-flow bioreactor for removing trace levels of atrazine. The effluent atrazine concentration was sustained below 30% of the influent concentration, <3 ppb, for 2 months.

Di-and trichloroisocyanuric acids are widely used as water disinfection agents, but cyanuric acid accumulates with repeated additions and must be removed to maintain free hypochlorite for disinfection. This study describes the development of methods for using a cyanuric acid-degrading enzyme contained within nonliving cells that were encapsulated within a porous silica matrix. Initially, three different bacterial cyanuric acid hydrolases were compared: TrzD from Acidovorax citrulli strain 12227, AtzD from Pseudomonas sp. strain ADP, and CAH from Moorella thermoacetica ATCC 39073. Each enzyme was expressed recombinantly in Escherichia coli and tested for cyanuric acid hydrolase activity using freely suspended or encapsulated cell formats. Cyanuric acid hydrolase activities differed by only a 2-fold range when comparing across the different enzymes with a given format. A practical water filtration system is most likely to be used with nonviable cells, and all cells were rendered nonviable by heat treatment at 70°C for 1 h. Only the CAH enzyme from the thermophile M. thermoacetica retained significant activity under those conditions, and so it was tested in a flowthrough system simulating a bioreactive pool filter. Starting with a cyanuric acid concentration of 10,000 M, more than 70% of the cyanuric acid was degraded in 24 h, it was completely removed in 72 h, and a respike of 10,000 M cyanuric acid a week later showed identical biodegradation kinetics. An experiment conducted with water obtained from municipal swimming pools showed the efficacy of the process, although cyanuric acid degradation rates decreased by 50% in the presence of 4.5 ppm hypochlorite. In total, these experiments demonstrated significant robustness of cyanuric acid hydrolase and the silica bead materials in remediation.C yanuric acid is a high-volume industrial chemical (1) and is not known to be a product of biosynthesis, but it is nonetheless biodegradable (2). Cyanuric acid is toxic in admixture with melamine, as was recently discovered when crude melamine containing cyanuric acid was added to pet food, and more than 1,000 animals died from kidney failure (3, 4). Cyanuric acid is also formed during the bacterial metabolism of s-triazine herbicides and as a by-product of disinfection processes (5). In the latter example, di-or trichloroisocyanuric acid is frequently used to disinfect outdoor swimming pools, and their decomposition leaves cyanuric acid (6), but high cyanuric acid levels impair the disinfection process, leaving people vulnerable to viral, bacterial, and protozoal infections. The cyanuric acid must be removed to sustain disinfection, and there are currently no efficient means to accomplish this. A typical remedy is to drain and refill pools, leading to downtime for the pool, disposal issues, and increased freshwater usage.Cyanuric acid is very stable below 300°C (7), but it can be degraded at ambient temperature in soil and water via a specific enzyme that has been identified within a limited number of bacteria (8). Those bac...