Identifying bioaugmentation candidates for bioremediation of polycyclic aromatic hydrocarbons in contaminated estuarine sediment of the Elizabeth River, VA, USA.

Estuarine sediments near former creosoting facilities along the Elizabeth River (Virginia, USA) are contaminated by polycyclic aromatic hydrocarbons (PAHs). In this study, we interrogated the bacterial community of the Elizabeth River with both culture-based and culture-independent methods to identify potential candidates for bioremediation of these contaminants. DNA-based stable isotope probing (SIP) experiments with phenanthrene and fluoranthene using sediment from the former Republic Creosoting site identified relevant PAH-degrading bacteria within the Azoarcus, Hydrogenophaga, and Croceicoccus genera. Targeted cultivation of PAH-degrading bacteria from the same site recovered 6 PAH-degrading strains, including one strain highly similar to Hydrogenophaga sequences detected in SIP experiments. Other isolates were most similar to organisms within the Novosphingobium, Sphingobium, Stenotrophomonas, and Alcaligenes genera. Lastly, we performed 16S rRNA gene amplicon microbiome analyses of sediment samples from four sites, including Republic Creosoting, with varying concentrations of PAHs. Analysis of these data showed a striking divergence of the microbial community at the highly contaminated Republic Creosoting site from less contaminated sites with the enrichment of several bacterial clades including those affiliated with the Pseudomonas genus. Sequences within the microbiome libraries similar to SIP-derived sequences were generally found at high relative abundance, while the Croceicoccus sequence was present at low to moderate relative abundance. These results suggest that Azoarcus and Hydrogenophaga strains might be good target candidates for biostimulation, while Croceicoccus spp. might be good targets for bioaugmentation in these sediments. Furthermore, this study demonstrates the value of culture-based and culture-independent methods in identifying promising bacterial candidates for use in a precision bioremediation scheme. KEY POINTS: • This study highlights the importance of using multiple strategies to identify promising bacterial candidates for use in a precision bioremediation scheme. • We used both selective cultivation techniques and DNA-based stable isotope probing to identify bacterial degraders of prominent PAHs at a historically contaminated site in the Elizabeth River, VA, USA. • Azoarcus and Hydrogenophaga strains might be good target candidates for biostimulation in Elizabeth River sediments, while Croceicoccus spp. might be good targets for bioaugmentation.

NlpI-mediated modulation of outer membrane vesicle production through peptidoglycan dynamics in Escherichia coli.

Outer membrane vesicles (OMVs) are ubiquitously secreted from the outer membrane (OM) of Gram-negative bacteria. These heterogeneous structures are composed of OM filled with periplasmic content from the site of budding. By analyzing mutants that have vesicle production phenotypes, we can gain insight into the mechanism of OMV budding in wild-type cells, which has thus far remained elusive. In this study, we present data demonstrating that the hypervesiculation phenotype of the nlpI deletion mutant of Escherichia coli correlates with changes in peptidoglycan (PG) dynamics. Our data indicate that in stationary phase cultures the nlpI mutant exhibits increased PG synthesis that is dependent on spr, consistent with a model in which NlpI controls the activity of the PG endopeptidase Spr. In log phase, the nlpI mutation was suppressed by a dacB mutation, suggesting that NlpI regulates penicillin-binding protein 4 (PBP4) during exponential growth. The data support a model in which NlpI negatively regulates PBP4 activity during log phase, and Spr activity during stationary phase, and that in the absence of NlpI, the cell survives by increasing PG synthesis. Further, the nlpI mutant exhibited a significant decrease in covalent outer membrane (OM-PG) envelope stabilizing cross-links, consistent with its high level of OMV production. Based on these results, we propose that one mechanism wild-type Gram-negative bacteria can use to modulate vesiculation is by altering PG-OM cross-linking via localized modulation of PG degradation and synthesis.

Residues of heat-labile enterotoxin involved in bacterial cell surface binding.

Enterotoxigenic Escherichia coli (ETEC) is a leading cause of traveler's diarrhea worldwide. One major virulence factor released by this pathogen is the heat-labile enterotoxin LT, which upsets the balance of electrolytes in the intestine. After export, LT binds to lipopolysaccharide (LPS) on the bacterial surface. Although the residues responsible for LT's binding to its host receptor are known, the portion of the toxin which mediates LPS binding has not been defined previously. Here, we describe mutations in LT that impair the binding of the toxin to the external surface of E. coli without altering holotoxin assembly. One mutation in particular, T47A, nearly abrogates surface binding without adversely affecting expression or secretion in ETEC. Interestingly, T47A is able to bind mutant E. coli expressing highly truncated forms of LPS, indicating that LT binding to wild-type LPS may be due primarily to association with an outer core sugar. Consequently, we have identified a region of LT distinct from the pocket involved in eukaryotic receptor binding that is responsible for binding to the surface of E. coli.