Utilization of lasso peptides for biodegradation of polycyclic aromatic hydrocarbons.

Many microbial genes involved in degrading recalcitrant environmental contaminants such as polycyclic aromatic hydrocarbons (PAHs) have been identified and characterized. However, all molecular mechanisms required for PAH utilization have not yet been elucidated. In this work, we demonstrate the proposed involvement of lasso peptides in the utilization of the PAH phenanthrene in Sphingomonas BPH. Transpositional mutagenesis of Sphingomonas BPH with the miniTn5 transposon yielded 3 phenanthrene utilization deficient mutants, #257, #1778, and #1782. In mutant #1782, Tn5 had inserted into the large subunit of the naph/bph dioxygenase gene. In mutant #1778, Tn5 had inserted into the B2 protease gene of a lasso peptide cluster. This finding is the first report on the role of lasso peptides in PAH utilization. Our studies also demonstrate that interruption of the lasso peptide cluster resulted in a significant increase in the amount of biosurfactant produced in the presence of glucose when compared to the wild-type strain. Collectively, these results suggest that the mechanisms Sphingomonas BPH utilizes to degrade phenanthrene are far more complex than previously understood and that the #1778 mutant may be a good candidate for bioremediation when glucose is applied as an amendment due to its higher biosurfactant production.

Bioremediation of copper in sediments from a constructed wetland ex situ with the novel bacterium Cupriavidus basilensis SRS.

The H-02 constructed wetland was designed to remove metals (primarily copper and zinc) to treat building process water and storm water runoff from multiple sources associated with the Tritium Facility at the DOE-Savannah River Site, Aiken, SC. The concentration of Cu and Zn in the sediments has increased over the lifetime of the wetland and is a concern. A bioremediation option was investigated at the laboratory scale utilizing a newly isolated bacterium of the copper metabolizing genus Cupriavidus isolated from Tim's Branch Creek, a second-order stream that eventually serves as a tributary to the Savannah River, contaminated with uranium and other metals including copper, nickel, and mercury. Cupriavidus basilensis SRS is a rod-shaped, gram-negative bacterium which has been shown to have predatory tendencies. The isolate displayed resistance to the antibiotics ofloxacin, tetracycline, ciprofloxacin, select fungi, as well as Cu2+ and Zn2+. Subsequent ribosomal sequencing demonstrated a 100% confidence for placement in the genus Cupriavidus and a 99.014% match to the C. basilensis type strain. When H-02 wetland samples were inoculated with Cupriavidus basilensis SRS samples showed significant (p < 0.05) decrease in Cu2+ concentrations and variability in Zn2+ concentrations. Over the 72-h incubation there were no significant changes in the inoculate densities (106-108 cells/ML) indicating Cupriavidus basilensis SRS resiliency in this environment. This research expands our understanding of the Cupriavidus genus and demonstrates the potential for Cupriavidus basilensis SRS to bioremediate sites impacted with heavy metals, most notably copper.

Bioremediation of Hexanoic Acid and Phenanthrene in Oil Sands Tailings by the Microbial Consortium BioTiger™.

Polycyclic aromatic hydrocarbons (PAHs) and naphthenic acids (NAs) are toxic contaminants of environmental concern found in process water and mature fine tailings, or tailings, from the oil sands industry. BioTiger™, a patented microbial consortium of twelve natural environmental isolates, was found to cometabolically biodegrade the NA hexanoic acid and the PAH phenanthrene in the presence of tailings. Hexanoamide was found to be produced and consumed during cometabolism of hexanoic acid. Mechanistic analysis demonstrated three of the BioTiger™ strains generated biosurfactants with the bacterial adhesion to hydrocarbons assay, seven with the methylene blue active substances assay, and nine with a hemolysis assay. Serial transfers of the BioTiger™ consortium demonstrated the stability of hexanoic acid degradation over several generations. The results demonstrate that BioTiger™ cometabolically biodegrades combinations of phenanthrene and hexanoic acid in tailings. This work reveals the potential for in situ bioremediation of tailings with this natural microbial consortium.

Iron and heme utilization in Porphyromonas gingivalis.

Porphyromonas gingivalis is a Gram-negative anaerobic bacterium associated with the initiation and progression of adult periodontal disease. Iron is utilized by this pathogen in the form of heme and has been shown to play an essential role in its growth and virulence. Recently, considerable attention has been given to the characterization of various secreted and surface-associated proteins of P. gingivalis and their contribution to virulence. In particular, the properties of proteins involved in the uptake of iron and heme have been extensively studied. Unlike other Gram-negative bacteria, P. gingivalis does not produce siderophores. Instead it employs specific outer membrane receptors, proteases (particularly gingipains), and lipoproteins to acquire iron/heme. In this review, we will focus on the diverse mechanisms of iron and heme acquisition in P. gingivalis. Specific proteins involved in iron and heme capture will be described. In addition, we will discuss new genes for iron/heme utilization identified by nucleotide sequencing of the P. gingivalis W83 genome. Putative iron- and heme-responsive gene regulation in P. gingivalis will be discussed. We will also examine the significance of heme/hemoglobin acquisition for the virulence of this pathogen.

Lysine-specific gingipain K and heme/hemoglobin receptor HmuR are involved in heme utilization in Porphyromonas gingivalis.

We have previously reported on the identification and characterization of the Porphyromonas gingivalis A7436 strain outer membrane receptor HmuR, which is involved in the acquisition of hemin and hemoglobin. We demonstrated that HmuR interacts with the lysine- (Kgp) and arginine- (HRgpA) specific proteases (gingipains) and that Kgp and HRgpA can bind and degrade hemoglobin. Here, we report on the physiological significance of the HmuR-Kgp complex in heme utilization in P. gingivalis through the construction and characterization of a defined kgp mutant and a hmuR kgp double mutant in P. gingivalis A7436. The P. gingivalis kgp mutant exhibited a decreased ability to bind both hemin and hemoglobin. Growth of this strain with hemoglobin was delayed and its ability to utilize hemin as a sole iron source was diminished as compared to the wild type strain. Inactivation of both the hmuR and kgp genes resulted in further decreased ability of P. gingivalis to bind hemoglobin and hemin, as well as diminished ability to utilize either hemin or hemoglobin as a sole iron source. Collectively, these in vivo results further confirmed that both HmuR and Kgp are involved in the utilization of hemin and hemoglobin in P. gingivalis A7436.