Growth of Polychlorinated-Biphenyl-Degrading Bacteria in the Presence of Biphenyl and Chlorobiphenyls Generates Oxidative Stress and Massive Accumulation of Inorganic Polyphosphate

Abstract

Inorganic polyphosphate (polyP) plays a significant role in increasing bacterial cell resistance to unfavorable environmental conditions and in regulating different biochemical processes. Using transmission electron microscopy of the polychlorinated biphenyl (PCB)-degrading bacterium Pseudomonas sp. strain B4 grown in defined medium with biphenyl as the sole carbon source, we observed large and abundant electron-dense granules at all stages of growth and following a shift from glucose to biphenyl or chlorobiphenyls. Using energy dispersive X-ray analysis and electron energy loss spectroscopy with an integrated energy-filtered transmission electron microscope, we demonstrated that these granules were mainly composed of phosphate. Using sensitive enzymatic methods to quantify cellular polyP, we confirmed that this polymer accumulates in PCB-degrading bacteria when they grow in the presence of biphenyl and chlorobiphenyls. Concomitant increases in the levels of the general stress protein GroEl and reactive oxygen species were also observed in chlorobiphenyl-grown cells, indicating that these bacteria adjust their physiology with a stress response when they are confronted with compounds that serve as carbon and energy sources and at the same time are chemical stressors.Polyphosphate (polyP) is a ubiquitous linear polymer consisting of hundreds of orthophosphate residues (P i ) linked by high-energy phosphoanhydride bonds. The best-known enzymes involved in the metabolism of polyP in bacteria are the polyphosphate kinase (PPK) that catalyzes the reversible conversion of the terminal phosphate of ATP into polyP and the exopolyphosphatase that processively hydrolyzes the terminal residues of polyP to liberate P i (15).The involvement of polyP in the regulation of both enzyme activities and expression of large group of genes is the basis of survival for different bacteria, including pathogens, under stress conditions and of adaptation to the stationary growth phase (reviewed in reference 16). Mutant bacterial cells that lack polyP survive poorly during growth in the stationary phase and are less resistant to heat, oxidants, osmotic challenge, antibiotics, and UV radiation (6,13,24,25,35). polyP accumulation in response to nutrient deprivation has also been reported in the genus Pseudomonas, and recent studies have demonstrated that PPK is essential in Pseudomonas aeruginosa not only for various forms of motility (26, 27) but also for biofilm development, quorum sensing, production of virulence factors, and virulence in the burned-mouse pathogenesis model (28).Chlorinated biphenyls (CBs) and polychlorinated biphenyls (PCBs) belong to one of the most widely distributed classes of chlorinated chemicals in the environment (33, 34). The toxicities and carcinogenicities of some PCB congeners make them a serious environmental and health problem (14). For cleanup of large areas of PCB-contaminated soils and aquatic environments bioremediation seems to be a promising approach (22). Although many genetic, enzymological, and bioch...

Cell Envelope Perturbation Induces Oxidative Stress and Changes in Iron Homeostasis inVibrio cholerae

“…Next, we aimed to measure the level of ROS in exponentially growing cells by using the oxidative-stress-sensitive fluorescent probe H2DCFDA (10,13,20). The level of ROS-generated fluorescence was four-to sevenfold greater in the T2S mutants than in the wild-type strain (Fig.…”

“…One hundred microliters of mid-log-phase cultures (OD 600 , approximately 0.5) was uniformly distributed on LB agar plates that were either left unsupplemented or supplemented with the iron chelator 2,2Ј-dipyridyl (100 and 150 M; Sigma-Aldrich) or with polymyxin B sulfate (100, 200, and 400 U/ml) as indicated in the text. Sterile filter paper discs (diameter, 7 mm) were placed on the surface of the agar and impregnated with 5 l of H 2 O 2 (44, 88, 176, 440, and 880 mM; Fisher) or streptonigrin (10,20,40,60,80, and 100 g/ml; Sigma-Aldrich) for V. cholerae and 5 l of 8.8 M H 2 O 2 or 100, 200, 400, 600, 800, or 1,000 g/ml of streptonigrin for E. coli. The zone of growth inhibition around the discs was recorded after 24 h of incubation at 37°C.…”

“…The level of intracellular oxidation was measured in vivo by using the molecular probe 2Ј,7Ј-dichlorodihydrofluorescein diacetate (H2DCFDA; Cayman Chemical, Ann Arbor, MI) (10,13). Bacterial cultures were grown in LB medium to the mid-log phase of growth (OD 600 , approximately 0.5), and the probe was added to a final concentration of 10 M. The cells were incubated in the dark at 37°C for 1 h, subsequently harvested, washed with 50 mM Tris-HCl buffer (pH 8.0), and disrupted by sonication in the same buffer.…”

In vivo monitoring of the potassium channel KcsA in Streptomyces lividans hyphae using immuno-electron microscopy and energy-filtering transmission electron microscopy

“…The spatial resolution is in the range of 1 nm, when evaluating a series of energy-filtered images around an edge (Hofer et al, 1999). EELS analysis has been adopted to investigate small biological samples (Somlyo & Shuman, 1982), including granules of vacuoles (Bucking et al, 1998), the apical hair membrane within the cochlea of the guinea pig (Heinrich et al, 1998), root cells of Allium (Liu & Kottke, 2003), polyphosphate granules in bacteria (Chavez et al, 2004), and spores of bacilli and different structures of mycorrhizal fungi (Kottke, 1991;Cruz, 2004).…”

The early stage of polyphosphate accumulation in <i>saccharomyces cerevisiae</i>: comparative study by extraction and DAPI staining

“…Inorganic polyphosphates (PolyP) are the linear polymers containing a few to several hundred orthophosphate (P i ) residues, which are linked by energy-rich phosphoanhydride bonds and perform in living cells a set of functions including participation in stress response, membrane transport, and gene expression regulation [1][2][3][4][5]. PolyP plays the key role in the processes of biomineralization and phosphorus homeostasis [6,7].…”