Rhodococci are common soil heterotrophs that possess diverse functional enzymatic activities with economic and ecological significance. In this study, the correlation between gene expression and biological removal of the water contaminant N-nitrosodimethylamine (NDMA) is explored. NDMA is a hydrophilic, potent carcinogen that has gained recent notoriety due to its environmental persistence and emergence as a widespread micropollutant in the subsurface environment. In this study, we demonstrate that Rhodococcus sp. strain RHA1 can constitutively degrade NDMA and that activity toward this compound is enhanced by approximately 500-fold after growth on propane. Transcriptomic analysis of RHA1 and reverse transcriptase quantitative PCR assays demonstrate that growth on propane elicits the upregulation of gene clusters associated with (i) the oxidation of propane and (ii) the oxidation of substituted benzenes. Deletion mutagenesis of prmA, the gene encoding the large hydroxylase component of propane monooxygenase, abolished both growth on propane and removal of NDMA. These results demonstrate that propane monooxygenase is responsible for NDMA degradation by RHA1 and explain the enhanced cometabolic degradation of NDMA in the presence of propane.Recently recognized as a drinking water contaminant (19), N-nitrosodimethylamine (NDMA) is now closely monitored by municipal water providers to minimize human exposure (3,6,20). Concern has developed due to NDMA's potent mutagenicity and carcinogenicity (11) coupled with increasing awareness of its presence as a groundwater contaminant associated with liquid rocket propellants, certain industrial processes, and chlorine-based water reuse projects (19,20,23). The combination of high subsurface mobility coupled with poor attenuation by volatilization, sorption, and abiotic and biological processes (19) has resulted in groundwater plumes that contain measurable quantities of NDMA following decades and miles of subsurface propagation (31). Despite its recalcitrance in groundwater, it has recently been shown that NDMA can be attenuated in wastewater treatment systems (23) and soils (2, 5, 32), presumably through the involvement of microorganisms. This dichotomy between persistence and potential biodegradability necessitates a more detailed understanding of the biochemical mechanisms that contribute to NDMA degradation.Microorganisms grown on substrates such as propane, methane, and toluene have been shown to rapidly oxidize NDMA in the laboratory (7,25). In these cases, evidence from inhibition and induction experiments along with observations of requisite oxygen consumption suggests that propane monooxygenases (PrMO), soluble methane monooxygenases (sMMO), and toluene monooxygenases (TMO) are most likely involved in these transformations. In addition, experiments with Escherichia coli clones expressing TMO inserts confirmed the role of toluene 4-monooxygenase (T4MO) in NDMA oxidation, while cupric selection for soluble rather than particulate MMO confirmed the role of sMMO (25). T...

Rhodococcus jostii RHA1 is a soil-residing actinomycete with many favorable metabolic capabilities that make it an ideal candidate for the bioremediation of contaminated soils. Arguably the most basic requirement for life is water, yet some nonsporulating bacteria, like RHA1, can survive lengthy droughts. Here we report the first transcriptomic analysis of a gram-positive bacterium during desiccation. Filtered RHA1 cells incubated at either low relative humidity (20%), as an air-drying treatment, or high relative humidity (100%), as a control, were transcriptionally profiled over a comprehensive time series. Also, the morphology of RHA1 cells was characterized by cryofixation scanning electron microscopy during each treatment. Desiccation resulted in a transcriptional response of approximately 8 times more differentially regulated genes than in the control (819 versus 106 genes, respectively). Genes that were differentially expressed during only the desiccation treatment primarily had expression profiles that were maximally up-regulated upon complete drying of the cells. The microarray expression ratios for some of the highly up-regulated genes were verified by reverse transcriptase quantitative PCR. These genes included dps1, encoding an oxidative stress protection protein which has not previously been directly associated with desiccation, and the two genes encoding sigma factors SigF1 and SigF3, possibly involved in the regulatory response to desiccation. RHA1 cells also induced the biosynthetic pathway for the compatible solute ectoine. These desiccation-specific responses represent the best candidates for important mechanisms of desiccation resistance in RHA1.Fluctuation in water availability is a fundamental stress challenging soil-residing microorganisms, and desiccation tolerance is a key adaptation of many such organisms. The structural integrity and proper functioning of many proteins and other cellular macromolecules depend upon interactions with water molecules. The mechanisms by which microorganisms adapt to water limitation have been best studied in cyanobacteria. Many cyanobacteria mitigate water loss by synthesizing extracellular polysaccharides (EPS) to create a barrier between themselves and the dry environment (31). The second main strategy employed by cyanobacteria for retaining water during air drying is to increase their intracellular solute concentrations to equilibrate them with those of their increasingly hypertonic surroundings (41). The molecules either imported or synthesized for this purpose are referred to as compatible solutes because, even at high concentrations, they permit cellular machinery to function (32). In a microarray study of a desiccated cyanobacterial species, Anabaena sp. strain PCC7120, the importance of compatible solute production as a major water stress response was identified (21). Other, concurrent responses included the up-regulation of genes associated with protein stabilization (heat shock and chaperone proteins) and with countering oxidative threats (probable...

Elevated plasma concentrations of lipoprotein(a) (Lp(a)) are a risk factor for a variety of atherosclerotic disorders including coronary heart disease. In the current study, we report that incubation of cultured human umbilical vein or coronary artery endothelial cells with Lp(a) elicits a dramatic rearrangement of the actin cytoskeleton characterized by increased central stress fiber formation and redistribution of focal adhesions. These effects are mediated by the apolipoprotein(a) (apo(a)) component of Lp(a) since incubation of apo(a) with the cells evoked similar cytoskeletal rearrangements, while incubation with low density lipoprotein had no effect. Apo(a) also produced a time-dependent increase in transendothelial permeability. The cytoskeletal rearrangements evoked by apo(a) were abolished by C3 transferase, which inhibits Rho, and by Y-27632, an inhibitor of Rho kinase. In addition to actin cytoskeleton remodeling, apo(a) was found to cause VE-cadherin disruption and focal adhesion molecule reorganization in a Rho-and Rho kinase-dependent manner. Cell-cell contacts were found to be regulated by Rho and Rac but not Cdc42. Apo(a) caused a transient increase in the extent of myosin light chain phosphorylation. Finally apo(a) did not evoke increases in intracellular calcium levels, although the effects of apo(a) on the cytoskeleton were found to be calcium-dependent. We conclude that the apo(a) component of Lp(a) activates a Rho/Rho kinasedependent intracellular signaling cascade that results in increased myosin light chain phosphorylation with attendant rearrangements of the actin cytoskeleton. We propose that the resultant increase in endothelial permeability caused by Lp(a) may help explain the atherosclerotic risk posed by elevated concentrations of this lipoprotein.The vascular endothelium acts as a pivotal regulator in vessel wall homeostasis by forming a selective barrier between components of the blood and extravascular tissues. Increasing evidence suggests that atypical endothelial function is a key event in the initial stages of atherosclerosis development. More specifically, enhanced endothelial cell permeability and the expression of a procoagulant, antifibrinolytic, and proinflammatory phenotype by the endothelium is thought to be a crucial event in the onset of this disease (1). Various physiological agents have been identified that elicit some manifestations of endothelial dysfunction. These include growth factors, inflammatory cytokines such as tumor necrosis factor-␣ (2), vasoactive substances such as thrombin (3), and mildly oxidized, but not native, low density lipoprotein (LDL) 1 (4). Many of these agents alter the permeability of the endothelium by stimulating cell contraction, thereby increasing the size of intercellular gaps and facilitating entry of inflammatory cells and atherogenic lipoproteins. The mechanism by which these agents are able to alter properties of the endothelium has received much attention. Endothelial cell contraction is mediated by interactions between actin filam...

Oxysterols from steroid autooxidation have numerous harmful effects, but their biodegradation is poorly understood. Microarrays were used to study mineralization of the most common oxysterol, 7-ketocholesterol (7KC), by Rhodococcus jostii RHA1. Growth on 7KC versus growth on cholesterol resulted in 363 differentially expressed genes, including upregulation of two large gene clusters putatively encoding steroid catabolism. Despite this difference, 7KC degradation required key genes involved in cholesterol degradation, indicating a common catabolic route.

ABSTRACTRhodococcus jostiiRHA1, a catabolically diverse soil actinomycete, is highly resistant to long-term nutrient starvation. After 2 years of carbon starvation, 10% of the bacterial culture remained viable. To study the molecular basis of such resistance, we monitored the abundance of about 1,600 cytosolic proteins during a 2-week period of carbon source (benzoate) starvation. Hierarchical cluster analysis elucidated 17 major protein clusters and showed that most changes occurred during transition to stationary phase. We identified 196 proteins. A decrease in benzoate catabolic enzymes correlated with benzoate depletion, as did induction of catabolism of alternative substrates, both endogenous (lipids, carbohydrates, and proteins) and exogenous. Thus, we detected a transient 5-fold abundance increase for phthalate, phthalate ester, biphenyl, and ethyl benzene catabolic enzymes, which coincided with at least 4-fold increases in phthalate and biphenyl catabolic activities. Stationary-phase cells demonstrated an ∼250-fold increase in carbon monoxide dehydrogenase (CODH) concurrent with a 130-fold increase in CODH activity, suggesting a switch to CO or CO2utilization. We observed two phases of stress response: an initial response occurred during the transition to stationary phase, and a second response occurred after the cells had attained stationary phase. Although SigG synthesis was induced during starvation, a ΔsigGdeletion mutant showed only minor changes in cell survival. Stationary-phase cells underwent reductive cell division. The extreme capacity of RHA1 to survive starvation does not appear to involve novel mechanisms; rather, it seems to be due to the coordinated combination of earlier-described mechanisms.