Biotransformation of nitrobenzene by bacteria containing toluene degradative pathways.

Nonpolar nitroaromatic compounds have been considered resistant to attack by oxygenases because of the electron withdrawing properties of the nitro group. We have investigated the ability of seven bacterial strains containing toluene degradative pathways to oxidize nitrobenzene. Cultures were induced with toluene vapor prior to incubation with nitrobenzene, and products were identified by high-performance liquid chromatography and gas chromatography-mass spectrometry. Pseudomonas cepacia G4 and a strain of Pseudomonas harboring the TOL plasmid (pTN2) did not transform nitrobenzene. Cells of Pseudomonas putida F1 and Pseudomonas sp. strain JS150 converted nitrobenzene to 3-nitrocatechol. Transformation of nitrobenzene in the presence of 18O2 indicated that the reaction in JS150 involved the incorporation of both atoms of oxygen in the 3-nitrocatechol, which suggests a dioxygenase mechanism. P. putida 39/D, a mutant strain of P. putida F1, converted nitrobenzene to a compound tentatively identified as cis-1,2-dihydroxy-3-nitrocyclohexa-3,5-diene. This compound was rapidly converted to 3-nitrocatechol by cells of strain JS150. Cultures of Pseudomonas mendocina KR-1 converted nitrobenzene to a mixture of 3- and 4-nitrophenol (10 and 63%, respectively). Pseudomonas pickettii PKO1 converted nitrobenzene to 3- and 4-nitrocatechol via 3- and 4-nitrophenol. The nitrocatechols were slowly degraded to unidentified metabolites. Nitrobenzene did not serve as an inducer for the enzymes that catalyzed its oxidation. These results indicate that the nitrobenzene ring is subject to initial attack by both mono- and dioxygenase enzymes.     

Kaposi's sarcoma-associated herpesvirus ORF54/dUTPase downregulates a ligand for the NK activating receptor NKp44

Kaposi's sarcoma-associated herpesvirus (KSHV) establishes long-term latent infection in humans and can cause cancers in endothelial and B cells. A functioning immune system is vital for restricting viral proliferation and preventing KSHV-dependent neoplasms. While natural killer (NK) lymphocytes are known to target virus-infected cells for destruction, their importance in the anti-KSHV immune response is not currently understood. Activating receptors on NK cells recognize ligands on target cells, including the uncharacterized ligand(s) for NKp44, termed NKp44L. Here we demonstrate that several NK ligands are affected when KSHV-infected cells are induced to enter the lytic program. We performed a screen of most of the known KSHV genes and found that the product of the ORF54 gene could downregulate NKp44L. The ORF54-encoded protein is a dUTPase; however, dUTPase activity is neither necessary nor sufficient for the downregulation of NKp44L. In addition, we find that ORF54 can also target proteins of the cytokine receptor family and the mechanism of downregulation involves perturbation of membrane protein trafficking. The ORF54-related proteins of other human herpesviruses do not possess this activity, suggesting that the KSHV homolog has evolved a novel immunoregulatory function and that the NKp44-NKp44L signaling pathway contributes to antiviral immunity.     

Tuberculosis treatment adherence and fatality in Spain

Background

The adherence to long tuberculosis (TB) treatment is a key factor in TB control programs. Always some patients abandon the treatment or die. The objective of this study is to identify factors associated with defaulting from or dying during antituberculosis treatment.

Methods

Prospective study of a large cohort of TB cases diagnosed during 2006-2007 by 61 members of the Spanish Society of Pneumology and Thoracic Surgery (SEPAR). Predictive factors of completion outcome (cured plus completed treatment vs. defaulters plus lost to follow-up) and fatality (died vs. the rest of patients) were based on logistic regression, calculating odds ratios (OR) and 95% confidence intervals (CI).

Results

Of the 1490 patients included, 29.7% were foreign-born. The treatment outcomes were: cured 792 (53.2%), completed treatment 540 (36.2%), failure 2 (0.1%), transfer-out 33 (2.2%), default 27 (1.8%), death 27 (1.8%), lost to follow-up 65 (4.4%), other 4 (0.3%). Completion outcome reached 93.5% and poor adherence was associated with: being an immigrant (OR = 2.03; CI:1.06-3.88), living alone (OR = 2.35; CI:1.05-5.26), residents of confined institutions (OR = 4.79; CI:1.74-13.14), previous treatment (OR = 2.93; CI:1.44-5.98), being an injecting drug user (IDU) (OR = 9.51; CI:2.70-33.47) and treatment comprehension difficulties (OR = 2.93; CI:1.44-5.98). Case fatality was 1.8% and it was associated with the following variables: age 50 or over (OR = 10.88; CI:1.12-105.01), retired (OR = 12.26;CI:1.74-86.04), HIV-infected (OR = 9.93; CI:1.48-66.34), comprehension difficulties (OR = 4.07; CI:1.24-13.29), IDU (OR = 23.59; CI:2.46-225.99) and Directly Observed Therapy (DOT) (OR = 3.54; CI:1.07-11.77).

Conclusion

Immigrants, those living alone, residents of confined institutions, patients treated previously, those with treatment comprehension difficulties, and IDU patients have poor adherence and should be targeted for DOT. To reduce fatality rates, stricter monitoring is required for patients who are retired, HIV-infected, IDU, and those with treatment comprehension difficulties.     

Investigations into the life cycle of the bacterial predator Bdellovibrio bacteriovorus 109J at an interface by atomic force microscopy

Atomic force microscopy was used to image Bdellovibrio bacteriovorus 109J, a gram-negative bacterial predator that consumes a variety of other gram-negative bacteria. In predator-prey communities grown on filters at hydrated air-solid interfaces, repeated cycles of hunting, invasion, growth, and lysis occurred readily even though the cells were limited to near two-dimensional movement. This system allowed us to image the bacteria directly without extensive preparation or modification, and many of the cells remained alive during imaging. Presented are images of the life cycle in two species of prey organisms, both Escherichia coli (a small prey bacterium that grows two-dimensionally on a surface) and Aquaspirillum serpens (a large prey bacterium that grows three-dimensionally on a surface), including high-resolution images of invaded prey cells called bdelloplasts. We obtained evidence for multiple invasions per prey cell, as well as significant heterogeneity in morphology of bdellovibrios. Mutant host-independent bdellovibrios were observed to have flagella and to excrete a coating that causes the predators to clump together on a surface. Most interestingly, changes in the texture of the cell surface membranes were measured during the course of the invasion cycle. Thus, coupled with our preparation method, atomic force microscopy allowed new observations to be made about Bdellovibrio at an interface. These studies raise important questions about the ways in which bacterial predation at interfaces (air-solid or liquid-solid) may be similar to or different from predation in solution.     

Bacterial conversion of hydroxylamino aromatic compounds by both lyase and mutase enzymes involves intramolecular transfer of hydroxyl groups

Hydroxylamino aromatic compounds are converted to either the corresponding aminophenols or protocatechuate during the bacterial degradation of nitroaromatic compounds. The origin of the hydroxyl group of the products could be the substrate itself (intramolecular transfer mechanism) or the solvent water (intermolecular transfer mechanism). The conversion of hydroxylaminobenzene to 2-aminophenol catalyzed by a mutase from Pseudomonas pseudoalcaligenes JS45 proceeds by an intramolecular hydroxyl transfer. The conversions of hydroxylaminobenzene to 2- and 4-aminophenol by a mutase from Ralstonia eutropha JMP134 and to 4-hydroxylaminobenzoate to protocatechuate by a lyase from Comamonas acidovorans NBA-10 and Pseudomonas sp. strain 4NT were proposed, but not experimentally proved, to proceed by the intermolecular transfer mechanism. GC-MS analysis of the reaction products formed in H(2)(18)O did not indicate any (18)O-label incorporation during the conversion of hydroxylaminobenzene to 2- and 4-aminophenols catalyzed by the mutase from R. eutropha JMP134. During the conversion of 4-hydroxylaminobenzoate catalyzed by the hydroxylaminolyase from Pseudomonas sp. strain 4NT, only one of the two hydroxyl groups in the product, protocatechuate, was (18)O labeled. The other hydroxyl group in the product must have come from the substrate. The mutase in strain JS45 converted 4-hydroxylaminobenzoate to 4-amino-3-hydroxybenzoate, and the lyase in Pseudomonas strain 4NT converted hydroxylaminobenzene to aniline and 2-aminophenol but not to catechol. The results indicate that all three types of enzyme-catalyzed rearrangements of hydroxylamino aromatic compounds proceed via intramolecular transfer of hydroxyl groups.     

Phylogenetic and kinetic diversity of aerobic vinyl chloride-assimilating bacteria from contaminated sites

Aerobic bacteria that grow on vinyl chloride (VC) have been isolated previously, but their diversity and distribution are largely unknown. It is also unclear whether such bacteria contribute to the natural attenuation of VC at chlorinated-ethene-contaminated sites. We detected aerobic VC biodegradation in 23 of 37 microcosms and enrichments inoculated with samples from various sites. Twelve different bacteria (11 Mycobacterium strains and 1 Nocardioides strain) capable of growth on VC as the sole carbon source were isolated, and 5 representative strains were examined further. All the isolates grew on ethene in addition to VC and contained VC-inducible ethene monooxygenase activity. The Mycobacterium strains (JS60, JS61, JS616, and JS617) all had similar growth yields (5.4 to 6.6 g of protein/mol), maximum specific growth rates (0.17 to 0.23 day(-1)), and maximum specific substrate utilization rates (9 to 16 nmol/min/mg of protein) with VC. The Nocardioides strain (JS614) had a higher growth yield (10.3 g of protein/mol), growth rate (0.71 day(-1)), and substrate utilization rate (43 nmol/min/mg of protein) with VC but was much more sensitive to VC starvation. Half-velocity constant (K(s)) values for VC were between 0.5 and 3.2 micro M, while K(s) values for oxygen ranged from 0.03 to 0.3 mg/liter. Our results indicate that aerobic VC-degrading microorganisms (predominantly Mycobacterium strains) are widely distributed at sites contaminated with chlorinated solvents and are likely to be responsible for the natural attenuation of VC.     

A Novel 2-Aminomuconate Deaminase in the Nitrobenzene Degradation Pathway of Pseudomonas pseudoalcaligenes JS45

2-Aminomuconate, an intermediate in the metabolism of tryptophan in mammals, is also an intermediate in the biodegradation of nitrobenzene by Pseudomonas pseudoalcaligenes JS45. Strain JS45 hydrolyzes 2-aminomuconate to 4-oxalocrotonic acid, with the release of ammonia, which serves as the nitrogen source for growth of the microorganism. As an initial step in studying the novel deamination mechanism, we report here the purification and some properties of 2-aminomuconate deaminase. The purified enzyme migrates as a single band with a molecular mass of 16.6 kDa in 15% polyacrylamide gel electrophoresis under denaturing conditions. The estimated molecular mass of the native enzyme was 100 kDa by gel filtration and 4 to 20% gradient nondenaturing polyacrylamide gel electrophoresis, suggesting that the enzyme consists of six identical subunits. The enzyme was stable at room temperature and exhibited optimal activity at pH 6.6. The K_m for 2-aminomuconate was approximately 67 μM, and the V_max was 125 μmol · min⁻¹ · mg⁻¹. The N-terminal amino acid sequence of the enzyme did not show any significant similarity to any sequence in the databases. The purified enzyme converted 2-aminomuconate directly to 4-oxalocrotonate, rather than 2-hydroxymuconate, which suggests that the deamination was carried out via an imine intermediate.