Genomic and functional analyses of the 2-aminophenol catabolic pathway and partial conversion of its substrate into picolinic acid in Burkholderia xenovorans LB400.

2-aminophenol (2-AP) is a toxic nitrogen-containing aromatic pollutant. Burkholderia xenovorans LB400 possess an amn gene cluster that encodes the 2-AP catabolic pathway. In this report, the functionality of the 2-aminophenol pathway of B. xenovorans strain LB400 was analyzed. The amnRJBACDFEHG cluster located at chromosome 1 encodes the enzymes for the degradation of 2-aminophenol. The absence of habA and habB genes in LB400 genome correlates with its no growth on nitrobenzene. RT-PCR analyses in strain LB400 showed the co-expression of amnJB, amnBAC, amnACD, amnDFE and amnEHG genes, suggesting that the amn cluster is an operon. RT-qPCR showed that the amnB gene expression was highly induced by 2-AP, whereas a basal constitutive expression was observed in glucose, indicating that these amn genes are regulated. We propose that the predicted MarR-type transcriptional regulator encoded by the amnR gene acts as repressor of the amn gene cluster using a MarR-type regulatory binding sequence. This report showed that LB400 resting cells degrade completely 2-AP. The amn gene cluster from strain LB400 is highly identical to the amn gene cluster from P. knackmussi strain B13, which could not grow on 2-AP. However, we demonstrate that B. xenovorans LB400 is able to grow using 2-AP as sole nitrogen source and glucose as sole carbon source. An amnBA (-) mutant of strain LB400 was unable to grow with 2-AP as nitrogen source and glucose as carbon source and to degrade 2-AP. This study showed that during LB400 growth on 2-AP this substrate was partially converted into picolinic acid (PA), a well-known antibiotic. The addition of PA at lag or mid-exponential phase inhibited LB400 growth. The MIC of PA for strain LB400 is 2 mM. Overall, these results demonstrate that B. xenovorans strain LB400 posses a functional 2-AP catabolic central pathway, which could lead to the production of picolinic acid.

Integrated microfluidic magnetic immunosensor for quantification of human serum IgG antibodies to Helicobacter pylori.

In this paper, we have developed and characterized a microfluidic magnetic immunosensor coupled to a gold electrode for the rapid and sensitive quantification of human serum IgG antibodies to Helicobacter pylori. This microorganism cause peptic ulcers and chronic gastritis, affecting around the 10% of the world population. The sensor was completely automated and the antibodies detection in serum samples was carried out using a non-competitive immunoassay based on the use of purified H. pylori antigens that are immobilized on magnetic microspheres 3-aminopropyl-modified. The magnetic microbeads were injected into microchannel devices and manipulated for an external removable magnet. The IgG antibodies in human serum sample are allowed to react immunologically with the immobilized antigens, and the bounded antibodies are quantified by alkaline phosphatase (AP) enzyme-labeled second antibodies specific to human IgG. The p-aminophenyl phosphate (p-APP) was converted to p-aminophenol (p-AP) by AP and an electroactive product was detected on gold layer electrode at 0.250V. The response current obtained from the product of enzymatic reaction is directly proportional to the activity of the enzyme and, consequently, to the amount of IgG antibodies to H. pylori in serum samples. The electrochemical detection can be done within 1min and total assay time was 25min. The calculated detection limits for electrochemical detection and the ELISA procedure were 0.37 and 2.1UmL(-1), respectively, and the within- and between-assay coefficients of variation were below 5%. Our results indicate the potential usefulness of our fabricated microbiochip for the early assessment of human serum immunoglobulin G (IgG) antibodies to H. pylori.

Immuno-column for on-line quantification of human serum IgG antibodies to Helicobacter pylori in human serum samples.

This study report an human serum IgG antibodies to Helicobacter pylori quantitation procedure based on the multiple use of an immobilized H. pylori antigen on an immuno-column incorporated into an a flow-injection (FI) analytical system. The immuno-adsorbent column was prepared by packing 3-aminopropyl-modified controlled-pore glass (APCPG) covalently linking H. pylori antigens in a 3-cm of Teflon tubing (0.5 i.d.). Antibodies in the serum sample are allowed to react immunologically with the immobilized H. pylori antigen, and the bound antibodies are quantified by alkaline phosphatase (AP) enzyme-labeled second antibodies specific to human IgG. p-Aminophenyl phosphate (pAPP) was converted to p-aminophenol (pAP) by AP and an electroactive product was quantified on glassy carbon electrode (GCE) modified with multiwall carbon nanotubes (MWCNT) (GCE-CNTs) at 0.30 V. The total assay time was 25 min. The calculated detection limits for amperometric detection and the ELISA procedure are 0.62 and 1.8 UmL(-1), respectively. Reproducibility assays were made using repetitive standards of H. pylori-specific antibody and the intra- and inter-assay coefficients of variation were below 5%. The immuno-affinity method showed higher sensitivity and lower time-consumed, demonstrate its potential usefulness for early assessment of human serum immunoglobulin G (IgG) antibodies to H. pylori.