Measuring mass transfer processes of octane with the help of an alkSalkB::gfp-tagged Escherichia coli.

Diffusion of octane from oily droplets in different microscale settings was measured using Escherichia coli expressing the stable green fluorescent protein (GFP) from the alkB promoter of Pseudomonas oleovorans. GFP fluorescence intensities were determined quantitatively at the single-cell level after 1.0 or 2.5 h incubation and compared with different calibration series using known concentrations of octane. By immobilizing the E. coli sensor cells on the bottom glass plate of a microscope flow chamber, it was possible to monitor the diffusion process for octane in aqueous solution as a function of time and distance from non-aqueous phase droplets of octane alone or oily octane mixtures. When a gas phase was included in the flow chambers, octane transport could be demonstrated from the oily mixtures to the cells through both gas and liquid phase. Assays of non-immobilized sensor cells in microdroplets in the presence or absence of soil particles incubated with octane through the vapour phase revealed a slight reduction in the total amount of induced E. coli cells in the presence of soil. Our results indicate the power of using GFP-marked single-cell biosensors in determining microscale bioavailability of organic pollutants.

Transcriptional organization and dynamic expression of the hbpCAD genes, which encode the first three enzymes for 2-hydroxybiphenyl degradation in Pseudomonas azelaica HBP1.

Pseudomonas azelaica HBP1 degrades the toxic substance 2-hydroxybiphenyl (2-HBP) by means of three enzymes that are encoded by structural genes hbpC, hbpA, and hbpD. These three genes form a small noncontiguous cluster. Their expression is activated by the product of regulatory gene hbpR, which is located directly upstream of the hbpCAD genes. The HbpR protein is a transcription activator and belongs to the so-called XylR/DmpR subclass within the NtrC family of transcriptional activators. Transcriptional fusions between the different hbp intergenic regions and the luxAB genes of Vibrio harveyi in P. azelaica and in Escherichia coli revealed the existence of two HbpR-regulated promoters; one is located in front of hbpC, and the other one is located in front of hbpD. Northern analysis confirmed that the hbpC and hbpA genes are cotranscribed, whereas the hbpD gene is transcribed separately. No transcripts comprising the entire hbpCAD cluster were detected, indicating that transcription from P(hbpC) is terminated after the hbpA gene. E. coli mutant strains lacking the structural genes for the RNA polymerase sigma(54) subunit or for the integration host factor failed to express bioluminescence from P(hbpC)- and P(hbpD)-luxAB fusions when a functional hbpR gene was provided in trans. This pointed to the active role of sigma(54) and integration host factor in transcriptional activation from these promoters. Primer extension analysis revealed that both P(hbpC) and P(hbpD) contain the typical motifs at position -24 (GG) and -12 (GC) found in sigma(54)-dependent promoters. Analysis of changes in the synthesis of the hbp mRNAs, in activities of the 2-HBP pathway enzymes, and in concentrations of 2-HBP intermediates during the first 4 h after induction of continuously grown P. azelaica cells with 2-HBP demonstrated that the specific transcriptional organization of the hbp genes ensured smooth pathway expression.

HbpR, a new member of the XylR/DmpR subclass within the NtrC family of bacterial transcriptional activators, regulates expression of 2-hydroxybiphenyl metabolism in Pseudomonas azelaica HBP1.

The regulation of 2-hydroxybiphenyl and 2,2'-dihydroxybiphenyl degradation in Pseudomonas azelaica is mediated by the regulatory gene, hbpR. The hbpR gene encodes a 63-kDa protein belonging to the NtrC family of prokaryotic transcriptional activators and having the highest homology to members of the XylR/DmpR subclass. Disruption of the hbpR gene in P. azelaica and complementation in trans showed that the HbpR protein was the key regulator for 2-hydroxybiphenyl metabolism. Induction experiments with P. azelaica and Escherichia coli containing luxAB-based transcriptional fusions revealed that HbpR activates transcription from a promoter (P(hbpC)) in front of the first gene for 2-hydroxybiphenyl degradation, hbpC, and that 2-hydroxybiphenyl itself is the direct effector for HbpR-mediated activation. Of several compounds tested, only the pathway substrates 2-hydroxybiphenyl and 2,2'-dihydroxybiphenyl and structural analogs like 2-aminobiphenyl and 2-hydroxybiphenylmethane were effectors for HbpR activation. HbpR is therefore, to our knowledge, the first regulator of the XylR/DmpR class that recognizes biaromatic but not monoaromatic structures. Analysis of a spontaneously occurring mutant, P. azelaica HBP1 Prp, which can grow with the non-wild-type effector 2-propylphenol, revealed a single mutation in the hbpR gene (T613C) leading to a Trp-->Arg substitution at amino acid residue 205. P. azelaica HBP1 derivative strains without a functional hbpR gene constitutively expressed the genes for 2-hydroxybiphenyl degradation when complemented in trans with the hbpR-T613C gene. This suggests the importance of this residue, which is conserved among all members of the XylR/DmpR subclass, for interdomain repression.

Development and characterization of a whole-cell bioluminescent sensor for bioavailable middle-chain alkanes in contaminated groundwater samples.

A microbial whole-cell biosensor was developed, and its potential to measure water-dissolved concentrations of middle-chain-length alkanes and some related compounds by bioluminescence was characterized. The biosensor strain Escherichia coli DH5 alpha(pGEc74, pJAMA7) carried the regulatory gene alkS from Pseudomonas oleovorans and a transcriptional fusion of PalkB from the same strain with the promoterless luciferase luxAB genes from Vibrio harveyi on two separately introduced plasmids. In standardized assays, the biosensor cells were readily inducible with octane, a typical inducer of the alk system. Light emission after induction periods of more than 15 min correlated well with octane concentration. In well-defined aqueous samples, there was a linear relationship between light output and octane concentrations between 24 and 100 nM. The biosensor responded to middle-chain-length alkanes but not to alicyclic or aromatic compounds. In order to test its applicability for analyzing environmentally relevant samples, the biosensor was used to detect the bioavailable concentration of alkanes in heating oil-contaminated groundwater samples. By the extrapolation of calibrated light output data to low octane concentrations with a hyperbolic function, a total inducer concentration of about 3 nM in octane equivalents was estimated. The whole-cell biosensor tended to underestimate the alkane concentration in the groundwater samples by about 25%, possibly because of the presence of unknown inhibitors. This was corrected for by spiking the samples with a known amount of an octane standard. Biosensor measurements of alkane concentrations were further verified by comparing them with the results of chemical analyses.