Marco C. M. Jaspers

Measurement of Biologically Available Naphthalene in Gas and Aqueous Phases by Use of a Pseudomonas putida Biosensor

ABSTRACT Genetically constructed microbial biosensors for measuring organic pollutants are mostly applied in aqueous samples. Unfortunately, the detection limit of most biosensors is insufficient to detect pollutants at low but environmentally relevant concentrations. However, organic pollutants with low levels of water solubility often have significant gas-water partitioning coefficients, which in principle makes it possible to measure such compounds in the gas rather than the aqueous phase. Here we describe the first use of a microbial biosensor for measuring organic pollutants directly in the gas phase. For this purpose, we reconstructed a bioluminescent Pseudomonas putida naphthalene biosensor strain to carry the NAH7 plasmid and a chromosomally inserted gene fusion between the sal promoter and the luxAB genes. Specific calibration studies were performed with suspended and filter-immobilized biosensor cells, in aqueous solution and in the gas phase. Gas phase measurements with filter-immobilized biosensor cells in closed flasks, with a naphthalene-contaminated aqueous phase, showed that the biosensor cells can measure naphthalene effectively. The biosensor cells on the filter responded with increasing light output proportional to the naphthalene concentration added to the water phase, even though only a small proportion of the naphthalene was present in the gas phase. In fact, the biosensor cells could concentrate a larger proportion of naphthalene through the gas phase than in the aqueous suspension, probably due to faster transport of naphthalene to the cells in the gas phase. This led to a 10-fold lower detectable aqueous naphthalene concentration (50 nM instead of 0.5 μM). Thus, the use of bacterial biosensors for measuring organic pollutants in the gas phase is a valid method for increasing the sensitivity of these valuable biological devices.

Microbial biogeography of drinking water: patterns in phylogenetic diversity across space and time

In this study, we collected water from different locations in 32 drinking water distribution networks in the Netherlands and analysed the spatial and temporal variation in microbial community composition by high-throughput sequencing of 16S rRNA gene amplicons. We observed that microbial community compositions of raw source and processed water were very different for each distribution network sampled. In each network, major differences in community compositions were observed between raw and processed water, although community structures of processed water did not differ substantially from end-point tap water. End-point water samples within the same distribution network revealed very similar community structures. Network-specific communities were shown to be surprisingly stable in time. Biofilm communities sampled from domestic water metres varied distinctly between households and showed no resemblance to planktonic communities within the same distribution networks. Our findings demonstrate that high-throughput sequencing provides a powerful and sensitive tool to probe microbial community composition in drinking water distribution systems. Furthermore, this approach can be used to quantitatively compare the microbial communities to match end-point water samples to specific distribution networks. Insight in the ecology of drinking water distribution systems will facilitate the development of effective control strategies that will ensure safe and high-quality drinking water.

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.

Prokaryotic Whole-Cell Living Bioreporters Expressing Bioluminescence Upon the Presence of Bioavailable Concentrations of Specific Pollutants

Bioremediation has gained a lot of support for the clean up of contaminated soils and sediments because of its elegance and cost-effectiveness compared to chemical and physical methods (for reviews, see (7, 30)). Bioremediation is based on the capaCity of many indigenous microorganisms to degrade polluting compounds (13, 39, 43), although many different factors determine the actual microbial activity in the environment (43). For example, (macro) physicochemical parameters (e.g., redox potential, pH, temperature, salinity, moisture content, availability of nutrients, the presence and nature of surfaces) modulate microbial activity. A key factor influencing the rates of biodegradation in the field is the extent to which a compound is accessible (bioavailable) to the microorganisms.

The Use of Whole-Cell Living Biosensors to Determine the Bioavailability of Pollutants to Microorganisms

To study the bioavailability of pollutants in a direct way, whole-cell living biosensors can be used. These are genetically constructed microorganisms, which upon sensing (bioavailable) pollutant concentrations express an easy detectable signal and may or may not degrade the pollutant as well. Biosensors are constructed by combining a sensor element (the regulatory protein) with a reporter gene fused to an inducible promoter. The most suitable reporter genes for the usage in biosensors are those coding for bioluminescent or fluorescent proteins like the luciferase and the Green Fluorescent Protein. Biosensors which are used to determine the bioavailability of pollutants in the environment should be sensitive, respond in a quantitative manner and be selective. Bioreportes should not be considered as an alternative for traditional chemical analyses but regarded as a valuable extension to these well-established techniques. By using both techniques, a better control in bioremediation processes may be obtained.