The Role of Physical, Chemical, and Microbial Heterogeneity on the Field-Scale Transport and Attachment of Bacteria

A field-scale bacterial transport experiment was conducted at the Narrow Channel Focus Area of the South Oyster field site located in Oyster, Virginia. The goal of the field experiment was to determine the relative influence of subsurface heterogeneity and microbial population parameters on flow direction, velocity, and attachment of bacteria at the field scale. The field results were compared with results from laboratory-scale column experiments to develop a method for predicting field-scale bacterial transport. The field site is a shallow, sandy, unconfined, aerobic aquifer that has been characterized by geophysical, sedimentological, and hydrogeological methods. Comamonas sp. strain DA001 and a conservative tracer, bromide (Br), were injected into an area of high permeability for 12 hours. The Br and bacterial concentrations in the groundwater were monitored for 1 week at 192 sampling ports spaced over a 2-m vertical zone located from 0.5 to 7 m down-gradient of the injection well. The bacterial and Br plume was observed to move past 95 sampling ports. The densely characterized field site enabled the comparison of variations in DA001 transport to the aquifer properties. The velocity of the injected plume was correlated with geophysical estimates of hydraulic conductivity. The bacterial and Br plume appeared to follow flow paths not coincident with the hydraulic gradient but through a zone of higher permeability located off the flow axis. The amount of breakthrough of the bacteria was similar in both the high and low permeability layers with only a weak correlation between the observed hydraulic conductivity and amount of bacterial breakthrough. The uniformity in the observed attachment rates across varying grain sizes could be explained by heterogeneity of microbial properties within the single strain of injected bacteria. Application of colloid filtration theory to the field data indicated that variations in the microbial population were described by a lognormal distribution of the collision efficiency (a). Core-scale studies were used to predict the a distribution and field-scale transport distances of DA001. In sandy aquifers, physical heterogeneity may play a secondary role in controlling field-scale bacterial transport, and future research should focus on the microbial factors affecting transport

Nucleotide sequence and initial functional characterization of the clcR gene encoding a LysR family activator of the clcABD chlorocatechol operon in Pseudomonas putida.

The 3-chlorocatechol operon clcABD is central to the biodegradative pathway of 3-chlorobenzoate. The clcR regulatory gene, which activates the clcABD operon, was cloned from the region immediately upstream of the operon and was shown to complement an insertion mutation for growth on 3-chlorobenzoate. ClcR activated the clcA promoter, which controls expression of the clcABD operon, in trans by 14-fold in an in vivo promoter probe assay in Pseudomonas putida when cells were incubated with 15 mM 3-chlorobenzoic acid. Specific binding of ClcR to the clcR-clcA intergenic promoter region was observed in a gel shift assay. Nucleotide sequence analysis of the clcR gene predicts a polypeptide of 32.5 kDa, which was confirmed by using specific in vivo 35S labeling of the protein from a T7 promoter-controlled ATG fusion construct. ClcR shares high sequence identity with the LysR family of bacterial regulator proteins and has especially high homology to a subgroup of the family consisting of TcbR (57% amino acid sequence identity), TfdS, CatR, and CatM. ClcR was shown to autoregulate its own production in trans to 35% of unrepressed levels but partially relieved this autorepression under conditions that induced transcription at the clcA promoter. Several considerations indicate that the clcR-clcABD locus is most similar to the tcbR-tcbCDEF regulon

Roles of CatR and cis,cis-muconate in activation of the catBC operon, which is involved in benzoate degradation in Pseudomonas putida.

In Pseudomonas putida, the catBC operon encodes enzymes involved in benzoate degradation. Previous studies have determined that these enzymes are induced when P. putida is grown in the presence of benzoate. Induction of the enzymes of the catBC operon requires an intermediate of benzoate degradation, cis,cis-muconate, and a regulatory protein, CatR. It has been determined that CatR binds to a 27-bp region of the catBC promoter in the presence or absence of inducer. We have called this the repression binding site. In this study, we used a gel shift assay to demonstrate that the inducer, cis,cis-muconate, increases the affinity of CatR for the catBC promoter region by 20-fold. Furthermore, in the absence of cis,cis-muconate, CatR forms two complexes in the gel shift assay. The inducer cis,cis-muconate confers specificity primarily for the formation of complex 2. DNase I footprinting showed that an additional 27 bp of the catBC promoter region is protected by CatR in the presence of cis,cis-muconate. We have named this second binding site the activation binding site. Methylation interference footprinting determined that in the presence or absence of inducer, five G nucleotides of the catBC promoter region were necessary for CatR interaction with the repression binding site, while a single G residue was important for CatR interaction with the activation binding site in the presence of cis,cis-muconate. Using polymerase chain reaction-generated constructs, we found that the binding of CatR to the repression binding site is independent of the activation binding site. However, binding of CatR to the activation binding site required an intact repression binding site