Spatiotemporal Analysis of Microbiological Contamination in New York State Produce Fields following Extensive Flooding from Hurricane Irene, August 2011.

Although flooding introduces microbiological, chemical, and physical hazards onto croplands, few data are available on the spatial extent, patterns, and development of contamination over time postflooding. To address this paucity of information, we conducted a spatially explicit study of Escherichia coli and Salmonella contamination prevalence and genetic diversity in produce fields after the catastrophic flooding that occurred in New England during 2011. Although no significant differences were detected between the two participating farms, both random forest and logistic regression revealed changes in the spatial pattern of E. coli contamination in drag swab samples over time. Analyses also indicated that E. coli detection was associated with changes in farm management to remediate the land after flooding. In particular, E. coli was widespread in drag swab samples at 21 days postflooding, but the spatial pattern changed by 238 days postflooding such that E. coli was then most prevalent in close proximity to surface water features. The combined results of several population genetics analyses indicated that over time postflooding E. coli populations on the farms (i) changed in composition and (ii) declined overall. Salmonella was primarily detected in surface water features, but some Salmonella strains were isolated from soil and drag swab samples at 21 and 44 days postflooding. Although postflood contamination and land management responses should always be evaluated in the context of each unique farm landscape, our results provide quantitative data on the general patterns of contamination after flooding and support the practice of establishing buffer zones between flood-contaminated cropland and harvestable crops in produce fields.

A LuxR-type repressor of Burkholderia cenocepacia inhibits transcription via antiactivation and is inactivated by its cognate acylhomoserine lactone.

Burkholderia cenocepacia is an opportunistic human pathogen that encodes two LuxI-type acylhomoserine lactone (AHL) synthases and three LuxR-type AHL receptors. Of these, cepI and cepR form a cognate synthase/receptor pair, as do cciI and cciR, while cepR2 lacks a genetically linked AHL synthase gene. Another group showed that a cepR2 mutant overexpressed a cluster of linked genes that appear to direct the production of a secondary metabolite. We found that these same genes were upregulated by octanoylhomoserine lactone (OHL), which is synthesized by CepI. These data suggest that several cepR2-linked promoters are repressed by CepR2 and that CepR2 is antagonized by OHL. Fusions of two divergent promoters to lacZ were used to confirm these hypotheses, and promoter resections and DNase I footprinting assays revealed a single CepR2 binding site between the two promoters. This binding site lies well upstream of both promoters, suggesting an unusual mode of repression. Adjacent to the cepR2 gene is a gene that we designate cepS, which encodes an AraC-type transcription factor. CepS is essential for expression of both promoters, regardless of the CepR2 status or OHL concentration. CepS therefore acts downstream of CepR2, and CepR2 appears to function as a CepS antiactivator.

Saturation mutagenesis of a CepR binding site as a means to identify new quorum-regulated promoters in Burkholderia cenocepacia.

Burkholderia cenocepacia is an opportunistic pathogen of humans that encodes two genes that resemble the acylhomoserine lactone synthase gene luxI of Vibrio fischeri and three genes that resemble the acylhomoserine lactone receptor gene luxR. Of these, CepI synthesizes octanoylhomoserine lactone (OHL), while CepR is an OHL-dependent transcription factor. In the current study we developed a strategy to identify genes that are directly regulated by CepR. We systematically altered a CepR binding site (cep box) upstream of a target promoter to identify nucleotides that are essential for CepR activity in vivo and for CepR binding in vitro. We constructed 34 self-complementary oligonucleotides containing altered cep boxes, and measured binding affinity for each. These experiments allowed us to identify a consensus CepR binding site. Several hundred similar sequences were identified, some of which were adjacent to probable promoters. Several such promoters were fused to a reporter gene with and without intact cep boxes. This allowed us to identify four new regulated promoters that were induced by OHL, and that required a cep box for induction. CepR-dependent, OHL-dependent expression of all four promoters was reconstituted in Escherichia coli. Purified CepR bound to each of these sites in electrophoretic mobility shift assays.