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We have identified in Pseudomonas aeruginosa strain JB2 a novel cluster of mobile genes encoding degradation of hydroxy-and halo-aromatic compounds. Nineteen open reading frames were located and, based on sequence similarities, were putatively identified as encoding a ring hydroxylating oxygenase (hybABCD), an ATP-binding cassette-type transporter, an extradiol ring-cleavage dioxygenase, transcriptional regulatory proteins, enzymes mediating chlorocatechol degradation, and transposition functions. Expression of hybABCD in Escherichia coli cells effected stoichiometric transformation of 2-hydroxybenzoate (salicylate) to 2,5-dihydroxybenzoate (gentisate). This activity was predicted from sequence similarity to functionally characterized genes, nagAaGHAb from Ralstonia sp. strain U2 (S. L. Fuenmayor, M. Wild, A. L. Boyes, and P. A. Williams, J. Bacteriol. 180:2522-2530, 1998), and is the second confirmed example of salicylate 5-hydroxylase activity effected by an oxygenase outside the flavoprotein group. Growth of strain JB2 or Pseudomonas huttiensis strain D1 (an organism that had acquired the 2-chlorobenzoate degradation phenotype from strain JB2) on benzoate yielded mutants that were unable to grow on salicylate or 2-chlorobenzoate and that had a deletion encompassing hybABCD and the region cloned downstream. The mutants' inability to grow on 2-chlorobenzoate suggested the loss of additional genes outside of, but contiguous with, the characterized region. Pulsed-field gel electrophoresis revealed a plasmid of >300 kb in strain D1, but no plasmids were detected in strain JB2. Hybridization analyses confirmed that the entire 26-kb region characterized here was acquired by strain D1 from strain JB2 and was located in the chromosome of both organisms. Further studies to delineate the element's boundaries and functional characteristics could provide new insights into the mechanisms underlying evolution of bacterial genomes in general and of catabolic pathways for anthropogenic pollutants in particular.Lateral gene transfer between bacteria can potentially affect a variety of processes in soil, including the biodegradation of organic pollutants (7,8,10,12,17,27,32,55,56,61). Acquisition of catabolic genes can enhance contaminant biodegradation by increasing the diversity of organisms able to effect at least partial transformation of a compound or expanding on existing pathways so that degradation is more extensive or complete (mineralization). Pathway complementation is exemplified by strains engineered to possess the upper biphenyl degradation pathway as well as the lower chlorobenzoate and chlorocatechol pathways, resulting in an enhanced ability to mineralize polychlorinated biphenyls (18,25,35,46). Similar hybrid pathways could evolve naturally in the environment by lateral gene transfer and affect the activity of microbial communities mediating polychlorinated biphenyl (PCB) biodegradation, but relatively little is known about their occurrence.The recovery of PCB-mineralizing strains from bioreactors or soil inoc...

The hepatotoxic cyclic heptapeptide toxins of cyanobacteria, collectively termed microcystins, are potent inhibitors of protein phosphatases PP1 and PP2A. The structure of microcystins resembles small, cyclic peptide secondary metabolites from fungi and eubacteria. Many of these metabolites are manufactured via a nonribosomal thio‐template mechanism. We submit evidence that microcystin is synthesized by a similar mechanism. The organism used in this study was Microcystis aeruginosa PCC7820. Using the traditional ATP‐32PPi exchange assay for thio‐template activity, we found activity in the presence of the substrate d‐amino acids occurring in microcystin. Thio‐template mechanisms are known to be unaffected by protein synthesis inhibitors such as chloramphenicol. We subjected cultures in exponential and stationary growth to chloramphenicol and monitored culture health versus toxicity. Although the health of the treated cultures declined, the toxicity of the remaining cells increased. We developed an in vitro assay to measure microcystin synthesis in cell lysates in the presence of chloramphenicol. By supplementing the lysates with ATP and the substrate amino acids present in microcystin, we detected a fourfold increase in total microcystins over the course of 20 min.

A large‐scale screening program was initiated to evaluate laboratory‐cultured blue‐green algae (cyanobacteria) as a source of novel compounds with inhibitory activity against certain serine/threonine protein phosphatases. Over 1600 extracts, representing 816 cyanophyte strains with broad habitat and taxonomic diversity, were screened. Inhibitors were identified in extracts produced from all orders tested except Chamaesiphonales, and the family Stigonemataceae was found to contain proportionally more inhibitors as compared to the total screen.