Genetically Modified Vibrio harveyi Strains as Potential Bioindicators of Mutagenic Pollution of Marine Environments

Abstract

For biodetection of mutagenic pollution of marine environments, an organism naturally occurring in these habitats should be used. We found that marine bacterium Vibrio harveyi may be an appropriate bioindicator of mutagenic pollution. For positive selection of mutants, we developed a simple method for isolation of V. harveyi mutants resistant to neomycin. We constructed genetically modified V. harveyi strains that produce significantly more neomycin-resistant mutants upon treatment with low concentrations of mutagens than the wild-type counterpart. The sensitivity of the mutagenicity test with the V. harveyi strains is at least comparable to (if not higher than) that of the commonly used Ames test, which uses Salmonella enterica serovar Typhimurium strains. Therefore, we consider that the V. harveyi strains described in this report could be used as potential bioindicators of mutagenic pollution of marine environments.Mutagenic pollution of natural environments seems to be a general and serious problem (see, for instance, information provided by the U.S. Environmental Protection Agency [6]) that has been extensively investigated (the Environmental Mutagen Information Center database contains over 20,000 citations to literature on agents that have been tested for mutagenic activity; see http://www.nlm.nih.gov/pubs/factsheets /emicfs.html). This problem also concerns marine habitats. Therefore, detection of the presence of mutagens in the environment is important. This is not an easy procedure, as mutagenic components usually occur in natural habitats at low concentrations. Moreover, mutagens are only a fraction of contaminating chemicals in natural environments. Thus, biological mutagenicity tests seem to be more sensitive and accurate than chemical analyses.The most commonly used mutagenicity test is that described by Ames (1) and subsequently modified by Ames and coworkers (2, 13). In this test, a series of genetically modified Salmonella enterica serovar Typhimurium strains are used. The presence of mutations in his genes allows positive selection of his ϩ revertants on minimal agar plates. A deletion of the uvrB gene in most of these tester strains ensures a higher efficiency of mutagenesis due to inactivation of one of the bacterial DNA repair systems (2). Moreover, these bacteria bear the rfa mutation, which causes a partial loss of the lipopolysaccharide barrier that coats the surface of the bacteria and increases permeability to large molecules (including some mutagens) that do not penetrate the normal cell wall (2). Some of these strains harbor, in addition, plasmid pKM101, which contains the mucA and mucB genes, responsible for the enhancement of an error-prone DNA repair system (14,22,25).Although the Ames test is very useful for detecting mutagens under laboratory conditions, we considered that for monitoring of marine environments, an organism that naturally lives in these habitats should be used. Vibrio harveyi is a freeliving bacterium found in diverse marine environments (18,19). Moreover, it is ea...

Cloning and characterization of the groE heat-shock operon of the marine bacterium Vibrio harveyi

“…Its use as a bioindicator of environmental pollution is under current investigation, especially for monitoring the presence of mutagenic substances. V. harveyi cells are highly sensitive to various mutagens and hypersensitive strains have been constructed, for use in a modified Ames test (Czyż et al, 2000). However, non-mutagenic toxic substances would not be detected in this type of test.…”

Chemicals used for maintenance of wood rafts in mussel farms: evaluation of their potential toxic risk to mussel culture

“…The Vibrio harveyi strains used in the mutagenicity assay were kindly provided by Dr. G. Wegrzyn (University of Gdansk, Poland): wild-type BB7 strain (Belas et al 1982); BB7X, a strain that is very sensitive to UV irradiation and that bears the Tn5TpMSC insertion (Czyz et al 2000, 2001, Sikora et al 2006; and analogous strains called BB7M and BB7XM bearing plasmid pAB91273 that contains mucA and mucB genes, responsible for enhanced errorproneDNA repair, in addition to ampicillin and chloramphenicol resistance genes (Czyz et al 2000).…”

“…The mutagenicity assay was performed according to Czyz et al (2000) slightly modified by Ruiz et al (2013). The culture medium used was BOSS broth (Klein et al 1995), hydrated with artificial marine water (Marine BOSS Broth).…”

“…For this reason, other mutagenicity assays using marine bacteria have been developed, such as the one based upon detection of neomycin-resistant mutants of Vibrio harveyi. The effectiveness of this assay has been compared to the Ames test, showing the highest sensitivity in samples with sea wa ter (Czyz et al 2000, 2002, Słoczynska et al 2010, Ruiz et al 2013.…”

Impaired chromosome partitioning and synchronization of DNA replication initiation in an insertional mutant in the Vibrio harveyi cgtA gene coding for a common GTP-binding protein

“…However, recently a viable insertional mutant in the cgtA gene of Vibrio har eyi was isolated [11,12] and underwent preliminary characterization [2]. This mutant reveals multiple phenotypes : it is unable to grow in minimal media, its survival in physiological saline is dramatically reduced, it is more mutagenic upon treatment with Abbreviations used : DAPI, 4h,6-diaminidino-2-phenylindole ; TCA, trichloracetic acid.…”