Isolation of a naturally-occurring nickel resistance plasmid from a rare hypersaline estuary (Laguna Madre, Texas, USA) for potential use as a bio-indicator of metal contamination

Abstract

Metal-resistant bacteria were isolated from sediments of the Laguna Madre, a rare hypersaline estuary impacted by many anthropogenic compounds, including various metals and metalloids. Bacteria were initially isolated on nutrient agar supplemented with NaCl; random isolates (n = 100) were tested for metal resistance toward zinc, nickel, chromium, and cadmium using a pour plate disc assay. Metal-resistant cultures were assayed for plasmids that contained naturally-occurring heavy metal resistance genes. Putative metal-resistance plasmids were tested for metal-resistance efficacy by transforming a metal-sensitive strain of Escherichia coli. Polymerase Chain Reaction (PCR) primers were designed to detect cnrA, part of a nickel-cobalt resistance gene cluster, and restriction endonuclease digests were performed to detect restriction sites within the plasmid. Results showed that many bacterial isolates tested were resistant toward most of the metals used in this study. Among tested bacteria cultures, 34 were resistant to zinc, 64 were resistant to chromium, and 51 resistant to cadmium. Only 8 cultures were resistant to nickel; however, most bacteria were found to be resistant to more than one metal. Several plasmids were found from the bacteria isolates. One plasmid, designated pDZ5, was isolated from a bacterium identified as Bacillus pumilus by 16S rRNA sequencing. Plasmid pDZ5 conferred nickel resistance to the metal-sensitive E. coli strain and was found to contain cnrA as confirmed by PCR amplification. Plasmid pDZ5 was successfully cut with restriction enzymes for potential ligation with reporter genes. The presence, abundance and expression of pDZ5 may prove to be a useful bio-indicator of metal contamination, specifically nickel pollution, in the Laguna Madre due to the fewer number of bacteria that were nickelresistant compared to other metals.

A Novel Bacterial-Mediated Method for the Differentiation of Aedes spp. Larvae in Mixed-Nursery Assays and Its Application in the Determination of Vegetative Lysinibacillus sphaericus Biocontrol Efficiency in Invasive Vector Eradication

“…In recent years, biotechnological exploits such as expression vectors have been thoroughly implemented and used across different fields in both industry and research applications, ranging from enabling protein expression in target organisms, to the creation of transgenic organisms [34,35]. Modified bacteria like some Escherichia coli strains possess plasmids containing fluorescent reporters like red fluorescent protein (mRFP) and are of great use as they can serve as easily distinguishable colorimetric bioindicators for environmental contaminants, which is the case of leftover antibiotics from both pharmaceutical and agricultural applications [36]. Despite the success expression vectors have had in other areas, they have not yet been employed in the control of arboviral diseases and, more specifically, in the differentiation of public health-relevant mosquito species, as is the case of Ae.…”