Biochemical characterization of a multiple heavy metal, pesticides and phenol resistant Pseudomonas fluorescens strain

Abstract

Pseudomonas fluorescens SM1 isolate was found to be resistant to some major water pollutants namely Cd²⁺, Cr⁶⁺, Cu²⁺, Ni²⁺, Pb²⁺, BHC, 2,4-D, mancozeb and phenols up to a concentration four times to the normal levels occurring in the highly pollulated regions. Curing experiment brought about the loss of one or more resistance markers indicating the plasmid born resistance. Plasmid profile of SM1 strain showed the presence of one DNA band of 43.6 kb. This Plasmid was isolated from SM1 strain and introduced into Escherichia coli DH5α with a transformation frequency of 6.7 × 10⁻⁴ transformants/recipient cell. The test SM1 strain was also capable of biotransforming Cr(VI) to Cr(III) which is less toxic compounds. Present studies further indicated that the test SM1 strain was not only resistant to 2,4-D, phenols and catechol but also capable of bioremediating these toxicants quite efficiently. Moreover, studies with inhibitors like sodium azide, 2,4-DNP and chloramphenicol suggested that the major mechanism for the bioremediation of the heavy metals other than Cr⁶⁺ would be the biosorption process.

Introduction

Pseudomonas fluorescens belongs to a group of common non-pathogenic saprophytes that colonize soil, water and plant surface environments. Physiological and genetic features of Pseudomonas make them a promising agent for utilization in biotechnology, agriculture and environmental bioremediation applications. The ability to biodegrade hazardous chemical wastes is an interesting feature of P. fluorescens (Appanna and Hamel, 1996, Barathi and Vasudevan, 2003). Heavy metals, pesticides, as well as phenols all pose a serious threat to living systems. They can react with proteins, nucleic acids and phospholipids, and thus arrest cellular proliferation (Frausto da Silva and Williams, 1993).

Environmental pollution by heavy metals as a result of fossil fuel burning and industrial discharges has been increasing. In the case of water bodies, the continued influx of pollution load is aggravated in summers when the water evaporates and thereby increasing metal content. During this process, many bacteria acquire metal tolerance (Ghosh et al., 2000). Moreover, bacteria can also degrade and detoxify a wide variety of hazardous compounds. Many of these bacteria also carry plasmids (Silver and Misra, 1988, Mergeay, 1991). The role of plasmids in Pseudomonas sp. in the biotransformation of certain heavy metals, pesticides and phenolics is also well documented (Rajini-Rani and Mahadevan, 1992, Deshpande et al., 2001, Thakur et al., 2001).

The ability of a genetic marker for transfer from one bacterium to another through conjugation and/or transformation provides a good presumptive evidence for the involvement of plasmid, particularly if the frequency of transfer is high. Moreover, loss of certain genetic markers as a result of treatment of bacterial cell to plasmid curing agents also suggests for the plasmidial nature of the marker (Mesas et al., 2004).

Some heavy metal resistance determinants move from plasmid to chromosome (or in the reverse direction). This makes plasmid encoding heavy metal resistance an important aspect of environmental research. These plasmids can be the source of resistance genes for cloning purpose which have potential use in biotechnology such as the manufacture of biosensors and bioremediation processes (Collard et al., 1994). In view of the above the present paper deals with the biochemical characterization of the P. fluorescens SM1 with particular reference to the probable role of plasmid mediated resistance to major water pollutants.