Preparation of a DNA gene probe for detection of mercury resistance genes in gram-negative bacterial communities

Abstract

A DNA gene probe was prepared to study genetic change mechanisms responsible for adaptation to mercury in natural bacterial communities. The probe was constructed from a 2.6-kilobase NcoI-EcoRI DNA restriction fragment which spans the majority of the mercury resistance operon (mer) in the R-factor R100. The range of specificity of this gene probe was defined by hybridization to the DNA of a wide variety of mercury-resistant bacteria previously shown to possess the mercuric reductase enzyme. All of the tested gram-negative bacteria had DNA sequences homologous to the mer probe, whereas no such homologies were detected in DNA of the gram-positive strains, Thus, the mer probe can be utilized to study gene flow processes in gram-negative bacterial communities.

Environmental significance of the potential for mer(Tn21)-mediated reduction of Hg2+ to Hg0 in natural waters

“…The hypothesis that DNA sequences coding for Hg2+ resistance and volatilization in saline water and freshwater communities had no homology to the characterized mer operon was tested by hybridization of randomly selected representative strains with a mer(Tn2l) DNA probe. Gram-positive strains were excluded from this analysis because their Hg2+ resistance genes bear little homology with mer(Tn21) (30,35) and homology was not sufficient for hybrid formation under the stringency conditions used in this study (8). However, only a minority of the resistant strains were gram positive (ranging from 7.5% in the Vortex Spring community to 1.9% in the coastal marine community).…”

“…Aquatic microbial communities adapt to Hg2+ by rapidly volatilizing (and thus eliminating) it from their immediate environment (7). Because Hg2+ reduction to volatile elemental mercury (Hgo) is a well-established resistance mechanism in pure bacterial cultures (35), its role in adaptation and volatilization by the aquatic communities was investigated by DNA-DNA colony hybridization with a DNA probe constructed of a gramnegative mer operon, mer(Tn2J) (8). Although resistance was widely distributed among aerobic .ulturable heterotrophs of adapted communities, DNA sequences homologous to mer were rarely (if at all) detected (7).…”

“…(iii) Mercuric mercury resistance and volatilization was mediated by bacterial genes that were not homologous to the characterized mer operon. The DNA sequences of mer operons originating in three gram-negative bacteria have been shown to have a high degree of homology (30), and this homology was sufficient to result in cross-hybridization with mer(Tn2l) (8). However, Hg2+-resistant strains that do not hybridize with this probe have been isolated from different environments (2, 10; N. Hamlett, personal communication), and NADPH-dependent mercuric reductase activity has been found in three mer(Tn2l)-negative marine caulobacters (22).…”

Genetic diversity within mer genes directly amplified from communities of noncultivated soil and sediment bacteria

“…This comprehensive approach should pro-Correspondence: K. D. Bruce vide a more representative assessment of the diversity of specific genes in natural bacterial populations. To date, most molecular techniques used for this purpose have involved the direct extraction of DNA (Trevors 1992), followed by DNA cloning (Schmidt et al 1991f, DNA hybridization using specific probes (Barkay et al 1985), polymerase chain readion (PCR) amplification (Britschgi & Giovannoni 1991) or a combination of these approaches.…”

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“…Первые работы, в которых использовали зонды ДНК для иденти фикации индивидуальных микрорганизмов или группы функциональ но близких микроорганизмов, по-прежнему основывались на необхо димости выделять живой организм на селективных средах и культи вировать его для получения и анализа ДНК [18,19,[21][22][23][24][25][26][27][28]. Серий ные разведения накопительной культуры микроорганизмов или разве дения природных образцов воды либо экстрактов почвы высевали на нитроцеллюлозные фильтры, которые помещали на селективные или неселективные питательные среды.…”