Biotransformation von Thiomersal durch natürlich vorkommende, quecksilberresistente Bakterien und genetisch modifizierte Mikroorganismen

Thiomersal (TH), an aryl-alkyl-organomercurial bactericide, has been used for vaccine production and preventing bacterial contamination. Presently, there is no remediation for wastewater contaminated with TH available. Therefore, this study was conducted to assess the feasibility of aerobic treatment of TH contaminated solutions. The potentialities for TH detoxification were determined by naturally mercury resistant isolates and two genetically engineered microorganisms. Generally, all isolates showed resistance up to 2 ppm TH, however with different transformation rates (8−443 ng Hg/ml/min). Ps. putida Spi3 proved in this study to be an excellent strain for TH detoxification. It reduces very high concentrations of TH up to 140 ppm. Analyzes showed that the optimal TH transformation was determined at 30°C and pH 7. Additionally, the results of the growth experiments showed that the cells at lag phase exhibited the highest TH transformation rates, followed by those at the exponential phase and stationary phase. The genetic structure of mercury resistant operons from all environmental strains was studied with special attention to organomercurial lyase (MerB). All sequenced strains carried multiple mer operons. They all harbour a narrow spectrum resistance operon (merNS) beside their resistance to TH that resulted in the presence of a broad spectrum resistant operon (merBS) including merB gene. The strain Ps. putida Spi3, however, carried four mer operons, one conferring merNS and three merBS. Analysis showed that the three merB genes are located on different mer operons. Two merB genes were mapped upstream of regulatory genes (merD). In contrast, the gene arrangement of the third merB gene is peculiar; merB3 is mapped between the merR and merT gene and has its own promoter and ribosomal binding site that may account for Spi3’s extremely high resistance to TH. These four mer operons constitute Spi3’s high resistant to mercuric ions and organomercurials.Thiomersal (TH) ist eine organische Quecksilberverbindung, die u. a. als Desinfektionsmittel zur Impfstoffherstellung verwendet wird. Die dabei anfallenden Abwässer sind stark mit TH belastet. Bis zum heutigen Tag gibt es keine Aufbereitungsmöglichkeit des Abwassers. Ziel dieser Arbeit ist die Entwicklung einer neuen Technologie zur Behandlung der Abwässer durch natürlich vorkommende Bakterien und genetisch modifizierte Mikroorganismen. Alle untersuchten Stämme waren in der Lage, 2 mg/l TH umzuwandeln, wobei sehr unterschiedlichen Transformationsraten (8−443 ng Hg/ml/min) gemessen wurden. Insbesondere Ps. putida Spi3 zeigte eine hervorragende TH-Resistenz, sogar bis Konzentrationen von 140 mg/l. Die optimale Temperatur für die Transformationsaktivität lag bei 30°C bei pH 7. Weiterhin zeigten die Untersuchungen eine höhere Transformationsrate der Zellen in der ersten Phase des Wachstums, gefolgt von der mittleren logarithmischen Phase. In der stationären Phase wurden die niedrigsten Transformationsraten ermittelt. Zur Aufklärung der unterschiedlichen Resistenzen wurde die genetische Struktur des mer-Operons untersucht. Der Resistenzmechanismus beruht auf Aktivitäten von zwei durch das mikrobielle mer-Operon codierten Enzymen, Quecksilberreduktase und Quecksilberlyase, die in der Lage sind, Organoquecksilberverbindungen und ionisches Quecksilber in metallisches Quecksilber zu überführen. Die Analysen zeigten, daß alle Mikroorganismen multiple mer Operons mit jeweils eigenen Regulatorgenen und Operator-/Promotorregion besaßen. Die Operons wiesen nicht nur Resistenz gegenüber ionische Quecksilberverbindungen (mer RTPAD) sondern auch gegenüber organischen Verbindungen (mer RTPABD) auf. Die hohe mikrobielle TH-Resistenz von Ps. putida Spi3 hingegen beruhte auf dem Vorhandensein von vier verschiedene mer Operons, wobei drei mer Operons jeweils das Organoquecksilber spaltende Enzym, MerB, codierten

Functional profiling of mercuric reductase (mer A) genes in biofilm communities of a technical scale biocatalyzer

BACKGROUND: Bacterial mercury resistance is based on enzymatic reduction of ionic mercury to elemental mercury and has recently been demonstrated to be applicable for industrial wastewater clean-up. The long-term monitoring of such biocatalyser systems requires a cultivation independent functional community profiling method targeting the key enzyme of the process, the merA gene coding for the mercuric reductase. We report on the development of a profiling method for merA and its application to monitor changes in the functional diversity of the biofilm community of a technical scale biocatalyzer over 8 months of on-site operation. RESULTS: Based on an alignment of 30 merA sequences from Gram negative bacteria, conserved primers were designed for amplification of merA fragments with an optimized PCR protocol. The resulting amplicons of approximately 280 bp were separated by thermogradient gelelectrophoresis (TGGE), resulting in strain specific fingerprints for mercury resistant Gram negative isolates with different merA sequences. The merA profiling of the biofilm community from a technical biocatalyzer showed persistence of some and loss of other inoculum strains as well as the appearance of new bands, resulting in an overall increase of the functional diversity of the biofilm community. One predominant new band of the merA community profile was also detected in a biocatalyzer effluent isolate, which was identified as Pseudomonas aeruginosa. The isolated strain showed lower mercury reduction rates in liquid culture than the inoculum strains but was apparently highly competitive in the biofilm environment of the biocatalyzer where moderate mercury levels were prevailing. CONCLUSIONS: The merA profiling technique allowed to monitor the ongoing selection for better adapted strains during the operation of a biocatalyzer and to direct their subsequent isolation. In such a way, a predominant mercury reducing Ps. aeruginosa strain was identified by its unique mercuric reductase gene