An RND transporter in the monoterpene metabolism of Castellaniella defragrans

The betaproteobacterium Castellaniella defragrans 65Phen grows on monoterpenes at concentrations toxic to many bacteria. Tolerance mechanisms include modifications of the membrane fatty acid composition and the mineralization of monoterpenes. In this study, we characterized an efflux transporter associated to the monoterpene metabolism. The inner-membrane transporter AmeD (apolar monoterpene efflux) affiliated to the HAE3 (hydrophobe/amphiphile efflux) family within the Resistance-Nodulation-Division (RND) superfamily. RND pumps of the HAE3 family are known for transporting substrates into the periplasm. AmeD is co-expressed with the outer membrane protein AmeA and the periplasmic proteins AmeB and AmeC, suggesting an export channel into the environment similar to HAE1-type RND exporters. Proteins AmeABCD are encoded within a genetic island involved in the metabolism of acyclic and cyclic monoterpenes. The deletion of ameABCD translated into a decrease in tolerance to monoterpenes in liquid cultures. The addition of acetate as cosubstrate in limonene-containing cultures partially alleviated monoterpene toxicity in the deletion mutant. Accumulation of Nile Red in cells of C. defragrans required dissipation of the proton motive force with carbonyl cyanide m-chlorophenylhydrazone (CCCP). Cells lacking AmeABCD accumulated more Nile Red, suggesting an export function of the proteins. Our observations suggest that the tetrapartite RND transporter AmeABCD acts as an exporter during monoterpene detoxification in C. defragrans

The oxygen-independent metabolism of cyclic monoterpenes in Castellaniella defragrans 65Phen

BACKGROUND: The facultatively anaerobic betaproteobacterium Castellaniella defragrans 65Phen utilizes acyclic, monocyclic and bicyclic monoterpenes as sole carbon source under oxic as well as anoxic conditions. A biotransformation pathway of the acyclic β-myrcene required linalool dehydratase-isomerase as initial enzyme acting on the hydrocarbon. An in-frame deletion mutant did not use myrcene, but was able to grow on monocyclic monoterpenes. The genome sequence and a comparative proteome analysis together with a random transposon mutagenesis were conducted to identify genes involved in the monocyclic monoterpene metabolism. Metabolites accumulating in cultures of transposon and in-frame deletion mutants disclosed the degradation pathway. RESULTS: Castellaniella defragrans 65Phen oxidizes the monocyclic monoterpene limonene at the primary methyl group forming perillyl alcohol. The genome of 3.95 Mb contained a 70 kb genome island coding for over 50 proteins involved in the monoterpene metabolism. This island showed higher homology to genes of another monoterpene-mineralizing betaproteobacterium, Thauera terpenica 58Eu(T), than to genomes of the family Alcaligenaceae, which harbors the genus Castellaniella. A collection of 72 transposon mutants unable to grow on limonene contained 17 inactivated genes, with 46 mutants located in the two genes ctmAB (cyclic terpene metabolism). CtmA and ctmB were annotated as FAD-dependent oxidoreductases and clustered together with ctmE, a 2Fe-2S ferredoxin gene, and ctmF, coding for a NADH:ferredoxin oxidoreductase. Transposon mutants of ctmA, B or E did not grow aerobically or anaerobically on limonene, but on perillyl alcohol. The next steps in the pathway are catalyzed by the geraniol dehydrogenase GeoA and the geranial dehydrogenase GeoB, yielding perillic acid. Two transposon mutants had inactivated genes of the monoterpene ring cleavage (mrc) pathway. 2-Methylcitrate synthase and 2-methylcitrate dehydratase were also essential for the monoterpene metabolism but not for growth on acetate. CONCLUSIONS: The genome of Castellaniella defragrans 65Phen is related to other genomes of Alcaligenaceae, but contains a genomic island with genes of the monoterpene metabolism. Castellaniella defragrans 65Phen degrades limonene via a limonene dehydrogenase and the oxidation of perillyl alcohol. The initial oxidation at the primary methyl group is independent of molecular oxygen

One size does not fit all:A systematic review of training outcomes on residential youth care professionals' skills

Despite the importance of training residential youth care professionals to increase their professional competences and, by doing so, enabling them to cope with the many stressors encountered during their work – such as the serious behaviour problems of the adolescents with whom they work – little attention has been paid so far to the influence of training on the behaviour and skills of residential professionals. This study aims to gain greater insight into the effects of training on the skills of these professionals. We conducted a systematic literature review using the PsycINFO, Eric, SocIndex and Academic Search Premier databases. Within the twelve studies retrieved, the outcomes of nine different training programmes were examined. These nine training programmes include The Solution Strategy (TSS), (Professional) Skills for Residential Child Care Workers (PSRCCW/SRCCW), Therapeutic Crisis Intervention (for Youth Care Workers) (TCIYCW/TCI), Child Teacher Relationship Training (CTRT), a Brief Training Program (BTP), Social Skills Training (SST), Behavior Analysis Services Program (BASP), Mental Health Training (MHT), and a Brief Training Manual (BTM). Our results demonstrate that positive changes in professional skills can be obtained after a training course. TSS (in both studies), PSFCCW, SRCCW, TCI, CTRT, BTP, SST, BASP and BTM programmes are associated with positive outcomes at the individual or organisational level. However, two studies also found negative outcomes related to decreases in the perceived recognition of youth care workers’ value to the agency and a decrease in the knowledge and skills that they learned through training after six months (TSS [Hickey, 1994] and PSRCCW). In addition, other outcomes were also found. The most common ‘other’ outcome was a variable training effect on the professionals’ skills. Although we did find positive (and some negative) training outcomes on the professionals’ skills, none of the studies specified the training elements which caused these changes. More thorough empirical studies using an experimental or repeated case study design are needed to identify effective training elements which could further improve the effectiveness of interventions targeting youths in residential youth care