Plasmid transfer between introduced and indigenous bacteria in leaf litter, soil and vermicompost as affected by soil invertebrates

 Plasmid transfer between introduced strains of Pseudomonas stutzeri JM302 (pLV1013), Pseudomonas putida PAW340 (pLV1017), Pseudomonas aeruginosa PAO5 (RP4), and Enterobacter cloacae MF10 (RP4), all of them harbouring genetically modified or naturally occurring plasmids and bacteria indigenous to oak leaf litter, soddy-podzolic soil or vermicompost was monitored using non-sterile laboratory microcosms inhabited by the millipede Pachyiulus flavipes, the woodlouse Armadillidium vulgare, or the earthworms Aporrectodea caliginosa and Eisenia fetida, respectively. Plasmid transfer from genetically modified Streptococcus faecalis JH2-2 (pAMβ₁) to Bacillus thuringiensis var. israelensis 1-5 under similar conditions was also followed. A recombinant conjugative plasmid (pLV1017) encoding resistance to antibiotics and expressing catechol 2,3-dioxygenase was transferred into two facultatively anaerobic gram-negative bacteria, isolated from the excrement of A. vulgare. Presumed transconjugants of the facultatively anerobic gram-negative bacteria given above were isolated from the leaves and soil (four strains) and the excrement of the invertebrates (11 strains). They carried the same plasmids and exhibited the same enzymatic activity as the respective inoculant strains; for most of them, however, DNA fingerprints were slightly different. Transfer from S. faecalis JH2-2 to B. thuringiensis var. israelensis of a naturally occurring plasmid, pAMb₁, encoding resistance to erythromycin was observed in vermicompost. Received: 18 November 1997     

Dephosphorylation of the calcium pump coupled to counterion occlusion

P-type ATPases extract energy by hydrolysis of adenosine triphosphate (ATP) in two steps, formation and breakdown of a covalent phosphoenzyme intermediate. This process drives active transport and countertransport of the cation pumps. We have determined the crystal structure of rabbit sarcoplasmic reticulum Ca2+ adenosine triphosphatase in complex with aluminum fluoride, which mimics the transition state of hydrolysis of the counterion-bound (protonated) phosphoenzyme. On the basis of structural analysis and biochemical data, we find this form to represent an occluded state of the proton counterions. Hydrolysis is catalyzed by the conserved Thr-Gly-Glu-Ser motif, and it exploits an associative nucleophilic reaction mechanism of the same type as phosphoryl transfer from ATP. On this basis, we propose a general mechanism of occluded transition states of Ca2+ transport and H+ countertransport coupled to phosphorylation and dephosphorylation, respectively.