Role of the mitochondrial DnaJ homolog Mdj1p as a chaperone for mitochondrially synthesized and imported proteins.

Mdj1p, a DnaJ homolog in the mitochondria of Saccharomyces cerevisiae, is involved in the folding of proteins in the mitochondrial matrix. In this capacity, Mdj1p cooperates with mitochondrial Hsp70 (mt-Hsp70). Here, we analyzed the role of Mdj1p as a chaperone for newly synthesized proteins encoded by mitochondrial DNA and for nucleus-encoded proteins as they enter the mitochondrial matrix. A series of conditional mutants of mdj1 was constructed. Mutations in the various functional domains led to a partial loss of Mdj1p function. The mutant Mdj1 proteins were defective in protecting the tester protein firefly luciferase against heat-induced aggregation in isolated mitochondria. The mitochondrially encoded var1 protein showed enhanced aggregation after synthesis in mdj1 mutant mitochondria. Mdj1p and mt-Hsp70 were found in a complex with nascent polypeptide chains on mitochondrial ribosomes. Mdj1p was not found to interact with translocation intermediates of imported proteins spanning the two membranes and exposing short segments into the matrix, in accordance with the lack of requirement of Mdj1p in the mt-Hsp70-mediated protein import into mitochondria. On the other hand, precursor proteins in transit which had further entered the matrix were found in a complex with Mdj1p. Our results suggest that Mdj1p together with mt-Hsp70 plays an important role as a chaperone for mitochondrially synthesized polypeptide chains emerging from the ribosome and for translocating proteins at a late import step.     

Localization and organization of phenol degradation genes ofPseudomonas putida strain H

The genetic organization of the DNA region encoding the phenol degradation pathway ofPseudomonas putida H has been investigated. This strain can utilize phenol or some of its methylated derivatives as its sole source of carbon and energy. The first step in this process is the conversion of phenol into catechol. Catechol is then further metabolized via themeta-cleavage pathway into TCA cycle intermediates. Genes encoding these enzymes are clustered on the plasmid pPGH1. A region of contiguous DNA spanning about 16 kb contains all of the genetic information necessary for inducible phenol degradation. The analysis of mutants generated by insertion of transposons and cassettes indicates that all of the catabolic genes are contained in a single operon. This codes for a multicomponent phenol hydroxylase andmeta-cleavage pathway enzymes. Catabolic genes are subject to positive control by the gene product(s) of a second locus.     

Ovarian mesothelial and extramesothelial cells in interactive culture

Summary The ovarian mesothelium (OM) represents the tissue of origin of ovarian epithelial cancer. To gain insight into the regulation of this tissue, OM organoids and submesothelial ovarian stromal cells (SC) were isolated from New Zealand White rabbits by a stepwise tissue dispersal technique, while granulosa cells (GC) were aspirated from mature follicles (14±4 groups/animal). OM and SC dispersal were sequentially accomplished by: a) 1-h incubation in collagenase type I (300 U/ml), gentle scraping of the ovarian surface, and 1 g sedimentation of OM organoids (equivalent to 0.93±0.40 × 10⁶ cells/animal) on 5% bovine serum albumin (BSA); b) 2-h incubation in pronase-collagenase (0.5%–300 U/ml) under periodical resuspension and gentle scraping of SC (1.40±0.25 × 10⁶/animal) from OM-denuded ovaries. After a week-long in vitro expansion, OM cells (OMC) were cultured alone and with SC or GC within monocameral vessels or bicameral transfilter vessels in serumless, fibronectinrich (4μg/ml) HL-1 medium. After 7 d of contact cell-cell interaction, cytokeratin-positive OMC became surrounded by fibroblastoid, vimentin-positive SC or by cytokeratin and vimentin-weakly positive GC. Filter-bound OMC humorally interacting with underlying SC or GC displayed a biphasic, epithelioid and spindle, morphology with universal cytokeratin expression. Bromo-2′-deoxyuridine (BrdU) immunoperoxidase revealed mean cell proliferation indices of 14.88% for OMC cultured alone, 11.21% and 19.39% for OMC cultured with GC or SC in monocameral dishes, and 15.25% or 22.47% for OMC cultured in bicameral vessels over GC or SC, respectively. This model provides an experimental tool for investigating the unexplored role of stromal-mesothelial interaction in OM pathobiology.     

Retrotransfer kinetics of R300B by pQKH6, a conjugative plasmid from river epilithon

Abstract The kinetics of conjugation, retrotransfer and mobilisation were studied at 5–15 min intervals between strains of Pseudomonas putida using plasmid pQKH6, isolated from river epilithon, and R300B. Transconjugants from the direct conjugation of pQKH6 and mobilisation of R300B by pQKH6 appeared rapidly, reaching maximum densities within 30–60 min of the start of both filter and liquid mating experiments. However, retrotransconjugants only appeared after a delay. This delay was short (approx. 45–60 min) in filter mating and much longer (2–5 h) for liquid mating experiments. Attempts at predicting the time course of retrotransconjugant development from (1) numbers of transconjugants from the conjugation and mobilisation experiments and (ii) mathematical models based on a mass action approach, both failed to reproduce the observed delay. It was concluded that retrotransfer did not proceed by either a one-step mechanism occuring early in conjugation or two separate conjugation and mobilisation steps. The clear demonstration of a delay in retrotransconjugant formation implies that a new mechanism must be sought. The likely importance of retrotransfer in the environment is discussed.     

Targeting of proteins to the thylakoids by bipartite presequences: CFoII is imported by a novel, third pathway.

The CFoII subunit of the ATP synthase is an integral component of the thylakoid membrane which is synthesized in the cytosol with a bipartite, lumen-targeting presequence similar in structural terms to those of imported lumenal proteins such as plastocyanin. This presequence is shown to possess a terminal cleavage site for the thylakoidal processing peptidase, but no intermediate site for the stromal processing peptidase. The integration of CFoII into the thylakoid membrane of Pisum sativum has been analysed using in vitro assays for the import of proteins into intact chloroplasts or isolated thylakoids. Efficient integration into thylakoids is observed in the light and dark, and the integration process does not require the presence of either stromal extracts or nucleoside triphosphates. The uncoupler nigericin inhibits integration only very slightly, indicating that the thylakoidal delta pH does not play a significant role in the integration mechanism. In each of these respects, the requirements for CFoII integration differ notably from those determined for integration of the light-harvesting chlorophyll-binding protein of photosystem II. The integration mechanism also differs significantly from the two mechanisms involved in the translocation of lumenal proteins across the thylakoid membrane, since one of these processes requires the presence of stromal protein factors and ATP, and the other mechanism is dependent on the thylakoidal delta pH. This conclusion is reinforced by the finding that saturation of the translocation system for the precursor to the lumenal 23 kDa oxygen-evolving complex protein does not affect integration of CFoII into thylakoids.(ABSTRACT TRUNCATED AT 250 WORDS)     

Anaerobic Degradation of Propionate by a Mesophilic Acetogenic Bacterium in Coculture and Triculture with Different Methanogens

A mesophilic acetogenic bacterium (MPOB) oxidized propionate to acetate and CO₂ in cocultures with the formate- and hydrogen-utilizing methanogens Methanospirillum hungatei and Methanobacterium formicicum. Propionate oxidation did not occur in cocultures with two Methanobrevibacter strains, which grew only with hydrogen. Tricultures consisting of MPOB, one of the Methanobrevibacter strains, and organisms which are able to convert formate into H₂ plus CO₂ (Desulfovibrio strain G11 or the homoacetogenic bacterium EE121) also degraded propionate. The MPOB, in the absence of methanogens, was able to couple propionate conversion to fumarate reduction. This propionate conversion was inhibited by hydrogen and by formate. Formate and hydrogen blocked the energetically unfavorable succinate oxidation to fumarate involved in propionate catabolism. Low formate and hydrogen concentrations are required for the syntrophic degradation of propionate by MPOB. In triculture with Methanospirillum hungatei and the aceticlastic Methanothrix soehngenii, propionate was degraded faster than in biculture with Methanospirillum hungatei, indicating that low acetate concentrations are favorable for propionate oxidation as well.     

Sequence divergence in the ORF6 gene of Mycoplasma pneumonia.

The ORF6 gene product of Mycoplasma pneumoniae is involved in a yet-unknown manner in the adhesion of the bacterium to its host cell. Part of the ORF6 gene is a repetitive DNA sequence (RepMP5), about 1,900 bp long. Seven additional similar copies of RepMP5 are dispersed on the genome. In the independently isolated strains M. pneumoniae M129 and FH, the RepMP5 copies residing in the ORF6 gene are not identical. Two conserved regions, ranging from nucleotides 1 to 799 and from nucleotide 1795 to the end of the gene, border a variable region, ranging from nucleotides 800 to 1794. This variable region differs in DNA sequence and by 201 bp. Analysis of RepMP5 copies outside the ORF6 gene showed that both M. pneumoniae M129 and M. pneumoniae FH carry a RepMP5 copy on a 6-kbp EcoRI fragment which has the same DNA sequence as the variable region of RepMP5 in the M. pneumoniae FH ORF6 gene. According to these data, a switch from the M. pneumoniae M129 ORF6 gene to the M. pneumoniae FH ORF6 gene could take place by gene conversion.