The nature of the transfer inhibitor of several F-like plasmids

Summary A model is proposed for the nature of the transfer inhibitor of F-like plasmids, and is shown to be applicable to six of these. In this model, the transfer inhibitor has two components. One, the product of a gene called fin (previously fi or i) is relatively non-specific. The other, called a P product, can be recognised by its slow synthesis or action in a newly-infected cell, and is relatively specific. An approximate location for traP, the cistron coding for the P product of Flac, has been found.Supported by a George Murray Fellowship from the University of Adelaide, Australia. Present address: Laboratory of Genetics, Department of Biochemistry, University of Oxford.     

Microbial metabolism of alkylbenzene sulphonates. Bacterial metabolism of undecylbenzene-p-sulphonate and dodecylbenzene-p-sulphonate

1. A study was made of the biodegradation of alkylbenzene sulphonate homologues, one of the major components of commercially marketed detergents. A Bacillus species was elected for growth on alkylbenzene sulphonate homologues as the sole source of carbon and sulphur. 2. The results from both whole-cell and cell-free systems indicated that the alkyl, aryl and sulphonate moieties of alkylbenzene sulphonate homologues were all further metabolized by the Bacillus species. 3. The alkyl side chain, after a presumed initial oxidation of the terminal methyl group, was subsequently oxidized by a beta-oxidation pathway. Three enzymes of the beta-oxidation pathway, i.e. acyl-CoA synthetase, acyl-CoA dehydrogenase and beta-hydroxyacyl-CoA dehydrogenase, were identified in cell-free extracts of the detergent-grown Bacillus species. The substrate specificity of acyl-CoA synthetase indicated activity towards several alkylbenzene sulphonate homologues. 4. The sulphonate moiety was released as sulphite by a desulphonating enzyme. Some kinetic properties of this enzyme were determined. The sulphite was subsequently metabolized to either sulphate or adenosine 5'-sulphatophosphate. Two enzymes involved in sulphite metabolism, i.e. sulphite-cytochrome c reductase and adenosine 5'-sulphatophosphate-cytochrome c reductase were detected in cell-free extracts of undecylbenzene-p-sulphonate-grown Bacillus species. 5. The combined results of continuous sampling programmes monitored by both t.l.c. and sulphite appearance in the growth medium indicated that desulphonation of the aromatic moiety was the likely first step in the overall biodegradation of several alkylbenzene sulphonate homologues. 6. The presence of p-hydroxyphenylpropionate, p-hydroxybenzoate and 3,4-dihydroxybenzoate in cells after growth on several alkylbenzene sulphonate homologues containing an odd number of carbon atoms in the side chain was confirmed by g.l.c. and t.l.c. analysis. Cells grown on several homologues containing an even number of carbon atoms in the side chain were shown to contain p-hydroxyphenylacetate and 3,4-dihydroxyphenylacetate. 7. The aromatic nucleus obtained from undecylbenzene-p-sulphonate was further metabolized by an oxidation sequence involving an ;ortho-cleavage' route. 8. An overall metabolic pathway for the biodegradation of various alkylbenzene sulphonate homologues by this Bacillus species is proposed.     

Butane 2,3-diol production by Aeromonas hydrophila grown on starch

Summary The conversion of soluble starch to butane 2,3-diol by Aeromonas hydrophila was investigated. The diol was produced optimally at pH 6.2 to 5.0. A significantly higher yield of the diol occurred in media buffered with potassium rather than sodium phosphates. The addition of relatively low concentrations of sodium acetate ( 1g/l) to the starch-based growth medium caused substantial increases in the yield of the diol, although no obvious proportional relationship between the amount of acetate added and the enhanced yield of the diol was recorded. The addition of 5g/l of sodium acetate caused severe growth inhibition and decreased the amount of butane 2,3-diol produced.     

Characterization of IS46, an insertion sequence found on two IncN plasmids.

The IncN plasmids R46 and N3 each contain two copies of an insertion sequence which we denote IS46. This insertion sequence has single PstI and SalI restriction sites and is 0.81 kilobases long. All four copies of IS46 were capable of forming cointegrates, although the DNA between the insertion sequences, which in each case carries a tetracycline resistance gene, was not transposable in the form of a compound transposon. IS46-mediated cointegrates resolved in Rec+ but not in RecA- cells. Recombination between two copies of IS46, causing an inversion, accounts for the existence of two distinct forms of R46. IS46-mediated deletions were probably responsible for the formation of the plasmid pKM101 from R46. IS46 was not homologous to IS1 but did show homology with IS15.     

Overproduction, purification and characterization of the F traT protein

Summary A lambda transducing phage (ED110) which carries the sex factor F surface exclusion genes, traS and traT, was characterized by both genetic and physiochemical techniques. The transducing segment consists of 5.2 kilobases of F tra DNA, and carries the carboxy-terminal onehalf of the upstream traG gene, as well as traS, traT, and the adjacent downstream gene traD. These tra proteins could be identified in infected UV-irradiated cells, and the major part of their synthesis was found to occur from the phage's late promoter p_R under Q control.Lysogens for ED110 were induced and found to greatly overproduce the traT gene product (TraTp), an outer membrane protein normally found in about 20,000 copies per cell, to levels which exceeded the major outer membrane proteins. This led to the development of a simple purification procedure for TraTp, the most important step of which was the construction of an appropriate ompB derivative to eliminate the major outer membrane porin proteins, which have several physical properties in common with TraTp.Purified TraTp was added to mixtures of donor and recipient cells and found to inhibit mating. The specificity of this assay was demonstrated by using an R100-1 donor, which responds to a heterologous surface exclusion system, and by using an altered TraTp containing a missense amino acid substitution.A mechanism by which TraTp mediates surface exclusion is proposed.     

Cloning and genetic analysis of tra cistrons of the Tra 2/Tra 3 region of plasmid RP1

Transfer-defective mutants of the 10.4-kb Tra 2/Tra 3 region of RP1 were identified by their ability to be complemented by clones carrying all or part of this region. The respective mutations occurred in six cistrons whose order (traA, B, E, R, P, Q) and location were determined by deletion and insertion mapping. The cistrons occupy a minimum of 5.5 kb with the most distal, traA, spanning the 28.0-kb map position and traR the KpnI site at map position 24.1 kb. Each cistron is expressed independently, as Tn5 or Tn504 insertions in any one cistron do not affect the other five. The phenotypes controlled by each cistron suggest that all contribute to pilus biosynthesis/function while three (traB, R, and P) also contribute to surface exclusion. Given the occurrence of tra cistrons in the "silent" region between Tra 2 and Tra 3 we propose that the epithet "Tra 2" should be used to describe this entire region.     

Biotransformation of cycloalkanediones by microorganisms; Stereoselective reduction of (±)-camphorquinone

Summary Washed-cell suspensions of cyclohexanol-grownAcinetobacter sp. NCIB 9871 have been shown to promote regio- and stereo-selective reduction of (±)-camphorquinone to yield two chiral diastereoisomericexo-hydroxyketones.     

Characterisation of transfer-deficient mutants of the R100-1TcS plasmid pDU202, caused by insertion of Tn10

Molecular Genetics and Genomics - The transposition of Tn10 from the E. coli chromosome to pDU202 (a TcS deletion mutant of R100-1) was selected by a mating technique: it took place at a frequency...