Detection of Plasmid Transfer from Pseudomonas fluorescens to Indigenous Bacteria in Soil by Using Bacteriophage phiR2f for Donor Counterselection

The transfer of a genetically marked derivative of plasmid RP4, RP4p, from Pseudomonas fluorescens to members of the indigenous microflora of the wheat rhizosphere was studied by using a bacteriophage that specifically lyses the donor strain and a specific eukaryotic marker on the plasmid. Transfer of RP4p to the wheat rhizosphere microflora was observed, and the number of transconjugants detected was approximately 10 transconjugants per g of soil when 10 donor cells per g of soil were added; transfer in the corresponding bulk soil was slightly above the limit of detection. All of the indigenous transconjugants which we analyzed contained a 60-kb plasmid and were able to transfer this plasmid to a Nx RpP. fluorescens recipient strain. The indigenous transconjugants were identified as belonging to Pseudomonas spp., Enterobacter spp., Comamonas spp., and Alcaligenes spp.     

Characterization of caulobacters isolated from wastewater treatment systems.

Caulobacters are generally assumed to be found only in environments of low organic content; however, we readily isolated strains from a variety of sewage treatment system designs and locations, and 33 distinct strains were characterized. Most were morphologically similar, having the crescent-shaped cell body, short stalk, and hexagonally packed, paracrystalline surface (S) layer characteristic of several Caulobacter crescentus laboratory strains. Upon closer examination, they were distinguishable on the basis of protein band profiles on polyacrylamide gel electrophoresis, gross colony characteristics, or holdfast composition or by DNA restriction fragment length polymorphism analysis with flagellin and S-layer gene probes. Most of the isolates contained one or more high-molecular-weight plasmids and were resistant to a number of antibiotics, characteristics generally not shared with caulobacters isolated from other sources. Six of the 33 strains were retained because they did not fit the typical isolate profile; these strains are overrepresented in our collection compared with their relative proportion in wastewater treatment systems. By colony hybridization and restriction fragment length polymorphism analysis, all of these and one typical isolate showed less homology than the others to the surface array gene of a laboratory strain (C. crescentus CB15), and three hybridized less strongly with the flagellin gene from the same strain. In sum, although the strains were distinguishable, caulobacters from the wastewater treatment systems we examined were relatively homogenous, were similar to characterized laboratory strains, and, with exceptions, could probably be reliably detected as a group by gene probes derived from C. crescentus strains.     

Identification and characterisation of an Acinetobacter sp. capable of assimilation of a range of cyano-metal complexes,free cyanide ions and simple organic nitriles

Summary An Acinetobacter sp. strain RFB1 was shown to be capable of degrading a wide range of cyano-metal complexes, simple cyanide salts and simple organic nitrile compounds. The enzymatic activity responsible for this degradation was located in an extracellular lipid complex. This complex could not be resolved into the constitutive components under standard conditions without loss of activity. Offsprint requests to: I. Finnegan     

Localization and quantification of extracellular polymers in methanogenic granular sludge

Summary Extracellular polymers were localized and quantitatively analysed in methanogenic granular sludge cultivated on either propionate or ethanol in laboratory upflow anaerobic sludge-blanket (UASB) reactors. Electron microscopical analysis of ultrathin sections of the two sludge types stained with ruthenium red revealed the presence of extracellular polymers with different densities and structures. For quantification, granular sludge from a large-scale UASB reactor at a liquid sugar plant was also included in this study. A three-step physical disintegration procedure was used to extract water-soluble extracellular material from the granules. After each disintegration step the extracts were analysed for polysaccharides and proteins. Cell damage and thus the contribution of intracellular proteins and polysaccharides was estimated simultaneously by the determination of free DNA and free ATP in the extracts. After two extraction steps, up to 3.5 mg polysaccharides/g organic material and 5.5 mg protein/g organic material were extracted, whereas no significant increase in DNA was detected. The role of extracellular polymers in granular stability is discussed. Offsprint requests to: A. J. B. Zehnder     

A comparison of techniques for isolation of the outer membrane proteins of Haemophilus influenzae type b

We compared several rapid techniques used for extraction of outer membrane proteins from gram-negative enteric bacteria to Haemophilus influenzae type b. After lysis of cells with a French press, the inner and outer membranes were separated by isopycnic centrifugation. Each membrane was identified by density, morphology, enzymatic activity, and susceptibility to solid-phase iodination of intact cells. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, we identified 10 polypeptides which were enriched in the outer membrane band compared to the inner membrane band. Using these proteins, we compared the polypeptide pattern of outer membranes with that obtained by (1) selective solubilization with sodium dodecyl-beta-D-maltoside, octyl-beta-D-glucopyranoside, Triton X-100, sodium, or cholamidopropyl dimethylaminopropanesulfonate; (2) extraction with chaotropic agents and heat; and (3) differential centrifugation of vesicles shed during transition from log growth phase to stationary growth phase. There were definable differences between the polypeptide pattern of membranes obtained with each rapid technique compared to the polypeptide pattern of isolated outer membranes. The polypeptide pattern of lithium extracts and the Triton X-100 insoluble fractions of total membranes most closely approximated the polypeptide pattern of isopycnically isolated outer membranes. Depending on the outer membrane protein sought, one of these rapid techniques can be utilized when a rapid method of outer membrane protein isolation is required.     

Kinetics of oxygen and carbon monoxide binding to liver fluke (Dicrocoelium dendriticum) hemoglobin. An extreme case?

The kinetics of oxygen and carbon monoxide binding to the monomeric liver fluke (Dicrocoelium dendriticum) hemoglobin have been studied. The ligand association rates are approximately 1 X 10(8) and approximately 3 X 10(8) M-1 s-1, respectively, for CO and O2 and show no pH dependence. On the contrary the ligand dissociation rates decrease by lowering the pH below 7, the pK of the transition being around 5.5. These findings, together with spectroscopic properties of the protein, are discussed in relation to the fact that, in this hemoglobin, the distal histidine is replaced by a glycine.     

The amino terminal half of the MS2-coded lysis protein is dispensable for function: implications for our understanding of coding region overlaps.

We have asked whether genetic overlaps only evolve to provide extra coding capacity in genomes of restricted size. As a model system we have used the lysis gene of the RNA bacteriophage MS2. This gene overlaps with the distal part of the coat protein gene and with the proximal part of the replicase gene. Using recombinant DNA procedures we have determined whether either of the two overlaps codes for amino acids that are not essential for the function of the 75 amino acid long lysis protein. We find that the first 40 amino acids of the lysis protein are dispensable for function. Thus all of the genetic information essential to the synthesis of the active C-terminal peptide lies within the overlap with the replicase gene, whereas all dispensable residues are encoded in the overlap with the coat protein gene and in the intercistronic region. This suggests that the overlap with the coat protein gene is not required for extra coding capacity but serves to regulate the expression of the lysis gene. Comparative sequence analysis is consistent with this idea.     

Metabolic and chemical properties of basic proteins isolated from nuclei of rat liver and thymus gland

1. The effects of alkylating agents and disulphides on the thiol-containing proteins of nuclei from rat thymus and liver were studied. Three protein fractions were examined: histones extracted with 50mm- and 250mm-hydrochloric acid and the residual protein. None of the reagents selectively reacted with any one of the protein fractions. 2. Amino acid uptake in vitro into the histones of nuclei from rat thymus was analysed by preparative electrophoresis of the proteins extracted with 50mm- and 250mm-hydrochloric acid. After 1hr. at 37° the greater incorporation was into the proteins extracted with 50mm-hydrochloric acid. 3. Preparative electrophoresis was used to study the relative thiol contents of the proteins of the 50mm-hydrochloric acid extract from thymus nuclei by labelling the histones in vitro with ¹⁴C-labelled N-ethylmaleimide. 4. The capacity of the proteins extracted from rat thymus with 50mm- and 250mm-hydrochloric acid, and of the components from these extracts separated by preparative electrophoresis, to combine with DNA and to depress DNA-dependent RNA synthesis was studied. The histones extracted with 50mm-hydrochloric acid were more lysine-rich than those extracted with 250mm-hydrochloric acid. Wide variations were found in the abilities of the separated components to depress RNA synthesis.     

Lipid production of revertants of Ufa mutants from the oleaginous yeast Apiotrichum curvatum

Summary From six unsaturated fatty acid auxotrophs (Ufa mutants) of the oleaginous yeast Apiotrichum curvatum blocked in the conversion of stearic to oleic acid, were isolated revertants able to grow in the absence of unsaturated fatty acids, in a search for strains that can produce cocoa butter equivalents. A broad range in the percentage of saturated fatty acids (%SFA) was observed in the lipids of individual revertants (varying from 27%–86% SFA), compared with the wild-type (44% SFA). Further analysis of fatty acid composition indicated that: (i) not all six Ufa mutants had the same genetic background and (ii) one specific Ufa mutation could be reverted in more than one way. Revertants that produced lipids with a %SFA>56%, were examined further. These strains were cultivated for 50 generations and half of them produced lipids with high %SFA after that time and were defined as stable. The viability of revertant strains with extremely high %SFA (>80%) may be explained by our finding that polar lipids, which are part of yeast membranes, contained much more polyunsaturated fatty acids and a significantly lower %SFA than neutral (storage) lipids. One revertant (R25.75) was selected that was able to produce lipids in whey permeate at a rate comparable with wild-type A. curvatum and with a fatty acid composition and congelation curve comparable with cocoa butter. Offprint requests to: A. Ykema