Cellular localization of Type I restriction-modification enzymes is family dependent.

Cellular localization of Type I restriction-modification enzymes EcoKI, EcoAI, and EcoR124I-the most frequently studied representatives of IA, IB, and IC families-was analyzed by immunoblotting of subcellular fractions isolated from Escherichia coli strains harboring the corresponding hsd genes. EcoR124I shows characteristics similar to those of EcoKI. The complex enzymes are associated with the cytoplasmic membrane via DNA interaction as documented by the release of the Hsd subunits from the membrane into the soluble fraction following benzonase treatment. HsdR subunits of the membrane-bound enzymes EcoKI and EcoR124I are accessible, though to a different extent, at the external surface of cytoplasmic membrane as shown by trypsinization of intact spheroplasts. EcoAI strongly differs from EcoKI and EcoR124I, since neither benzonase nor trypsin affects its association with the cytoplasmic membrane. Possible reasons for such a different organization are discussed in relation of the control of the restriction-modification activities in vivo.

Identification of the EcoKI and EcoR124I Type I restriction--modification enzyme subunits by non-equilibrium pH gradient two-dimensional gel electrophoresis.

Effectively optimized and reproducible procedure for monitoring the composition of type I restriction-modification endonucleases EcoKI and EcoR124I by non-equilibrium pH gradient two-dimensional (2-D) gel electrophoresis is described. Three subunits of the enzyme complex, which widely differ from one another in their isoelectric points and molar mass, were identified in crude cell extracts of E. coli. For the first time all three subunits of both EcoKI and EcoR124I were detected as distinct spots on a single 2-D gel. A sensitive immunoblotting procedure was suggested suitable for routine use in determining the identity of individual subunits. Potential application of this method for detailed studies of regulation of the function and stoichiometry of the enzyme complexes is discussed.

Localization of the type I restriction-modification enzyme EcoKI in the bacterial cell.

To localise the type I restriction-modification (R-M) enzyme EcoKI within the bacterial cell, the Hsd subunits present in subcellular fractions were analysed using immunoblotting techniques. The endonuclease (ENase) as well as the methylase (MTase) were found to be associated with the cytoplasmic membrane. HsdR and HsdM subunits produced individually were soluble, cytoplasmic polypeptides and only became membrane-associated when coproduced with the insoluble HsdS subunit. The release of enzyme from the membrane fraction following benzonase treatment indicated a role for DNA in this interaction. Trypsinization of spheroplasts revealed that the HsdR subunit in the assembled ENase was accessible to protease, while HsdM and HsdS, in both ENase and MTase complexes, were fully protected against digestion. We postulate that the R-M enzyme EcoKI is associated with the cytoplasmic membrane in a manner that allows access of HsdR to the periplasmic space, while the MTase components are localised on the inner side of the plasma membrane.

Two temperature-sensitive mutations in the DNA binding subunit of EcoKI with differing properties.

Two temperature-sensitive mutations in the hsdS gene, which encodes the DNA specificity subunit of the type IA restriction-modification system EcoKI, designated Sts1 (Ser(340)Phe) and Sts2 (Ala(204)Thr) had a different impact on restriction-modification functions in vitro and in vivo. The enzyme activities of the Sts1 mutant were temperature-sensitive in vitro and were reduced even at 30 degrees C (permissive temperature). Gel retardation assays revealed that the Sts1 mutant had significantly decreased DNA binding, which was temperature-sensitive. In contrast the Sts2 mutant did not show differences from the wild-type enzyme even at 42 degrees C. Unlike the HsdSts1 subunit, the HsdSts2 subunit was not able to compete with the wild-type subunit in assembly of the restriction enzyme in vivo, suggesting that the Sts2 mutation affects subunit assembly. Thus, it appears that these two mutations map two important regions in HsdS subunit responsible for DNA-protein and protein-protein interactions, respectively.

The effect of recA mutation on the expression of EcoKI and EcoR124I hsd genes cloned in a multicopy plasmid.

Type I restriction-modification (R-M) endonucleases are composed of three subunits--HsdR, required for restriction, and HsdM and HsdS which can produce a separate DNA methyltransferase. The HsdS subunit is required for DNA recognition. In this paper we describe the effect of cloned EcoKI and EcoR124I hsd genes on the resulting R-M phenotype. The variability in the expression of the wild type (wt) restriction phenotype after cloning of the wt hsd genes in a multicopy plasmid in Escherichia coli recA+ background suggests that the increased production of the restriction endonuclease from pBR322 is detrimental to the cell and this leads to the deletion of the cloned hsd genes from the hybrid plasmid and/or inactivation of the enzyme. The effect of a mutation in E. coli recA gene on the expression of R-M phenotype is described and discussed in relation to the role of the cell surface and the localization of the restriction endonuclease in the cell.

Intergeneric conjugal transfer of Escherichia coli/Methylobacterium sp. shuttle vector.

To develop a host-vector system for Methylobacterium sp. using a construct based on a small indigenous methylotrophic plasmid, the E. coli--Methylobacterium sp. shuttle vector pWUBR (12.7 kb, Apr, Tcr) was constructed by joining the E. coli plasmid pBR328 and the cryptic plasmid pWU7 (7.8 kb), isolated from the soil facultative methylotrophic bacterium, Methylobacterium sp. strain M17. Via mobilization by the pDPT51 R plasmid, belonging to the IncP-1 incompatibility group, plasmid pWUBR was transferred into the original host of cryptic plasmid pWU7, strain M17, where a competition between the introduced hybrid plasmid and the indigenous cryptic plasmid took place, and into the plasmidless Methylobacterium sp. strain R2b. The stability of pWUBR in Tcr methylotrophic transconjugants after 25 generations of growth under nonselective conditions was more than 90% in both hosts. The ability to replicate in R2b strain demonstrates that the host spectrum of pWUBR is not restricted to the original host of pWU7 and indicates the possibility to use the present system for other methylotrophs.

Characterization of new plasmids from methylotrophic bacteria.

Several tens of methanol-utilizing bacterial strains isolated from soil were screened for the presence of plasmids. From the obligate methylotroph Methylomonas sp. strain R103a plasmid pIH36 (36 kb) was isolated and its restriction map was constructed. In pink-pigmented facultative methylotrophs (PPFM), belonging to the genus Methylobacterium four plasmids were detected: plasmids pIB200 (200 kb) and pIB14 (14 kb) in the strain R15d and plasmids pWU14 (14 kb) and pWU7 (7.8 kb) in the strain M17. Because of the small size and the presence of several unique REN sites (HindIII, EcoRI, NcoI), plasmid pWU7 was chosen for the construction of a vector for cloning in methylotrophs. Cointegrates pKWU7A and pKWU7B were formed between pWU7 and the E. coli plasmid pK19 Kmr, which were checked for conjugative transfer from E. coli into the methylotrophic host.

An efficient method for isolation of plasmid DNA from methylotrophic bacteria.

A method, suitable for the isolation of closed circular plasmid DNA from methylotrophic bacteria is described. Improvement of cell lysis was achieved by butanol extraction of cells before application of the lytic agent. Using this method, cryptic plasmids of 7.8, 14, 36 and 200 kb were purified from soil-isolated methylotrophs.

Staining of fungal cell walls with fluorescent brighteners: flow-cytometric analysis.

Spore walls of Backusella lamprospora (Mucorales) were stained with ten fluorescent brighteners (FB) and the intensity of their fluorescence was determined. The fluorescence was most intense with Uvitex 2B (100%), other brighteners yielding lower fluorescence intensities: Blankophor BA 267% and BA 200% about 75%, Rylux BSU about 50%, other Rylux agents 10-30%. The agents most suitable for microscopic diagnostics of human and animal mycoses are Uvitex 2B, Blankophor BA 267% and BA 200%, Rylux BSU, and also Rylux BS and PRS. The regulation of excessive fluorescence of fungal cells during microscopic observation is discussed. For the purposes of microscopic diagnosis of human and animal mycosis Uvitex 2B, Blankophor BA 267% and BA 200%, Rylux BSU and, possibly, Rylux BS and PRS are recommended.

Transfer of plasmids from Escherichia coli and Pseudomonas aeruginosa to methylotrophic bacteria and their detection in the new hosts.

Several plasmids of incompatibility group P were transferred from Escherichia coli and Pseudomonas aeruginosa strains to Methylophilus methylotrophus and two other methylotrophs to test their recipient ability. The presence of plasmids in transconjugants was confirmed by electrophoretic analysis. Optimal conditions for detection of plasmid DNA in the strains tested based on alkaline lysis of cells at elevated temperature were established. Special behaviour of plasmids carrying the Mu phage in methylotrophic hosts is described.

Genetic transfers in Brevibacterium sp.

A positive genetic transfer by protoplast fusion was obtained in auxotrophic mutants Brevibacterium sp. M27 his and Brevibacterium sp. M27 arg. Transformation and protoplast fusion with liposomes (as genetic transfers in intact cells and their protoplasts by both the chromosomal and plasmid DNA) did not lead to transfer of the markers followed.

Isolation and characterization of the pMI10 plasmid from Bacillus subtilis, an alpha-amylase producer.

The plasmid DNA pMI10 (5310 bp) was isolated from the alpha-amylase producing strain B. subtilis A18. Thirteen restriction endonucleases were used to digest pMI10 DNA and the restriction map of pMI10 DNA was constructed by mapping PstI (1), HindII (2), BglI (2), BspRI (3) and HindIII (3) sites.

Isolation and characterization of mutants of the RP4 plasmid coding for increased resistance to ampicillin.

E. coli strain J53(RP4) was mutagenized with ethyl methanesulfonate and N-methyl-N'-nitro-N-nitrosoguanidine. Clones showing a two-to threefold increase in resistance to ampicillin were produced. This increase was not due to an increased number of RP4 copies per chromosome. The level of penicillinase activity was twice higher in comparison with the parental strain. No detectable changes were found in the region coding for the resistance to ampicillin on the plasmid by restriction analysis.

Transfer of liposome-encapsulated plasmid DNA to Bacillus subtilis protoplasts and calcium-treated Escherichia coli cells.

Protoplasts of Bacillus subtilis 168 trpC2 str and cells of Escherichia coli SK 1590 after treatment with calcium chloride were transformed to tetracycline resistance with the recombinant plasmid pUN82 entrapped in the reverse phase evaporation liposomes. Frequency of transfer was 4 X 10(-4)% in B. subtilis and 8 X 10(-6)% in E. coli.

Curing effect of clorobiocin on Escherichia coli plasmids.

Clorobiocin, an inhibitor of the gyrB subunit of DNA gyrase, was used for the curing of some Escherichia coli plasmids. Of the plasmids studied, ampicillin resistant R28K and a miniplasmid derived from R1drd-19 were effectively eliminated. We also succeeded in eliminating the ColA factor from E. coli strain B834(pBS103), which was resistant to the effect of currently used curing agents. Although a derivative of ColE1-pBR322 was effectively cured by clorobiocin, the ColE1 plasmid was resistant to its effect. The ColV plasmid determining virulence was effectively eliminated.

The mechanism of resistance to streptomycin in Escherichia coli. Functional analysis of the permeability barrier of cells harbouring the R1 drd-19Km- plasmid.

The mechanism of phenotypically altered SM resistance in mutants of Escherichia coli JC5455 (Rldrd-19Km-) lrs and JC5455 (pON5300) was compared with that of the standard strain JC5455 (Rldrd-19Km-). On analyzing the membrane polypeptides in polyacrylamide gel both mutants were found to possess a protein spectrum different from that of ths standard strain. Transport of D-xylose and L-arginine was the same in all strains, transport of L-proline was decreased in JC5455 (pON5300) which may indicate a mutational interference with energy metabolism. The basic uptake of dihydrostreptomycin was the same in all strains but there were differences after preincubation of cells with streptomycin or glucose. The increased resistance of JC5455 (Rldrd-19Km-) lrs may be due to observed quantitative differences in membrane polypeptides that might play a role in the binding and functional expression of aminoglycoside-3'adenylyl transferase which modifies streptomycin. The increased sensitivity toward streptomycin in JC5455 (pON5300) can be explained by a mutation due to N-methyl-N'-nitro-N-nitrosoguanidine in the host cell since this change of sensitivity to streptomycin could not be transferred by transformation into a nonmutagenized strain. The coincidence of inducibility of increased transport of streptomycin by this antibiotic and the altered frequency of reversion to high levels of streptomycin resistance in JC5455 (pON5300) and in the transformant JC5455 (pON5302) may indicate that the altered reversibility toward phenotypically high resistance to streptomycin is a property of pON5300 and is transferred by transformation.

Membrane mutation affecting energy-linked functions in Escherichia coli K 12.

A small-colony forming variant of Escherichia coli with a mutation in the ncf gene was analysed. The alternation of the protein composition in the cytoplasmic membrane and the interaction with K and E group colicins indicated a membrane mutation. The effect of this mutation on some membrane-bound processes, the activity of Mg2+-activated ATPase, the growth on different carbon sources and the active transport of amino acids, is described. This mutation does not exert any effect on the electron transport system.