The SalI (SalGI) restriction-modification system of Streptomyces albus G

The salIR and salM genes of Streptomyces albus G specify the SalGI (SalI) restriction enzyme and its cognate methyltransferase, respectively. These enzymes are responsible for restriction and modification of bacteriophages. Some phages carry genes that interfere with SalI-specific modification. The sal genes have been cloned in a Streptomyces host-vector system. Use of the cloned DNA as a hybridization probe reveals that sal mutants frequently arise from transposition of a DNA segment of approx. 1 kb into the sal genes. Some, but not all, other bacteria that produce SalGI isoschizomers contain nucleotide sequences that hybridize with sal DNA.     

Characterization of Rrh4273I, a restriction-modification system of Rhodococcus rhodochrous ATCC 4273 (Nocardia corallina) which recognizes the same sequence as the Streptomyces albus G SalI restriction-modification system

Rhodococcus rhodochrous ATCC 4275 (Nocardia corallina) has a restriction-modification system with the same recognition sequence, methylation site and cleavage site as the SalI restriction-modification system. Both the restriction endonuclease and the DNA-methyltransferase (DNA-MTase) have been partially purified and characterized. The nuclease has requirements of activity similar to SalI, and a native Mr of about 46,000. The DNA-MTase is a protein with an Mr of about 67,000. No DNA homology was detected between the cloned salI restriction-modification genes of Streptomyces albus and R. rhodochrous chromosomal DNA.     

The restriction-modification system in Streptomyces flavopersicus

To clone bifunctional vectors in streptomycetes, it was necessary to define the restriction-modification system ofStreptomyces flavopersicus. Plasmid DNA from bifunctional vectors pIJ699 and pXED3-13, isolated fromE. coli strains with different methylation systems:E. coli DH5α (dam ⁺ dcm ⁺),E. coli MB5386(dam dcm), E. coli CB51 (dam dcm ⁺),E. coli NM544 (dam ⁺ dcm), was used for transformation of protoplasts from strainS. flavopersicus. Restriction ofdcm-methylated DNA fromS. flavopersicus was established. As a host in the intermediate cloning strainE. coli NM544 (dam ⁺ dcm) should be used, as thedcm-transmethylase-dependent strainS. flavopersicus does not process DNA from this strain.     

Isolation and genetic structure of IS112, an insertion sequence responsible for the inactivation of the SalI restriction-modification system of Streptomyces albus G.

Summary IS112 is a transposable element identified in Streptomyces albus G by its frequent mutagenic insertion into the genes for the SalI restriction-modification system. IS112 is present in several copies in the genome of S. albus G. Homologous sequences were detected in other Streptomyces strains. Sequence analysis revealed that IS112 has a length of 883 by with a GC content of 67.4%. The copy that was isolated contained imperfect inverted repeats (16/20 match) at its ends and was flanked by a 2 by duplication at the target site, which was located within the gene (salIR) for the Sall endonuclease. A long open reading frame (ORF) encoding a putative polypeptide of 256-253 amino acids spans almost the entire sequence. Significant homology was detected between this polypeptide and that corresponding to ORFB of IS493, an insertion sequence recently isolated from Streptomyces lividans 66.

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A new restriction endonuclease from Streptomyces albus G.

A restriction endonuclease, SalI, has been partially purified from Streptomyces albus G. This enzyme cleaves adenovirus-2 DNA at three sites, bacteriophage λ DNA at two sites, but does not cleave simian virus 40 DNA or φX174 DNA. It recognizes the sequence

and cuts at the sites indicated by the arrows. An endonuclease (XamI) with similar specificity has also been isolated from Xanthomonas amaranthicola.     

Isolation of mutants of the obligate methanotroph Methylomonas albus defective in growth on methane

A strain of Methylomonas albus BG8WM, a type 1 obligate methanotroph, which had been maintained for 2 ycars by serial passage on solid medium containing methanol as a carbon source was found to mutate at a frequency of 10^-5-10^-6 to resistance to dichloromethane (DCM^R), the parental strain BG8 did not give rise to DCM^R colonies. DCM^R strains were no longer capable of growth on methane as a carbon cource and exhibited greatly reduced or undetectable methane mono-oxygenase activity. The mutants fell into three groups on the basis of SDS-PAGE analysis of the polypeptide profiles of the particulate fraction of cell extracts. One or more of four polypeptides of Mr 70,000, 50,000, 25,000 and 23,000 were implicated as being components of the methane mono-oxygenase. Spontaneous reversion to growth on methane and sensitivity to dichloromethane occurred at a frequency of about 10^-8. The loss of methane mono-oxygenase activity was not associated with loss of the resident 55 kb plasmid.Abbreviations DCM^R dichloromethane-resistant - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - NMS nitrate minimal salts medium     

Protein phosphorylation in Streptomyces albus

The phosphorylated proteins of Streptomyces albus, radioactively labeled with [32P]orthophosphate have been analyzed by gel electrophoresis and autoradiography. More than 10 protein species were found to be phosphorylated. With [32P]ATP as substrate cell free extracts phosphorylated endogenous proteins in vitro which were predominantly phosphorylated in vivo. From cell extract which exhibited active phosphorylated in vitro, a protein kinase has been partially purified. The kinase activity was identified in fractions corresponding to a 90 kDa protein.     

Restriction of a bacteriophage of Streptomyces albus G involving endonuclease SalI.

The bacteriophage Pa16, isolated from soil on Streptomyces albus G, was restricted when transferred from an alternative host back to S. albus G. Extracted unmodified Pa16 deoxyribonucleic acid was cleaved at a single site by a cell-free extract of S. albus G. Fractions cleaving Pal6 deoxyribonucleic acid contained the endonuclease SalI first described by J. Arrand, P. Myers, and R. J. Roberts (unpublished data). A mutant of S. albus G was isolated which was defective in both restriction and modification of Pal6. This mutant lacked SalI activity. It is concluded that SalI is the agent of restriction of Pal6 by S. albus G.     

Active-site-directed inactivators of the Zn2+-containing D-alanyl-D-alanine-cleaving carboxypeptidase of Streptomyces albus G

Several types of active-site-directed inactivators (inhibitors) of the Zn2+-containing D-alanyl-D-alanine-cleaving carboxypeptidase were tested. (i) Among the heavy-atom-containing compounds examined, K2Pt(C2O4)2 inactivates the enzyme with a second-order rate constant of about 6 X 10(-2)M-1 X S-1 and has only one binding site located close to the Zn2+ cofactor within the enzyme active site. (ii) Several compounds possessing both a C-terminal carboxylate function and, at the other end of the molecule, a thiol, hydroxamate or carboxylate function were also examined. 3-Mercaptopropionate (racemic) and 3-mercaptoisobutyrate (L-isomer) inhibit the enzyme competitively with a Ki value of 5 X 10 X 10(-9)M. (iii) Classical beta-lactam compounds have a very weak inhibitory potency. Depending on the structure of the compounds, enzyme inhibition may be competitive (and binding occurs to the active site) or non-competitive (and binding causes disruption of the protein crystal lattice). (iv) 6-beta-Iodopenicillanate inactivates the enzyme in a complex way. At high beta-lactam concentrations, the pseudo-first-order rate constant of enzyme inactivation has a limit value of 7 X 10(-4)S-1 X 6-beta-Iodopenicillanate binds to the active site just in front of the Zn2+ cofactor and superimposes histidine-190, suggesting that permanent enzyme inactivation is by reaction with this latter residue.     

Restriction mapping of plasmid pSa1, isolated from Streptomyces albus G strain

A novel plasmid designated pSa1 has been isolated from Streptomyces albus G strain producing SalGI restriction endonuclease. Molecular weight of the plasmid is 3.4 +/- 0.2 mD. The action of 12 restriction endonucleases on the plasmid DNA was studied. Restriction map of pSa1 DNA was established for SmaI, HindII, XbaI and KpnI endonucleases.     

The exocellular DD-carboxypeptidase-endopeptidase of Streptomyces albus G. Interaction with beta-lactam antibiotics.

Kinetically, the three-step model proposed for the interaction between beta-lactam antibiotics and the exocellular DD-carboxypeptidases-transpeptidases of Streptomyces R61 and Actinomadura R39 [Frère, Ghuysen & Iwatsubo (1975) Eur. J. Biochem. 57, 343--357; Fuad, Frère, Ghuysen, Duez & Iwatsubo (1976) Biochem. J. 155, 623--629] applies to the interaction between the much less penicillin-sensitive exocellular DD-carboxypeptidase-endopeptidase of Streptomyces albus G and at least phenoxymethylpenicillin, cephalothin and cephalosporin C. The penicillin resistance of the albus G enzyme is mainly due to the low efficiency with which the first reversible complex formed with the antibiotic (complex EI) undergoes transformation into a second more stable complex EI*. Analysis of the ternary interaction between enzyme, NalphaNepsilon-diacetyl-L-lysyl-D-alanyl-D-alanine (Ac2-L-Lys-D-Ala-D-Ala) and cephalosporin C indicates a non-competitive mechanism.