Cloning, sequencing, and overexpression of gene 16 of salmonella bacteriophage P22

Umlauf B, Dreiseikelmann B. Cloning, sequencing, and overexpression of gene 16 of salmonella bacteriophage P22. Virology. 1992;188(2):495-501.It has been suggested that gene product 16 of bacteriophage P22 forms a pore for DNA transfer and/or that it functions as a pilot protein guiding the DNA across the membrane. We have cloned gene 16 and determined the nucleotide sequence. Within the sequenced region there is an open reading frame that could encode a protein of 609 amino acids having a molecular weight of 64,366. The hydropathic plot of this protein does not reveal putative membrane-spanning regions as expected for a protein forming a membrane pore. Overproduction of gene product 16 in Escherichia coli was successful only in a mutant in which the La protease was inactivated. Gene 16 mutants of phage P22 were not able to infect recBCD mutants of Salmonella typhimurium nor was protein 16, synthesized in E. colifrom a plasmid, able to substitute for the pilot protein of phage T4. It seems that gene product 16 is not a pilot protein in the meaning of binding to the ends of linear DNA, thus protecting it from degradation by nucleases

The sim gene of Escherichia coli phage P1: nucleotide sequence and purification of the processed protein

Maillou J, Dreiseikelmann B. The sim gene of Escherichia coli phage P1: nucleotide sequence and purification of the processed protein. Virology. 1990;175(2):500-507.The sim gene of bacteriophage P1 causes exclusion of a superinfecting P1 phage. We determined the nucleotide sequence of a 1.9-kb DNA fragment that, in plasmids, causes Sim phenotype. There are two open reading frames within this region for proteins of 82 and 259 amino acids. A 1.3-kb fragment containing the larger open reading frame was inserted into an expression vector. Induced cells carrying the hybrid plasmid, termed pBD5, were not infected by phage P1 and produced a 24-kDa protein and, to a smaller extent, a 25-kDa protein. The 24-kDa protein was purified. Comparison of its amino-terminal amino acid sequence with the nucleotide sequence indicated that it is processed from a precursor protein by removal of a hydrophobic leader peptide of 20 amino acids. In vivo processing depends on secA gene function and is necessary for Sim interference with P1 infection. The data are discussed with respect to the function of the sim gene in superinfection exclusion

The effect of differential methylation by Escherichia coli of plasmid DNA and phage T7 and [lambda] DNA on the cleavage by restriction endonuclease MboI from Moraxella bovis

Dreiseikelmann B, Eichenlaub R, Wackernagel W. The effect of differential methylation by Escherichia coli of plasmid DNA and phage T7 and [lambda] DNA on the cleavage by restriction endonuclease MboI from Moraxella bovis. Biochimica et Biophysica Acta, Nucleic Acids and Protein Synthesis. 1979;562(3):418-428.The nucleotide sequence recognized and cleaved by the restriction endonuclease MboI is 5 ' GATC and is identical to the central tetranucleotide of the restriction sites of BamHI and BglII. Experiments on the restriction of DNA from Escherichia coli dam and dam + confirm the notion that GATC sequences are adenosyl-methylated by the dam function of E. coli and thereby are made refractory to cleavage by MboI. On the basis of this observation the degree of dam methylation of various DNAs was examined by cleavage with MboI and other restriction endonucleases. In plasmid DNA essentially all of the GATC sequences are methylated by the dam function. The DNA of phage [lambda] is only partially methylated. Extended methylation is observed in the DNA of a substitution mutant of [lambda], [lambda] gal 8bio 256, and in the [lambda] derived plasmid, [lambda]dv93, which is completely methylated. In contrast, phage T7 DNA is not methylated by dam. A suppression of dam methylation of T7 DNA appears to act only in cis since plasmid DNA replicated in a T7-infected cell is completely methylated. The results are discussed with respect to the participation of the dam methylase in different replication systems

The cl repressor of bacteriophage P1: isolation and characterization of the repressor protein

Dreiseikelmann B, Velleman M, Schuster H. The cl repressor of bacteriophage P1: isolation and characterization of the repressor protein. The Journal of Biochemistry. 1988;263(3):1391-1397.The c1 repressor gene of bacteriophage P1 is located on P1 DNA EcoRI fragment 7 (Sternberg, N. (1979) Virology 96, 129-142). Subfragments of P1 DNA EcoRI fragment 7 were cloned into expression vectors, and the c1 repressor protein from P1 wild-type phage and a revertant of a temperature-sensitive repressor mutant were overproduced in Escherichia coli and purified to near-homogeneity. The decreased electrophoretic mobility of P1 DNA BamHI fragment 9 in the presence of appropriate protein fractions was used as an assay for the repressor protein. Highly purified repressor migrates as a single polypeptide on denaturing sodium dodecyl sulfate-polyacrylamide gels, corresponding to a molecular weight of about 33,000. A molecular weight of about 63,000 for the native repressor molecule was calculated from determinations of the sedimentation coefficient, which was 2.6 s, and the Stokes radius, which was 55 A. Cross-linking the protein with glutaraldehyde yielded two bands. These data and a high frictional coefficient (2.1) suggest that the native repressor exists in solution as an asymmetric dimer molecule

Bacteriophages of freshwater Brevundimonas vesicularis isolates

Beilstein F, Dreiseikelmann B. Bacteriophages of freshwater Brevundimonas vesicularis isolates. RESEARCH IN MICROBIOLOGY. 2006;157(3):213-219.Nine strains of Brevundimonas vesicularis were isolated from surface water of three ponds in Bielefeld. Germany. With those strains as indicators seven bacteriophages with different host ranges were isolated. Molecular characterization showed that all phages contained linear double-stranded DNA with a similar genome size of about 37 kb. Restriction analysis and hybridization of phage DNAs revealed that three of these phages are closely related to each other. These phages had morphologies typical of the family Siphoviridae. Their genomes contained cohesive ends. Four phages were classified into the family of Podoviridae. Restriction analysis of the DNAs of these phages did not reveal any similarities. The DNA of these phages were terminally redundant. All phages were unable to transduce plasmids or market genes. (c) 2005 Elsevier SAS. All rights reserved

A functional plasmid-borne rrn operon in soil isolates belonging to the genus Paracoccus

Battermann A, Disse-Kromker C, Dreiseikelmann B. A functional plasmid-borne rrn operon in soil isolates belonging to the genus Paracoccus. Microbiology. 2003;149:3587-3593.Plasmid analysis of isolates from a small Paracoccus population revealed that all 15 representatives carried at least one endogenous plasmid of 23 or 15 kb in size, in addition to further plasmids of different sizes. It was shown by restriction analysis and hybridization that the 23 and 15 kb plasmids from the different isolates were identical or very similar to each other. By partial sequencing of pOL18/23, one of the 23 1 plasmids, a complete rrn operon with the structural genes for 16S, 23S and 5S rRNA, two genes for tRNA(Ile) and tRNA(Ala) within the spacer between the 16S and 23S rRNA genes, and a final tRNA(IMet) at the end of the operon were discovered. Expression of a green fluorescent protein gene (gfp) after insertion of a DNA fragment from the region upstream of the rRNA genes into a promoter-probe vector demonstrated that the rrn promoter region is functional. The rrn operon encoded by plasmid pOL18/23 is the first complete rrn operon sequenced from a strain of the genus Paracoccus, and only the second example of an rrn operon on a small plasmid

Development of a tomato plant resistant to Clavibacter michiganensis using the endolysin gene of bacteriophage CMP1 as a transgene

Wittmann J, Brancato C, Berendzen KW, Dreiseikelmann B. Development of a tomato plant resistant to Clavibacter michiganensis using the endolysin gene of bacteriophage CMP1 as a transgene. PLANT PATHOLOGY. 2016;65(3):496-502.The bacteriophage CMP1 endolysin gene (lys), encoding, a peptidase that was shown to effectively reduce Clavibacter michiganensis by specifically hydrolysing its murein, was transferred into tomato plants by Agrobacterium-mediated transformation. The presence of the gene was verified by PCR and the gene product was confirmed in immunoblots and stably expressed over three generations. Transgenic tomato plants did not show disease symptoms after infection with C.michiganensis subsp. michiganensis, despite the fact that small amounts of bacteria could still be identified in xylem sap and leaf extracts, although in significantly reduced amounts

Infectivity of various forms of bacteriophage T7 DNA

Wackernagel W, Seroka K, Dreiseikelmann B, Prell A. Infectivity of various forms of bacteriophage T7 DNA. In: Portolés A, ed. Modern trends in bacterial transformation and transfection. Amsterdam: North-Holland Publ. Co.; 1977: 185-192

Characterization and genome comparisons of three Achromobacter phages of the family Siphoviridae

Dreiseikelmann B, Bunk B, Sproeer C, Rohde M, Nimtz M, Wittmann J. Characterization and genome comparisons of three Achromobacter phages of the family Siphoviridae. ARCHIVES OF VIROLOGY. 2017;162(8):2191-2201.In this study, we present the characterization and genomic data of three Achromobacter phages belonging to the family Siphoviridae. Phages 83-24, JWX and JWF were isolated from sewage samples in Paris and Braunschweig, respectively, and infect Achromobacter xylosoxidans, an emerging nosocomial pathogen in cystic fibrosis patients. Analysis of morphology and growth parameters revealed that phages 83-24 and JWX have similar properties, both have nearly the same head and tail measurements, and both have a burst size between 85 and 100 pfu/cell. In regard to morphological properties, JWF had a much longer and more flexible tail compared to other phages. The linear double-stranded DNAs of all three phages are terminally redundant and not circularly permutated. The complete nucleotide sequences consist of 81,541 bp for JWF, 49,714 bp for JWX and 48,216 bp for 83-24. Analysis of the genome sequences showed again that phages JWX and 83-24 are quite similar. Comparison to the GenBank database via BLASTN revealed partial similarities to Roseobacter phage RDJL phi1 and Burkholderia phage BcepGomr. In contrast, BLASTN analysis of the genome sequence of phage JWF revealed only few similarities to non-annotated prophage regions in different strains of Burkholderia and Mesorhizobium