Integration of vector-containing Bacillus subtilis chromosomal DNA by a Campbell-like mechanism

Vosman, Ben; Kooistra, J; Olijve, Jos; Venema, Gerard

doi:10.1007/bf00331035

Cited by 20 publications

(13 citation statements)

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“…None of the clones had integrated the plasmid at the expected position in the mrp operon (data not shown). Integration of pMBR600 by homologous recombination (Campbell integration [29]) over the 0.6-kb PstIHindIII fragment would result in two new EcoRI fragments of 1.0 and 13.9 kb as compared with a 10.5-kb EcoRI fragment in the wild type. In 10 tetracycline-resistant clones examined, the 10.5-kb fragment was seen together with one or two other fragments (approximately 2.5, 3.0, and 9.5 kb), indicating that the plasmid had integrated outside the mrp region owing to some sequence homology between the vector and the staphylococcal genome.…”

Section: Resultsmentioning

confidence: 99%

Cloning and expression in Escherichia coli of genes encoding a multiprotein complex involved in secretion of proteins from Staphylococcus aureus

Adler

Arvidson

1988

J Bacteriol

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The genes encoding the multiprotein membrane-bound ribosomal protein (MBRP) complex (mrp genes), associated with membrane-bound ribosomes in Staphylococcus aureus, were cloned in Escherichia coli. All four components (molecular sizes 71, 60, 46, and 41 kilodaltons) of the MBRP complex were expressed from an 8.5-kilobase DNA fragment as judged by Western blot (immunoblot) analysis. The order of the individual genes within the cloned DNA fragment was determined by deletion mutagenesis and subcloning of various restriction fragments. Three RNAs, transcribed from the same DNA strand, were identified within the MBRP-coding region: one large RNA of approximately 5.9 kilobases, presumably coding for all four MBRP components, and two minor RNAs, coding for MBRP-71 and MBRP-60. The two minor RNAs seemed to be transcribed from promoters within the large transcription unit. Attempts to make insertional inactivations of the mrp genes with an internal 600-base-pair DNA fragment of the MBRP-coding region as a target were unsuccessful, presumably because such insertions are lethal.Earlier work in this laboratory (1), aiming at the identification of the components of the apparatus for protein secretion in Staphylococcus aureus, has shown that membrane-bound but not cytoplasmic ribosomes are associated with a multiprotein complex (the membrane-bound ribosomal protein complex [MBRP complex]) of four proteins (molecular sizes 71, 60, 46, and 41 kilodaltons [kDa]). The membrane-bound fraction of the MBRP complex is bound to the 50S subunit of the ribosome (1) and is located between the inner surface of the cytoplasmic membrane and the membrane-bound ribosome (3). A free pool of the complex was also found in the cytoplasm (3). The distribution of the complex between the cytoplasm and the membrane was found to vary depending on the rate of exoprotein production (3), strongly suggesting a role of the MBRP complex in protein secretion. A similar complex, immunologically related to the MBRP complex, has also been identified in Bacillus subtilis (2, 5, 6). As there is no in vitro translationsecretion system in S. aureus or B. subtilis available by which the role of the MBRP complex in secretion can be studied, we decided to use a genetic approach instead. In the present paper we describe the cloning and expression of the MBRP-coding genes (mrp genes) in Escherichia coli. The genetic organization is analyzed by deletion mutagenesis and determination of transcripts. We also present evidence that the mrp genes are essential. MATERIALS AND METHODSBacterial strains and cultivation conditions. S aureus V8 (ATCC 27733), from which the MBRP complex was originally isolated, was used as the source of the MBRP-coding genes. Restriction-weak S. aureus RN4220 (12) was the primary staphylococcal recipient of E. coli-propagated plasmids. Strain RN4220 has a decreased production of several extracellular proteins (unpublished results) similar to the decreased production in exp mutants (10). E. coli MC1061 (4) was the host in the cloning experiments and i...

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Section: Resultsmentioning

confidence: 99%

Cloning and expression in Escherichia coli of genes encoding a multiprotein complex involved in secretion of proteins from Staphylococcus aureus

Adler

Arvidson

1988

J Bacteriol

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“…influenzae and to investigate the usefulness of the technique to identify transformation genes not mutated in previous screens (Beattie & Setlow, 1971;Tomb et al, 1989;Gwinn et al, 1998;. Cassette mutagenesis techniques based on similar approaches have been described for other transformable organisms, such as Bacillus subtilis (Vosman et al, 1986) or yeast (Tettelin et al, 1995). The current study represents a detailed analysis of the cassette mutagenesis procedure in H .…”

Section: Discussionmentioning

confidence: 99%

Identification of Haemophilus influenzae Rd transformation genes using cassette mutagenesis

Dougherty¹,

Smith²

1999

Microbiology

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Genes required for natural transformation of Haemophilus influenzae R d were identified by a cassette mutagenesis protocol consisting of the following steps : random insertional mutagenesis, phenotypic screening, sequencing of genome sequence tags from the DNA flanking the insertion in the selected mutants and comparison of genome sequence tags to genomic sequence data. The cassette mutagenesis screen for transformation genes resulted in five distinct mutant classes, two of which have been identified in previous studies.Insertions in the three newly identified loci interrupted genes with predicted protein products homologous to a type IV pilin-like protein biogenesis operon, drug-eff lux transporters and a phospholipid-biosynthesis enzyme. The most significant finding of this screen is the requirement for type IV pilin-like proteins in genetic transformation of H. influenzae. These surface structures are utilized for DNA uptake in a number of Gram-positive and Gram-negative bacteria, and appear to be the common component among the systems for DNA binding. INTRODUCTIONHaemophilus inf'luenxae is a Gram-negative facultative anaerobe which resides in the human upper respiratory tract. Isolates are classified either as serotypes A-F or as non-typable, depending on the presence or absence, respectively, of type-specific capsular polysaccharide. H. influenxae is also capable of taking DNA from its environment and integrating it into the bacterial chromosome (Smith et al., 1981; Goodgal, 1982). This natural transformation process has been demonstrated for many strains of H . influenxae but has been best characterized using strain Rd (Alexander & Leidy, 1951 Abbreviations: sBHI, supplemented brain heart infusion media; Kan: kanamycin-resistance; GST, genome sequence tag; MIV, medium IV; Tfo-, transformation-deficient.formed cell per lo7 viable cells) in early exponential phase to about lo-* in late exponential phase. The addition of CAMP to early-phase cultures increases the frequency of transformation to lop4 (Wise et al., 1973;. Furthermore, under conditions of transient anaerobiosis (Goodgal & Herriott, 1961) or transfer to starvation medium (Herriott et al., 1970) transformation frequencies are optimized to approximately lop2. Transformation is also observed when H. influenxae is grown in diffusion chambers implanted into animals (Dargis et al., 1992), suggesting that transformation is not an artifact of in vitro cultivation.A number of genes required for DNA transformation have been identified in H . influenzae. These studies have used a variety of molecular methods to identify transformation genes, including complementation of transformation-deficient mutants derived by chemical mutagenesis , a 'poison-DNA' selection method (Beattie & Setlow, 1971), mini-Tn20 mutagenesis (Tomb et al., 1989), Tn916 mutagenesis (Gwinn et al., 1998) and other genetic selection techniques . It has been a goal of our laboratory to screens (e.g. hot-spotting) and yet retain the advantage of gene disruption with an antibiotic-resistance...

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“…ComK (2,3) is known to be a positively acting transcription factor, previously named CTF (competence transcription factor) (ref. 4 and D. van Sinderen, A. Luttinger, L.K., D.D., G. Venema, and L. Hamoen, unpublished work).…”

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confidence: 99%

Regulation of competence-specific gene expression by Mec-mediated protein-protein interaction in Bacillus subtilis.

Kong¹,

Dubnau²

1994

Proc. Natl. Acad. Sci. U.S.A.

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Integration of vector-containing Bacillus subtilis chromosomal DNA by a Campbell-like mechanism

Cited by 20 publications

References 28 publications

Cloning and expression in Escherichia coli of genes encoding a multiprotein complex involved in secretion of proteins from Staphylococcus aureus

Cloning and expression in Escherichia coli of genes encoding a multiprotein complex involved in secretion of proteins from Staphylococcus aureus

Identification of Haemophilus influenzae Rd transformation genes using cassette mutagenesis

Regulation of competence-specific gene expression by Mec-mediated protein-protein interaction in Bacillus subtilis.

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