Studies on the synthesis of plasmid-coded proteins and their control in Bacillus subtilis minicells

Shivakumar, A. G.; Hahn, Judith; Dubnau, David

doi:10.1016/0147-619x(79)90046-5

Cited by 77 publications

(69 citation statements)

References 21 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Consistent with this idea are the observations that Em stabilizes Bacillus megaterium polysomes in vivo, whereas other MLS antibiotics, such as spiramycin (a 16-membered-ring macrolide) and lincomycin, cause their rapid breakdown (4,7). Likewise, Em has been found to stabilize E. coli polysomes (22).…”

Section: Discussionsupporting

confidence: 68%

“…Exposure of cells containing ermC to nanomolar concentrations of erythromycin (Em), a macrolide antibiotic, results in about a 10-fold increase in the intracellular level of methylase protein during a 60-min period (22), and a model for regulation of ermC has been proposed (9,12). According to the model, ermC mRNA is transcribed constitutively, but only small amounts of methylase protein are produced due to sequestration of the methylase ribosome-binding site and initiator Met codon by mRNA secondary structure, as shown in Fig.…”

mentioning

confidence: 99%

See 1 more Smart Citation

The ermC leader peptide: amino acid alterations leading to differential efficiency of induction by macrolide-lincosamide-streptogramin B antibiotics

Mayford

Weisblum

1990

J Bacteriol

View full text Add to dashboard Cite

The inducibility of ermC by erythromycin, megalomicin, and celesticetin was tested with both wild-type ermC and several regulatory mutants altered in the 19-amino-acid-residue leader peptide, MGIFSIFVISTVHYQP NKK. In the model test system that was used, the ErmC methylase was translationally fused to 13-galactosidase. Mutational alterations that mapped in the interval encoding Phe-4 through Ile-9 of the leader peptide not only affected induction by individual antibiotics, but did so differentially. The subset of mutations that affected inducibility by the two macrolides erythromycin and megalomicin overlapped and were distinct from the subset of mutations that affected induction by celesticetin. These studies provide a model system for experimentally varying the relative efficiencies with which different antibiotics induce the expression of ermC. The possibility that antibiotics with inducing activity interact directly with the nascent leader peptide was tested by using a chemically synthesized decapeptide, MGIFSIFVIS-, attached at its C-terminus to a solid-phase support. This peptide, however, failed to bind erythromycin in vitro.The ermC gene encodes an inducible rRNA methylase that confers resistance to the macrolide-lincosamide-streptogramin B (MLS) group of procaryotic translation inhibitors (24). Exposure of cells containing ermC to nanomolar concentrations of erythromycin (Em), a macrolide antibiotic, results in about a 10-fold increase in the intracellular level of methylase protein during a 60-min period (22), and a model for regulation of ermC has been proposed (9, 12). According to the model, ermC mRNA is transcribed constitutively, but only small amounts of methylase protein are produced due to sequestration of the methylase ribosome-binding site and initiator Met codon by mRNA secondary structure, as shown in Fig. 1.In the presence of Em, an antibiotic-bound ribosome is thought to stall while translating the 19-amino-acid leader peptide located upstream from the methylase-coding region. Genetic (18) and biochemical (19) studies have suggested that the Em-bound ribosome stalls at leader peptide codons Val-8 and Ile-9, causing the ermC transcript to assume an alternative (active) conformation in which segments 2 and 3 become associated and the methylase translation initiation sequence becomes unpaired. This realignment of complementary segments allows ribosomes to initiate methylase translation at a higher rate than they can with mRNA in the repressed conformation shown.Inducible MLS resistance is found in a wide range of bacterial genera. The induction specificity, defined as the subset of MLS antibiotics that induce expression of a particular MLS resistance determinant, can differ depending on the organism (8, 11). Inducible MLS resistance determinants from several different organisms have been cloned and studied in detail, and they appear to be regulated by a translational attenuation mechanism (6, 11, 13). The molecular basis for the variability of induction specificity is not clear; however, i...

show abstract

Section: Discussionsupporting

confidence: 68%

mentioning

confidence: 99%

The ermC leader peptide: amino acid alterations leading to differential efficiency of induction by macrolide-lincosamide-streptogramin B antibiotics

Mayford

Weisblum

1990

J Bacteriol

View full text Add to dashboard Cite

show abstract

“…The use of lower antibiotic concentrations resulted in a progressive loss of recombinant organisms over the first five to ten generations, to give a population dominated ( > 90 %) by plasmid-free organisms. The plasmid-encoded Cmr in BC 1 is due to inducible chloramphenicol acetyltransferase (CAT) activity (Shivakumar et al, 1979). Although 15 mg Cm 1-l was considerably higher than the MIC for the plasmid-free strain BCO (Table l), the concentration of Cm in the culture may have been less than that in the reservoir because of its degradation by the CAT activity of the recombinant clones.…”

Section: Resultsmentioning

confidence: 98%

The Isolation of Strains of Bacillus subtilis Showing Improved Plasmid Stability Characteristics by Means of Selective Chemostat Culture

1988

View full text Add to dashboard Cite

A PUB 1 10-derived plasmid encoding chloramphenicol resistance, kanamycin resistance and high-temperature a-amylase showed a high degree of segregational instability when inserted into Bacillussuhtilis. In an attempt to obtain stable derivatives, the organism was grown in chemostat culture in the presence of chloramphenicol. It was periodically found necessary to increase the concentration of chloramphenicol in the medium feed in order to avoid plasmid loss. Strains were isolated after 19 and 160 generations, which showed high levels of plasmid stability. This characteristic appeared to be genotypic. No detectable difference in plasmid copy number was found between the original and the improved strains. The stability characteristics resided in the host, rather than in the plasmid. Stable isolates possessed elevated MICs for both chloramphenicol and kanamycin. Their maximum specific growth rates were higher than that of the original strain, and similar to that of the plasmid-free parent strain. INTRODUCTIONBacteria of the genus Bacillus are attractive hosts for the manufacture of many important enzymes. Unfortunately, instability of chimeric plasmids encoding commercially valuable foreign proteins may result in loss of the inserted gene during culture. In particular, PUB1 10-derived plasmids are prone to both structural and segregational instability when introduced into Bucillus suhtilis (Bron & Luxen, 1985;Joyet et al., 1984;Vehmaanpera & Korhola, 1986; Rabinovich et ul., 1985). Carrier et al. (1983) and Doi (1984) have suggested the use of agents, such as antibiotics, which disadvantage plasmid-free organisms in cultures and thus maximize the proportion of recombinant organisms in the population. This technique would be especially effective with continuous cultures, where disadvantaged organisms would tend to wash out. In continuous cultures, such a technique would not only ensure a high proportion of plasmid-containing organisms, but also favour the dominance of stable recombinant clones over less stable ones. This effect may be explained by considering the fate of the daughters of a cell in continuous culture. A simple heuristic model (Kubitschek, 1970) will be used for clarity, though our proposals also apply to the more realistic stochastic model. The heuristic model considers a population in which each cell represents a clone. In the absence of selection pressures, one daughter of each division event will be washed out of the culture vessel, and the other will remain to continue the cell line. In a system where selective pressure is applied against plasmidfree cells, the retained offspring of stable recombinant clones will always be resistant to the selective pressure, and thus able to continue the clone. With increasing degrees of instability, the probability of one or both of the daughters being plasmid-free increases. There will thus be an increasing probability that the retained daughter will be plasmid-free, disadvantaged, and unable to continue the cell line. Hence, as culture proceeds we would expe...

show abstract

“…The samples for slab gel electrophoresis were prepared by the method of Shivakumar et al 18 ) and subjected to SDS-polyacrylamide gel electrophoresis as described by Laemmli and Favre. 19 ) After electrophoresis, the gels were processed for fluorographyZO) and exposed at -80°C to Kodak X-ray film (X-Omat XRP-5).…”

Section: Incorporation Of a I4c-amino Acid Mixture Into Minicells Andmentioning

confidence: 99%

Identification of the Product of Sporulation Genespo0BinBacillus subtilis

Oda¹,

Yamada²,

Nomi³

et al. 1985

Agricultural and Biological Chemistry

View full text Add to dashboard Cite

Studies on the synthesis of plasmid-coded proteins and their control in Bacillus subtilis minicells

Cited by 77 publications

References 21 publications

The ermC leader peptide: amino acid alterations leading to differential efficiency of induction by macrolide-lincosamide-streptogramin B antibiotics

The ermC leader peptide: amino acid alterations leading to differential efficiency of induction by macrolide-lincosamide-streptogramin B antibiotics

The Isolation of Strains of Bacillus subtilis Showing Improved Plasmid Stability Characteristics by Means of Selective Chemostat Culture

Identification of the Product of Sporulation Genespo0BinBacillus subtilis

Contact Info

Product

Resources

About