Stabilization of a plasmid-encoded LacZ phenotype inBacillus subtilis

Recombinant plasmid pCED3 was structurally unstable inBacillus subtilis cultures grown in the presence of kanamycin to eliminate the effects of segregational instability. Analysis of 96 modified plasmids indicated that deletions in the plasmid occur at many different sites. The presence of plasmid pCED3 slowed the growth rate of theB. subtilis host. Cells that contained modified plasmids grew faster than the parental cells and took over the population. Two different methodologies were developed to reduce the cultural instability of the plasmid-directed LacZ⁺ phenotype. By growing the cells in a medium that supports a low growth rate, the growth rate ratio between modified and parental cells was reduced, resulting in a partial stabilization (40 generations) of the LacZ⁺ phenotype in the population [35]. Removal of a 4.77 kbEcoRI fragment (which consists primarily of the pBR322 replicon) from plasmid pCED3 produced a more stable plasmid derivative, designated pYS1. Cells harboring plasmid pYS1 grew faster than pCED3-bearing cells, although the level of activity of -galactosidase was similar in both strains. By combining the two approaches (i.e., growth of pYS1-bearing cells in a medium that supports low growth rate), the LacZ⁺ phenotype was stably maintained in the cell population for over 170 generations. Under these conditions, there was no detectable difference between the growth rates of cells bearing the pYS1 plasmid and further modified plasmids.     

Regulation of ACV synthetase activity in the beta-lactam biosynthetic pathway by carbon sources and their metabolites

The activity of -l--aminoadipyl)-l-cysteinyl-d-valine catalysis. Addition of l-cysteine to fermentation media increased -lactam production in both organisms and alleviated the negative carbon source regulation by glycerol in S. clavuligerus.     

Ammonium repression of cephalosporin production by Streptomyces clavuligerus

Production of beta-lactam antibiotics took place during growth of Streptomyces clavulgerus in chemically defined medium. The specific activities of isopenicillin N synthetase ("cyclase"), isopenicillin N epimerase, and deacetoxycephalosporin C synthetase ("expandase") increased during the exponential phase of growth. Specific cephalosporin productivity during fermentation followed a similar pattern, reaching a maximum near the end of the growth phase and decaying rapidly in the stationary phase. Ammonium chloride depressed cephalosporin production, presumably as a result of repression of cyclase and expandase formation, but not of epimerase. No inhibitory effects on enzyme activity by ammonium were found. Addition of tribasic magnesium phosphate [Mg3(PO4)2 X 8H2O] prevented the repression of cyclase and markedly stimulated cephalosporin production. Cephamycin C and, in smaller amounts, O-carbamoyldeacetylcephalosporin C were the only cephalosporins detected. Growth with ammonium resulted in lower titers of both compounds, and did not change the relative proportion of each. The correlation found between cephalosporin productivity and cyclase specific activity in different media suggests that formation of this enzyme may be the rate-limiting step in the pathway.     

Germination-initiated spores of Bacillus brevis Nagano retain their resistance properties.

Initiated spores and vegetative cells of the gramicidin S-producing Bacillus brevis Nagano were compared with respect to their resistance to various forms of stress (osmotic shock-starvation, exposure to ethanol, sonic oscillation, and heat). The resistance of initiated spores to all of these stress situations was considerably greater than that of vegetative cells and approached that of dormant spores. The period during which the initiated spores remained resistant to heat was extended by addition of gramicidin S. The antibiotic may therefore be of survival value to the species in nature by slowing down the development of initiated spores in the outgrowth phase of germination, thereby extending the period during which the cells are resistant to environmental stress.     

Isopenicillin N epimerase activity in a high cephalosporin-producing strain of Cephalosporium acremonium

Summary Isopenicillin N epimerase activity in Cephalosporium acremonium CW-19 is so labile that it has never been detected in sonic extracts. Prior to this work, it had only been obtained by the laborious protoplast lysate procedure. The present work shows that the enzyme is present in sonic extracts of a high cephalosporin-producing strain (C-10) of C. acremonium throughout the fermentation.     

Alternate Requirement for Vitamin B12 or Methionine in Mutants of Pseudomonas denitrificans, a Vitamin B12-producing Bacterium

Experiments are described which indicate that Pseudomonas denitrificans, an organism that overproduces vitamin B(12), uses the B(12) pathway exclusively for methionine synthesis.     

Dependence of Diaminopurine Utilization on the Mutational Site of Purine Auxotrophy in Bacillus subtilis II. Tracer Experiments

Tracer experiments were carried out in an attempt to explain why guanineless auxotrophs can use diaminopurine as a guanine replacement but nonexacting purine auxotrophs cannot do so. Cell suspensions of the nonexacting purineless Bacillus subtilis MB-1356 incorporated more radioactivity from diaminopurine-2-¹⁴C into nucleic acid than did guanineless B. subtilis MB-1517. The radioactivity in MB-1356 ribonucleic acid (RNA) was distributed in both adenine and guanine nucleotides, thus eliminating the possibility that the deamination of diaminopurine to guanine occurred predominantly on the level of nucleoside di- or triphosphates. Strain MB-1517 incorporated adenine-8-¹⁴C into nucleic acids extremely poorly. This correlated with results obtained with cell-free extracts; strain MB-1517 showed much less adenosine monophosphate (AMP) pyrophosphorylase activity than did MB-1356. Likewise, guanineless MB-1517 converted diaminopurine to its nucleotide much more slowly than did the nonexacting purine auxotroph. The results indicated that the lack of growth of nonexacting auxotrophs on diaminopurine alone is due not to an inability to convert the analogue to nucleic acid adenine but to the greater capacity of the nonexacting auxotrophs to convert diaminopurine to its 5′-ribonucleotide. Presumably, this compound, or a coenzyme analogue produced from it, inhibits growth of mutants which cannot make AMP de novo and only when the medium is devoid of adenine.     

Restriction endonuclease activity in Clostridium thermocellum and Clostridium thermosaccharolyticum

 Clostridium thermocellum cell extracts exhibit specific endonuclease activity with very little non-specific exonuclease activity at 55°C. The Dam methylation system of Escherichia coli offers complete protection from digestion by C. thermocellum ATCC 27405 cell extracts for all DNA tested (totaling >100 kb, insuring that most potential restriction sequences have been exposed). Based on both the Dam recognition sequence and the similarity of cell extract and MboI DNA digests, the C. thermocellum restriction enzyme recognition sequence appears to be 5′ GATC 3′. Cell extracts made from a second thermophile, C. thermosaccharolyticum ATCC 31960 do not exhibit specific endonuclease activity under the conditions tested. Genomic DNA from C. thermocellum exhibits a Dam⁺ phenotype while genomic DNA from C. thermosaccharolyticum exhibits a Dam^- phenotype. Received: 10 March 1995/Received revision: 4 September 1995/Accepted: 13 September 1995     

Evolution of the ATP-binding-cassette transmembrane transporters of vertebrates

The ATP-binding-cassette transmembrane transporters (ABC transporters) known from vertebrates belong to four major subfamilies: (1) the P- glycoproteins (Pgp); (2) the cystic fibrosis transmembrane conductance regulators (CFTR); (3) the Tap proteins encoded with the major histocompatibility complex of mammals; and (4) the peroxisomal membrane proteins. Both Pgp and CFTR have a structure suggesting a past internal gene duplication; a phylogenetic analysis indicated that these duplications occurred independently, while an independent tandem gene duplication occurred in the case of the Tap family. Both the Pgp and Tap proteins show evidence of relationship to bacterial ABC transporters lacking internal duplication, and both are significantly more closely related to the HlyB and MsbA families of transporters from purple bacteria than they are to ABC transporters from nonpurple bacteria. The simplest hypothesis to explain this observation is that eukaryotic Pgp and Tap genes are descended from a mitochondrial gene or genes that were subsequently translocated to the nuclear genome. The Pgp genes of eukaryotes are characterized by a remarkable degree of convergent evolution between the ATP-binding cassettes of their N- terminal and C-terminal halves, whereas no such convergence is seen between the two halves of CFTR genes or between the duplicated Tap genes. Exon 13 of the CFTR gene, which encodes a putative regulatory domain not found in other ABC transporters apart from CFTR, showed high levels of both synonymous and nonsynonymous difference in comparisons among different mammalian species, suggesting that this region is a mutational hot spot.