Expression of the Bacillus pumilus chloramphenicol acetyltransferase gene in Bacillus subtilis, achieved by the P-R-promotor of phage lambda

The possibility of expression of the Bacillus pumilus chloramphenicol acetyltransferase gene (cat) in Bacillus subtilis from the pR promoter of phage lambda has been investigated in this work. For this purpose, the plasmid pPL703 carrying the B. pumilus DNA segment with the cat gene lacking promoter has been combined with the plasmid pBM21 containing the pR promoter. The recombinant plasmid pEL1 is capable of providing the 60 mkg/ml chloramphenicol resistance in Bac. subtilis cells.     

Translational coupling in Escherichia coli of a heterologous Bacillus subtilis-Escherichia coli gene fusion.

The efficient expression in Escherichia coli of the Tn9-derived chloramphenicol acetyltransferase (EC 2.3.1.28) gene fused distal to the promoter and N terminus of the Bacillus subtilis aprA gene was dependent on the initiation of translation from the ribosome-binding site in the aprA gene.     

Bacillus subtilis rRNA promoters are growth rate regulated in Escherichia coli.

rRNA promoters from the rrnB locus of Bacillus subtilis and from the rrnB locus of Escherichia coli were fused to the gene for chloramphenicol acetyltransferase (CAT). The level of expression of CAT in E. coli showed growth rate dependence when the CAT gene was linked to either E. coli or B. subtilis tandem promoters. The downstream promoter of the tandem Bacillus pair showed growth rate regulation, while the upstream promoter did not, whereas for the E. coli tandem promoters, only the upstream promoter was growth rate regulated.     

Molecular cloning of cis-acting regulatory alleles of the Bacillus subtilis amyR region by using gene conversion transformation.

Three cis-acting alleles (gra-10, gra-5, and amyR2) of the Bacillus subtilis amyR promoter locus each cause catabolite repression-resistance of amyE-encoded alpha-amylase synthesis. The gra-10, gra-5, and amyR2 alleles were transferred from the chromosomes of their respective hosts to a plasmid carrying the amyR1-amyE+ gene by the process of gene conversion which is carried out during transformation of competent B. subtilis by plasmid clones carrying homologous DNA. The cloned amyR promoter regions containing the gra-10 and gra-5 mutations were shown to confer catabolite repression-resistance in cis to the synthesis of chloramphenicol acetyltransferase encoded by the cat-86 indicator gene when subcloned into the promoter-probe plasmid pPL603B. Implications concerning both the regulation of amyR utilization and the process of gene conversion in B. subtilis are discussed.     

Cloning restriction fragments that promote expression of a gene in Bacillus subtilis

Plasmid pPL603 (3.1 megadaltons) specifies neomycin resistance in Bacillus subtilis and contains a structural gene for chloramphenicol acetyltransferase. Cells harboring the plasmid cannot grow on solid media containing 10 microgram of chloramphenicol per ml. Cloning EcoRI (or EcoRI)-generated fragments of deoxyribonucleic acid from several sources into the single EcoRI site in plasmid pPL603, with subsequent selection of transformants of media containing 10 micrograms of chloramphenicol per ml, permits the identification of restriction fragments that promote expression of the chloramphenicol acetyltransferase gene.     

A 5''-flanking region of the chicken acetylcholine receptor alpha-subunit gene confers tissue specificity and developmental control of expression in transfected cells.

The 5' end and promoter region of the alpha-subunit gene of chicken muscle acetylcholine receptor was mapped and sequenced. It includes a TATA and a CAAT box and a potential Sp1-binding site. When inserted in front of the chloramphenicol acetyltransferase gene, this promoter (including 850 base pairs of upstream sequence) directed high transient chloramphenicol acetyltransferase expression in transfected mouse C2.7 myotubes but not in C2.7 myoblasts or nonmyogenic 3T6 cells.     

Restriction fragments that exert promoter activity during postexponential growth of Bacillus subtilis.

Two restriction fragments of Bacillus subtilis DNA were identified which caused the cat-86 gene present on the promoter cloning plasmid pPL703 to be activated predominantly during postexponential growth of host cells. The postexponential increase was observed in both sporulation-positive strains and in a spoOA mutant of B. subtilis. However, the postexponential increase in the cat-86 gene product, chloramphenicol acetyltransferase, was diminished or not observed when the plasmid-containing cells were grown in the presence of excess glucose. The promoter-containing fragment, designated as 33, was mapped to a site on the B. subtilis chromosome adjacent to hisA. The other fragment, 14, mapped to a site adjacent to ctrA. When present on a high-copy vector, both fragments caused a reduction in the sporulation frequency of host cells. Fragment 33 in high copy number conferred on B. subtilis cells three additional phenotypic changes: brown colony color, intracellular inclusions, and, in a protease-deficient mutant, the production of extracellular protease activity. These activities were observed only in postexponential-phase cultures.     

Isolation and characterization of Lactococcus lactis subsp. lactis promoters.

DNA fragments with promoter activity were isolated from the chromosome of Lactococcus lactis subsp. lactis. For the isolation, a promoter probe vector based on the cat gene was constructed, which allowed direct selection with chloramphenicol in Bacillus subtilis and L. lactis. Four of the putative promoters (P1, P2, P10, and P21) were analyzed further by sequencing, mapping of the 5' end of the mRNA, Northern (RNA blot) hybridization, and chloramphenicol acetyltransferase activity measurements. From these fragments, -10 and -35 regions resembling the consensus Escherichia coli sigma 70 and B. subtilis sigma 43 promoters were identified. Another set of promoters, together with a signal sequence, were also isolated from the same organism. These fragments promoted secretion of TEM beta-lactamase from L. lactis. When the two sets of promoters were compared, it was found that the ones isolated with the cat vector were more efficient (produced more mRNA). By changing the promoter part of the promoter-signal sequence fragment giving the best TEM beta-lactamase secretion into a more efficient one (P2), a 10-fold increase in enzyme production was obtained.     

Isolation and characterization of Lactococcus lactis subsp. lactis promoters.

DNA fragments with promoter activity were isolated from the chromosome of Lactococcus lactis subsp. lactis. For the isolation, a promoter probe vector based on the cat gene was constructed, which allowed direct selection with chloramphenicol in Bacillus subtilis and L. lactis. Four of the putative promoters (P1, P2, P10, and P21) were analyzed further by sequencing, mapping of the 5' end of the mRNA, Northern (RNA blot) hybridization, and chloramphenicol acetyltransferase activity measurements. From these fragments, -10 and -35 regions resembling the consensus Escherichia coli sigma 70 and B. subtilis sigma 43 promoters were identified. Another set of promoters, together with a signal sequence, were also isolated from the same organism. These fragments promoted secretion of TEM beta-lactamase from L. lactis. When the two sets of promoters were compared, it was found that the ones isolated with the cat vector were more efficient (produced more mRNA). By changing the promoter part of the promoter-signal sequence fragment giving the best TEM beta-lactamase secretion into a more efficient one (P2), a 10-fold increase in enzyme production was obtained.     

Identification of plant-induced genes of the bacterial pathogen Xanthomonas campestris pathovar campestris using a promoter-probe plasmid

A promoter-probe plasmid suitable for use in Xanthomonas campestris pathovar campestris (causal agent of crucifer black rot) was constructed by ligating a broad host range IncQ replicon into the promoter-probe plasmid pKK232-8, which contains a promoterless chloramphenicol acetyltransferase gene. Xanthomonas chromosomal DNA fragments were 'shotgun' cloned into a restriction site in front of this gene, and the resulting library was transferred en masse into Xanthomonas. Individual transconjugants possessing DNA insertions with promoter activity in plants were identified by virtue of their ability to infect chloramphenicol-treated turnip seedlings. Of 19 transconjugants identified in this way five were chloramphenicol resistant both in turnip seedlings and on agar plates. However the remaining 14 were only chloramphenicol resistant in planta, and thus apparently contained plant-inducible promoter fragments. Resistance to chloramphenicol was correlated with increased chloramphenicol acetyltransferase activity for the transconjugants assayed. The promoter fragments were used to isolate genomic clones from a library, and the role of the genes contained in these clones in pathogenicity is being investigated.