RESUMEN
Plant growth-promoting Pseudomonas B10 produces its yellow-green, fluorescent siderophore (microbial iron transport agent) pseudobactin under iron-limiting conditions. A structural gene encoding the 85,000-Da putative outer membrane receptor protein for ferric pseudobactin was identified in a gene bank from Pseudomonas B10 prepared with the broad host-range conjugative cosmid cloning vector pLAFR1. Transposon Tn5 mutagenesis of recombinant plasmid pJLM300 localized the functional gene to a region of approximately 2.4 kilobases consistent with the apparent molecular weight of the receptor protein. Mobilization of pJLM300 into Pseudomonas A124 and A225, whose growth was inhibited by Pseudomonas B10 or pseudobactin, rendered these strains no longer susceptible to iron starvation by pseudobactin because they were now able to transport ferric pseudobactin. Pseudobactin biosynthetic genes flanked this receptor gene on both sides and were on separate operons. Transposon Tn5 insertion mutants of Pseudomonas B10 lacking this receptor protein were generated by a marker exchange technique and were defective in ferric pseudobactin transport. Such mutants could be complemented in trans by pJLM300. The production of pseudobactin, the receptor protein, and four other outer membrane proteins in Pseudomonas B10 was coordinately regulated by the level of intracellular iron.
Asunto(s)
Proteínas Bacterianas , Clonación Molecular , Oligopéptidos/metabolismo , Pseudomonas/genética , Receptores de Superficie Celular/genética , Enzimas de Restricción del ADN/metabolismo , Desoxirribonucleasa EcoRI , Electroforesis en Gel de Poliacrilamida , Genes , Hierro/metabolismo , Peso Molecular , Pseudomonas/análisisRESUMEN
The gene encoding rat somatostatin has been isolated from a lambda phage gene library. Phage harboring the gene were identified by plaque hybridization using a nick-translated fragment derived from the cDNA for rat somatostatin. The transcriptional unit includes exons of 238 and 367 base pairs (bp) separated by one intron of 621 bp. The intron is located between the codons for Gln (-57) and Glu (-56) of prosomatostatin. Analysis of the nucleotide sequence 5' to the start of transcription reveals a number of sequences which may be involved in the expression of somatostatin. A variant of the "TATA" box, TTTAAA, lies 26 bp upstream from the start of transcription, and a sequence homologous to the "CAAT" box (GGCTAAT) is 92 bp upstream from the transcription start. A long alternating purine-pyrimidine stretch, (GT)25, which is similar to Z DNA-forming sequences in other genes, lies 628 bp 5' to the transcription start and is flanked by small repeats. Hybridization analysis shows that this region is highly repeated in the genome and that homologous sequences are located approximately 2 kilobase pairs downstream from the poly(A) addition site. Southern hybridization of the lambda clone with probes derived from brain or liver poly(A+) RNA demonstrates that another transcribed sequence lies about 7 kilobase pairs downstream from the poly(A) addition site of the rat somatostatin gene. Analysis of rat DNA suggests that there may be restriction-site polymorphisms in or near the gene or that additional somatostatin-hybridizing sequences may exist in the genome.