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Control by A-Factor of a Metalloendopeptidase Gene Involved in Aerial Mycelium Formation in Streptomyces griseus. Jun-ya Kato, 2002.In Streptomyces griseus, A-factor (2-isocapryloyl-3R-hydroxymethyl-  -butyrolactone) switches on aerial mycelium formation and secondary metabolite biosynthesis . An A-factor-dependent transcriptional activator, AdpA, activates multiple genes required for morphological development and secondary metabolism in a programmed manner . A region upstream of a zinc-containing metalloendopeptidase gene (sgmA) was found among the DNA fragments that had been isolated as AdpA-binding sites . The primary product of sgmA consisted of N-terminal pre, N-terminal pro, mature, and C-terminal pro regions . sgmA was transcribed in an AdpA-dependent manner, and its transcription was markedly enhanced at the timing of aerial mycelium formation . AdpA bound two sites in the region upstream of the sgmA promoter; one was at about nucleotide position -60 (A site) with respect to the transcriptional start point of sgmA, and the other was at about position -260 (B site), as determined by DNase I footprinting . Transcriptional analysis with mutated promoters showed that the A site was essential for the switching on of sgmA transcription and that the B site was necessary for the marked enhancement of transcription at the timing of aerial mycelium formation . Disruption of the chromosomal sgmA gene resulted in a delay in aerial hypha formation by half a day . SgmA is therefore suggested to be associated with the programmed morphological development of Streptomyces, in which this peptidase, perhaps together with other hydrolytic enzymes, plays a role in the degradation of proteins in substrate hyphae for reuse in aerial hypha formation .
Transcriptional Organization of the Pseudomonas putida tol-oprL Genes. María A. Llamas, 2003.Proteins of the Tol system play a key role in the maintenance of outer membrane integrity and cell morphology in gram-negative bacteria . In Pseudomonas putida, the seven genes, orf1, tolQ, tolR, tolA, tolB, oprL, and orf2, which encode the proteins of this complex, are clustered in a 5.8-kb region of chromosomal DNA . Analysis of polar mutations, reverse transcriptase PCR assays, and transcriptional fusion constructs with a promoterless lacZ gene revealed that the genes are arranged in two operons: orf1 tolQ tolR tolA tolB and oprL orf2 . We were also able to find a transcript that was initiated at the orf1 promoter and covered the two operons in a single mRNA . On the basis of the OprL protein level, we surmised that this transcript contributed only about 10 to 15% of the total OprL protein . Primer extension analysis identified the oprL orf2 operon promoter within the tolB gene, and the -10 and -35 regions exhibited some similarity to those of  70-recognized promoters . The transcription start point of orf1 was located 91 bp upstream of the orf1 start codon, and the -10/-35 region also exhibited
70 -10/-35 recognition sequences . The expression from both promoters in rich and minimal media was constitutive and was very little influenced by the growth phase or iron-deficient conditions . In addition, analyses of the ß-galactosidase activities of different translational fusion constructs revealed that translation of tolA and orf2 genes was dependent on the translation of their corresponding upstream genes (tolR and oprL, respectively) .
The Gene yjcG, Cotranscribed with the Gene acs, Encodes an Acetate Permease in Escherichia coli. Rosa Gimenez, 2003.We isolated an Escherichia coli mutant strain that suppresses the glycolate-negative phenotype of a strain deficient in both GlcA and LldP transporters of this compound . This suppressing phenotype was assigned to yjcG, a gene whose function was previously unknown, which was found to encode a membrane protein able to transport glycolate . On the basis of sequence similarity, the yjcG gene product was classified as a member of the sodium:solute symporter family . Northern experiments revealed that yjcG is cotranscribed with its neighbor, acs, encoding acetyl coenzyme A synthetase, which is involved in the scavenging acetate . The fortuitous presence of an IS2 element in acs, which impaired yjcG expression by polarity in our parental strain, allowed us to conclude that the alternative glycolate carrier became active after precise excision of IS2 in the suppressed strain . The finding that yjcG encodes a putative membrane carrier for glycolate and the cotranscription of yjcG with acs suggested that the primary function of the yjcG gene product (proposed gene name, actP) could be acetate transport and allowed us to define an operon involved in acetate metabolism . The time course of [1,2-14C]acetate uptake and the results of a concentration kinetics analysis performed with cells expressing ActP or cells deficient in ActP supported the the hypothesis that this carrier is an acetate transporter and suggested that there may be another transport system for this monocarboxylate .
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