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Pseudomonas aeruginosa PA2196 is a TetR family transcriptional repressor. In this study, the deletion of the PA2196 gene caused increased expression of the downstream gene curA (PA2197), which encodes for a NADPH-dependent curcumin/dihydrocurcumin reductase. The PA2196 gene was then identified as curR, and a DNA footprinting assay showed that CurR directly bound to the curA promoter at an imperfect 15-bp inverted repeat, 5'-TAGTTGA-C-TGGTCTA-3'. A curA promoter-lacZ fusion assay and site-directed mutagenesis further demonstrated that the identified CurR binding site plays a crucial role in curA repression by CurR. curA transcription was inducible by sodium hypochlorite (NaOCl) and N-ethylmaleimide (NEM) but not by hydrogen peroxide, organic hydroperoxide, or curcumin. The oxidation and alkylation of CurR by NaOCl and NEM, respectively, resulted in the inactivation of its DNA-binding activity, which induced curA expression. Under the tested conditions, the deletion of either curR or curA did not affect the survival of P. aeruginosa under NaOCl stress in the absence or presence of curcumin., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Burkholderia pseudomallei (Bp) is the causative agent of the life-threatening melioidosis in humans. The global transcription factor oxyR gene was isolated and characterized. It is located between recG, encoding a putative DNA helicase, and katG, encoding a putative catalase-peroxidase. oxyR is expressed as a monocistronic 1 kb mRNA and is induced by oxidative stress compounds. Northern, primer extension, and transcription reporter fusion analyses showed that oxyR mRNA is induced by 0.2 mM menadione, 2 mM paraquat, and 10 mM H(2)O(2). Two knockout mutants of oxyR were constructed, by single- and double-crossover recombination, and found to be hypersensitive to H(2)O(2) and paraquat. Bp lacking OxyR exhibited autoaggregation when cultured in liquid broth and an increased ability to form biofilms in minimal medium, but not in Luria-Bertani broth. The oxyR mutants also have a decreased level of extracellular protease activity. The altered phenotypes of oxyR deficient mutants were complemented when a copy of oxyR was transposed into the mutant chromosomes on the mini-Tn5 transposon.

The recA gene from the bacterium Xanthomonas oryzae pv. oryzae (Xoo), a rice pathogen, was cloned based on its ability to complement DNA repair defects of Escherichia coli recA- mutants. The Xoo recA was localized to a 1.3-kb Sau3AI-XhoI fragment and, when cloned into pBR322, specifies increased methylmethanesulfonate and mitomycin C resistance to E. coli recA mutants and allows lambda red- gam- to plaque on an E. coli recA- host. An E. coli recA- strain harboring a plasmid containing the Xoo recA-like gene was shown to produce a 40-kDa protein which cross-reacted with an anti-E. coli RecA antibody. A similar molecular mass protein to RecA has been detected in several Xanthomonas pathovars using an anti-E. coli RecA antibody. Furthermore, the cloned Xoo recA was shown to hybridize to genomic DNA from various Xanthomonas pathovars, but not to genomic DNA from other bacteria species under high-stringency hybridization conditions. These results indicate the isolation of the Xoo recA gene.

The complete nucleotide (nt) sequence of the small subunit ribosomal RNA-encoding gene of Opisthorchis viverrini reported in this study is the first nt sequence reported for a trematode. The gene is 1992 nt long and has a G + C content of 50.94%. It is made up of alternated constant and variable regions that are similar to the gene organization of other eukaryotes. It is also of interest to note an unexpectedly high degree of sequence homology between O. viverrini and human genes.

Plasmid pPL703 is a promoter-cloning plasmid for Bacillus subtilis consisting of the promoter-less cat-86 gene inserted between the EcoRI and BamHI sites of pUB110. The orientation of cat-86 in pPL703 is opposite to that of two major transcript species that occur within the pUB110 vector portion of pPL703. Therefore, transcripts initiated in cloned promoters which activate cat-86 expression presumably must terminate prior to entering the vector portion of pPL703 to permit stable maintenance of promoter-containing derivatives in host cells. We have identified an apparent Rho-independent transcription terminator 35 bp 3' to the cat-86 coding sequence. A restriction fragment spanning the terminator is 90% efficient in terminating transcription in both B. subtilis and Escherichia coli. The structure of the cat-86 transcription termination site is similar to Rho-independent termination sites identified in E. coli.

The ability to express cloned genes transiently is an important technique in the study of eukaryotic gene expression. Numerous useful expression vectors have been constructed for this purpose although many of them share several common drawbacks. In this paper I describe the construction and characterization of novel expression vectors pSVKII and pSVKIII which have 13 and 8 unique restriction sites, respectively, suitable for cloning genes. These vectors have phage M13 ori, which permits them to produce and package ss DNA molecules in Escherichia coli host, thus facilitating the sequencing and site directed mutagenesis of a cloned gene. Expression vector pSVKIII has a T7 phage promoter located between the SV40 promoter and the multiple cloning sites, which permits efficient transcription and in vitro translation of the cloned gene prior to in vivo studies.