Evidence that an additional mutation is required to tolerate insertional inactivation of the Streptomyces lividans recA gene.

In contrast to recA of other bacteria, the recA gene of Streptomyces lividans has been described as indispensable for viability (G. Muth, D. Frese, A. Kleber, and W. Wohlleben, Mol. Gen. Genet. 255:420-428, 1997.). Therefore, a closer analysis of this gene was performed to detect possible unique features distinguishing the Streptomyces RecA protein from the well-characterized Escherichia coli RecA protein. The S. lividans recA gene restored UV resistance and recombination activity of an E. coli recA mutant. Also, transcriptional regulation was similar to that of E. coli recA. Gel retardation experiments showed that S. lividans recA is also under control of the Streptomyces SOS repressor LexA. The S. lividans recA gene could be replaced only by simultaneously expressing a plasmid encoded recA copy. Surprisingly, the recA expression plasmid could subsequently be eliminated using an incompatible plasmid without the loss of viability. Besides being UV sensitive and recombination deficient, all the mutants were blocked in sporulation. Genetic complementation restored UV resistance and recombination activity but did not affect the sporulation defect. This indicated that all the recA mutants had suffered from an additional mutation, which might allow toleration of a recA deficiency.

Transcriptional analysis of the gene for glutamine synthetase II and two upstream genes in Streptomyces coelicolor A3(2).

The glutamine synthetase II (GSII, encoded by glnII) activity detectable in crude extracts from Streptomyces coelicolor is low compared to the activity of glutamine synthetase I (GSI, encoded by glnA) and to that of GSII from S. viridochromogenes. We have identified and sequenced a 3.9-kb BglII-BamHI fragment carrying the glutamine synthetase II gene (glnII) from S. coelicolor. Besides glnII, this region contains four ORFs (orf1-orf4). While homologues of orf1 and orf2 were also found in the glnII region of the S. viridochromogenes chromosome, this was not the case for orf3 and orf4, which encode a putative hydrolase and a transcriptional regulator (Ptr) of the MarR family, respectively. High-resolution S1 nuclease mapping showed that the S. coelicolor glnII gene is expressed from two overlapping promoters. The first comprises a vegetative promoter sequence and the second contains sequence elements that are recognized by Esigma31. Similar promoter structures were found upstream of the S. viridochromogenes glnII gene. The involvement of ptr in glnII regulation was studied by gel retardation assays. Recombinant Ptr interacted with the upstream region of ptr, but not with the promoter region of glnII. A ptr gene replacement mutant (S. coelicolor IP) was also constructed. RT-PCR analysis of RNA from wild-type S. coelicolor and the IP mutant demonstrated that expression of orf3 depends on Ptr. Thus, the difference in gene organization between S. coelicolor and S. viridochromogenes is not responsible for the difference in GSII activity.

Transcriptional and mutational analyses of the Streptomyces lividans recX gene and its interference with RecA activity.

The role of the 20,922-Da RecX protein and its interference with RecA activity were analyzed in Streptomyces lividans. The recX gene is located 220 bp downstream of recA. Transcriptional analysis by reverse transcriptase PCR demonstrated that recX and recA constitute an operon. While recA was transcribed at a basal level even under noninducing conditions, a recA-recX cotranscript was only detectable after induction of recA following DNA damage. The recA-recX cotranscript was less abundant than the recA transcript alone. The recX gene was inactivated by gene replacement. The resulting mutant had a clearly diminished colony size, but was not impaired in recombination activity, genetic instability, and resistance against UV irradiation. Expression of an extra copy of the S. lividans recA gene under control of the thiostrepton-inducible tipA promoter was lethal to the recX mutant, demonstrating that RecX is required to overcome the toxic effects of recA overexpression. Since inactivation of the recX gene did not influence transcription of recA, the putative function of the RecX protein might be the downregulation of RecA activity by interaction with the RecA protein or filament.

The adjunctive use of perfluoroperhydrophenanthrene (Vitreon) in diabetic vitrectomy.

OBJECTIVE: To evaluate the utility and efficacy of perfluoroperhydrophenanthrene in the management of retinal detachments secondary to severe proliferative diabetic retinopathy. PATIENTS AND METHODS: Forty consecutive patients with proliferative diabetic retinopathy and retinal detachments were entered into the study at nine participating clinical centers. Perfluoroperhydrophenanthrene (Vitreon) was used as an adjunct to pars plana vitrectomy and membranectomy. RESULTS: Preoperative diagnoses included combined traction and rhegmatogenous retinal detachments in 23 eyes (57.5%), traction retinal detachments in 13 eyes (32.5%), and recurrent rhegmatogenous retinal detachments in 4 eyes (10). Vitreous hemorrhage was present in 17 eyes (42.5%). Preoperative visual acuity ranged from light perception or hand motion in 28 eyes (70%) to 5/200 or greater in 12 eyes (30%). Vitreon was primarily used to flatten the retina following relaxing retinotomy in 12 eyes (30%), to displace subretinal fluid in a posterior-to-anterior direction without performing a drainage retinotomy in 15 eyes (37.5%), and to manage intraoperative complications such as iatrogenic tears in 8 (20%) and retinal dialysis in 5 eyes (12.5%). The retina flattened intraoperatively in all cases, facilitating administration of laser photocoagulation. Patients were followed for a minimum of six months (mean 13.2 months). At last follow up, the macula remained attached in 37 eyes (92.5%), including 31 (77.5%) in which the retina was totally attached. The retina remained detached in 3 eyes (7.5%). Visual acuity improved postoperatively in 20 patients (50%), was unchanged in 13 patients (32.5%), and worsened in 7 patients (17.5%). CONCLUSIONS: Perfluoroperhydrophenanthrene is a useful and effective intraoperative tool for the management of complex retinal detachments secondary to severe proliferative diabetic retinopathy.