Growth of [lambda] variants with added or altered promoters in N-limiting bacterial mutants: Evidence that an N recognition site lies in the PR promoter

Transcription of the [lambda] genome, initiating at the early rightward promoter (PR), traverses the cII-O-P operon and extends through the Q gene. In the absence of the [lambda] N function, this transcription is prematurely terminated at either of two termination sites, tR1 and tR2. The cII-O-P operon lies distal to tR1, but proximal to tR2. A number of mutations resulting in new promoter activities (e.g., c17 and ric5b) mapping distal to tR1, but proximal to tR2, have been isolated.Although phages carrying the c17 mutation grow in a normal Escherichia coli host, we find that [lambda] derivatives carrying this mutation will not grow in mutant E. coli K12 hosts, Nus-, which limit [lambda] growth by inhibiting the expression of N function. However, under the same conditions, a [lambda] phage containing only the normal [lambda] promoters grows significantly better in the Nus- hosts. Our studies demonstrate that under conditions of limited N expression, phage carrying the c17 mutation can express functions coded for by genes in the cII-O-P operon, but not endolysin, a function coded for by a gene distal to tR2. Thus, under conditions of low N activity, functions whose genes lie downstream from the c17 promoter without any intervening termination signals are expressed. On the other hand, functions whose genes lie downstream from this promoter with an intervening termination signal are not expressed.These results are consistent with a model of N action, which has N acting only with transcription initiating at a specific class of promoters (e.g., PR), c17 not being a member of this class. Although previous studies (Friedman and Ponce-Campos, 1975) have shown that the ric5b promoter is also not a member of the N-utilizing class of promoters, we find that [lambda]ric5b grows on Nus- hosts. This suggests that whereas c17 interferes with transcription from PR, ric5b does not show such an interference.We also find that [lambda] variants carrying two mutations v3 and vs326, which map in the OR - PR region, exhibit the same growth characteristics in Nus- hosts as phages carrying the c17 mutation. These observations imply that the combination of v3 and vs326 interfere with N-modification of transcription initiating at PR, and lead us to conclude that one site for N recognition is located within the PR promoter.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/21867/1/0000271.pd

Cooperative effects of bacterial mutations affecting [lambda] N gene expression : II. Isolation and characterization of mutations in the rif region

We report the isolation and initial characterization of a class of mutations, Snu, that map near the rif locus on the Escherichia coli chromosome. Snu mutations inhibit the growth of phage [lambda], an effect primarily seen when Snu mutations are combined with another class of mutations, nus. Nus mutations have previously been shown to inhibit the expression of the N gene product of [lambda], and the experiments reported here suggest that Snu mutations add to this inhibitory effect.One Snu mutation, Snu-9, was shown to cause bacterial growth to be temperature-sensitive. This suggests that, at least, some Snu mutations may be in genes coding for a function essential for bacterial growth. Since genes coding for the [beta] and [beta]' subunits of RNA polymerase map in this region, we recognize that Snu mutations might alter either of these subunits of RNA polymerase. Complementation studies demonstrate that Snu+ is dominant to Snu-, indicating that the mutant phenotype is due to the partial loss of a function necessary for full N expression.Although the hosts carrying Snu and nus mutations (called Supernus) severely restrict the growth of phage which express the N function of [lambda], they do not show any increased inhibitory effect on the growth of [lambda]immP22 and [lambda]imm21, phages which express N functions different from that of [lambda]. However, Supernus hosts do restrict the growth of a [lambda] variant that can grow well in bacteria carrying either component mutation, Snu- or nus-. The restrictive effect of the Supernus strain is far greater than would be expected if the restriction was due to an additive effect of the two component mutations. This implies that there might be an interaction between Snu and nus products and that the Supernus phenotype results from an interference with this interaction.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/21880/1/0000286.pd

Therapeutic Anticoagulation with Heparin in Critically Ill Patients with Covid-19.

BACKGROUND: Thrombosis and inflammation may contribute to morbidity and mortality among patients with coronavirus disease 2019 (Covid-19). We hypothesized that therapeutic-dose anticoagulation would improve outcomes in critically ill patients with Covid-19. METHODS: In an open-label, adaptive, multiplatform, randomized clinical trial, critically ill patients with severe Covid-19 were randomly assigned to a pragmatically defined regimen of either therapeutic-dose anticoagulation with heparin or pharmacologic thromboprophylaxis in accordance with local usual care. The primary outcome was organ support-free days, evaluated on an ordinal scale that combined in-hospital death (assigned a value of -1) and the number of days free of cardiovascular or respiratory organ support up to day 21 among patients who survived to hospital discharge. RESULTS: The trial was stopped when the prespecified criterion for futility was met for therapeutic-dose anticoagulation. Data on the primary outcome were available for 1098 patients (534 assigned to therapeutic-dose anticoagulation and 564 assigned to usual-care thromboprophylaxis). The median value for organ support-free days was 1 (interquartile range, -1 to 16) among the patients assigned to therapeutic-dose anticoagulation and was 4 (interquartile range, -1 to 16) among the patients assigned to usual-care thromboprophylaxis (adjusted proportional odds ratio, 0.83; 95% credible interval, 0.67 to 1.03; posterior probability of futility [defined as an odds ratio <1.2], 99.9%). The percentage of patients who survived to hospital discharge was similar in the two groups (62.7% and 64.5%, respectively; adjusted odds ratio, 0.84; 95% credible interval, 0.64 to 1.11). Major bleeding occurred in 3.8% of the patients assigned to therapeutic-dose anticoagulation and in 2.3% of those assigned to usual-care pharmacologic thromboprophylaxis. CONCLUSIONS: In critically ill patients with Covid-19, an initial strategy of therapeutic-dose anticoagulation with heparin did not result in a greater probability of survival to hospital discharge or a greater number of days free of cardiovascular or respiratory organ support than did usual-care pharmacologic thromboprophylaxis. (REMAP-CAP, ACTIV-4a, and ATTACC ClinicalTrials.gov numbers, NCT02735707, NCT04505774, NCT04359277, and NCT04372589.)