Augusto F. Lois

RETRACTED: DNA-PKcs Is Required for Activation of Innate Immunity by Immunostimulatory DNA

This article has been retracted at the request of the authors. Please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). Reason: After publication of the above article, we realized that the anti-IKKα (IB) loading controls presented in Figures 3A, 3B, 3C, and 4C are duplicate presentations of the same gel lanes and do not represent the correct controls for the individual experiments. In addition, the anti-IKKα (IB) loading control in the right panel of Figure 4C is an inadvertent duplication of the DNA-PKcs (IB) data in the left panel of Figure 5F. These errors in figure preparation limit the interpretability of the related experimental data in these figures, which are an essential component of the support for the main conclusions of the paper regarding the activation of IKK and NF-κB. We are therefore retracting this paper and apologize for these errors and for any inconvenience they may have caused. Despite these errors, we stand by the reproducibility of the experimental data and the conclusion, which has been reached by numerous subsequent studies, that IKK and NF-κB are required for activation of innate immunity. Dr. Lois was not reachable via any of the available contact information and therefore has not seen or agreed to the text of this retraction.

Mutants of the two-component regulatory protein FixJ of Rhizobium meliloti that have increased activity at the nifA promoter

FixL and FixJ belong to a two-component regulatory system in Rhizobium meliloti that induces the expression of numerous nitrogen-fixation genes during symbiosis with alfalfa. FixJ is a positive activator required for transcription of the regulatory genes nifA and fixK, while FixL is an oxygen-binding hemoprotein capable of regulating the phosphorylation status of both itself and FixJ, in response to oxygen availability. In this study, we isolated four FixJ mutants that display increased activity at the nifA promoter (PnifA) in Escherichia coli. All four mutants possess amino acid changes in a domain of FixJ that is conserved in other response regulator proteins, and all exhibit increased activity at PnifA in R. meliloti that is dependent on the presence of FixL. One of the mutant proteins, while less efficient at accepting phosphate from a truncated derivative of FixL (FixL*), nevertheless has a phosphorylated form that is more stable than the phosphorylated form of wild-type (wt) FixJ and is more resistant to the phosphatase activity of FixL*. The wt FixJ-phosphate was found to have a half-life of approximately 4 h, which makes it an unusually long-lived response regulator protein. The exceptional stability of wt FixJ-phosphate and the altered phosphorylation properties observed for the mutant are discussed in relation to signal transduction in the FixLJ system.

Behavior of Rhizobium meliloti in oxygen gradients

Rhizobium meliloti cells responded to an abrupt change in oxygen concentration by changing the cell speed (chemokinesis), but they did not alter the frequency at which swimming cells stopped briefly (aerotaxis). Changes in cell speed upon stimulation with oxygen coincided with changes in membrane potential. The cells did not form an aerotactic band in a spatial gradient of oxygen as do the cells of other bacterial species. The fixL and fixJ genes which encode a heme‐containing protein kinase that senses oxygen and a response regulator, respectively, were not involved in the behavior of R. meliloti in oxygen gradients.

Isolation of phosphorylation-deficient mutants of the Rhizobium meliloti two-component regulatory protein, FixJ.

Rhizobium meliloti FixL and FixJ are members of a symbiotically essential two‐component system that regulates nitrogen‐fixation genes in response to environmental oxygen concentrations. FixL is a membrane protein that is thought to relay information about oxygen availability to FixJ via a phosphotransfer mechanism. FixJ increases expression of the nifA and fixK genes by activating transcription of the nifA and fixK promoters (p‐nifA and p‐fixK, respectively). In this study, we examined the relationship between the in vivo activity of FixJ as a transcriptional regulator and its ability to be phosphorylated in vitro by the sensor FixL. FixJ mutants were isolated that showed decreased activity on p‐nifA in Escherichia coli. Most of the FixJ mutant proteins also showed decreased activity on the fixK promoter. These mutants were analysed in R. meliloti for activity on p‐nifA during vegetative growth, where similarities and differences were observed when compared with their phenotypes in E. coli. Three mutants showing significantly less activity in R. meliloti were examined for symbiotic activity in planta and were found to be ineffective. When these three mutant FixJ proteins were examined in vitro for their ability to be phosphorylated by FixL, two mutants were found to have a significantly decreased ability to accept phosphate from FixL. These findings are discussed in relation to signal transduction in the FixLJ system.

Oxygen Sensing by the Rhizobium Meliloti Two-Component Regulatory System, Fixlj

The soil bacterium Rhizobium meliloti fixes dinitrogen (N2) when it forms root nodules with its plant host, Medicago sativa (alfalfa). The formation of nitrogen fixing root nodules is a complex developmental process that requires signal exchange between the bacterium and the plant host. There are many bacterial genes involved in establishing a successful symbiosis including the nod, exo, ndv, nif and fix genes (see Long 1989 for review). The nif and fix genes are specifically required for the nitrogen fixation process and these genes are expressed in the later stages of nodule development. The expression of nif and fix genes, including the structural genes for nitrogenase, is induced ex planta in response to a decrease in oxygen concentration (Ditta, et. al., 1987). This induction of nif and fix gene expression by low oxygen is physiologically relevant because a low oxygen environment is maintained in the nodule to prevent inactivation of the highly oxygen sensitive nitrogenase enzyme.

Oxygen Sensing and Protein Phosphorylation by the Two-Component Regulatory FixLJ System from Rhizobium Meliloti

We have been studying the signal transduction mechanism mediated by the Rhizobium meliloti FixL and FixJ proteins in response to oxygen. FixL is a hemoprotein kinase that can sense oxygen tension and can respond to this signal by modifying its kinase activity. In vitro studies with a soluble truncated version of FixL (FixL*) demonstrated that low oxygen tension specifically augments the autophosphorylating and not the transfer activity to FixJ. FixL* also contains a phosphatase activity that is repressed under anaerobic conditions. Regulation of the phosphatase activity by oxygen is dependent on the phosphorylation state of the FixL* protein. These results demonstrate that oxygen regulates two opposing activities in the FixL protein, kinase and phosphatase, in a reciprocal manner and suggest a specific mechanism by which the oxygen tension within the nodule regulates transcription of the nitrogen fixation genes.