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RamA Is an Alternate Activator of the Multidrug Resistance Cascade in Enterobacter aerogenes. Renaud Chollet, 2004.Multidrug resistance (MDR) in Enterobacter aerogenes can be mediated by induction of MarA, which is triggered by certain antibiotics and phenolic compounds . In this study, we identified the gene encoding RamA, a 113-amino-acid regulatory protein belonging to the AraC-XylS transcriptional activator family, in the Enterobacter aerogenes ATCC 13048 type strain and in a clinical multiresistant isolate . Overexpression of RamA induced an MDR phenotype in drug-susceptible Escherichia coli JM109 and E . aerogenes ATCC 13048, as demonstrated by 2- to 16-fold-increased resistance to ß-lactams, tetracycline, chloramphenicol, and quinolones, a decrease in porin production, and increased production of AcrA, a component of the AcrAB-TolC drug efflux pump . We show that RamA enhances the transcription of the marRAB operon but is also able to induce an MDR phenotype in a mar-deleted strain . We demonstrate here that RamA is a transcriptional activator of the Mar regulon and is also a self-governing activator of the MDR cascade . Restricted Translocation across the Cell Wall Regulates Secretion of the Broad-Range Phospholipase C of Listeria monocytogenes. Aleksandra Snyder, 2003.The virulence of Listeria monocytogenes is directly related to its ability to spread from cell to cell without leaving the intracellular milieu . During cell-to-cell spread, bacteria become temporarily confined to secondary vacuoles . Among the bacterial factors involved in escape from these vacuoles is a secreted broad-range phospholipase C (PC-PLC), the activation of which requires processing of an N-terminal prodomain . Mpl, a secreted metalloprotease of Listeria, is involved in the proteolytic activation of PC-PLC . We previously showed that, during intracellular growth, bacteria maintain a pool of PC-PLC that is not accessible to antibodies and that is rapidly released in its active form in response to a decrease in pH . pH-regulated release of active PC-PLC is Mpl dependent . To further characterize the mechanism regulating secretion of PC-PLC, the bacterial localization of PC-PLC and Mpl was investigated . Both proteins were detected in the bacterial supernatant and lysate with no apparent changes in molecular weight . Extraction of bacteria-associated PC-PLC and Mpl required cell wall hydrolysis, but there was no indication that either protein was covalently bound to the bacterial cell wall . Results from pulse-chase experiments performed with infected macrophages indicated that the rate of synthesis of PC-PLC exceeded the rate of translocation across the bacterial cell wall and confirmed that the pool of PC-PLC associated with bacteria was efficiently activated and secreted upon acidification of the host cell cytosol . These data suggest that bacterially associated PC-PLC and Mpl localize at the cell wall-membrane interface and that translocation of PC-PLC across the bacterial cell wall is rate limiting, resulting in the formation of a bacterially associated pool of PC-PLC that would readily be accessible for activation and release into nascent secondary vacuoles . Signal Transduction Protein PII Is Required for NtcA-Regulated Gene Expression during Nitrogen Deprivation in the Cyanobacterium Synechococcus elongatus Strain PCC 7942. M. Fadi Aldehni, 2003.The transcription factor of the cyclic AMP receptor protein/FNR family, NtcA, and the PII signaling protein play central roles in global nitrogen control in cyanobacteria . A dependence on PII for NtcA-regulated transcription, however, has not been observed . In the present investigation, we examined alterations in gene expression following nitrogen deprivation in Synechococcus elongatus strain PCC 7942 and specifically the roles of NtcA and PII . Global changes in de novo protein synthesis following combined-nitrogen deprivation were visualized by in vivo [35S]methionine labeling and two-dimensional polyacrylamide gel electrophoresis analysis . Nearly all proteins whose synthesis responded specifically to combined-nitrogen deprivation in wild-type cells of S . elongatus failed to respond in PII- and NtcA-deficient mutants . One of the proteins whose synthesis was down-regulated in a PII- and NtcA-dependent manner was RbcS, the small subunit of RubisCO . Quantification of its mRNA revealed that the abundance of the rbcLS transcript following combined-nitrogen deprivation rapidly declined in wild-type cells but not in PII and NtcA mutant cells . To investigate further the relationship between PII and NtcA, fusions of the promotorless luxAB reporter genes to the NtcA-regulated glnB gene were constructed and these constructs were used to transform wild-type cells and PII- and NtcA- mutants . Determination of bioluminescence under different growth conditions showed that NtcA represses gene expression in the presence of ammonium in a PII-independent manner . By contrast, NtcA-dependent activation of glnB expression following combined-nitrogen deprivation was impaired in the absence of PII . Together, these results suggest that under conditions of combined-nitrogen deprivation, the regulation of NtcA-dependent gene expression requires the PII signal transduction protein .
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