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Abstract: Bacterial transcription activators regulate transcription by making essential protein–protein interactions with RNA polymerase, for example, with region 4 of the σ70 subunit (σ70 R4). Rob, SoxS, and MarA comprise a closely related subset of members of the AraC/XylS family of transcription factors that activate transcription of both class I and class II promoters. Recently, we showed that interactions between SoxS and σ70 R4 occlude the binding of σ70 R4 to the −35 promoter element of class II promoters. Although Rob shares many similarities with SoxS, it contains a C-terminal domain (CTD) that the other paralogs do not. Thus, a goal of this study was to determine whether Rob makes protein–protein interactions with σ70 R4 at class II promoters and, if so, whether the interactions occlude the binding of σ70 R4 to the −35 hexamer despite the presence of the CTD. We found that although Rob makes fewer interactions with σ70 R4 than SoxS, the two proteins make the same, unusual, position-dependent interactions. Importantly, we found that Rob occludes σ70 R4 from binding the −35 hexamer, just as does SoxS. Thus, the CTD does not substantially alter the way Rob interacts with σ70 R4 at class II promoters. Moreover, in contrast to inferences drawn from the co-crystal structure of Rob bound to robbox DNA, which showed that only one of Rob''s dual helix–turn–helix (HTH) DNA binding motifs binds a recognition element of the promoter''s robbox, we determined that the two HTH motifs each bind a recognition element in vivo. [Copyright &y& Elsevier]

Abstract: In Escherichia coli, Rob activates transcription of the SoxRS/MarA/Rob regulon. Previous work revealed that Rob resides in three to four immunostainable foci, that dipyridyl and bile salts are inducers of its activity, and that inducers bind to Rob''s C-terminal domain (CTD). We propose that sequestration inactivates Rob by blocking its access to the transcriptional machinery and that inducers activate Rob by mediating its dispersal, allowing interaction with RNA polymerase. To test “sequestration–dispersal” as a new mechanism for regulating the activity of transcriptional activators, we fused Rob''s CTD to SoxS and used indirect immunofluorescence microscopy to determine the effect of inducers on SoxS–Rob''s cellular localization. Unlike native SoxS, which is uniformly distributed throughout the cell, SoxS–Rob is sequestered without an inducer, but is rapidly dispersed when cells are treated with an inducer. In this manner, Rob''s CTD serves as an anti-sigma factor in regulating the co-sigma-factor-like activity of SoxS when fused to it. Rob''s CTD also protects its N-terminus from Lon protease, since Lon''s normally rapid degradation of SoxS is blocked in the chimera. Accordingly, Rob''s CTD has novel regulatory properties that can be bestowed on another E. coli protein. [Copyright &y& Elsevier]

Bacterial transcription activators regulate transcription by making essential protein-protein interactions with RNA polymerase, for example, with region 4 of the σ(70) subunit (σ(70) R4). Rob, SoxS, and MarA comprise a closely related subset of members of the AraC/XylS family of transcription factors that activate transcription of both class I and class II promoters. Recently, we showed that interactions between SoxS and σ(70) R4 occlude the binding of σ(70) R4 to the -35 promoter element of class II promoters. Although Rob shares many similarities with SoxS, it contains a C-terminal domain (CTD) that the other paralogs do not. Thus, a goal of this study was to determine whether Rob makes protein-protein interactions with σ(70) R4 at class II promoters and, if so, whether the interactions occlude the binding of σ(70) R4 to the -35 hexamer despite the presence of the CTD. We found that although Rob makes fewer interactions with σ(70) R4 than SoxS, the two proteins make the same, unusual, position-dependent interactions. Importantly, we found that Rob occludes σ(70) R4 from binding the -35 hexamer, just as does SoxS. Thus, the CTD does not substantially alter the way Rob interacts with σ(70) R4 at class II promoters. Moreover, in contrast to inferences drawn from the co-crystal structure of Rob bound to robbox DNA, which showed that only one of Rob's dual helix-turn-helix (HTH) DNA binding motifs binds a recognition element of the promoter's robbox, we determined that the two HTH motifs each bind a recognition element in vivo., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Methicillin-resistant Staphylococcus aureus (MRSA) in health-care settings results in life-threatening infections. We examined the incidence of MRSA at the combat support hospital located at the Ibn Sina Hospital in Baghdad, Iraq. We compiled isolate data from 2005 to 2009 characterizing antibiotic susceptibilities, annual trends, patient populations, infection sites, and hospital locations. Approximately 46.1% of S. aureus were MRSA, with increase in numbers of yearly isolates. MRSA was isolated in higher numbers from U.S. military personnel. Non-U.S. patient isolates displayed higher antibiotic susceptibility compared to U.S. military personnel isolates. Outpatient clinic, forward operating bases, and intermediate care ward 1 isolated the most MRSA. Common isolation sites were wound and skin cultures. Community-acquired MRSA was likely present in 291 out of 303 isolates based on antibiotic susceptibility. Our data suggests that most MRSA were community-acquired with limited nosocomial spread. We recommend increases in combat support hospital molecular lab capability to rapidly identify both MRSA categories.

Antimicrobial resistance is depleting the pharmacopeia of agents clinically useful against Gram-negative bacilli. As the number of active agents diminishes, accurate susceptibility testing becomes critical. We studied the susceptibilities of 107 isolates of the Acinetobacter baumannii-calcoaceticus complex to amikacin, gentamicin, and tobramycin using disk diffusion, Etest, as well as the Phoenix, Vitek 2, and MicroScan automated systems, and compared the results to those obtained by broth microdilution. Genes encoding aminoglycoside-modifying enzymes (AMEs) were detected by multiplex PCR, and clonal relationships were determined by pulsed-field gel electrophoresis. Tobramycin was the most active aminoglycoside (27.1% of isolates were susceptible). Disk diffusion and Etest tended to be more accurate than the Vitek 2, Phoenix, and MicroScan automated systems; but errors were noted with all methods. The Vitek 2 instrument incorrectly reported that more than one-third of the isolates were susceptible to amikacin (a very major error). Isolates were polyclonal, with 26 distinct strains, and carried multiple AME genes unrelated to the strain type. The presence of the ant(2")-Ia gene was statistically associated with resistance to each aminoglycoside. The AME genotype accounted for the resistance profile observed in a minority of isolates, suggesting the involvement of multiple resistance mechanisms. Hospital pharmacy records indicated the preferential use of amikacin over other aminoglycosides in the burn intensive care unit, where aminoglycoside resistance is prevalent. The resistance in that unit did not correlate with a predominant strain, AME genotype, or total annual aminoglycoside consumption. Susceptibility to tobramycin increased, even though susceptible isolates carried AME genotypes predicting the inactivation of tobramycin. Determination of the relative contribution of multiple concurrent resistance mechanisms may improve our understanding of aminoglycoside resistance in the Acinetobacter baumannii-calcoaceticus complex.