Role of Pseudomonas aeruginosa mifSR Two-Component System in Antibiotic Resistance and Biofilm Formation

Pseudomonas aeruginosa is an opportunistic pathogen found in a wide variety of environments. It is one of the leading causes of morbidity and mortality in cystic fibrosis patients, and one of the main sources of nosocomial infections in the United States. One of the most prominent features of this pathogen is its wide resistance to antibiotics. P. aeruginosa employs a variety of mechanisms including efflux pumps and the expression of B-lactamases to overcome antibiotic treatment. Two chromosomally encoded lactamases, ampC and poxB, have been identified in P. aeruginosa. Sequence analyses have shown the presence of a two-component system (TCS) called MifSR (MifS-Sensor and MifR-Response Regulator), immediately upstream of the poxAB operon. It is hypothesized that the MifSR TCS is involved in B-lactam resistance via the regulation of poxB. Recently, the response regulator MifR has been reported to play a crucial role in biofilm formation, a major characteristic of chronic infections and increased antibiotic resistance. In this study, mifR and mifSR deletion mutants were constructed, and compared to the wild type parent strain PAOl for differences in growth and B-lactam sensitivity. Results obtained thus far indicate that mifR and mifSR are not essential for growth, and do not confer B-lactam resistance under the conditions tested. This study is significant because biofilm formation and antibiotic resistance are two hallmarks of P. aeruginosa infections, and finding a link between these two may lead to the development of improved treatment strategies.

Mechanisms of Arsenic Detoxification and Resistance

Arsenic is a ubiquitous environmental toxic substance. As a consequence of continual exposure to arsenic, nearly every organism, from Escherichia coli to humans have evolved arsenic detoxification pathways. One of the pathways is extrusion of arsenic from inside the cells, thereby conferring resistance. The R773 arsRDABC operon in E. coli encodes an ArsAB efflux pump that confers resistance to arsenite. ArsA is the catalytic subunit of the pump, while ArsB forms the oxyanion conducting pathway. ArsD is an arsenite metallochaperone that binds arsenite and transfers it to ArsA. The interaction of ArsA and ArsD allows for resistance to As(III) at environmental concentrations. The interaction between ArsA ATPase and ArsD metallochaperone was examined. A quadruple mutant in the arsD gene encoding a K2A/K37A/K62A/K104A ArsD is unable to interact with ArsA. An error-prone mutagenesis approach was used to generate random mutations in the arsA gene that restored interaction with the quadruple arsD mutant in yeast two-hybrid assays. Three such mutants encoding Q56R, F120I and D137V ArsA were able to restore interaction with the quadruple ArsD mutant. Structural models generated by in silico docking suggest that an electrostatic interface favors reversible interaction between ArsA and ArsD. Mutations in ArsA that propagate changes in hydrogen bonding and salt bridges to the ArsA-ArsD interface also affect their interactions. The second objective was to examine the mechanism of arsenite resistance through methylation and subsequent volatilization. Microbial ArsM (As(III) S-adenosylmethyltransferase) catalyzes the formation of trimethylarsine as the volatile end product. The net result is loss of arsenic from cells. The gene for CrArsM from the eukaryotic green alga Chlamydomonas reinhardtii was chemically synthesized and expressed in E. coli. The purified protein catalyzed the methylation of arsenite into methyl-, dimethyl- and trimethyl products. Synthetic purified CrArsM was crystallized in an unliganded form. Biochemical and biophysical studies conducted on CrArsM sheds new light on the pathways of biomethylation. While in microbes ArsM detoxifies arsenic, the human homolog, hAS3MT, converts inorganic arsenic into more toxic and carcinogenic forms. An understanding of the enzymatic mechanism of ArsM will be critical in deciphering its parallel roles in arsenic detoxification and carcinogenesis.

Investigating the outcomes of two chronic disease self-management programs and understanding the correlates of completion for each program

Chronic disease affects 80% of adults over the age of 65 and is expected to increase in prevalence. To address the burden of chronic disease, self-management programs have been developed to increase self-efficacy and improve quality of life by reducing or halting disease symptoms. Two programs that have been developed to address chronic disease are the Chronic Disease Self-Management Program (CDSMP) and Tomando Control de su Salud (TCDS). CDSMP and TCDS both focus on improving participant self-efficacy, but use different curricula, as TCDS is culturally tailored for the Hispanic population. Few studies have evaluated the effectiveness of CDSMP and TCDS when translated to community settings. In addition, little is known about the correlation between demographic, baseline health status, and psychosocial factors and completion of either CDSMP or TCDS. This study used secondary data collected by agencies of the Healthy Aging Regional Collaborative from 10/01/2008–12/31/2010. The aims of this study were to examine six week differences in self-efficacy, time spent performing physical activity, and social/role activity limitations, and to identify correlates of program completion using baseline demographic and psychosocial factors. To examine if differences existed a general linear model was used. Additionally, logistic regression was used to examine correlates of program completion. Study findings show that all measures showed improvement at week six. For CDSMP, self-efficacy to manage disease (p = .001), self-efficacy to manage emotions (p = .026), social/role activities limitations (p = .001), and time spent walking (p = .008) were statistically significant. For TCDS, self-efficacy to manage disease (p = .006), social/role activities limitations (p = .001), and time spent walking (p = .016) and performing other aerobic activity (p = .005) were significant. For CDSMP, no correlates predicting program completion were found to be significant. For TCDS, participants who were male (OR=2.3, 95%CI: 1.15–4.66), from Broward County (OR=2.3, 95%CI: 1.27–4.25), or living alone (OR=2.0, 95%CI: 1.29-–3.08) were more likely to complete the program. CDSMP and TCDS, when implemented through a collaborative effort, can result in improvements for participants. Effective chronic disease management can improve health, quality of life, and reduce health care expenditures among older adults.

Hybrid approaches to estimating freeway travel times using point traffic detector data

The accurate and reliable estimation of travel time based on point detector data is needed to support Intelligent Transportation System (ITS) applications. It has been found that the quality of travel time estimation is a function of the method used in the estimation and varies for different traffic conditions. In this study, two hybrid on-line travel time estimation models, and their corresponding off-line methods, were developed to achieve better estimation performance under various traffic conditions, including recurrent congestion and incidents. The first model combines the Mid-Point method, which is a speed-based method, with a traffic flow-based method. The second model integrates two speed-based methods: the Mid-Point method and the Minimum Speed method. In both models, the switch between travel time estimation methods is based on the congestion level and queue status automatically identified by clustering analysis. During incident conditions with rapidly changing queue lengths, shock wave analysis-based refinements are applied for on-line estimation to capture the fast queue propagation and recovery. Travel time estimates obtained from existing speed-based methods, traffic flow-based methods, and the models developed were tested using both simulation and real-world data. The results indicate that all tested methods performed at an acceptable level during periods of low congestion. However, their performances vary with an increase in congestion. Comparisons with other estimation methods also show that the developed hybrid models perform well in all cases. Further comparisons between the on-line and off-line travel time estimation methods reveal that off-line methods perform significantly better only during fast-changing congested conditions, such as during incidents. The impacts of major influential factors on the performance of travel time estimation, including data preprocessing procedures, detector errors, detector spacing, frequency of travel time updates to traveler information devices, travel time link length, and posted travel time range, were investigated in this study. The results show that these factors have more significant impacts on the estimation accuracy and reliability under congested conditions than during uncongested conditions. For the incident conditions, the estimation quality improves with the use of a short rolling period for data smoothing, more accurate detector data, and frequent travel time updates.

Transcription-Coupled DNA Supercoiling in Escherichia Coli: Mechanisms and Biological Functions

Transcription by RNA polymerase can induce the formation of hypernegatively supercoiled DNA both in vivo and in vitro. This phenomenon has been explained by a “twin-supercoiled-domain” model of transcription where a positively supercoiled domain is generated ahead of the RNA polymerase and a negatively supercoiled domain behind it. In E. coli cells, transcription-induced topological change of chromosomal DNA is expected to actively remodel chromosomal structure and greatly influence DNA transactions such as transcription, DNA replication, and recombination. In this study, an IPTG-inducible, two-plasmid system was established to study transcription-coupled DNA supercoiling (TCDS) in E. coli topA strains. By performing topology assays, biological studies, and RT-PCR experiments, TCDS in E. coli topA strains was found to be dependent on promoter strength. Expression of a membrane-insertion protein was not needed for strong promoters, although co-transcriptional synthesis of a polypeptide may be required. More importantly, it was demonstrated that the expression of a membrane-insertion tet gene was not sufficient for the production of hypernegatively supercoiled DNA. These phenomenon can be explained by the “twin-supercoiled-domain” model of transcription where the friction force applied to E. coli RNA polymerase plays a critical role in the generation of hypernegatively supercoiled DNA. Additionally, in order to explore whether TCDS is able to greatly influence a coupled DNA transaction, such as activating a divergently-coupled promoter, an in vivo system was set up to study TCDS and its effects on the supercoiling-sensitive leu-500 promoter. The leu-500 mutation is a single A-to-G point mutation in the -10 region of the promoter controlling the leu operon, and the AT to GC mutation is expected to increase the energy barrier for the formation of a functional transcription open complex. Using luciferase assays and RT-PCR experiments, it was demonstrated that transient TCDS, “confined” within promoter regions, is responsible for activation of the coupled transcription initiation of the leu-500 promoter. Taken together, these results demonstrate that transcription is a major chromosomal remodeling force in E. coli cells.