A Mycobacterial Systems Resource for the Research Community.

Functional characterization of bacterial proteins lags far behind the identification of new protein families. This is especially true for bacterial species that are more difficult to grow and genetically manipulate than model systems such as Escherichia coli and Bacillus subtilis To facilitate functional characterization of mycobacterial proteins, we have established a Mycobacterial Systems Resource (MSR) using the model organism Mycobacterium smegmatis This resource focuses specifically on 1,153 highly conserved core genes that are common to many mycobacterial species, including Mycobacterium tuberculosis, in order to provide the most relevant information and resources for the mycobacterial research community. The MSR includes both biological and bioinformatic resources. The biological resource includes (i) an expression plasmid library of 1,116 genes fused to a fluorescent protein for determining protein localization; (ii) a library of 569 precise deletions of nonessential genes; and (iii) a set of 843 CRISPR-interference (CRISPRi) plasmids specifically targeted to silence expression of essential core genes and genes for which a precise deletion was not obtained. The bioinformatic resource includes information about individual genes and a detailed assessment of protein localization. We anticipate that integration of these initial functional analyses and the availability of the biological resource will facilitate studies of these core proteins in many Mycobacterium species, including the less experimentally tractable pathogens M. abscessus, M. avium, M. kansasii, M. leprae, M. marinum, M. tuberculosis, and M. ulceransIMPORTANCE Diseases caused by mycobacterial species result in millions of deaths per year globally, and present a substantial health and economic burden, especially in immunocompromised patients. Difficulties inherent in working with mycobacterial pathogens have hampered the development and application of high-throughput genetics that can inform genome annotations and subsequent functional assays. To facilitate mycobacterial research, we have created a biological and bioinformatic resource (https://msrdb.org/) using Mycobacterium smegmatis as a model organism. The resource focuses specifically on 1,153 proteins that are highly conserved across the mycobacterial genus and, therefore, likely perform conserved mycobacterial core functions. Thus, functional insights from the MSR will apply to all mycobacterial species. We believe that the availability of this mycobacterial systems resource will accelerate research throughout the mycobacterial research community.

A simple mechanism for co-dependence on two activators at an Escherichia coli promoter.

The Escherichia coli melAB promoter is co-dependent upon two transcription activators, MelR and the cyclic AMP receptor protein, CRP. In this study we demonstrate positive co-operativity between the binding of MelR and CRP at the melAB promoter, which provides a simple mechanism for its co-dependence. MelR binds to four sites, centred at positions -42.5, -62.5, -100.5 and -120.5 relative to the melAB transcription start point. When MelR is pre-bound, CRP is able to bind to a target located between MelR at positions -62.5 and -100.5. This increases the occupation of the two downstream sites for MelR, which is essential for transcription activation. We have identified residues within activating region 1 (AR1) of CRP that are important in transcription activation of the melAB promoter. At simple CRP-dependent promoters, the surface of CRP containing these residues is involved in contacting the RNA polymerase alpha subunit. Our results show that, at the melAB promoter, the surface of CRP containing AR1 contacts MelR rather than RNA polymerase. Thus, MelR and CRP activate transcription by a novel mechanism in which they bind co-operatively to adjacent sites and form a bacterial enhanceosome.

Transcription activation at the Escherichia coli melAB promoter: the role of MelR and the cyclic AMP receptor protein.

MelR is a melibiose-triggered transcription activator that belongs to the AraC family of transcription factors. Using purified Escherichia coli RNA polymerase and a cloned DNA fragment carrying the entire melibiose operon intergenic region, we have demonstrated in vitro open complex formation and activation of transcription initiation at the melAB promoter. This activation is dependent on MelR and melibiose. These studies also show that the cyclic AMP receptor protein (CRP) interacts with the melAB promoter and increases MelR-dependent transcription activation. DNAase I footprinting has been exploited to investigate the location of MelR-and CRP-binding sites at the melAB promoter. We showed previously that MelR binds to two identical 18 bp target sequences centred at position -100.5 (Site 1) and position -62.5 (Site 2). In this work, we show that MelR additionally binds to two other related 18 bp sequences: Site 1', centred at position -120.5, located immediately upstream of Site 1, and Site R, at position -238.5, which overlaps the transcription start site of the divergent melR promoter. MelR can bind to Site 1', Site 1, Site 2 and Site R, in both the absence and the presence of melibiose. However, in the presence of melibiose, MelR also binds to a fifth site (Site 2', centred at position -42.5) located immediately downstream of Site 2, and overlapping the -35 region of the melAB promoter. Additionally, although CRP is unable to bind to the melAB promoter in the absence of MelR, in the presence of MelR, it binds to a site located between MelR binding Site 1 and Site 2. Thus, tandem-bound MelR recruits CRP to the MelR. We propose that expression from the melAB promoter has an absolute requirement for MelR binding to Site 2'. Optimal expression of the melAB promoter requires Sites 1', Site 1, Site 2 and Site 2'; CRP acts as a 'bridge' between MelR bound at Sites 1' and 1 and at Sites 2 and 2', increasing expression from the melAB promoter. In support of this model, we show that improvement of the base sequence of Site 2' removes the requirement for Site 1' and Site 1, and short circuits the effects of CRP.

Repression of the Escherichia coli melR promoter by MelR: evidence that efficient repression requires the formation of a repression loop.

The Escherichia coli MelR protein is a transcription activator that, in the presence of melibiose, activates expression of the melAB operon by binding to four sites located just upstream of the melAB promoter. MelR is encoded by the melR gene, which is expressed from a divergent transcript that starts 237 bp upstream of the melAB promoter transcript start point. In a recent study, we have identified a fifth DNA site for MelR that overlaps the melR promoter transcript start and -10 region. Here we show that MelR binding to this site can downregulate expression from the melR promoter; thus, MelR autoregulates its own expression. Optimal repression of the melR promoter is observed in the absence of melibiose and requires one of the four other DNA sites for MelR at the melAB promoter. The two MelR binding sites required for this optimal repression are separated by 177 bp. We suggest that, in the absence of melibiose, MelR forms a loop between these two sites. We argue that, in the presence of melibiose, this loop is broken as the melAB promoter is activated. However, in the presence of melibiose, the melR promoter can still be partially repressed by MelR binding to the site that overlaps the transcript start and -10 region. Parallels with the Escherichia coli araC-araBAD regulatory region are discussed.

Postoperative pain control after lumbar spine fusion. Patient-controlled analgesia versus continuous epidural analgesia.

STUDY DESIGN: A prospective, randomized, double-blind clinical trial. OBJECTIVE: To compare the efficacy of postoperative continuous epidural analgesia versus patient-controlled analgesia in patients undergoing lumbar fusion. SUMMARY OF BACKGROUND DATA: Controversy remains regarding the optimal strategy for postoperative pain control. METHODS: Fifty-four patients were divided into two treatment groups. There was no difference between the groups with respect to age, levels fused, estimated blood loss, and use of spinal instrumentation. Patient-controlled analgesia or epidural analgesia was administered in a double-blind manner for a 3-day postoperative course. Each patient received both an epidural and a patient-controlled analgesia delivery system; 26 received the epidural active agent and 28 received patient-controlled analgesia. Postoperative time to liquids and solid food, ambulation, length of stay, and side effects was recorded. Pain was evaluated by a visual analog scale on postoperative days 1, 2, and 3. RESULTS: Results showed no difference between the groups with reference to diet, ambulation, length of stay, and visual analog scale scores. Minor side effects occurred in 71% of patients in both groups. No major complications occurred. Epidural catheter dislodgment occurred in 11% of patients. The total cost for epidural analgesia was approximately $550 more than that for patient-controlled analgesia for a 3-day postoperative course. CONCLUSIONS: These data suggest that there is no clinical advantage of epidural opiate/local anesthetic analgesia over systemic opiate by patient-controlled analgesia for spinal fusion patients. However, possible technical limitations (namely, the low dosage of bupivacaine and placement of the catheter tip) may have prevented adequate delivery of anesthetic to the involved segments. Although the incidence of side effects is similar, cost factors and a high incidence of epidural catheter dislodgment favor use of patient-controlled analgesia.