The PshX subunit of the photochemical reaction center from Heliobacterium modesticaldum acts as a low-energy antenna.

The anoxygenic phototrophic bacterium Heliobacterium modesticaldum contains a photochemical reaction center protein complex (called the HbRC) consisting of a homodimer of the PshA polypeptide and two copies of a newly discovered polypeptide called PshX, which is a single transmembrane helix that binds two bacteriochlorophyll g molecules. To assess the function of PshX, we produced a ∆pshX strain of Hbt. modesticaldum by leveraging the endogenous Hbt. modesticaldum Type I-A CRISPR-Cas system to aid in mutant selection. We optimized this system by separating the homologous recombination and CRISPR-based selection steps into two plasmid transformations, allowing for markerless gene replacement. Fluorescence and low-temperature absorbance of the purified HbRC from the wild-type and ∆pshX strains showed that the bacteriochlorophylls bound by PshX have the lowest site energies in the entire HbRC. This indicates that PshX acts as a low-energy antenna subunit, participating in entropy-assisted uphill energy transfer toward the P800 special bacteriochlorophyll g pair. We further discuss the role that PshX may play in stability of the HbRC, its conservation in other heliobacterial species, and the evolutionary pressure to produce and maintain single-TMH subunits in similar locations in other reaction centers.

Deletion of the cytochrome bc complex from Heliobacterium modesticaldum results in viable but non-phototrophic cells.

The heliobacteria, a family of anoxygenic phototrophs, possess the simplest known photosynthetic apparatus. Although they are photoheterotrophs in the light, the heliobacteria can also grow chemotrophically via pyruvate metabolism in the dark. In the heliobacteria, the cytochrome bc complex is responsible for oxidizing menaquinol and reducing cytochrome c553 in the electron flow cycle used for phototrophy. However, there is no known electron acceptor for the mobile cytochrome c553 other than the photochemical reaction center. We have, therefore, hypothesized that the cytochrome bc complex is necessary for phototrophy, but unnecessary for chemotrophic growth in the dark. We used a two-step method for CRISPR-based genome editing in Heliobacterium modesticaldum to delete the genes encoding the four major subunits of the cytochrome bc complex. Genotypic analysis verified the deletion of the petCBDA gene cluster encoding the catalytic components of the complex. Spectroscopic studies revealed that re-reduction of cytochrome c553 after flash-induced photo-oxidation was over 100 times slower in the ∆petCBDA mutant compared to the wild-type. Steady-state levels of oxidized P800 (the primary donor of the photochemical reaction center) were much higher in the ∆petCBDA mutant at every light level, consistent with a limitation in electron flow to the reaction center. The ∆petCBDA mutant was unable to grow phototrophically on acetate plus CO2 but could grow chemotrophically on pyruvate as a carbon source similar to the wild-type strain in the dark. The mutants could be complemented by reintroduction of the petCBDA gene cluster on a plasmid expressed from the clostridial eno promoter.

Using the Endogenous CRISPR-Cas System of Heliobacterium modesticaldum To Delete the Photochemical Reaction Center Core Subunit Gene.

In Heliobacterium modesticaldum, as in many Firmicutes, deleting genes by homologous recombination using standard techniques has been extremely difficult. The cells tend to integrate the introduced plasmid into the chromosome by a single recombination event rather than perform the double recombination required to replace the targeted locus. Transformation with a vector containing only a homologous recombination template for replacement of the photochemical reaction center gene pshA produced colonies with multiple genotypes, rather than a clean gene replacement. To address this issue, we required an additional means of selection to force a clean gene replacement. In this study, we report the genetic structure of the type I-A and I-E CRISPR-Cas systems from H. modesticaldum, as well as methods to leverage the type I-A system for genome editing. In silico analysis of the CRISPR spacers revealed a potential consensus protospacer adjacent motif (PAM) required for Cas3 recognition, which was then tested using an in vivo interference assay. Introduction of a homologous recombination plasmid that carried a miniature CRISPR array targeting sequences in pshA (downstream of a naturally occurring PAM sequence) produced nonphototrophic transformants with clean replacements of the pshA gene with ∼80% efficiency. Mutants were confirmed by PCR, sequencing, optical spectroscopy, and growth characteristics. This methodology should be applicable to any genetic locus in the H. modesticaldum genome.IMPORTANCE The heliobacteria are the only phototrophic members of the largely Gram-positive phylum Firmicutes, which contains medically and industrially important members, such as Clostridium difficile and Clostridium acetobutylicum Heliobacteria are of interest in the study of photosynthesis because their photosynthetic system is unique and the simplest known. Since their discovery in the early 1980s, work on the heliobacteria has been hindered by the lack of a genetic transformation system. The problem of introducing foreign DNA into these bacteria has been recently rectified by our group; however, issues still remained for efficient genome editing. The significance of this work is that we have characterized the endogenous type I CRISPR-Cas system in the heliobacteria and leveraged it to assist in genome editing. Using the CRISPR-Cas system allowed us to isolate transformants with precise replacement of the pshA gene encoding the main subunit of the photochemical reaction center.

A Molecular Biology Tool Kit for the Phototrophic Firmicute Heliobacterium modesticaldum.

The heliobacteria are members of the bacterial order Clostridiales and form the only group of phototrophs in the phylum Firmicutes Several physiological and metabolic characteristics make them an interesting subject of investigation, including their minimalist photosynthetic system, nitrogen fixation abilities, and ability to reduce toxic metals. While the species Heliobacterium modesticaldum is an excellent candidate as a model system for the family Heliobacteriaceae, since an annotated genome and transcriptomes are available, studies in this organism have been hampered by the lack of genetic tools. We adapted techniques for genetic manipulation of related clostridial species for use with H. modesticaldum Five heliobacterial DNA methyltransferase genes were expressed in an Escherichia coli strain engineered as a conjugative plasmid donor for broad-host-range plasmids. Premethylation of the shuttle vectors before conjugation into H. modesticaldum is absolutely required for production of transconjugant colonies. The introduced shuttle vectors are maintained stably and can be recovered using a modified minipreparation procedure developed to inhibit endogenous DNase activity. Furthermore, we describe the formulation of various growth media, including a defined medium for metabolic studies and isolation of auxotrophic mutants.IMPORTANCE Heliobacteria are anoxygenic phototrophic bacteria with the simplest known photosynthetic apparatus. They are unique in using bacteriochlorophyll g as their main pigment and lacking a peripheral antenna system. Until now, research on this organism has been hampered by the lack of a genetic transformation system. Without such a system, gene knockouts, site-directed mutations, and gene expression studies cannot be performed to help us further understand or manipulate the organism. Here we report the genetic transformation of a heliobacterium, which should enable future genetic studies in this unique phototrophic organism.

Light-driven quinone reduction in heliobacterial membranes.

Photosynthetic reaction centers (RCs) evolved > 3 billion years ago and have diverged into Type II RCs reducing quinones and Type I RCs reducing soluble acceptors via iron-sulfur clusters. Photosystem I (PSI), the exemplar Type I RC, uses modified menaquinones as intermediate electron transfer cofactors, but it has been controversial if the Type I RC of heliobacteria (HbRC) uses its two bound menaquinones in the same way. The sequence of the quinone-binding site in PSI is not conserved in the HbRC, and the recently solved crystal structure of the HbRC does not reveal a quinone in the analogous site. We found that illumination of heliobacterial membranes resulted in reduction of menaquinone to menaquinol, suggesting that the HbRC can perform a function thought restricted to Type II RCs. Experiments on membranes and live cells are consistent with the hypothesis that the HbRC preferentially reduces soluble electron acceptors (e.g., ferredoxins) in low light, but switches to reducing lipophilic quinones in high light, when the soluble acceptor pool becomes full. Thus, the HbRC may represent a functional evolutionary intermediate between PSI and the Type II RCs.

Double reduction of plastoquinone to plastoquinol in photosystem 1.

In Photosystem 1 (PS1), phylloquinone (PhQ) acts as a secondary electron acceptor from chlorophyll ec(3) and also as an electron donor to the iron-sulfur cluster F(X). PS1 possesses two virtually equivalent branches of electron transfer (ET) cofactors from P(700) to F(X), and the lifetime of the semiquinone intermediate displays biphasic kinetics, reflecting ET along the two different branches. PhQ in PS1 serves only as an intermediate in ET and is not normally fully reduced to the quinol form. This is in contrast to PS2, in which plastoquinone (PQ) is doubly reduced to plastoquinol (PQH(2)) as the terminal electron acceptor. We purified PS1 particles from the menD1 mutant of Chlamydomonas reinhardtii that cannot synthesize PhQ, resulting in replacement of PhQ by PQ in the quinone-binding pocket. The magnitude of the stable flash-induced P(700)(+) signal of menD1 PS1, but not wild-type PS1, decreased during a train of laser flashes, as it was replaced by a ~30 ns back-reaction from the preceding radical pair (P(700)(+)A(0)(-)). We show that this process of photoinactivation is due to double reduction of PQ in the menD1 PS1 and have characterized the process. It is accelerated at lower pH, consistent with a rate-limiting protonation step. Moreover, a point mutation (PsaA-L722T) in the PhQ(A) site that accelerates ET to F(X) ~2-fold, likely by weakening the sole H-bond to PhQ(A), also accelerates the photoinactivation process. The addition of exogenous PhQ can restore activity to photoinactivated PS1 and confer resistance to further photoinactivation. This process also occurs with PS1 purified from the menB PhQ biosynthesis mutant of Synechocystis PCC 6803, demonstrating that it is a general phenomenon in both prokaryotic and eukaryotic PS1.

The association between guideline-based treatment instructions at the point of discharge and lower 1-year mortality in Medicare patients after acute myocardial infarction: the American College of Cardiology's Guidelines Applied in Practice (GAP) initiative in Michigan.

BACKGROUND: The American College of Cardiology's Guidelines Applied in Practice (GAP) initiative for acute myocardial infarction (AMI) has been shown to increase the use of guideline-based therapies and improve outcomes in patients with AMI. It is unknown whether hospitals that are more successful in using the standard discharge contract--a key component of GAP that emphasizes guideline-based medications, lifestyle modification, and follow-up planning--experience a proportionally greater improvement in patient outcomes. METHODS: Medicare patients treated for AMI in all 33 participating GAP hospitals in Michigan were enrolled. We aggregated the hospitals into 3 tertiles based on the rates of discharge contract use: 0% to 8.4% (tertile 1), >8.4% to 38.0% (tertile 2), and >38.0% to 61.1% (tertile 3). We analyzed 1-year follow-up mortality both pre- and post-GAP and compared the mortality decline post-GAP with discharge contract use according to tertile. RESULTS: There were 1368 patients in the baseline (pre-GAP) cohort and 1489 patients in the post-GAP cohort. After GAP implementation, mortality at 1 year decreased by 1.2% (P = .71), 1.2% (P = .68), and 6.0% (P = .03) for tertiles 1, 2, and 3, respectively. After multivariate adjustment, discharge contract use was significantly associated with decreased 1-year mortality in tertile 2 (odds ratio 0.43, 95% CI 0.22-0.84) and tertile 3 (odds ratio 0.45, 95% CI 0.27-0.75). CONCLUSIONS: Increased hospital utilization of the standard discharge contract as part of the GAP program is associated with decreased 1-year mortality in Medicare patient populations with AMI. Hospital efforts to promote adherence to guideline-based care tools such as the discharge contract used in GAP may result in mortality reductions for their patient populations at 1 year.

Sex differences in the application of evidence-based therapies for the treatment of acute myocardial infarction: the American College of Cardiology's Guidelines Applied in Practice projects in Michigan.

BACKGROUND: Studies have shown that women with acute myocardial infarction (AMI) are less likely to receive evidence-based care compared with men. The American College of Cardiology's AMI Guidelines Applied in Practice (GAP) program has been shown to increase the rates of evidence-based medicine use and reduce mortality in patients with AMI. The objective of this study was to investigate the relative benefits of the GAP program in men and women. METHODS: By using a predesign-postdesign, standard orders, and a discharge tool to improve evidence-based indicator rates and long-term mortality in patients with AMI in Michigan, this study compared the success of GAP in men vs women. Logistic regression was used to develop predictive models for death at 30 days and 1 year in men and women. RESULTS: Use of evidence-based care, including use of beta-blockers and aspirin in men and women at hospital discharge and lipid-lowering agent use in men, was higher in the post-GAP sample (P<.01 for all). Use of the discharge tool promoted by the GAP program was independently protective against death at 1 year in women (adjusted odds ratio, 0.46; 95% confidence interval, 0.27-0.79), and a trend existed for similar results in men (adjusted odds ratio, 0.62; 95% confidence interval, 0.36-1.06). However, the tool was used slightly less often with women (27.9% vs 33.96%; P=.003). CONCLUSIONS: The GAP program increased the use of evidence-based therapies in male and female patients. In addition, the GAP discharge tool may decrease mortality rates at 1 year in patients with AMI; however, the tool was used less often with women. Greater use of the GAP discharge tool in women might narrow the post-MI sex mortality gap.

Guideline-based standardized care is associated with substantially lower mortality in medicare patients with acute myocardial infarction: the American College of Cardiology's Guidelines Applied in Practice (GAP) Projects in Michigan.

OBJECTIVES: We sought to assess the impact of the American College of Cardiology's Guidelines Applied in Practice (GAP) project for acute myocardial infarction (AMI) care, encompassing 33 acute-care hospitals in southeastern Michigan, on rates of mortality in Medicare patients treated in Michigan. BACKGROUND: The GAP project increases the use of evidence-based therapies in patients with AMI. It is unknown whether GAP also can reduce the rate of mortality in patients with AMI. METHODS: Using a before (n = 1,368) and after GAP implementation (n = 1,489) cohort study, 2,857 Medicare patients with AMI were studied to assess the influence of the GAP program on mortality. Multivariate models tested the independent impact of GAP after controlling for other conditions on in-hospital, 30-day, and one-year mortality. RESULTS: Average patient age was 76 years, 48% were women, and 16% represented non-white minorities. The rate of mortality decreased after GAP for each interval studied: hospital, 10.4% versus 13.6%; 30-day, 16.7% versus 21.6%; and one-year, 33.2% versus 38.3%; all p < 0.02. After multivariate adjustment, GAP correlated with a 21% to 26% reduction in mortality, particularly at 30 days (odds ratio of GAP to baseline 0.74; 95% confidence interval [CI] 0.59 to 0.94; p = 0.012) and one year (odds ratio 0.78; 95% CI 0.64 to 0.95; p = 0.013), particularly in the patients for whom a standard discharge tool was used (1-year mortality, odds ratio 0.53; 95% CI 0.36 to 0.76; p = 0.0006). CONCLUSIONS: Embedding AMI guidelines into practice was associated with improved 30-day and one-year mortality. This benefit is most marked when patients are cared for using standardized, evidence-based clinical care tools.

Improved quality of stroke care for hospitalized Medicare beneficiaries in Michigan.

BACKGROUND AND PURPOSE: We reported previously that acute ischemic stroke patients encountered delays in obtaining neuroimaging and receiving thrombolysis, and that deep venous thrombosis prophylaxis was used only in a minority of eligible patients. We investigated whether these and other measures improved after a quality improvement initiative. METHODS: Medicare fee-for-service ischemic stroke and transient ischemic attack discharges in 136 acute care hospitals in Michigan were identified by International Classification of Diseases, 9th Revision, Clinical Modification codes. Only patients with stroke symptoms persisting for >1 hour and present on arrival were included in the analysis. Seven quality indicators were abstracted from chart review at baseline (discharges between July 1, 1998, and June 30, 1999) and at remeasurement (discharges between January 1, 2001, and June 30, 2001) after an intensive quality improvement initiative throughout Michigan hospitals. Quality indicators were compared at baseline and remeasurement. RESULTS: Indicators of care were determined in 5146 patients at baseline and 4980 patients on remeasurement. Four quality-of-care indicators showed significant improvement on remeasurement: antithrombotic prescribed at discharge (81.9 baseline versus 83.7% remeasurement; P=0.026), avoidance of sublingual nifedipine in patients with acute ischemic stroke (97.1 versus 99.7%; P<0.0001), documentation of a computed tomography (CT)/MRI during hospitalization (98.0 versus 99.1%; P=0.024), and appropriate deep venous thrombosis prophylaxis (13.8 versus 26.9%; P<0.0001). Time to CT/MRI did not significantly change, but time to thrombolysis improved (113 versus 88.5 minutes; P=0.045). CONCLUSIONS: Improvement occurred in several indicators of quality of care in Michigan Medicare beneficiaries presenting with acute stroke symptoms.

Differences in quality of care among patients hospitalized with atrial fibrillation as primary or secondary cause for admission.

OBJECTIVE: Several factors have been linked to the variation in the quality of care for patients with atrial fibrillation (AF). Whether hospitalization primarily for AF (primary diagnosis of AF) as opposed to another primary diagnosis but having concomitant AF (secondary diagnosis of AF) impacts quality of care for AF is not known. Accordingly, we sought to evaluate the differences in quality of care of Medicare patients admitted with primary diagnosis versus secondary diagnosis of AF. DESIGN AND SETTING: We studied a random sample of Medicare fee-for-service discharges from Michigan's acute care hospitals over a 1-year period with a primary or secondary diagnosis of AF (ICD-9-CM 427.31). Main outcome measure. Warfarin use at the time of discharge. RESULTS: Of 5993 patients in the study, 772 had a primary diagnosis of AF and 5221 had a secondary diagnosis of AF. Patients with a secondary diagnosis of AF were older, more likely to be male, and less likely to be hypertensive. Patients with a secondary diagnosis of AF 'ideal' for anticoagulation (n = 1648) were less likely to receive warfarin compared with 'ideal' patients with primary diagnosis of AF (n = 363) (52.6% versus. 59.8%, P < 0.001). Adherence to test indicators was lower in patients with secondary diagnosis of AF. CONCLUSION: Secondary diagnosis of AF rather than AF as a primary diagnosis appears to account for most Medicare patients with AF admitted to hospitals. Whereas quality of care is lower in patients with secondary diagnosis of AF, opportunity for quality improvement exists for both groups of patients with AF.

Enhancing quality of care for acute myocardial infarction: shifting the focus of improvement from key indicators to process of care and tool use: the American College of Cardiology Acute Myocardial Infarction Guidelines Applied in Practice Project in Michigan: Flint and Saginaw Expansion.

OBJECTIVES: This project evaluated if by focusing on process changes and tool use rather than key indicator rates, the use of evidence-based therapies in patients with acute myocardial infarction (AMI) would increase. BACKGROUND: The use of tools designed to improve quality of care in the American College of Cardiology AMI Guidelines Applied in Practice Pilot Project resulted in improved adherence to evidence-based therapies for patients, but overall, tool use was modest. METHODS: The current project, implemented in five hospitals, was modeled after the previous project, but with greater emphasis on tool use. This allowed early identification of barriers to tool use and strategies to overcome barriers. Main outcome measures were AMI quality indicators in pre-measurement (January 1, 2001 to June 30, 2001) and post-measurement (December 15, 2001 to March 31, 2002) samples. RESULTS: One or more tools were used in 93% of patients (standard orders = 82%, and discharge document = 47%). Tool use was associated with significantly higher adherence to most discharge quality indicator rates with increases in aspirin, angiotensin-converting enzyme inhibitors, and smoking cessation and dietary counseling. Patients undergoing coronary artery bypass grafting (CABG) had low rates of discharge indicators. Patients undergoing percutaneous coronary revascularization were more likely to receive evidence-based therapies. CONCLUSIONS: These data validate the results of the pilot project that quality of AMI care can be improved through the use of guideline-based tools. Identifying and overcoming barriers to tool use led to substantially higher rates of tool use. The low rates of adherence to quality indicators in patients undergoing CABG suggest that these patients should be particularly targeted for quality improvement efforts.

Acute ischemic stroke in hospitalized medicare patients: evaluation and treatment.

BACKGROUND AND PURPOSE: This study describes several quality indicators of care in hospitalized stroke patients in Michigan from 1998 to 1999. SUMMARY OF REPORT: Median times from admission to head CT/MRI (89.5 minutes) and thrombolysis (113 minutes) exceeded recommended guidelines. Deep venous thrombosis prophylaxis was used in only 13.8% of eligible patients. CONCLUSIONS: Timing for brain imaging and acute ischemic stroke symptom onset need to be better documented, along with more provider education for routine deep venous thrombosis prophylaxis.

A rapid-cycle collaborative model to promote guidelines for acute myocardial infarction.

BACKGROUND: This American College of Cardiology (ACC) Acute Myocardial Infarction (AMI) Guidelines Applied in Practice (GAP) collaborative in Michigan represented ACC's third initiative, in partnership with local health care coalitions and the Michigan Peer Review Organization. The GAP Pilot Project formed the basis for this project, which supported caregivers' efforts to improve their processes and consistently apply the evidence-based guidelines for AMI care. THE SOUTHEAST MICHIGAN EXPANSION PROJECT: The Institute for Healthcare Improvement (IHI) Breakthrough Series model of improvement was modified to merge the GAP Pilot Project's design with a rapid-cycle quality improvement model. The collaborative included learning sessions that focused on five phases--planning, tool implementation, monitoring tool use, remeasurement, and results--and on increasing tool use rates in each phase. CONCLUSIONS: Building on the work of two previous efforts, the ACC AMI GAP projects yielded substantial collective knowledge. Developing and fostering a collaborative culture allowed hospital teams to avoid barriers or overcome them successfully based on others' experiences and collectively solve problems, and it shortened the learning curve and accelerated QI.

Taking the national guideline for care of acute myocardial infarction to the bedside: developing the guideline applied in practice (GAP) initiative in Southeast Michigan.

BACKGROUND: The Guideline Applied in Practice (GAP) program was developed in 2000 to improve the quality of care by improving adherence to clinical practice guidelines. For the first GAP project, the American College of Cardiology (ACC) partnered with the Southeast Michigan Quality Forum Cardiovascular Subgroup and the Michigan Peer Review Organization (MPRO) to develop interventions that might facilitate the use of the ACC/AHA Acute Myocardial Infarction (AMI) guideline in the practice setting. Ten Michigan hospitals participated in implementing the project, which began in March 2000. DESIGNING THE PROJECT: The project developed a multifaceted intervention aimed at key players in the care delivery triangle: the physician, nurse, and patient. Intervention components included a project kick-off presentation and dinner, creation and implementation of a customized tool kit, identification and assignment of local nurse and physician opinion leaders, grand rounds site visits, and measurement before and after the intervention. IMPLEMENTING THE PROJECT: The GAP project experience suggests that hospitals are enthusiastic about partnering with ACC to improve quality of care; partners can work together to develop a program for guideline implementation; rapid-cycle implementation is possible with the GAP model; guidelines and quality indicators for AMI are well accepted; and hospitals can adapt the national guideline for care into usable tools focused on physicians, nurses, and patients. DISCUSSION: Important structure and process changes--both of which are required for successful QI efforts--have been demonstrated in this project. Ultimately, the failure or success of this initiative will depend on an indication that the demonstrated improvement in the quality indicators is sustained over time.