Polynucleobacter necessarius, a model for genome reduction in both free-living and symbiotic bacteria.

We present the complete genomic sequence of the essential symbiont Polynucleobacter necessarius (Betaproteobacteria), which is a valuable case study for several reasons. First, it is hosted by a ciliated protist, Euplotes; bacterial symbionts of ciliates are still poorly known because of a lack of extensive molecular data. Second, the single species P. necessarius contains both symbiotic and free-living strains, allowing for a comparison between closely related organisms with different ecologies. Third, free-living P. necessarius strains are exceptional by themselves because of their small genome size, reduced metabolic flexibility, and high worldwide abundance in freshwater systems. We provide a comparative analysis of P. necessarius metabolism and explore the peculiar features of a genome reduction that occurred on an already streamlined genome. We compare this unusual system with current hypotheses for genome erosion in symbionts and free-living bacteria, propose modifications to the presently accepted model, and discuss the potential consequences of translesion DNA polymerase loss.

Impact of Lowered Inpatient Correctional Bedtime Insulin Dosing on Glycemic Outcomes of Veterans.

Purpose: This study evaluated glycemic outcomes for hospitalized patients after reduction in bedtime correctional insulin dosing. Methods: This was a retrospective, single-center analysis of a protocol change that reduced bedtime correctional insulin scale. Comparable cohorts pre- and post-protocol change were created which included patients who were ordered correctional insulin with at least 1 blood glucose (BG) reading. The primary outcome was number of nocturnal hypoglycemia readings. Secondary outcomes included, but were not limited to, mean fasting BG, BG within various ranges, and length of stay. Results: 3 percent of patients in the post-protocol change group (N = 100) experienced nocturnal hypoglycemia compared to 6% of patients in the pre-change group (N = 100) (P = .507). There were no significant differences in BG ranges <110 mg/dL, <140 mg/dL, 140 to 180 mg/dL, and >180 mg/dL. However, 19% of patients in the post-protocol change group had BG of >250 mg/dL as compared to 9% in the pre-change group (P = .033). Mean fasting BG was higher in the post-protocol change group compared to the pre-change group (156.5 mg/dL vs 139.3 mg/dL [P = .002]), as was hospital length of stay (5.17 vs 4.6 days, [P = .024]). Conclusions: A decreased bedtime correctional insulin scale had mixed results with more patients achieving goal fasting BG but also more patients experiencing BG > 250 mg/dL and longer length of stay. Larger prospective studies are required to evaluate the safety and efficacy of this type of intervention and its long-term impact.

Complete genome sequence of Francisella philomiragia ATCC 25017.

Francisella philomiragia is a saprophytic gammaproteobacterium found only occasionally in immunocompromised individuals and is the nearest neighbor to the causative agent of tularemia and category A select agent Francisella tularensis. To shed insight into the key genetic differences and the evolution of these two distinct lineages, we sequenced the first complete genome of F. philomiragia strain ATCC 25017, which was isolated as a free-living microorganism from water in Bear River Refuge, Utah.

Complete genome sequence of the plant growth-promoting endophyte Burkholderia phytofirmans strain PsJN.

Burkholderia phytofirmans PsJN(T) is able to efficiently colonize the rhizosphere, root, and above-ground plant tissues of a wide variety of genetically unrelated plants, such as potatoes, canola, maize, and grapevines. Strain PsJN shows strong plant growth-promoting effects and was reported to enhance plant vigor and resistance to biotic and abiotic stresses. Here, we report the genome sequence of this strain, which indicates the presence of multiple traits relevant for endophytic colonization and plant growth promotion.

Genome of Ochrobactrum anthropi ATCC 49188 T, a versatile opportunistic pathogen and symbiont of several eukaryotic hosts.

Ochrobactrum anthropi is a common soil alphaproteobacterium that colonizes a wide spectrum of organisms and is being increasingly recognized as an opportunistic human pathogen. Potentially life-threatening infections, such as endocarditis, are included in the list of reported O. anthropi infections. These reports, together with the scant number of studies and the organism's phylogenetic proximity to the highly pathogenic brucellae, make O. anthropi an attractive model of bacterial pathogenicity. Here we report the genome sequence of the type strain O. anthropi ATCC 49188, which revealed the presence of two chromosomes and four plasmids.

Multiple genome sequences reveal adaptations of a phototrophic bacterium to sediment microenvironments.

The bacterial genus Rhodopseudomonas is comprised of photosynthetic bacteria found widely distributed in aquatic sediments. Members of the genus catalyze hydrogen gas production, carbon dioxide sequestration, and biomass turnover. The genome sequence of Rhodopseudomonas palustris CGA009 revealed a surprising richness of metabolic versatility that would seem to explain its ability to live in a heterogeneous environment like sediment. However, there is considerable genotypic diversity among Rhodopseudomonas isolates. Here we report the complete genome sequences of four additional members of the genus isolated from a restricted geographical area. The sequences confirm that the isolates belong to a coherent taxonomic unit, but they also have significant differences. Whole genome alignments show that the circular chromosomes of the isolates consist of a collinear backbone with a moderate number of genomic rearrangements that impact local gene order and orientation. There are 3,319 genes, 70% of the genes in each genome, shared by four or more strains. Between 10% and 18% of the genes in each genome are strain specific. Some of these genes suggest specialized physiological traits, which we verified experimentally, that include expanded light harvesting, oxygen respiration, and nitrogen fixation capabilities, as well as anaerobic fermentation. Strain-specific adaptations include traits that may be useful in bioenergy applications. This work suggests that against a backdrop of metabolic versatility that is a defining characteristic of Rhodopseudomonas, different ecotypes have evolved to take advantage of physical and chemical conditions in sediment microenvironments that are too small for human observation.

Impact of Intersession Course Remediation on NAPLEX/PCOA Scores in an Accelerated Doctor of Pharmacy Program.

Objective. To compare Pharmacy Curriculum Outcomes Assessment (PCOA) scaled scores and North American Pharmacist Licensure Examination (NAPLEX) pass rates in students who completed a two-week intersession remediation or repeated a course with the scaled scores and pass rates of students who did not require any form of didactic remediation and did not have to repeat a course. Methods. Data examined for this study included NAPLEX/PCOA scores, NAPLEX pass/fail status, and remediation history for students at one college of pharmacy. Students from the graduating classes of 2016, 2017, and 2018 were organized into four groups: non-remediation, one course remediation and no repeats, more than one course remediation and no repeats, and one or more course repeats. Differences were analyzed using linear regression, logistic regression, and Pearson correlations. Results. The PCOA scores for students in the remediation groups were significantly lower than scores for students in the non-remediation groups, with a reduction of 37.8 to 50.9 points from the expected non-remediators' score. The NAPLEX scores for students who remediated more than one course or repeated one or more courses were 16-20 points lower compared to students who did not remediate. The likelihood of a student failing the NAPLEX was also not significantly lower for students who remediated one course but was significantly lower for other remediation groups. Conclusion. Although single course remediation in this curricular model appears to have minimal impact on NAPLEX outcomes and may be an acceptable intervention for many students, additional support and interventions may be warranted for students who qualify for remediation in multiple courses and/or for repeating a course.

Impact of a Journal Club Elective Course on Student Learning Measures.

Objective. To assess the impact of a journal club elective course on measures of student's longitudinal performance throughout an accelerated three-year Doctor of Pharmacy (PharmD) curriculum. Methods. Students were separated into two groups (those who did and did not complete a journal club elective). The following primary and secondary student outcomes were assessed using hierarchical linear regression analysis: score on the Pharmacy Curriculum Outcomes Assessment (PCOA), overall course grade in the Pharmacotherapeutics IV course, overall course grade in the Research Design and Literature Evaluation II course, and average grade on acute/ambulatory care advanced pharmacy practice experiences (APPEs). Results. One hundred ninety-seven students were included in the study (73 students who completed the journal club elective and 124 students who did not). After controlling for baseline confounders, enrollment in the journal club elective was associated with students scoring 24.5 points higher on their overall scaled score on the PCOA. Enrollment in the journal club elective also appeared to add 2% to a student's overall grade in each of the courses and APPEs. All results were statistically significant. Conclusion. An elective journal club course can significantly improve multiple objective measures of pharmacy student learning. Components of this course, such as reading primary literature, presenting a journal club, learning from peers, and scaffolding of pharmacotherapeutic concepts are important elements to consider when designing a journal club curriculum.

Complete genome sequence of Nitrosospira multiformis, an ammonia-oxidizing bacterium from the soil environment.

The complete genome of the ammonia-oxidizing bacterium Nitrosospira multiformis (ATCC 25196(T)) consists of a circular chromosome and three small plasmids totaling 3,234,309 bp and encoding 2,827 putative proteins. Of the 2,827 putative proteins, 2,026 proteins have predicted functions and 801 are without conserved functional domains, yet 747 of these have similarity to other predicted proteins in databases. Gene homologs from Nitrosomonas europaea and Nitrosomonas eutropha were the best match for 42% of the predicted genes in N. multiformis. The N. multiformis genome contains three nearly identical copies of amo and hao gene clusters as large repeats. The features of N. multiformis that distinguish it from N. europaea include the presence of gene clusters encoding urease and hydrogenase, a ribulose-bisphosphate carboxylase/oxygenase-encoding operon of distinctive structure and phylogeny, and a relatively small complement of genes related to Fe acquisition. Systems for synthesis of a pyoverdine-like siderophore and for acyl-homoserine lactone were unique to N. multiformis among the sequenced genomes of ammonia-oxidizing bacteria. Gene clusters encoding proteins associated with outer membrane and cell envelope functions, including transporters, porins, exopolysaccharide synthesis, capsule formation, and protein sorting/export, were abundant. Numerous sensory transduction and response regulator gene systems directed toward sensing of the extracellular environment are described. Gene clusters for glycogen, polyphosphate, and cyanophycin storage and utilization were identified, providing mechanisms for meeting energy requirements under substrate-limited conditions. The genome of N. multiformis encodes the core pathways for chemolithoautotrophy along with adaptations for surface growth and survival in soil environments.

Comparative genome analysis of Burkholderia phytofirmans PsJN reveals a wide spectrum of endophytic lifestyles based on interaction strategies with host plants.

Burkholderia phytofirmans PsJN is a naturally occurring plant-associated bacterial endophyte that effectively colonizes a wide range of plants and stimulates their growth and vitality. Here we analyze whole genomes, of PsJN and of eight other endophytic bacteria. This study illustrates that a wide spectrum of endophytic life styles exists. Although we postulate the existence of typical endophytic traits, no unique gene cluster could be exclusively linked to the endophytic lifestyle. Furthermore, our study revealed a high genetic diversity among bacterial endophytes as reflected in their genotypic and phenotypic features. B. phytofirmans PsJN is in many aspects outstanding among the selected endophytes. It has the biggest genome consisting of two chromosomes and one plasmid, well-equipped with genes for the degradation of complex organic compounds and detoxification, e.g., 24 glutathione-S-transferase (GST) genes. Furthermore, strain PsJN has a high number of cell surface signaling and secretion systems and harbors the 3-OH-PAME quorum-sensing system that coordinates the switch of free-living to the symbiotic lifestyle in the plant-pathogen R. solanacearum. The ability of B. phytofirmans PsJN to successfully colonize such a wide variety of plant species might be based on its large genome harboring a broad range of physiological functions.

Evaluation of the effectiveness of cholecalciferol in long-term care elderly patients with hypovitaminosis D.

OBJECTIVE: To study the efficacy and safety of cholecalciferol (vitamin D3) 2,000 units daily for 12 weeks in elderly patients with hypovitaminosis D. Secondarily, to postulate causes for those patients who did not return to normal 25-hydroxy vitamin D (25-OHD) levels. DESIGN: One-group, pretest post-test. Retrospective chart review. Data collected from Computerized Patient Record System. SETTING: Long-term care facility at Central Arkansas Veterans Healthcare System. PATIENTS, PARTICIPANTS: Potential study subjects were elderly patients (≥ 65 years of age) treated with total daily dose of cholecalciferol 2,000 units daily by mouth, for at least 12 weeks' duration period between July 1, 2007, and July 31, 2009, with documented 25-OHD levels before and after treatment. INTERVENTIONS: None. MAIN OUTCOME MEASURES: Efficacy of cholecalciferol was assessed by categorizing patients as responders (25-OHD levels ≥ 30 ng/mL) and nonresponders (25-OHD levels < 30 ng/mL). Safety of vitamin D3 was determined by measuring prevalence of hypercalcemia. Patient demographics, disease states, and medications that can affect 25-OHD levels between responders and nonresponders of vitamin D treatment were assessed. RESULTS: 24 patients were included in analysis. 58.3% (14) of patients responded to cholecalciferol treatment and 41.7% (10) of patients did not. The mean 25-OHD level after treatment was 38.7 ng/mL in responders and 26.4 ng/mL in non responders. Twelve patients (50%) developed hypercalcemia. CONCLUSION: The present study has shown that some long-term care elderly patients respond to three months of vitamin D supplementation. The reason why some patients did not respond cannot be determined from this study.

Cytochromes P4501 (CYP1): catalytic activities and inducibility by diesel exhaust particle extract and benzo[a]pyrene in intact human lung ex vivo.

Catalytic activities of CYP1A1, CYP1A2 and CYP1B1, and inducibility of the activities were studied in intact human lung samples (from 23 human subjects) ex vivo. The activities [as measured by ethoxyresorufin O-deethylase (EROD), methoxyresorufin O-demethylase (MROD), and 3-cyano-7-ethoxycoumarin O-deethylase (CECOD)] were present in the lungs and were induced by benzo[a]pyrene (B[a]P) and diesel exhaust particle extract (DEE) but with extensive inter-subject variability. All three activities were substantially inhibited (>or=75%) by the CYP1 inhibitor alpha-naphthoflavone, whereas only MROD and CECOD were substantially inhibited by the CYP1A2-preferential inhibitor fluvoxamine. None of the three activities was substantially inhibited by the CYP1B1-preferential inhibitor tetramethoxystilbene, indicating lack of involvement of CYP1B1 in the activities in the intact lung. CYP1A1 and CYP1A2 proteins were present in the lungs (by western blot analysis), also with extensive inter-subject variability, and were induced by B[a]P or DEE more extensively than by the combination of B[a]P and DEE (B[a]P+DEE). CYP1B1 was also present in the lungs and its level varied extensively between subjects. In contrast with CYP1A and CYP1A2 levels, CYP1B1 level was not significantly altered by B[a]P or DEE treatment and was diminished more extensively by treatment with B[a]P+DEE. The findings point to the potential usefulness of the intact lung for assessing in situ xenobiotic biotransformation reactions as well as the CYP1 specificity of the reactions ex vivo. The findings also suggest MROD and CECOD as potential markers of CYP1A2 activity in the intact lung.

Whole-genome analysis of the methyl tert-butyl ether-degrading beta-proteobacterium Methylibium petroleiphilum PM1.

Methylibium petroleiphilum PM1 is a methylotroph distinguished by its ability to completely metabolize the fuel oxygenate methyl tert-butyl ether (MTBE). Strain PM1 also degrades aromatic (benzene, toluene, and xylene) and straight-chain (C(5) to C(12)) hydrocarbons present in petroleum products. Whole-genome analysis of PM1 revealed an approximately 4-Mb circular chromosome and an approximately 600-kb megaplasmid, containing 3,831 and 646 genes, respectively. Aromatic hydrocarbon and alkane degradation, metal resistance, and methylotrophy are encoded on the chromosome. The megaplasmid contains an unusual t-RNA island, numerous insertion sequences, and large repeated elements, including a 40-kb region also present on the chromosome and a 29-kb tandem repeat encoding phosphonate transport and cobalamin biosynthesis. The megaplasmid also codes for alkane degradation and was shown to play an essential role in MTBE degradation through plasmid-curing experiments. Discrepancies between the insertion sequence element distribution patterns, the distributions of best BLASTP hits among major phylogenetic groups, and the G+C contents of the chromosome (69.2%) and plasmid (66%), together with comparative genome hybridization experiments, suggest that the plasmid was recently acquired and apparently carries the genetic information responsible for PM1's ability to degrade MTBE. Comparative genomic hybridization analysis with two PM1-like MTBE-degrading environmental isolates (approximately 99% identical 16S rRNA gene sequences) showed that the plasmid was highly conserved (ca. 99% identical), whereas the chromosomes were too diverse to conduct resequencing analysis. PM1's genome sequence provides a foundation for investigating MTBE biodegradation and exploring the genetic regulation of multiple biodegradation pathways in M. petroleiphilum and other MTBE-degrading beta-proteobacteria.

Whole-genome analysis of the ammonia-oxidizing bacterium, Nitrosomonas eutropha C91: implications for niche adaptation.

Analysis of the structure and inventory of the genome of Nitrosomonas eutropha C91 revealed distinctive features that may explain the adaptation of N. eutropha-like bacteria to N-saturated ecosystems. Multiple gene-shuffling events are apparent, including mobilized and replicated transposition, as well as plasmid or phage integration events into the 2.66 Mbp chromosome and two plasmids (65 and 56 kbp) of N. eutropha C91. A 117 kbp genomic island encodes multiple genes for heavy metal resistance, including clusters for copper and mercury transport, which are absent from the genomes of other ammonia-oxidizing bacteria (AOB). Whereas the sequences of the two ammonia monooxygenase and three hydroxylamine oxidoreductase gene clusters in N. eutropha C91 are highly similar to those of Nitrosomonas europaea ATCC 19718, a break of synteny in the regions flanking these clusters in each genome is evident. Nitrosomonas eutropha C91 encodes four gene clusters for distinct classes of haem-copper oxidases, two of which are not found in other aerobic AOB. This diversity of terminal oxidases may explain the adaptation of N. eutropha to environments with variable O(2) concentrations and/or high concentrations of nitrogen oxides. As with N. europaea, the N. eutropha genome lacks genes for urease metabolism, likely disadvantaging nitrosomonads in low-nitrogen or acidic ecosystems. Taken together, this analysis revealed significant genomic variation between N. eutropha C91 and other AOB, even the closely related N. europaea, and several distinctive properties of the N. eutropha genome that are supportive of niche specialization.

Amaranth decoloration by Trametes versicolor in a rotating biological contacting reactor.

Sequential batch and continuous operation of a rotating biological contacting (RBC) reactor and the effects of dissolved oxygen on the decoloration of amaranth by Trametes versicolor were evaluated. Amaranth belongs to the group of azo dyes which are potential carcinogens and/or mutagens that can be transformed into toxic aryl amines under anaerobic conditions. Cultivation of T. versicolor in a stirred tank reactor was found to be unsuitable for amaranth decoloration due to significant biomass fouling and increase in medium viscosity. Assuming that decoloration follows first-order kinetics, amaranth was decolorized more rapidly when T. versicolor was immobilized on jute twine in a RBC reactor operated either in a sequential batch (k=0.25 h(-1)) or in a continuous (0.051 h(-1)) mode compared to a stirred tank reactor (0.015 h(-1)). Oxygen was found to be essential for decoloration with the highest decoloration rates occurring at oxygen saturation. Although longer retention times resulted in more decoloration when the RBC was operated in the continuous mode (about 33% amaranth decoloration), sequential batch operation gave better results (>95%) under similar nutrient conditions. Our data indicate that the fastest decoloration should occur in the RBC using nitrogen-free Kirk's medium with 1 g/l glucose in sequential batch operation at rotational speeds and/or aeration rates which maintain oxygen saturation in the liquid phase.