Bacteriophage infection has profound effects on bacterial biology. Clustered regular interspaced short palindromic repeats (CRISPRs) and cas (CRISPR-associated) genes are found in most archaea and many bacteria and have been reported to play a role in resistance to bacteriophage infection. We observed that lysogenic infection of Pseudomonas aeruginosa PA14 with bacteriophage DMS3 inhibits biofilm formation and swarming motility, both important bacterial group behaviors. This inhibition requires the CRISPR region in the host. Mutation or deletion of five of the six cas genes and one of the two CRISPRs in this region restored biofilm formation and swarming to DMS3 lysogenized strains. Our observations suggest a role for CRISPR regions in modifying the effects of lysogeny on P. aeruginosa.Bacteriophages are probably best known for their role as tools used to study bacteria. Phages have also served as important models for the study of mechanisms of transcription, recombination, and transposition (8). Bacteriophages also shape microbial populations both by impacting the size and structure of bacterial communities and through the transfer of genetic material between bacterial strains (6, 39, 52). It is estimated that phages can lyse as many as 20% of all bacterial cells daily; therefore, these infectious particles can have a profound impact on the evolution of microbes (53).While some bacteriophage infections are primarily lytic, temperate or lysogenic bacteriophages often integrate into the bacterial genome as a prophage causing a chronic infection of the host bacterium (52). In some cases, genes carried by a bacteriophage confer a new function upon a bacterium, typically not directly related to the phage life cycle, through a process known as lysogenic conversion. Lysogenic conversion likely supports phage survival indirectly by increasing the fitness of the host microbe, thus promoting the continued persistence of the phage genome within the host population. Examples of this phenomenon include the phage-mediated introduction of secreted virulence factors such as cholera toxin (30), altered lipopolysaccharide profile (35), and improved adhesion to epithelial cells (50).Here we report that infection of Pseudomonas aeruginosa PA14 by phage DMS3 results in lysogenized strains unable to form a biofilm or undergo swarming motility-two key group behaviors of this bacterium. Furthermore, we show that the loss of biofilm formation and swarming motility requires clustered regular interspaced short palindromic repeats (CRISPRs) and five of six cas (CRISPR-associated) genes. Our data suggest a complex interaction between microbe and bacteriophage impacts the group behaviors of P. aeruginosa. MATERIALS AND METHODSBacterial and phage culture preparation. Strains and plasmids used in this study are shown in Table 1. Overnight cultures were streaked from glycerol stocks stored at Ϫ80°C onto lysogeny broth (LB) agar (1.5%) and incubated overnight at 37°C to isolated single colonies. These colonies were then used to inoculate plankt...

No abstract

In most well-studied rod-shaped bacteria, peptidoglycan is primarily crosslinked by penicillin-binding proteins (PBPs). However, in mycobacteria, crosslinks formed by L,D-transpeptidases (LDTs) are highly abundant. To elucidate the role of these unusual crosslinks, we characterized Mycobacterium smegmatis cells lacking all LDTs. We find that crosslinks generate by LDTs are required for rod shape maintenance specifically at sites of aging cell wall, a byproduct of polar elongation. Asymmetric polar growth leads to a non-uniform distribution of these two types of crosslinks in a single cell. Consequently, in the absence of LDT-mediated crosslinks, PBP-catalyzed crosslinks become more important. Because of this, Mycobacterium tuberculosis (Mtb) is more rapidly killed using a combination of drugs capable of PBP- and LDT- inhibition. Thus, knowledge about the spatial and genetic relationship between drug targets can be exploited to more effectively treat this pathogen.

Rod-shaped mycobacteria expand from their poles, yet d-amino acid probes label cell wall peptidoglycan in this genus at both the poles and sidewall. We sought to clarify the metabolic fates of these probes. Monopeptide incorporation was decreased by antibiotics that block peptidoglycan synthesis or l,d-transpeptidation and in an l,d-transpeptidase mutant. Dipeptides complemented defects in d-alanine synthesis or ligation and were present in lipid-linked peptidoglycan precursors. Characterizing probe uptake pathways allowed us to localize peptidoglycan metabolism with precision: monopeptide-marked l,d-transpeptidase remodeling and dipeptide-marked synthesis were coincident with mycomembrane metabolism at the poles, septum and sidewall. Fluorescent pencillin-marked d,d-transpeptidation around the cell perimeter further suggested that the mycobacterial sidewall is a site of cell wall assembly. While polar peptidoglycan synthesis was associated with cell elongation, sidewall synthesis responded to cell wall damage. Peptidoglycan editing along the sidewall may support cell wall robustness in pole-growing mycobacteria.

We describe the structure and optimization of the Open Force Field 1.0.0 small molecule force field, code-named Parsley. Parsley uses the SMIRKS-native Open Force Field (SMIRNOFF) parameter assignment formalism in which parameter types are assigned directly by chemical perception, in contrast to traditional atom type-based approaches. This method provides a natural means to incorporate increasingly diverse chemistry without needlessly increasing force field complexity. In this work, we present essentially a full optimization of the valence parameters in the force field. The optimization was carried out with the ForceBalance tool and was informed by reference quantum chemical data that include torsion potential energy profiles, optimized gas-phase structures, and vibrational frequencies. These data were computed and are maintained with QCArchive, an open-source and freely available distributed computing and database software ecosystem. Tests of the resulting force field against compounds and data types outside the training set show improvements in optimized geometries and conformational energetics and demonstrate that Parsley's accuracy for liquid properties is similar to that of other general force fields. <br>

Pituitary surgery is often accompanied by disturbances of osmoregulation, which may result from manipulation or vascular alterations of the neurohypophysis. These disturbances can manifest as postoperative hypernatremia or hyponatremia. Postoperative hypernatremia is typically caused by diabetes insipidus due to deficient antidiuretic hormone (ADH) secretion. Hyponatremia after pituitary surgery is typically due to syndrome of inappropriate antidiuretic hormone secretion (SIADH) and is typically associated with a euvolemic state. There are, however, rare cases of cerebral salt wasting and the associated contraction of extracellular volume that have been reported after pituitary surgery.2 Hyponatremia after pituitary surgery can be profoundly symptomatic with symptoms including headache, dizziness, nausea, and vomiting, which can lead to costly readmissions after otherwise successful elective pituitary surgeries. 3,4,11 Previous studies have suggested tumor size or patient age as risk factors for postoperative hyponatremia after pituitary surgery, 11 but these studies have been based on small sample sizes of 88-150 patients and did not adequately represent the range of typical pathologies operated on during pituitary surgery. Furthermore, prior studies did not focus on the contribution of other factors such as Department of Neurosurgery and California Center for Pituitary Disorders, University of California, San Francisco, CaliforniaObject. Syndrome of inappropriate antidiuretic hormone secretion-induced hyponatremia is a common morbidity after pituitary surgery that can be profoundly symptomatic and cause costly readmissions. The authors calculated the frequency of postoperative hyponatremia after 1045 consecutive operations and determined the efficacy of interventions correcting hyponatremia.Methods. The authors performed a retrospective review of 1045 consecutive pituitary surgeries in the first 946 patients treated since forming a dedicated pituitary center 5 years ago. Patients underwent preoperative and daily inpatient sodium checks, with outpatient checks as needed.Results. Thirty-two patients presented with hyponatremia; 41% of these patients were symptomatic. Postoperative hyponatremia occurred after 165 operations (16%) a mean of 4 days after surgery (range 0-28 days); 19% of operations leading to postoperative hyponatremia were associated with postoperative symptoms (38% involved dizziness and 29% involved nausea/vomiting) and 15% involved readmission for a mean of 5 days (range 1-20 days). In a multivariate analysis including lesion size, age, sex, number of prior pituitary surgeries, surgical approach, pathology, lesion location, and preoperative hypopituitarism, only preoperative hypopituitarism predicted postoperative hyponatremia (p = 0.006). Of patients with preoperative hyponatremia, 59% underwent medical correction preoperatively and 56% had persistent postoperative hyponatremia. The mean correction rates were 0.4 mEq/L/hr (no treatment; n = 112), 0.5 mEq/L/hr (free water restriction; n = 24),...

The available tools for conditional gene expression in Plasmodium falciparum are limited. Here, to enable reliable control of target gene expression, we build a system to efficiently modulate translation. We overcame several problems associated with other approaches for regulating gene expression in P. falciparum. Specifically, our system functions predictably across several native and engineered promoter contexts, and affords control over reporter and native parasite proteins irrespective of their subcellular compartmentalization. Induction and repression of gene expression are rapid, homogeneous, and stable over prolonged periods. To demonstrate practical application of our system, we used it to reveal direct links between antimalarial drugs and their native parasite molecular target. This is an important out come given the rapid spread of resistance, and intensified efforts to efficiently discover and optimize new antimalarial drugs. Overall, the studies presented highlight the utility of our system for broadly controlling gene expression and performing functional genetics in P. falciparum.

Background and Purpose— To identify the specific post-endovascular stroke therapy (EVT) peak systolic blood pressure (SBP) threshold that best discriminates good from bad functional outcomes (a priori hypothesized to be 160 mm Hg), we conducted a prospective, multicenter, cohort study with a prespecified analysis plan. Methods— Consecutive adult patients treated with EVT for an anterior ischemic stroke were enrolled from November 2017 to July 2018 at 12 comprehensive stroke centers accross the United States. All SBP values within 24 hours post-EVT were recorded. Using Youden index, the threshold of peak SBP that best discriminated primary outcome of dichotomized 90-day modified Rankin Scale score (0–2 versus 3–6) was identified. Association of this SBP threshold with the outcomes was quantified using multiple logistic regression. Results— Among 485 enrolled patients (median age, 69 [interquartile range, 57–79] years; 51% females), a peak SBP of 158 mm Hg was associated with the largest difference in the dichotomous modified Rankin Scale score (absolute risk reduction of 19%). Having a peak SBP >158 mm Hg resulted in an increased likelihood of modified Rankin Scale score 3 to 6 (odds ratio, 2.24 [1.52–3.29], P <0.01; adjusted odds ratio, 1.29 [0.81–2.06], P =0.28, after adjustment for prespecified variables). Conclusions— A peak post-EVT SBP of 158 mm Hg was prospectively identified to best discriminate good from bad functional outcome. Those with a peak SBP >158 had an increased likelihood of having a bad outcome in unadjusted, but not in adjusted analysis. The observed effect size was similar to prior studies. This finding should undergo further testing in a future randomized trial of goal-targeted post-EVT antihypertensive treatment.