bluuebunny/crossref_metadata_2025_split huggingface dataset:
Role of Bacillus subtilis DNA Glycosylase MutM in Counteracting Oxidatively Induced DNA Damage and in Stationary-Phase-Associated Mutagenesis
Authors: Gómez-Marroquín Martha , Vidales Luz E. , Debora Bernardo N. , Santos-Escobar Fernando , Obregón-Herrera Armando , Robleto Eduardo A. , Pedraza-Reyes Mario 
Published: April 2015

ABSTRACT

Reactive oxygen species (ROS) promote the synthesis of the DNA lesion 8-oxo-G, whose mutagenic effects are counteracted in distinct organisms by the DNA glycosylase MutM. We report here that in
Bacillus subtilis
,
mutM
is expressed during the exponential and stationary phases of growth. In agreement with this expression pattern, results of a Western blot analysis confirmed the presence of MutM in both stages of growth. In comparison with cells of a wild-type strain, cells of
B. subtilis
lacking MutM increased their spontaneous mutation frequency to Rif
r
and were more sensitive to the ROS promoter agents hydrogen peroxide and 1,1′-dimethyl-4,4′-bipyridinium dichloride (Paraquat). However, despite MutM's proven participation in preventing ROS-induced-DNA damage, the expression of
mutM
was not induced by hydrogen peroxide, mitomycin C, or NaCl, suggesting that transcription of this gene is not under the control of the RecA, PerR, or σ
B
regulons. Finally, the role of MutM in stationary-phase-associated mutagenesis (SPM) was investigated in the strain
B. subtilis
YB955 (
hisC952 metB5 leuC427
). Results revealed that under limiting growth conditions, a
mutM
knockout strain significantly increased the amount of stationary-phase-associated
his
,
met
, and
leu
revertants produced. In summary, our results support the notion that the absence of MutM promotes mutagenesis that allows nutritionally stressed
B. subtilis
cells to escape from growth-limiting conditions.

IMPORTANCE
The present study describes the role played by a DNA repair protein (MutM) in protecting the soil bacterium
Bacillus subtilis
from the genotoxic effects induced by reactive oxygen species (ROS) promoter agents. Moreover, it reveals that the genetic inactivation of
mutM
allows nutritionally stressed bacteria to escape from growth-limiting conditions, putatively by a mechanism that involves the accumulation and error-prone processing of oxidized DNA bases.
Regulation by the Modulation of Gene Expression Variability
Authors: Dubnau David 
Published: April 2015

ABSTRACT

Classically, transcription is regulated so that the average expression per cell changes, often with a distribution that extends across the population. Roggiani and Goulian (
M. Roggiani and M. Goulian
, J. Bacteriol. 197:1976–1987, 2015, doi:
http://dx.doi.org/10.1128/JB.00074-15
) have shown that this is what happens when the
torCAD
operon of
Escherichia coli
is induced anaerobically by the addition of trimethylamine-
N
-oxide (TMAO). However, when the same inducer is added to aerobically growing cells, only a subset of the cells respond, although the mean expression per cell is similar to that obtained anaerobically. Thus, in the presence of oxygen, the variance but not the expression mean is altered. The regulation of gene expression variance appears to be due to noise in the phosphorelay that governs
torCAD
transcription.
Basal Body Structures Differentially Affect Transcription of RpoN- and FliA-Dependent Flagellar Genes in Helicobacter pylori
Authors: Tsang Jennifer , Hoover Timothy R. 
Published: April 2015

ABSTRACT

Flagellar biogenesis in
Helicobacter pylori
is regulated by a transcriptional hierarchy governed by three sigma factors, RpoD (σ
80
), RpoN (σ
54
), and FliA (σ
28
), that temporally coordinates gene expression with the assembly of the flagellum. Previous studies showed that loss of flagellar protein export apparatus components inhibits transcription of flagellar genes. The FlgS/FlgR two-component system activates transcription of RpoN-dependent genes though an unknown mechanism. To understand better the extent to which flagellar gene regulation is coupled to flagellar assembly, we disrupted flagellar biogenesis at various points and determined how these mutations affected transcription of RpoN-dependent (
flaB
and
flgE
) and FliA-dependent (
flaA
) genes. The MS ring (encoded by
fliF
) is one of the earliest flagellar structures assembled. Deletion of
fliF
resulted in the elimination of RpoN-dependent transcripts and an ∼4-fold decrease in
flaA
transcript levels. FliH is a cytoplasmic protein that functions with the C ring protein FliN to shuttle substrates to the export apparatus. Deletions of
fliH
and genes encoding C ring components (
fliM
and
fliY
) decreased transcript levels of
flaB
and
flgE
but had little or no effect on transcript levels of
flaA
. Transcript levels of
flaB
and
flgE
were elevated in mutants where genes encoding rod proteins (
fliE
and
flgBC
) were deleted, while transcript levels of
flaA
was reduced ∼2-fold in both mutants. We propose that FlgS responds to an assembly checkpoint associated with the export apparatus and that FliH and one or more C ring component assist FlgS in engaging this flagellar structure.

IMPORTANCE
The mechanisms used by bacteria to couple transcription of flagellar genes with assembly of the flagellum are poorly understood. The results from this study identified components of the
H. pylori
flagellar basal body that either positively or negatively affect expression of RpoN-dependent flagellar genes. Some of these basal body proteins may interact directly with regulatory proteins that control transcription of the
H. pylori
RpoN regulon, a hypothesis that can be tested by examining protein-protein interactions
in vitro
.
Recoding of the Vesicular Stomatitis Virus L Gene by Computer-Aided Design Provides a Live, Attenuated Vaccine Candidate
Authors: Wang Bingyin , Yang Chen , Tekes Gergely , Mueller Steffen , Paul Aniko , Whelan Sean P. J. , Wimmer Eckard 
Published: April 2015

ABSTRACT

Codon pair bias (CPB), which has been observed in all organisms, is a neglected genomic phenomenon that affects gene expression. CPB results from synonymous codons that are paired more or less frequently in ORFeomes regardless of codon bias. The effect of an individual codon pair change is usually small, but when it is amplified by large-scale genome recoding, strikingly altered biological phenotypes are observed. The utility of codon pair bias in the development of live attenuated vaccines was recently demonstrated by recodings of poliovirus (a positive-strand RNA virus) and influenza virus (a negative-strand segmented RNA virus). Here, the L gene of vesicular stomatitis virus (VSV), a nonsegmented negative-sense RNA virus, was partially recoded based on codon pair bias. Totals of 858 and 623 silent mutations were introduced into a 5′-terminal segment of the viral L gene (designated L1) to create sequences containing either overrepresented or underrepresented codon pairs, designated L1
sdmax
and L1
min
, respectively. Analysis revealed that recombinant VSV containing the L1
min
sequence could not be recovered, whereas the virus with the sdmax sequence showed a modest level of attenuation in cell culture. More strikingly, in mice the L1
sdmax
virus was almost as immunogenic as the parental strain but highly attenuated. Taken together, these results open a new road to attain a balance between VSV virulence and immunogenicity, which could serve as an example for the attenuation of other negative-strand, nonsegmented RNA viruses.

IMPORTANCE
Vesicular stomatitis virus (VSV) is the prototypic rhabdovirus in the order
Mononegavirales
. A wide range of human pathogens belong to this family. Using a unique computer algorithm and large-scale genome synthesis, we attempted to develop a live attenuated vaccine strain for VSV, which could be used as an antigen delivery platform for humans. Recombinant VSVs with distinct codon pair biases were rationally designed, constructed, and analyzed in both cell culture and an animal model. One such recombinant virus, L1
sdmax
, contained extra overrepresented codon pairs in its L gene open reading frame (ORF) and showed promise as an effective vaccine candidate because of a favorable balance between virulence and immunogenicity. Our study not only contributes to the understanding of the underlying mechanism of codon pair bias but also may facilitate the development of live attenuated vaccines for other viruses in the order
Mononegavirales
.
Distinct Roles of the Repeat-Containing Regions and Effector Domains of the Vibrio vulnificus Multifunctional-Autoprocessing Repeats-in-Toxin (MARTX) Toxin
Authors: Kim Byoung Sik , Gavin Hannah E. , Satchell Karla J. F. 
Published: April 2015

ABSTRACT

Vibrio vulnificus
is a seafood-borne pathogen that destroys the intestinal epithelium, leading to rapid bacterial dissemination and death. The most important virulence factor is the multifunctional-autoprocessing repeats-in-toxin (MARTX) toxin comprised of effector domains in the center region flanked by long repeat-containing regions which are well conserved among MARTX toxins and predicted to translocate effector domains. Here, we examined the role of the repeat-containing regions using a modified
V. vulnificus
MARTX (MARTX
Vv
) toxin generated by replacing all the internal effector domains with β-lactamase (Bla). Bla activity was detected in secretions from the bacterium and also in the cytosol of intoxicated epithelial cells. The modified MARTX
Vv
toxin without effector domains retained its necrotic activity but lost its cell-rounding activity. Further, deletion of the carboxyl-terminal repeat-containing region blocked toxin secretion from the bacterium. Deletion of the amino-terminal repeat-containing region had no effect on secretion but completely abolished translocation and necrosis. Neither secretion nor translocation was affected by enzymatically inactivating the cysteine protease domain of the toxin. These data demonstrate that the amino-terminal and carboxyl-terminal repeat-containing regions of the MARTX
Vv
toxin are necessary and sufficient for the delivery of effector domains and epithelial cell lysis
in vitro
but that effector domains are required for other cytopathic functions. Furthermore, Ca
2+
-dependent secretion of the modified MARTX
Vv
toxin suggests that nonclassical RTX-like repeats found in the carboxyl-terminal repeat-containing region are functionally similar to classical RTX repeats found in other RTX proteins.

IMPORTANCE
Up to 95% of deaths from seafood-borne infections in the United States are due solely to one pathogen,
V. vulnificus
. Among its various virulence factors, the MARTX
Vv
toxin has been characterized as a critical exotoxin for successful pathogenesis of
V. vulnificus
in mouse infection models. Similarly to MARTX toxins of other pathogens, MARTX
Vv
toxin is comprised of repeat-containing regions, central effector domains, and an autoprocessing cysteine protease domain. Yet how each of these regions contributes to essential activities of the toxins has not been fully identified for any of MARTX toxins. Using modified MARTX
Vv
toxin fused with β-lactamase as a reporter enzyme, the portion(s) responsible for toxin secretion from bacteria, effector domain translocation into host cells, rapid host cell rounding, and necrotic host cell death was identified. The results are relevant for understanding how MARTX
Vv
toxin serves as both a necrotic pore-forming toxin and an effector delivery platform.
The Glycerol-Dependent Metabolic Persistence of Pseudomonas putida KT2440 Reflects the Regulatory Logic of the GlpR Repressor
Authors: Nikel Pablo I. , Romero-Campero Francisco J. , Zeidman Joshua A. , Goñi-Moreno Ángel , de Lorenzo Víctor 
Published: April 2015

ABSTRACT

The growth of the soil bacterium
Pseudomonas putida
KT2440 on glycerol as the sole carbon source is characterized by a prolonged lag phase, not observed with other carbon substrates. We examined the bacterial growth in glycerol cultures while monitoring the metabolic activity of individual cells. Fluorescence microscopy and flow cytometry, as well as the analysis of the temporal start of growth in single-cell cultures, revealed that adoption of a glycerol-metabolizing regime was not the result of a gradual change in the whole population but rather reflected a time-dependent bimodal switch between metabolically inactive (i.e., nongrowing) and fully active (i.e., growing) bacteria. A transcriptional Φ(
glpD-gfp
) fusion (a proxy of the glycerol-3-phosphate [G3P] dehydrogenase activity) linked the macroscopic phenotype to the expression of the
glp
genes. Either deleting
glpR
(encoding the G3P-responsive transcriptional repressor that controls the expression of the
glpFKRD
gene cluster) or altering G3P formation (by overexpressing
glpK
, encoding glycerol kinase) abolished the bimodal
glpD
expression. These manipulations eliminated the stochastic growth start by shortening the otherwise long lag phase. Provision of
glpR
in
trans
restored the phenotypes lost in the
ΔglpR
mutant. The prolonged nongrowth regime of
P. putida
on glycerol could thus be traced to the regulatory device controlling the transcription of the
glp
genes. Since the physiological agonist of GlpR is G3P, the arrangement of metabolic and regulatory components at this checkpoint merges a positive feedback loop with a nonlinear transcriptional response, a layout fostering the observed time-dependent shift between two alternative physiological states.

IMPORTANCE
Phenotypic variation is a widespread attribute of prokaryotes that leads,
inter alia
, to the emergence of persistent bacteria, i.e., live but nongrowing members within a genetically clonal population. Persistence allows a fraction of cells to avoid the killing caused by conditions or agents that destroy most growing bacteria (e.g., some antibiotics). Known molecular mechanisms underlying the phenomenon include genetic changes, epigenetic variations, and feedback-based multistability. We show that a prolonged nongrowing state of the bacterial population can be brought about by a distinct regulatory architecture of metabolic genes when cells face specific nutrients (e.g., glycerol).
Pseudomonas putida
may have adopted the resulting carbon source-dependent metabolic bet hedging as an advantageous trait for exploring new chemical and nutritional landscapes. Defeating such naturally occurring adaptive features of environmental bacteria is instrumental in improving the performance of these microorganisms as whole-cell catalysts in a bioreactor setup.
Rapid Genome Assembly and Comparison Decode Intrastrain Variation in Human Alphaherpesviruses
Authors: Parsons Lance R. , Tafuri Yolanda R. , Shreve Jacob T. , Bowen Christopher D. , Shipley Mackenzie M. , Enquist L. W. , Szpara Moriah L. 
Published: April 2015

ABSTRACT

Herpes simplex virus (HSV) is a widespread pathogen that causes epithelial lesions with recurrent disease that manifests over a lifetime. The lifelong aspect of infection results from latent viral infection of neurons, a reservoir from which the virus reactivates periodically. Recent work has demonstrated the breadth of genetic variation in globally distributed HSV strains. However, the amount of variation or capacity for mutation within one strain has not been well studied. Here we developed and applied a streamlined new approach for assembly and comparison of large DNA viral genomes such as HSV-1. This viral genome assembly (VirGA) workflow incorporates a combination of
de novo
assembly, alignment, and annotation strategies to automate the generation of draft genomes for large viruses. We applied this approach to quantify the amount of variation between clonal derivatives of a common parental virus stock. In addition, we examined the genetic basis for syncytial plaque phenotypes displayed by a subset of these strains. In each of the syncytial strains, we found an identical DNA change, affecting one residue in the gB (UL27) fusion protein. Since these identical mutations could have appeared after extensive
in vitro
passaging, we applied the VirGA sequencing and comparison approach to two clinical HSV-1 strains isolated from the same patient. One of these strains was syncytial upon first culturing; its sequence revealed the same gB mutation. These data provide insight into the extent and origin of genome-wide intrastrain HSV-1 variation and present useful methods for expansion to
in vivo
patient infection studies.

IMPORTANCE
Herpes simplex virus (HSV) infects more than 70% of adults worldwide, causing epithelial lesions and recurrent disease that manifests over a lifetime. Prior work has demonstrated that HSV strains vary from country to country and between individuals. However, the amount of variation within one strain has not been well studied. To address this, we developed a new approach for viral genome assembly (VirGA) and analysis. We used this approach to quantify the amount of variation between sister clones of a common parental virus stock and to determine the basis of a unique fusion phenotype displayed by several variants. These data revealed that while sister clones of one HSV stock are more than 98% identical, these variants harbor enough genetic differences to change their observed characteristics. Comparative genomics approaches will allow us to explore the impacts of viral inter- and intrastrain diversity on drug and vaccine efficacy.
Which determinants predict tibiofemoral and patellofemoral osteoarthritis after anterior cruciate ligament injury? A systematic review
Authors: van Meer Belle L , Meuffels Duncan E , van Eijsden Wilbert A , Verhaar Jan A N , Bierma-Zeinstra Sita M A , Reijman Max 
Published: April 2015

BackgroundAnterior cruciate ligament (ACL) injury is an important risk factor for development of knee osteoarthritis (OA). To identify those ACL injured patients at increased risk for knee OA, it is necessary to understand risk factors for OA.AimTo summarise the evidence for determinants of (1) tibiofemoral OA and (2) patellofemoral OA in ACL injured patients.MethodsMEDLINE, EMBASE, Web of Science and CINAHL databases were searched up to 20 December 2013. Additionally, reference lists of eligible studies were manually and independently screened by two reviewers. 2348 studies were assessed for the following main inclusion criteria: ≥20 patients; ACL injured patients treated operatively or non-operatively; reporting OA as outcome; description of relationship between OA outcome and determinants; and a follow-up period ≥2 years. Two reviewers extracted the data, assessed the risk of bias and performed a best-evidence synthesis.ResultsSixty-four publications were included and assessed for quality. Two studies were classified as low risk of bias. Medial meniscal injury/meniscectomy showed moderate evidence for influencing OA development (tibiofemoral OA and compartment unspecified). Lateral meniscal injury/meniscectomy showed moderate evidence for no relationship (compartment unspecified), as did time between injury and reconstruction (tibiofemoral and patellofemoral OA).ConclusionsMedial meniscal injury/meniscectomy after ACL rupture increased the risk of OA development. In contrast, it seems that lateral meniscal injury/meniscectomy has no relationship with OA development. Our results suggest that time between injury and reconstruction does not influence patellofemoral and tibiofemoral OA development. Many determinants showed conflicting and limited evidence and no determinant showed strong evidence.
Practice guidelines for sedation and analgesia management of critically ill children: a pilot study evaluating guideline impact and feasibility in the PICU
Authors: Keogh Samantha J , Long Debbie A , Horn Desley V 
Published: April 2015

AimsThe aim of this study was to develop and implement guidelines for sedation and analgesia management in the paediatric intensive care unit (PICU) and evaluate the impact, feasibility and acceptability of these as part of a programme of research in this area and as a prelude to future trial work.MethodThis pilot study used a pre–post design using a historical control.SettingTwo PICUs at different hospitals in an Australian metropolitan city.ParticipantsPatients admitted to the PICU and ventilated for ≥24 h, aged more than 1 month and not admitted for seizure management or terminal care.InterventionGuidelines for sedation and analgesia management for critically ill children including algorithm and assessment tools.Outcome variablesIn addition to key outcome variables (ventilation time, medication dose and duration, length of stay), feasibility outcomes data (recruitment, data collection, safety) were evaluated. Guideline adherence was assessed through chart audit and staff were surveyed about merit and the use of guidelines.ResultsThe guidelines were trialled for a total of 12 months on 63 patients and variables compared with the historical control group (n=75). Analysis revealed differences in median Morphine infusion duration between groups (pretest 3.63 days (87 h) vs post-test 2.83 days (68 h), p=0.05) and maximum doses (pretest 120 μg/kg/h vs post-test 97.5 μg/kg/h) with no apparent change to ventilation duration. Chart audit revealed varied use of tools, but staff were positive about the guidelines and their use in practice.ConclusionsThe sedation guidelines impacted on the duration and dosage of agents without any apparent impact on ventilation duration or length of stay. Furthermore, the guidelines appeared to be feasible and acceptable in clinical practice. The results of the study have laid the foundation for follow-up studies in withdrawal from sedation, point prevalence and longitudinal studies of sedation practices as well as drug trial work.
Educational interventions for improving control of blood pressure in patients with hypertension: a systematic review protocol
Authors: Ribeiro Cibele D , Resqueti Vanessa R , Lima Íllia , Dias Fernando A L , Glynn Liam , Fregonezi Guilherme A F 
Published: April 2015

IntroductionThe aim of this review is to evaluate the effectiveness of educational interventions on improving the control of blood pressure in patients with hypertension.MethodsRandomised controlled trials including patients over 18 years of age, regardless of sex and ethnicity, with a diagnosis of hypertension (either treated or not treated with antihypertensive medications) will be assessed in our analysis. We will electronically search four databases: MEDLINE, CINAHL, PEDro and ScienceDirect. There will be no language restrictions in the search for studies. The data will be extracted independently by two authors using predefined criteria. Disagreements will be resolved between the authors. The risk of bias will be assessed using the Cochrane risk of bias tool. After searching and screening of the studies, we will run a meta-analysis of the included randomised controlled trials. We will summarise the results as risk ratio for dichotomous data and mean differences for continuous data.Ethics and disseminationThe review will be published in a journal. The findings from the review will also be disseminated electronically and at conference presentations.Trial registration numberPROSPERO CRD4201401071.