bluuebunny/crossref_metadata_2025_split huggingface dataset:
The iron–sulfur cluster biosynthesis protein SUFB is required for chlorophyll synthesis, but not phytochrome signaling
Authors: Hu Xueyun , Page Mike T. , Sumida Akihiro , Tanaka Ayumi , Terry Matthew J. , Tanaka Ryouichi 
Published: December 2016

SummaryProteins that contain iron–sulfur (Fe–S) clusters play pivotal roles in various metabolic processes such as photosynthesis and redox metabolism. Among the proteins involved in the biosynthesis of Fe–S clusters in plants, the SUFB subunit of the SUFBCD complex appears to be unique because SUFB has been reported to be involved in chlorophyll metabolism and phytochrome‐mediated signaling. To gain insights into the function of the SUFB protein, we analyzed the phenotypes of two SUFB mutants, laf6 and hmc1, and RNA interference (RNAi) lines with reduced SUFB expression. When grown in the light, the laf6 and hmc1 mutants and the SUFB RNAi lines accumulated higher levels of the chlorophyll biosynthesis intermediate Mg‐protoporphyrin IX monomethylester (Mg‐proto MME), consistent with the impairment of Mg‐proto MME cyclase activity. Both SUFC‐ and SUFD‐deficient RNAi lines accumulated the same intermediate, suggesting that inhibition of Fe‐S cluster synthesis is the primary cause of this impairment. Dark‐grown laf6 seedlings also showed an increase in protoporphyrin IX (Proto IX), Mg‐proto, Mg‐proto MME and 3,8‐divinyl protochlorophyllide a (DV‐Pchlide) levels, but this was not observed in hmc1 or the SUFB RNAi lines, nor was it complemented by SUFB overexpression. In addition, the long hypocotyl in far‐red light phenotype of the laf6 mutant could not be rescued by SUFB overexpression and segregated from the pale‐green SUFB‐deficient phenotype, indicating it is not caused by mutation at the SUFB locus. These results demonstrate that biosynthesis of Fe–S clusters is important for chlorophyll biosynthesis, but that the laf6 phenotype is not due to a SUFB mutation.
In Vivo
Fitness Adaptations of Colistin-Resistant Acinetobacter baumannii Isolates to Oxidative Stress
Authors: Jones Crystal L. , Singh Shweta S. , Alamneh Yonas , Casella Leila G. , Ernst Robert K. , Lesho Emil P. , Waterman Paige E. , Zurawski Daniel V. 
Published: December 2016

ABSTRACT

The loss of fitness in colistin-resistant (CR)
Acinetobacter baumannii
was investigated using longitudinal isolates from the same patient. Early CR isolates were outcompeted by late CR isolates for growth in broth and survival in the lungs of mice. Fitness loss was associated with an increased susceptibility to oxidative stress since early CR strains had reduced
in vitro
survival in the presence of hydrogen peroxide and decreased catalase activity compared to that of late CR and colistin-susceptible (CS) strains.
Nonnucleoside Reverse Transcriptase Inhibitors Reduce HIV-1 Production from Latently Infected Resting CD4
+
T Cells following Latency Reversal
Authors: Zerbato Jennifer M. , Tachedjian Gilda , Sluis-Cremer Nicolas 
Published: December 2016

ABSTRACT

Therapeutic strategies that target the latent HIV-1 reservoir in resting CD4
+
T cells of infected individuals are always administered in the presence of combination antiretroviral therapy. Using a primary cell of HIV-1 latency, we evaluated whether different antiviral drug classes affected latency reversal (as assessed by extracellular virus production) by anti-CD3/CD28 monoclonal antibodies or bryostatin 1. We found that the nonnucleoside reverse transcriptase inhibitors efavirenz and rilpivirine significantly decreased HIV-1 production, by ≥1 log.
Analysis of Ribonucleotide 5′-Triphosphate Analogs as Potential Inhibitors of Zika Virus RNA-Dependent RNA Polymerase by Using Nonradioactive Polymerase Assays
Authors: Lu Gaofei , Bluemling Gregory R. , Collop Paul , Hager Michael , Kuiper Damien , Gurale Bharat P. , Painter George R. , De La Rosa Abel , Kolykhalov Alexander A. 
Published: December 2016

ABSTRACT

Zika virus (ZIKV) is an emerging human pathogen that is spreading rapidly through the Americas and has been linked to the development of microcephaly and to a dramatically increased number of Guillain-Barré syndrome cases. Currently, no vaccine or therapeutic options for the prevention or treatment of ZIKV infections exist. In the study described in this report, we expressed, purified, and characterized full-length nonstructural protein 5 (NS5) and the NS5 polymerase domain (NS5pol) of ZIKV RNA-dependent RNA polymerase. Using purified NS5, we developed an
in vitro
nonradioactive primer extension assay employing a fluorescently labeled primer-template pair. Both purified NS5 and NS5pol can carry out
in vitro
RNA-dependent RNA synthesis in this assay. Our results show that Mn
2+
is required for enzymatic activity, while Mg
2+
is not. We found that ZIKV NS5 can utilize single-stranded DNA but not double-stranded DNA as a template or a primer to synthesize RNA. The assay was used to compare the efficiency of incorporation of analog 5′-triphosphates by the ZIKV polymerase and to calculate their discrimination versus that of natural ribonucleotide triphosphates (rNTPs). The 50% inhibitory concentrations for analog rNTPs were determined in an alternative nonradioactive coupled-enzyme assay. We determined that, in general, 2′-C-methyl- and 2′-C-ethynyl-substituted analog 5′-triphosphates were efficiently incorporated by the ZIKV polymerase and were also efficient chain terminators. Derivatives of these molecules may serve as potential antiviral compounds to be developed to combat ZIKV infection. This report provides the first characterization of ZIKV polymerase and demonstrates the utility of
in vitro
polymerase assays in the identification of potential ZIKV inhibitors.
In Vitro
Activities of MCB3681 and Eight Comparators against Clostridium difficile Isolates with Known Ribotypes and Diverse Geographical Spread
Authors: Freeman Jane , Pilling Sally , Vernon Jonathan , Wilcox Mark H. 
Published: December 2016

ABSTRACT

Treatments for
Clostridium difficile
infection remain limited, despite the introduction of fidaxomicin, and development of new agents is necessary. We determined the
in vitro
susceptibilities of 199 prevalent or emerging
Clostridium difficile
PCR ribotypes to MCB3681, a novel investigational quinolonyl-oxazolidinone, and 8 comparators (metronidazole, vancomycin, fidaxomicin, moxifloxacin, ciprofloxacin, clindamycin, tigecycline, and linezolid). MCB3681 showed good activity against
C. difficile
with no evidence of MCB3681 resistance in isolates showing either moxifloxacin or linezolid resistance or both moxifloxacin and linezolid resistance.
Real-Time Monitoring of
nfxB
Mutant Occurrence and Dynamics in Pseudomonas aeruginosa Biofilm Exposed to Subinhibitory Concentrations of Ciprofloxacin
Authors: Zaborskyte Greta , Andersen Jens Bo , Kragh Kasper Nørskov , Ciofu Oana 
Published: December 2016

ABSTRACT

Biofilm infections caused by
Pseudomonas aeruginosa
are frequently treated with ciprofloxacin (CIP); however, resistance rapidly develops. One of the primary resistance mechanisms is the overexpression of the MexCD-OprJ pump due to a mutation in
nfxB
, encoding the transcriptional repressor of this pump. The aim of this study was to investigate the effect of subinhibitory concentrations of CIP on the occurrence of
nfxB
mutants in the wild-type PAO1 flow cell biofilm model. For this purpose, we constructed fluorescent reporter strains (PAO1 background) with an
mCherry
tag for constitutive red fluorescence and chromosomal transcriptional fusion between the P

mexCD

promoter and
gfp
leading to green fluorescence upon mutation of
nfxB
. We observed a rapid development of
nfxB
mutants by live confocal laser scanning microscopy (CLSM) imaging of the flow cell biofilm (reaching 80 to 90% of the whole population) when treated with 1/10 minimal biofilm inhibitory concentration of CIP for 24 h and 96 h. Based on the observed developmental stages, we propose that
nfxB
mutants emerged
de novo
in the biofilm during CIP treatment from filamentous cells, which might have arisen due to the stress responses induced by CIP. Identical
nfxB
mutations were found in fluorescent colonies from the same flow cell biofilm, especially in 24-h biofilms, suggesting selection and clonal expansion of the mutants during biofilm growth. Our findings point at the significant role of high-enough antibiotic dosages or appropriate combination therapy to avoid the emergence of resistant mutants in biofilms.
FlrA Represses Transcription of the Biofilm-Associated
bpfA
Operon in Shewanella putrefaciens
Authors: Cheng Yuan-Yuan , Wu Chao , Wu Jia-Yi , Jia Hui-Ling , Wang Ming-Yu , Wang Huan-Yu , Zou Si-Min , Sun Rui-Rui , Jia Rong , Xiao Ya-Zhong 
Published: December 2016

ABSTRACT

Manipulation of biofilm formation in
Shewanella
is beneficial for application to industrial and environmental biotechnology. BpfA is an adhesin largely responsible for biofilm formation in many
Shewanella
species. However, the mechanism underlying BpfA production and the resulting biofilm remains vaguely understood. We previously described the finding that BpfA expression is enhanced by DosD, an oxygen-stimulated diguanylate cyclase, under aerobic growth. In the present work, we identify FlrA as a critical transcription regulator of the
bpfA
operon in
Shewanella putrefaciens
CN32 by transposon mutagenesis. FlrA acted as a repressor of the operon promoter by binding to two boxes overlapping the −10 and −35 sites recognized by σ
70
. DosD regulation of the expression of the
bpfA
operon was mediated by FlrA, and cyclic diguanylic acid (c-di-GMP) abolished FlrA binding to the operon promoter. We also demonstrate that FlhG, an accessory protein for flagellum synthesis, antagonized FlrA repression of the expression of the
bpfA
operon. Collectively, this work demonstrates that FlrA acts as a central mediator in the signaling pathway from c-di-GMP to BpfA-associated biofilm formation in
S. putrefaciens
CN32.

IMPORTANCE
Motility and biofilm are mutually exclusive lifestyles, shifts between which are under the strict regulation of bacteria attempting to adapt to the fluctuation of diverse environmental conditions. The FlrA protein in many bacteria is known to control motility as a master regulator of flagellum synthesis. This work elucidates its effect on biofilm formation by controlling the expression of the adhesin BpfA in
S. putrefaciens
CN32 in response to c-di-GMP. Therefore, FlrA plays a dual role in controlling motility and biofilm formation in
S. putrefaciens
CN32. The cooccurrence of
flrA
,
bpfA
, and the FlrA box in the promoter region of the
bpfA
operon in diverse
Shewanella
strains suggests that
bpfA
is a common mechanism that controls biofilm formation in this bacterial species.
Detection of ESKAPE Bacterial Pathogens at the Point of Care Using Isothermal DNA-Based Assays in a Portable Degas-Actuated Microfluidic Diagnostic Assay Platform
Authors: Renner Lars D. , Zan Jindong , Hu Linda I. , Martinez Manuel , Resto Pedro J. , Siegel Adam C. , Torres Clint , Hall Sara B. , Slezak Tom R. , Nguyen Tuan H. , Weibel Douglas B. 
Published: December 2016

ABSTRACT

An estimated 1.5 billion microbial infections occur globally each year and result in ∼4.6 million deaths. A technology gap associated with commercially available diagnostic tests in remote and underdeveloped regions prevents timely pathogen identification for effective antibiotic chemotherapies for infected patients. The result is a trial-and-error approach that is limited in effectiveness, increases risk for patients while contributing to antimicrobial drug resistance, and reduces the lifetime of antibiotics. This paper addresses this important diagnostic technology gap by describing a low-cost, portable, rapid, and easy-to-use microfluidic cartridge-based system for detecting the ESKAPE (
Enterococcus faecium
,
Staphylococcus aureus
,
Klebsiella pneumoniae
,
Acinetobacter baumannii
,
Pseudomonas aeruginosa
, and
Enterobacter
spp.) bacterial pathogens that are most commonly associated with antibiotic resistance. The point-of-care molecular diagnostic system consists of a vacuum-degassed microfluidic cartridge preloaded with lyophilized recombinase polymerase amplification (RPA) assays and a small portable battery-powered electronic incubator/reader. The isothermal RPA assays detect the targeted ESKAPE pathogens with high sensitivity (e.g., a limit of detection of ∼10 nucleic acid molecules) that is comparable to that of current PCR-based assays, and they offer advantages in power consumption, engineering, and robustness, which are three critical elements required for the point-of-care setting.

IMPORTANCE
This paper describes a portable system for rapidly identifying bacteria in resource-limited environments; we highlight the capabilities of the technology by detecting different pathogens within the ESKAPE collection, which cause nosocomial infections. The system is designed around isothermal DNA-based assays housed within an autonomous plastic cartridge that are designed with the end user in mind, who may have limited technological training. Displaying excellent sensitivity and specificity, the assay systems that we demonstrate may enable future diagnoses of bacterial infection to guide the development of effective chemotherapies and may have a role in areas beyond health where rapid detection is valuable, including in industrial processing and manufacturing, food security, agriculture, and water quality testing.
Novel Cadmium Resistance Determinant in Listeria monocytogenes
Authors: Parsons Cameron , Lee Sangmi , Jayeola Victor , Kathariou Sophia 
Published: December 2016

ABSTRACT

Listeria monocytogenes
is a foodborne pathogen that can cause severe disease (listeriosis) in susceptible individuals. It is ubiquitous in the environment and often exhibits resistance to heavy metals. One of the determinants that enables
Listeria
to tolerate exposure to cadmium is the
cadAC
efflux system, with CadA being a P-type ATPase. Three different
cadA
genes (designated
cadA1
to
cadA3
) were previously characterized in
L. monocytogenes
. A novel putative cadmium resistance gene (
cadA4
) was recently identified through whole-genome sequencing, but experimental confirmation for its involvement in cadmium resistance is lacking. In this study, we characterized
cadA4
in
L. monocytogenes
strain F8027, a cadmium-resistant strain of serotype 4b. By screening a mariner-based transposon library of this strain, we identified a mutant with reduced tolerance to cadmium and that harbored a single transposon insertion in
cadA4
. The tolerance to cadmium was restored by genetic complementation with the cadmium resistance cassette (
cadA4C
), and enhanced cadmium tolerance was conferred to two unrelated cadmium-sensitive strains via heterologous complementation with
cadA4C
. Cadmium exposure induced
cadA4
expression, even at noninhibitory levels. Virulence assessments in the
Galleria mellonella
model suggested that a functional
cadA4
suppressed virulence, potentially promoting commensal colonization of the insect larvae. Biofilm assays suggested that
cadA4
inactivation reduced biofilm formation. These data not only confirm
cadA4
as a novel cadmium resistance determinant in
L. monocytogenes
but also provide evidence for roles in virulence and biofilm formation.

IMPORTANCE
Listeria monocytogenes
is an intracellular foodborne pathogen causing the disease listeriosis, which is responsible for numerous hospitalizations and deaths every year. Among the adaptations that enable the survival of
Listeria
in the environment are the abilities to persist in biofilms, grow in the cold, and tolerate toxic compounds, such as heavy metals. Here, we characterized a novel determinant that was recently identified on a larger mobile genetic island through whole-genome sequencing. This gene (
cadA4
) was found to be responsible for cadmium detoxification and to be a divergent member of the Cad family of cadmium efflux pumps. Virulence assessments in a
Galleria mellonella
model suggested that
cadA4
may suppress virulence. Additionally,
cadA4
may be involved in the ability of
Listeria
to form biofilms. Beyond the role in cadmium detoxification, the involvement of
cadA4
in other cellular functions potentially explains its retention and wide distribution in
L. monocytogenes
.
Whole-Genome Analysis of Antimicrobial-Resistant and Extraintestinal Pathogenic Escherichia coli in River Water
Authors: Gomi Ryota , Matsuda Tomonari , Matsumura Yasufumi , Yamamoto Masaki , Tanaka Michio , Ichiyama Satoshi , Yoneda Minoru 
Published: December 2016

ABSTRACT

Contamination of surface waters by antimicrobial-resistant bacteria and pathogenic bacteria is a great concern. In this study, 531
Escherichia coli
isolates obtained from the Yamato River in Japan were evaluated phenotypically for resistance to 25 antimicrobials. Seventy-six isolates (14.3%) were multidrug resistant (MDR), 66 (12.4%) were nonsusceptible to one or two classes of agents, and 389 (73.3%) were susceptible. We performed whole-genome sequencing of selected strains by using Illumina technology. In total, the genome sequences of 155 strains were analyzed for antibiotic resistance determinants and phylogenetic characteristics. More than 50 different resistance determinants, including acquired resistance genes and chromosomal resistance mutations, were detected. Among the sequenced MDR strains (
n
= 66), sequence type 155 (ST155) complex (
n
= 9), ST10 complex (
n
= 9), and ST69 complex (
n
= 7) were prevalent. Among extraintestinal pathogenic
E. coli
(ExPEC) strains (
n
= 58), clinically important clonal groups, namely, ST95 complex (
n
= 18), ST127 complex (
n
= 8), ST12 complex (
n
= 6), ST14 complex (
n
= 6), and ST131 complex (
n
= 6), were prevalent, demonstrating the clonal distribution of environmental ExPEC strains. Typing of the
fimH
(type 1 fimbrial adhesin) gene revealed that ST131 complex strains carried
fimH22
or
fimH41
, and no strains belonging to the
fimH30
subgroup were detected. Fine-scale phylogenetic analysis and virulence gene content analysis of strains belonging to the ST95 complex (one of the major clonal ExPEC groups causing community-onset infections) revealed no significant differences between environmental and clinical strains. The results indicate contamination of surface waters by
E. coli
strains belonging to clinically important clonal groups.

IMPORTANCE
The prevalence of antimicrobial-resistant and pathogenic
E. coli
strains in surface waters is a concern because surface waters are used as sources for drinking water, irrigation, and recreational purposes. In this study, MDR and ExPEC strains in river water were characterized by genomic sequencing and analysis. We detected more than 50 resistance determinants and identified clonal groups specific to MDR and ExPEC strains. This study showed contamination of surface waters by
E. coli
strains belonging to clinically important clonal groups. Overall, this study advances our understanding of environmental MDR and ExPEC strains.
Unexpected Diversity and High Abundance of Putative Nitric Oxide Dismutase (Nod) Genes in Contaminated Aquifers and Wastewater Treatment Systems
Authors: Zhu Baoli , Bradford Lauren , Huang Sichao , Szalay Anna , Leix Carmen , Weissbach Max , Táncsics András , Drewes Jörg E. , Lueders Tillmann 
Published: December 2016

ABSTRACT

It has recently been suggested that oxygenic dismutation of NO into N
2
and O
2
may occur in the anaerobic methanotrophic “
Candidatus
Methylomirabilis oxyfera” and the alkane-oxidizing gammaproteobacterium HdN1. It may represent a new pathway in microbial nitrogen cycling catalyzed by a putative NO dismutase (Nod). The formed O
2
enables microbes to employ aerobic catabolic pathways in anoxic habitats, suggesting an ecophysiological niche space of substantial appeal for bioremediation and water treatment. However, it is still unknown whether this physiology is limited to “
Ca
. Methylomirabilis oxyfera” and HdN1 and whether it can be coupled to the oxidation of electron donors other than alkanes. Here, we report insights into an unexpected diversity and remarkable abundance of
nod
genes in natural and engineered water systems. Phylogenetically diverse
nod
genes were recovered from a range of contaminated aquifers and N-removing wastewater treatment systems. Together with
nod
genes from “
Ca
. Methylomirabilis oxyfera” and HdN1, the novel environmental
nod
sequences formed no fewer than 6 well-supported phylogenetic clusters, clearly distinct from canonical NO reductase (quinol-dependent NO reductase [qNor] and cytochrome
c
-dependent NO reductase [cNor]) genes. The abundance of
nod
genes in the investigated samples ranged from 1.6 × 10
7
to 5.2 × 10
10
copies · g
−1
(wet weight) of sediment or sludge biomass, accounting for up to 10% of total bacterial 16S rRNA gene counts. In essence, NO dismutation could be a much more widespread physiology than currently perceived. Understanding the controls of this emergent microbial capacity could offer new routes for nitrogen elimination or pollutant remediation in natural and engineered water systems.

IMPORTANCE
NO dismutation into N
2
and O
2
is a novel process catalyzed by putative NO dismutase (Nod). To date, only two bacteria, the anaerobic methane-oxidizing bacterium “
Ca
. Methylomirabilis oxyfera” and the alkane-oxidizing gammaproteobacterium HdN1, are known to harbor
nod
genes. In this study, we report efficient molecular tools that can detect and quantify a wide diversity of
nod
genes in environmental samples. A surprisingly high diversity and abundance of
nod
genes were found in contaminated aquifers as well as wastewater treatment systems. This evidence indicates that NO dismutation may be a much more widespread physiology in natural and man-made environments than currently perceived. The molecular tools presented here will facilitate further studies on these enigmatic microbes in the future.