bluuebunny/crossref_metadata_2025_split huggingface dataset:
Methanogens Are Major Contributors to Nitrogen Fixation in Soils of the Florida Everglades
Authors: Bae Hee-Sung , Morrison Elise , Chanton Jeffrey P. , Ogram Andrew 
Published: January 2018

ABSTRACT

The objective of this study was to investigate the interaction of the nitrogen (N) cycle with methane production in the Florida Everglades, a large freshwater wetland. This study provides an initial analysis of the distribution and expression of N-cycling genes in Water Conservation Area 2A (WCA-2A), a section of the marsh that underwent phosphorus (P) loading for many years due to runoff from upstream agricultural activities. The elevated P resulted in increased primary productivity and an N limitation in P-enriched areas. Results from quantitative real-time PCR (qPCR) analyses indicated that the N cycle in WCA-2A was dominated by
nifH
and
nirK
/
S
, with an increasing trend in copy numbers in P-impacted sites. Many
nifH
sequences (6 to 44% of the total) and
nifH
transcript sequences (2 to 49%) clustered with the methanogenic
Euryarchaeota
, in stark contrast to the proportion of core gene sequences representing
Archaea
(≤0.27% of SSU rRNA genes) for the WCA-2A microbiota. Notably, archaeal
nifH
gene transcripts were detected at all sites and comprised a significant proportion of total
nifH
transcripts obtained from the unimpacted site, indicating that methanogens are actively fixing N
2
. Laboratory incubations with soils taken from WCA-2A produced
nifH
transcripts with the production of methane from H
2
plus CO
2
and acetate as electron donors and carbon sources. Methanogenic N
2
fixation is likely to be an important, although largely unrecognized, route through which fixed nitrogen enters the anoxic soils of the Everglades and may have significant relevance regarding methane production in wetlands.

IMPORTANCE
Wetlands are the most important natural sources of the greenhouse gas methane, and much of that methane emanates from (sub)tropical peatlands. Primary productivity in these peatlands is frequently limited by the availability of nitrogen or phosphorus; however, the response to nutrient limitations of microbial communities that control biogeochemical cycling critical to ecosystem function may be complex and may be associated with a range of processes, including methane production. We show that many, if not most, of the methanogens in the peatlands of the Florida Everglades possess the
nifH
gene and actively express it for N
2
fixation coupled with methanogenesis. These findings indicate that archaeal N
2
fixation would play crucial role in methane emissions and overall N cycle in subtropical wetlands suffering N limitation.
Novel Cell Wall Hydrolase CwlC from Bacillus thuringiensis Is Essential for Mother Cell Lysis
Authors: Chen Xiaomin , Gao Tantan , Peng Qi , Zhang Jie , Chai Yunrong , Song Fuping 
Published: January 2018

ABSTRACT

In this study, a sporulation-specific gene (tentatively named
cwlC
) involved in mother cell lysis in
Bacillus thuringiensis
was characterized. The encoded CwlC protein consists of an N-terminal
N
-acetylmuramoyl-
l
-alanine amidase (Mur
N
Ac-LAA) domain and a C-terminal amidase02 domain. The recombinant histidine-tagged CwlC proteins purified from
Escherichia coli
were able to directly bind to and digest the
B. thuringiensis
cell wall. The CwlC point mutations at the two conserved glutamic acid residues (Glu-24 and Glu-140) shown to be critical for the catalytic activity in homologous amidases resulted in a complete loss of cell wall lytic activity, suggesting that CwlC is an
N
-acetylmuramoyl-
l
-alanine amidase. Results of transcriptional analyses indicated that
cwlC
is transcribed as a monocistronic unit and that its expression is dependent on sporulation sigma factor K (σ
K
). Deletion of
cwlC
completely blocked mother cell lysis during sporulation without impacting the sporulation frequency, Cry1Ac protein production, and insecticidal activity. Taken together, our data suggest that CwlC is an essential cell wall hydrolase for
B. thuringiensis
mother cell lysis during sporulation. Engineered
B. thuringiensis
strains targeting
cwlC
, which allows the crystal inclusion to remain encapsulated in the mother cell at the end of sporulation, may have the potential to become more effective biological control agents in agricultural applications since the crystal inclusion remains encapsulated in the mother cell at the end of sporulation.

IMPORTANCE
Mother cell lysis has been well studied in
Bacillus subtilis
, which involves three distinct yet functionally complementary cell wall hydrolases. In this study, a novel cell wall hydrolase, CwlC, was investigated and found to be essential for mother cell lysis in
Bacillus thuringiensis
. CwlC of
B. thuringiensis
only shows 9 and 21% sequence identity with known
B. subtilis
mother cell hydrolases CwlB and CwlC, respectively, suggesting that mechanisms of mother cell lysis may differ between
B. subtilis
and
B. thuringiensis
. The
cwlC
gene deletion completely blocked the release of spores and crystals from the mother cell without affecting insecticidal activity. This may provide a new effective strategy for crystal encapsulation against UV light inactivation.
Small GTPase Immunity-Associated Proteins Mediate Resistance to Toxoplasma gondii Infection in Lewis Rat
Authors: Kim Chi Yong , Zhang Xuejin , Witola William H. 
Published: January 2018

ABSTRACTRats vary in their susceptibilities toToxoplasma gondiiinfection depending on the rat strain. Compared to theT. gondii-susceptible Brown Norway (BN) rat, the Lewis (LEW) rat is extremely resistant toT. gondii. Thus, these two rat strains are ideal models for elucidating host mechanisms that are important for host resistance toT. gondiiinfection. Therefore, in our efforts to unravel molecular factors directing the protective early innate immune response in the LEW rat, we performed RNA sequencing analysis of the LEW versus BN rat with or withoutT. gondiiinfection. We identified three candidate smallGTPaseimmunity-associatedproteins (GIMAPs) that were upregulated (false discovery rate, 0.05) in the LEW rat in response toT. gondiiinfection. Subsequently, we engineeredT. gondii-susceptible NR8383 rat macrophage cells for overexpression of LEW rat-derived candidate GIMAP 4, 5, and 6. By immunofluorescence analysis we observed that GIMAP 4, 5, and 6 inT. gondii-infected NR8383 cells each colocalized with GRA5, a parasite parasitophorous vacuole membrane (PVM) marker protein, suggesting their translocation to the PVM. Interestingly, overexpression of each candidate GIMAP inT. gondii-infected NR8383 cells induced translocation of LAMP1, a lysosome marker protein, to theT. gondiisurface membrane. Importantly, overexpression of GIMAP 4, 5, or 6 individually inhibited intracellularT. gondiigrowth, with GIMAP 4 having the highest inhibitory effect. Together, our findings indicate that upregulation of GIMAP 4, 5, and 6 contributes to the robust refractoriness of the LEW rat toT. gondiithrough induction of lysosomal fusion to the otherwise nonfusogenic PVM.
Shining the Light on Cyclic di-GMP Dark Matter
Authors: Waters Christopher M. 
Published: January 2018

ABSTRACT

Bacterial cyclic di-GMP signaling networks often consist of dozens of components, and the majority of these components have no observable function. Dahlstrom et al. (J. Bacteriol. 200:e00703-17, 2018,
https://doi.org/10.1128/JB.00703-17
) explored the function of every component of the
Pseudomonas fluorescens
cyclic di-GMP network under 188 different growth conditions and identified activities for 80% of the network. They further demonstrated that multiple mechanisms function in tandem to control the activity of the network in different environments.
Survival of Anaerobic Fe
2+
Stress Requires the ClpXP Protease
Authors: Bennett Brittany D. , Redford Kaitlyn E. , Gralnick Jeffrey A. 
Published: January 2018

ABSTRACT

Shewanella oneidensis
strain MR-1 is a versatile bacterium capable of respiring extracellular, insoluble ferric oxide minerals under anaerobic conditions. The respiration of iron minerals results in the production of soluble ferrous ions, which at high concentrations are toxic to living organisms. It is not fully understood how Fe
2+
is toxic to cells anaerobically, nor is it fully understood how
S. oneidensis
is able to resist high levels of Fe
2+
. Here we describe the results of a transposon mutant screen and subsequent deletion of the genes
clpX
and
clpP
in
S. oneidensis
, which demonstrate that the protease ClpXP is required for anaerobic Fe
2+
resistance. Many cellular processes are known to be regulated by ClpXP, including entry into stationary phase, envelope stress response, and turnover of stalled ribosomes. However, none of these processes appears to be responsible for mediating anaerobic Fe
2+
resistance in
S. oneidensis
. Protein trapping studies were performed to identify ClpXP targets in
S. oneidensis
under Fe
2+
stress, implicating a wide variety of protein targets.
Escherichia coli
strains lacking
clpX
or
clpP
also display increased sensitivity to Fe
2+
anaerobically, indicating Fe
2+
resistance may be a conserved role for the ClpXP protease system. Hypotheses regarding the potential role(s) of ClpXP during periods of high Fe
2+
are discussed. We speculate that metal-containing proteins are misfolded under conditions of high Fe
2+
and that the ClpXP protease system is necessary for their turnover.

IMPORTANCE
Prior to the evolution of cyanobacteria and oxygenic photosynthesis, life arose and flourished in iron-rich oceans. Today, aqueous iron-rich environments are less common, constrained to low-pH conditions and anaerobic systems such as stratified lakes and seas, digestive tracts, subsurface environments, and sediments. The latter two ecosystems often favor dissimilatory metal reduction, a process that produces soluble Fe
2+
from iron oxide minerals. Dissimilatory metal-reducing bacteria must therefore have mechanisms to tolerate anaerobic Fe
2+
stress, and studying resistance in these organisms may help elucidate the basis of toxicity.
Shewanella oneidensis
is a model dissimilatory metal-reducing bacterium isolated from metal-rich sediments. Here we demonstrate a role for ClpXP, a protease system widely conserved in bacteria, in anaerobic Fe
2+
resistance in both
S. oneidensis
and
Escherichia coli
.
HilE Regulates HilD by Blocking DNA Binding in Salmonella enterica Serovar Typhimurium
Authors: Grenz Jesse R. , Cott Chubiz Jessica E. , Thaprawat Pariyamon , Slauch James M. 
Published: January 2018

ABSTRACT

The
Salmonella
type three secretion system (T3SS), encoded in the
Salmonella
pathogenicity island 1 (SPI1) locus, mediates the invasion of the host intestinal epithelium. SPI1 expression is dependent upon three AraC-like regulators: HilD, HilC, and RtsA. These regulators act in a complex feed-forward loop to activate each other and
hilA
, which encodes the activator of the T3SS structural genes. HilD has been shown to be the major integration point of most signals known to activate the expression of the SPI1 T3SS, acting as a switch to control induction of the system. HilE is a negative regulator that acts upon HilD. Here we provide genetic and biochemical data showing that HilE specifically binds to HilD but not to HilC or RtsA. This protein-protein interaction blocks the ability of HilD to bind DNA as shown by both an
in vivo
reporter system and an
in vitro
gel shift assay. HilE does not affect HilD dimerization, nor does it control the stability of the HilD protein. We also investigated the role of HilE during the infection of mice using competition assays. Although deletion of
hilE
does not confer a phenotype, the
hilE
mutation does suppress the invasion defect conferred by loss of FliZ, which acts as a positive signal controlling HilD protein activity. Together, these data suggest that HilE functions to restrict low-level HilD activity, preventing premature activation of SPI1 until positive inputs reach a threshold required to fully induce the system.

IMPORTANCE
Salmonella
is a leading cause of gastrointestinal and systemic disease throughout the world. The SPI1 T3SS is required for
Salmonella
to induce inflammatory diarrhea and to gain access to underlying tissue. A complex regulatory network controls expression of SPI1 in response to numerous physiological inputs. Most of these signals impinge primarily on HilD translation or activity. The system is triggered when HilD activity crosses a threshold that allows efficient activation of its own promoter. This threshold is set by HilE, which binds to HilD to prevent the inevitable minor fluctuations in HilD activity from inappropriately activating the system. The circuit also serves as a paradigm for systems that must integrate numerous environmental parameters to control regulatory output.
PhoPR Positively Regulates
whiB3
Expression in Response to Low pH in Pathogenic Mycobacteria
Authors: Feng Lipeng , Chen Shiyun , Hu Yangbo 
Published: January 2018

ABSTRACT

During infection,
Mycobacterium tuberculosis
colonizes macrophages or necrotic granulomas, in which low pH is one of the major challenges. The PhoPR two-component regulatory system and the cytosolic redox sensor WhiB3 both play important roles in the response to low pH by
M. tuberculosis
. However, whether close association exists between PhoPR and WhiB3 remains unclear. In this study, the positive regulation of
whiB3
by PhoPR in mycobacteria was characterized. We observed that the expression patterns of the
whiB3
gene under acidic conditions are different among mycobacterial species, suggesting that the regulation of
whiB3
differs among mycobacteria. A sequence analysis of the
whiB3
promoters (
whiB3p
) from
M. tuberculosis
and two closely related species, namely,
M. marinum
and
M. smegmatis
, showed that the
whiB3p
regions from
M. tuberculosis
and
M. marinum
contain a new type of PhoP box that is absent in the
M. smegmatis
whiB3p
. Direct binding of PhoP to
whiB3p
from
M. tuberculosis
and
M. marinum
but not that from
M. smegmatis
was validated by
in vitro
protein-DNA binding assays. The direct activation of
whiB3
by PhoPR under acidic conditions was further verified by reverse transcription-quantitative PCR (qRT-PCR) analysis in
M. marinum
. Moreover, mutating the residues important for the phosphorylation pathway of PhoPR in
M. marinum
abolished the activation of
whiB3
expression by PhoPR under acidic conditions, suggesting that low pH triggers the phosphorylation of PhoPR, which in turn activates the transcription of
whiB3
. Since the PhoP box was only identified in
whiB3p
of pathogenic mycobacteria, we suggest that the PhoPR-
whiB3
regulatory pathway may have evolved to facilitate mycobacterial infection.

IMPORTANCE
The low pH in macrophages is an important barrier for infection by microbes. The PhoPR two-component regulatory system is required for the response to low pH and plays a role in redox homeostasis in
Mycobacterium tuberculosis
. WhiB3, a cytosolic redox-sensing transcriptional regulator, is also involved in these processes. However, there is no direct evidence to demonstrate the regulation of WhiB3 by PhoPR. In this study, we found that PhoPR directly activates
whiB3
expression in response to low pH. An atypical PhoP box in the
whiB3
promoters has been identified and is only found in pathogenic mycobacteria, which suggests that the PhoPR-
whiB3
regulatory pathway may facilitate mycobacterial infection. This study provides novel information for further characterization of the PhoPR regulon.
NS1 Protein of 2009 Pandemic Influenza A Virus Inhibits Porcine NLRP3 Inflammasome-Mediated Interleukin-1 Beta Production by Suppressing ASC Ubiquitination
Authors: Park Hong-Su , Liu GuanQun , Thulasi Raman Sathya N. , Landreth Shelby L. , Liu Qiang , Zhou Yan 
Published: January 2018

ABSTRACT
The inflammasome represents a molecular platform for innate immune regulation and controls proinflammatory cytokine production. The NLRP3 inflammasome is comprised of NLRP3, ASC, and procaspase-1. When the NLRP3 inflammasome is activated, it causes ASC speck formation and caspase-1 activation, resulting in the maturation of interleukin-1β (IL-1β). The NLRP3 inflammasome is regulated at multiple levels, with one level being posttranslational modification. Interestingly, ubiquitination of ASC has been reported to be indispensable for the activation of the NLRP3 inflammasome. Influenza A virus (IAV) infection induces NLRP3 inflammasome-dependent IL-1β secretion, which contributes to the host antiviral defense. However, IAVs have evolved multiple antagonizing mechanisms, one of which is executed by viral NS1 protein to suppress the NLRP3 inflammasome. In this study, we compared IL-1β production in porcine alveolar macrophages in response to IAV infection and found that the 2009 pandemic H1N1 induced less IL-1β than swine influenza viruses (SIVs). Further study revealed that the NS1 C terminus of pandemic H1N1 but not that of SIV was able to significantly inhibit NLRP3 inflammasome-mediated IL-1β production. This inhibitory function was attributed to impaired ASC speck formation and suppression of ASC ubiquitination. Moreover, we identified two target lysine residues, K110 and K140, which are essential for both porcine ASC ubiquitination and NLRP3 inflammasome-mediated IL-1β production. These results revealed a novel mechanism by which the NS1 protein of the 2009 pandemic H1N1 suppresses NLRP3 inflammasome activation.

IMPORTANCE
Influenza A virus (IAV) infection activates the NLRP3 inflammasome, resulting in the production of IL-1β, which contributes to the host innate immune response. ASC, an adaptor protein of NLRP3, forms specks that are critical for inflammasome activation. Here, we report that the NS1 C terminus of the 2009 pandemic H1N1 has functions to suppress porcine IL-1β production by inhibiting ASC speck formation and ASC ubiquitination. Furthermore, the ubiquitination sites on porcine ASC were identified. The information gained here may contribute to an in-depth understanding of porcine inflammasome activation and regulation in response to different IAVs, helping to further enhance our knowledge of innate immune responses to influenza virus infection in pigs.
α4β7+CD4+Effector/Effector Memory T Cells Differentiate into Productively and Latently Infected Central Memory T Cells by Transforming Growth Factor β1 during HIV-1 Infection
Authors: Cheung Ka-Wai , Wu Tongjin , Ho Sai Fan , Wong Yik Chun , Liu Li , Wang Hui , Chen Zhiwei 
Published: January 2018

ABSTRACTHIV-1 transmission occurs mainly through mucosal tissues. During mucosal transmission, HIV-1 preferentially infects α4β7+gut-homing CCR7−CD4+effector/effector memory T cells (TEM) and results in massive depletion of these cells and other subsets of TEMin gut-associated lymphoid tissues. However, besides being eliminated by HIV-1, the role of TEMduring the early stage of infection remains inconclusive. Here, usingin vitro-induced α4β7+gut-homing TEM(α4β7+TEM), we found that α4β7+TEMdifferentiated into CCR7+CD4+central memory T cells (TCM). This differentiation was HIV-1 independent but was inhibited by SB431542, a specific transforming growth factor β (TGF-β) receptor I kinase inhibitor. Consistently, TEM-to-TCMdifferentiation was observed in α4β7+TEMstimulated with TGF-β1 (TGF-β). The TCMproperties of the TGF-β-induced TEM-derived TCM(α4β7+TCM) were confirmed by their enhanced CCL19 chemotaxis and the downregulation of surface CCR7 upon T cell activationin vitro. Importantly, the effect of TGF-β on TCMdifferentiation also held in TEMdirectly isolated from peripheral blood. To investigate the significance of the TGF-β-dependent TEM-to-TCMdifferentiation in HIV/AIDS pathogenesis, we observed that both productively and latently infected α4β7+TCMcould differentiate from α4β7+TEMin the presence of TGF-β during HIV-1 infection. Collectively, this study not only provides a new insight for the plasticity of TEMbut also suggests that the TGF-β-dependent TEM-to-TCMdifferentiation is a previously unrecognized mechanism for the formation of latently infected TCMafter HIV-1 infection.IMPORTANCEHIV-1 is the causative agent of HIV/AIDS, which has led to millions of deaths in the past 30 years. Although the implementation of highly active antiretroviral therapy has remarkably reduced the HIV-1-related morbidity and mortality, HIV-1 is not eradicated in treated patients due to the presence of latent reservoirs. Besides, the pathogenesis in CD4 T cells early after infection still remains elusive. Immediately after HIV-1 mucosal infection, CD4 T cells are preferentially infected and depleted. However, in addition to being depleted, the other roles of the CD4 T cells, especially the effector/effector memory T cells (TEM), in disease progression are not completely understood. The significance of this study is in revealing a novel mechanism for the formation of latently HIV-1-infected central memory CD4 T cells, a major latent reservoir from CD4 TEMafter infection. Our findings suggest previously unrecognized roles of CD4 TEMin HIV-1 pathogenesis.
HIV-1 Envelope Glycoproteins from Diverse Clades Differentiate Antibody Responses and Durability among Vaccinees
Authors: Yates Nicole L. , deCamp Allan C. , Korber Bette T. , Liao Hua-Xin , Irene Carmela , Pinter Abraham , Peacock James , Harris Linda J. , Sawant Sheetal , Hraber Peter , Shen Xiaoying , Rerks-Ngarm Supachai , Pitisuttithum Punnee , Nitayapan Sorachai , Berman Phillip W. , Robb Merlin L. , Pantaleo Giuseppe , Zolla-Pazner Susan , Haynes Barton F. , Alam S. Munir , Montefiori David C. , Tomaras Georgia D. 
Published: January 2018

ABSTRACTInduction of broadly cross-reactive antiviral humoral responses with the capacity to target globally diverse circulating strains is a key goal for HIV-1 immunogen design. A major gap in the field is the identification of diverse HIV-1 envelope antigens to evaluate vaccine regimens for binding antibody breadth. In this study, we define unique antigen panels to map HIV-1 vaccine-elicited antibody breadth and durability. Diverse HIV-1 envelope glycoproteins were selected based on genetic and geographic diversity to cover the global epidemic, with a focus on sexually acquired transmitted/founder viruses with a tier 2 neutralization phenotype. Unique antigenicity was determined by nonredundancy (Spearman correlation), and antigens were clustered using partitioning around medoids (PAM) to identify antigen diversity. Cross-validation demonstrated that the PAM method was better than selection by reactivity and random selection. Analysis of vaccine-elicited V1V2 binding antibody in longitudinal samples from the RV144 clinical trial revealed the striking heterogeneity among individual vaccinees in maintaining durable responses. These data support the idea that a major goal for vaccine development is to improve antibody levels, breadth, and durability at the population level. Elucidating the level and durability of vaccine-elicited binding antibody breadth needed for protection is critical for the development of a globally efficacious HIV vaccine.IMPORTANCEThe path toward an efficacious HIV-1 vaccine will require characterization of vaccine-induced immunity that can recognize and target the highly genetically diverse virus envelope glycoproteins. Antibodies that target the envelope glycoproteins, including diverse sequences within the first and second hypervariable regions (V1V2) of gp120, were identified as correlates of risk for the one partially efficacious HIV-1 vaccine. To build upon this discovery, we experimentally and computationally evaluated humoral responses to define envelope glycoproteins representative of the antigenic diversity of HIV globally. These diverse envelope antigens distinguished binding antibody breadth and durability among vaccine candidates, thus providing insights for advancing the most promising HIV-1 vaccine candidates.