bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Small Protein HemP Is a Transcriptional Activator for the Hemin Uptake Operon in Burkholderia multivorans ATCC 17616
Authors: Sato Takuya , Nonoyama Shouta , Kimura Akane , Nagata Yuji , Ohtsubo Yoshiyuki , Tsuda Masataka 
Published: June 2017

ABSTRACT

Iron and heme play very important roles in various metabolic functions in bacteria, and their intracellular homeostasis is maintained because high concentrations of free forms of these molecules greatly facilitate the Fenton reaction-mediated production of large amounts of reactive oxygen species that severely damage various biomolecules. The ferric uptake regulator (Fur) from
Burkholderia
multivorans
ATCC 17616 is an iron-responsive global transcriptional regulator, and its
fur
deletant exhibits pleiotropic phenotypes. In this study, we found that the phenotypes of the
fur
deletant were suppressed by an additional mutation in
hemP
. The transcription of
hemP
was negatively regulated by Fur under iron-replete conditions and was constitutive in the
fur
deletant. Growth of a
hemP
deletant was severely impaired in a medium containing hemin as the sole iron source, demonstrating the important role of HemP in hemin utilization. HemP was required as a transcriptional activator that specifically binds the promoter-containing region upstream of a Fur-repressive
hmuRSTUV
operon, which encodes the proteins for hemin uptake. A
hmuR
deletant was still able to grow using hemin as the sole iron source, albeit at a rate clearly lower than that of the wild-type strain. These results strongly suggested (i) the involvement of HmuR in hemin uptake and (ii) the presence in ATCC 17616 of at least part of other unknown hemin uptake systems whose expression depends on the HemP function. Our
in vitro
analysis also indicated high-affinity binding of HemP to hemin, and such a property might modulate transcriptional activation of the
hmu
operon.

IMPORTANCE
Although the
hmuRSTUV
genes for the utilization of hemin as a sole iron source have been identified in a few
Burkholderia
strains, the regulatory expression of these genes has remained unknown. Our analysis in this study using
B. multivorans
ATCC 17616 showed that its HemP protein is required for expression of the
hmuRSTUV
operon, and the role of HemP in betaproteobacterial species was elucidated for the first time, to our knowledge, in this study. The HemP protein was also found to have two additional properties that have not been reported for functional homologues in other species; one is that HemP is able to bind to the promoter-containing region of the
hmu
operon to directly activate its transcription, and the other is that HemP is also required for the expression of an unknown hemin uptake system.
Survival of Highly Pathogenic Avian Influenza H5N1 Virus in Tissues Derived from Experimentally Infected Chickens
Authors: Yamamoto Yu , Nakamura Kikuyasu , Mase Masaji 
Published: June 2017

ABSTRACT

Eurasian lineage highly pathogenic avian influenza (HPAI) H5N1 virus has been a severe threat to the poultry industry since its emergence in 1996. The carcass or tissues derived from infected birds may present the risk of the virus spreading to humans, animals, and the surrounding environment. In this study, we investigated the survival of the virus in feather, muscle, and liver tissues collected from six chickens (
Gallus gallus
) experimentally infected with HPAI H5N1 virus. The tissues were stored at +4°C or +20°C, and viral isolation was performed at different times for 360 days. The maximum periods for viral survival were observed in samples stored at +4°C in all tissue types and were 240 days in feather tissues, 160 days in muscle, and 20 days in liver. The viral infectivity at +20°C was maintained for a maximum of 30 days in the feather tissues, 20 days in muscle, and 3 days in liver. The viral inactivation rates partly overlapped in the feather and muscle tissues at the two temperatures. The virus was inactivated rapidly in the liver. Our experimental results indicate that the tissue type and temperature can greatly influence the survival of HPAI H5N1 virus in the tissues of infected chickens.

IMPORTANCE
Highly pathogenic avian influenza virus of the H5N1 subtype can cause massive losses of poultry, and people need to handle a large number of chicken carcasses contaminated with the virus at outbreak sites. This study evaluated how long the virus can keep its infectivity in the three types of tissues derived from chickens infected with the virus. Our experimental results indicate that the virus can survive in tissues for a specific period of time depending on the tissue type and temperature. Our results are valuable for better understanding of viral ecology in the environment and for reducing the risk of the virus spreading via bird tissues contaminated with the virus.
Stress Survival Islet 2, Predominantly Present in Listeria monocytogenes Strains of Sequence Type 121, Is Involved in the Alkaline and Oxidative Stress Responses
Authors: Harter Eva , Wagner Eva Maria , Zaiser Andreas , Halecker Sabrina , Wagner Martin , Rychli Kathrin 
Published: June 2017

ABSTRACT

The foodborne pathogen
Listeria monocytogenes
is able to survive a variety of stress conditions leading to the colonization of different niches like the food processing environment. This study focuses on the hypervariable genetic hot spot
lmo0443
to
lmo0449
haboring three inserts: the stress survival islet 1 (SSI-1), the single-gene insert
LMOf2365_0481
, and two homologous genes of the nonpathogenic species
Listeria innocua
:
lin0464
, coding for a putative transcriptional regulator, and
lin0465
, encoding an intracellular PfpI protease. Our prevalence study revealed a different distribution of the inserts between human and food-associated isolates. The
lin0464-lin0465
insert was predominantly found in food-associated strains of sequence type 121 (ST121). Functional characterization of this insert showed that the putative PfpI protease Lin0465 is involved in alkaline and oxidative stress responses but not in acidic, gastric, heat, cold, osmotic, and antibiotic stresses. In parallel, deletion of
lin0464
decreased survival under alkaline and oxidative stresses. The expression of both genes increased significantly under oxidative stress conditions independently of the alternative sigma factor σ
B
. Furthermore, we showed that the expression of the protease gene
lin0465
is regulated by the transcription factor
lin0464
under stress conditions, suggesting that
lin0464
and
lin0465
form a functional unit. In conclusion, we identified a novel stress survival islet 2 (SSI-2), predominantly present in
L. monocytogenes
ST121 strains, beneficial for survival under alkaline and oxidative stresses, potentially supporting adaptation and persistence of
L. monocytogenes
in food processing environments.

IMPORTANCE
Listeria monocytogenes
strains of ST121 are known to persist for months and even years in food processing environments, thereby increasing the risk of food contamination and listeriosis. However, the molecular mechanism underlying this remarkable niche-specific adaptation is still unknown. Here, we demonstrate that the genomic islet SSI-2, predominantly present in
L. monocytogenes
ST121 strains, is beneficial for survival under alkaline and oxidative stress conditions, which are routinely encountered in food processing environments. Our findings suggest that SSI-2 is part of a diverse set of molecular determinants contributing to niche-specific adaptation and persistence of
L. monocytogenes
ST121 strains in food processing environments.
Heterologous Expression of the Clostridium carboxidivorans CO Dehydrogenase Alone or Together with the Acetyl Coenzyme A Synthase Enables both Reduction of CO
2
and Oxidation of CO by Clostridium acetobutylicum
Authors: Carlson Ellinor D. , Papoutsakis Eleftherios T. 
Published: June 2017

ABSTRACT

With recent advances in synthetic biology, CO
2
could be utilized as a carbon feedstock by native or engineered organisms, assuming the availability of electrons. Two key enzymes used in autotrophic CO
2
fixation are the CO dehydrogenase (CODH) and acetyl coenzyme A (acetyl-CoA) synthase (ACS), which form a bifunctional heterotetrameric complex. The CODH/ACS complex can reversibly catalyze CO
2
to CO, effectively enabling a biological water-gas shift reaction at ambient temperatures and pressures. The CODH/ACS complex is part of the Wood-Ljungdahl pathway (WLP) used by acetogens to fix CO
2
, and it has been well characterized in native hosts. So far, only a few recombinant CODH/ACS complexes have been expressed in heterologous hosts, none of which demonstrated
in vivo
CO
2
reduction. Here, functional expression of the
Clostridium carboxidivorans
CODH/ACS complex is demonstrated in the solventogen
Clostridium acetobutylicum
, which was engineered to express CODH alone or together with the ACS. Both strains exhibited CO
2
reduction and CO oxidation activities. The CODH reactions were interrogated using isotopic labeling, thus verifying that CO was a direct product of CO
2
reduction, and vice versa. CODH apparently uses a native
C. acetobutylicum
ferredoxin as an electron carrier for CO
2
reduction. Heterologous CODH activity depended on actively growing cells and required the addition of nickel, which is inserted into CODH without the need to express the native Ni insertase protein. Increasing CO concentrations in the gas phase inhibited CODH activity and altered the metabolite profile of the CODH-expressing cells. This work provides the foundation for engineering a complete and functional WLP in nonnative host organisms.

IMPORTANCE
Functional expression of CO dehydrogenase (CODH) from
Clostridium carboxidivorans
was demonstrated in
C. acetobutylicum
, which is natively incapable of CO
2
fixation. The expression of CODH, alone or together with the
C. carboxidivorans
acetyl-CoA synthase (ACS), enabled
C. acetobutylicum
to catalyze both CO
2
reduction and CO oxidation. Importantly, CODH exhibited activity in both the presence and absence of ACS.
13
C-tracer studies confirmed that the engineered
C. acetobutylicum
strains can reduce CO
2
to CO and oxidize CO during growth on glucose.
The Irr and RirA Proteins Participate in a Complex Regulatory Circuit and Act in Concert To Modulate Bacterioferritin Expression in Ensifer meliloti 1021
Authors: Costa Daniela , Amarelle Vanesa , Valverde Claudio , O'Brian Mark R. , Fabiano Elena 
Published: June 2017

ABSTRACT

In this work we found that the
bfr
gene of the rhizobial species
Ensifer meliloti
, encoding a bacterioferritin iron storage protein, is involved in iron homeostasis and the oxidative stress response. This gene is located downstream of and overlapping the
smc03787
open reading frame (ORF). No well-predicted RirA or Irr boxes were found in the region immediately upstream of the
bfr
gene although two presumptive RirA boxes and one presumptive Irr box were present in the putative promoter of
smc03787
. We demonstrate that
bfr
gene expression is enhanced under iron-sufficient conditions and that Irr and RirA modulate this expression. The pattern of
bfr
gene expression as well as the response to Irr and RirA is inversely correlated to that of
smc03787
. Moreover, our results suggest that the small RNA SmelC759 participates in RirA- and Irr-mediated regulation of
bfr
expression and that additional unknown factors are involved in iron-dependent regulation.

IMPORTANCE
E. meliloti
belongs to the
Alphaproteobacteria
, a group of bacteria that includes several species able to associate with eukaryotic hosts, from mammals to plants, in a symbiotic or pathogenic manner. Regulation of iron homeostasis in this group of bacteria differs from that found in the well-studied
Gammaproteobacteria
. In this work we analyzed the effect of
rirA
and
irr
mutations on
bfr
gene expression. We demonstrate the effect of an
irr
mutation on iron homeostasis in this bacterial genus. Moreover, results obtained indicate a complex regulatory circuit where multiple regulators, including RirA, Irr, the small RNA SmelC759, and still unknown factors, act in concert to balance
bfr
gene expression.
Different Amplicon Targets for Sequencing-Based Studies of Fungal Diversity
Authors: De Filippis Francesca , Laiola Manolo , Blaiotta Giuseppe , Ercolini Danilo 
Published: June 2017

ABSTRACT

Target-gene amplicon sequencing is the most exploited high-throughput sequencing application in microbial ecology. The targets are taxonomically relevant genes, with 16S rRNA being the gold standard for bacteria. As for fungi, the most commonly used target is the internal transcribed spacer (ITS). However, the uneven ITS length among species may promote preferential amplification and sequencing and incorrect estimation of their abundance. Therefore, the use of different targets is desirable. We evaluated the use of three different target amplicons for the characterization of fungal diversity. After an
in silico
primer evaluation, we compared three amplicons (the ITS1-ITS2 region [ITS1-2], 18S ribosomal small subunit RNA, and the D1/D2 domain of the 26S ribosomal large subunit RNA), using biological samples and a mock community of common fungal species. All three targets allowed for accurate identification of the species present. Nevertheless, high heterogeneity in ITS1-2 length was found, and this caused an overestimation of the abundance of species with a shorter ITS, while both 18S and 26S amplicons allowed for more reliable quantification. We demonstrated that ITS1-2 amplicon sequencing, although widely used, may lead to an incorrect evaluation of fungal communities, and efforts should be made to promote the use of different targets in sequencing-based microbial ecology studies.

IMPORTANCE
Amplicon-sequencing approaches for fungi may rely on different targets affecting the diversity and abundance of the fungal species. An increasing number of studies will address fungal diversity by high-throughput amplicon sequencing. The description of the communities must be accurate and reliable in order to draw useful insights and to address both ecological and biological questions. By analyzing a mock community and several biological samples, we demonstrate that using different amplicon targets may change the results of fungal microbiota analysis, and we highlight how a careful choice of the target is fundamental for a thorough description of the fungal communities.
Flow Cytometry Is a Powerful Tool for Assessment of the Viability of Fungal Conidia in Metalworking Fluids
Authors: Vanhauteghem D. , Demeyere K. , Callaert N. , Boelaert A. , Haesaert G. , Audenaert K. , Meyer E. 
Published: June 2017

ABSTRACT

Fungal contamination of metalworking fluids (MWF) is a dual problem in automated processing plants because resulting fungal biofilms obstruct cutting, drilling, and polishing machines. Moreover, some fungal species of MWF comprise pathogens such as
Fusarium solani
. Therefore, the development of an accurate analytical tool to evaluate conidial viability in MWF is important. We developed a flow cytometric method to measure fungal viability in MWF using
F. solani
as the model organism. To validate this method, viable and dead conidia were mixed in several proportions and flow was cytometrically analyzed. Subsequently, we assessed the fungicidal activity of two commercial MWF using flow cytometry (FCM) and compared it with microscopic analyses and plating experiments. We evaluated the fungal growth in both MWF after 7 days using quantitative PCR (qPCR) to assess the predictive value of FCM. Our results showed that FCM distinguishes live from dead conidia as early as 5 h after exposure to MWF, whereas the microscopic germination approach detected conidial viability much later and less accurately. At 24 h, microscopic analyses of germinating conidia and live/dead analyses by FCM correlated well, although the former consistently underestimated the proportion of viable conidia. In addition, the reproducibility and sensitivity of the flow cytometric method were high and allowed assessment of the fungicidal properties of two commercial MWF. Importantly, the obtained flow cytometric results on viability of
F. solani
conidia at both early time points (5 h and 24 h) correlated well with fungal biomass measurements assessed via a qPCR methodology 7 days after the start of the experiment.

IMPORTANCE
This result shows the predictive power of flow cytometry (FCM) in assessing the fungicidal capacity of MWF formulations. It also implies that FCM can be implemented as a rapid detection tool to estimate the viable fungal load in an industrial processing matrix (MWF).
Novel Antibiotic Resistance Determinants from Agricultural Soil Exposed to Antibiotics Widely Used in Human Medicine and Animal Farming
Authors: Lau Calvin Ho-Fung , van Engelen Kalene , Gordon Stephen , Renaud Justin , Topp Edward 
Published: June 2017

ABSTRACT

Antibiotic resistance has emerged globally as one of the biggest threats to human and animal health. Although the excessive use of antibiotics is recognized as accelerating the selection for resistance, there is a growing body of evidence suggesting that natural environments are “hot spots” for the development of both ancient and contemporary resistance mechanisms. Given that pharmaceuticals can be entrained onto agricultural land through anthropogenic activities, this could be a potential driver for the emergence and dissemination of resistance in soil bacteria. Using functional metagenomics, we interrogated the “resistome” of bacterial communities found in a collection of Canadian agricultural soil, some of which had been receiving antibiotics widely used in human medicine (macrolides) or food animal production (sulfamethazine, chlortetracycline, and tylosin) for up to 16 years. Of the 34 new antibiotic resistance genes (ARGs) recovered, the majority were predicted to encode (multi)drug efflux systems, while a few share little to no homology with established resistance determinants. We characterized several novel gene products, including putative enzymes that can confer high-level resistance against aminoglycosides, sulfonamides, and broad range of beta-lactams, with respect to their resistance mechanisms and clinical significance. By coupling high-resolution proteomics analysis with functional metagenomics, we discovered an unusual peptide, PPP
AZI 4
, encoded within an alternative open reading frame not predicted by bioinformatics tools. Expression of the proline-rich PPP
AZI 4
can promote resistance against different macrolides but not other ribosome-targeting antibiotics, implicating a new macrolide-specific resistance mechanism that could be fundamentally linked to the evolutionary design of this peptide.

IMPORTANCE
Antibiotic resistance is a clinical phenomenon with an evolutionary link to the microbial pangenome. Genes and protogenes encoding specialized and potential resistance mechanisms are abundant in natural environments, but understanding of their identity and genomic context remains limited. Our discovery of several previously unknown antibiotic resistance genes from uncultured soil microorganisms indicates that soil is a significant reservoir of resistance determinants, which, once acquired and “repurposed” by pathogenic bacteria, can have serious impacts on therapeutic outcomes. This study provides valuable insights into the diversity and identity of resistance within the soil microbiome. The finding of a novel peptide-mediated resistance mechanism involving an unpredicted gene product also highlights the usefulness of integrating proteomics analysis into metagenomics-driven gene discovery.
Indole-Induced Reversion of Intrinsic Multiantibiotic Resistance in Lysobacter enzymogenes
Authors: Han Yong , Wang Yan , Yu Yameng , Chen Haotong , Shen Yuemao , Du Liangcheng 
Published: June 2017

ABSTRACT

Lysobacter
species are a group of environmental bacteria that are emerging as a new source of antibiotics. One characteristic of
Lysobacter
is intrinsic resistance to multiple antibiotics, which had not been studied. To understand the resistance mechanism, we tested the effect of blocking two-component regulatory systems (TCSs) on the antibiotic resistance of
Lysobacter enzymogenes
, a prolific producer of antibiotics. Upon treatment with LED209, an inhibitor of the widespread TCS QseC/QseB,
L. enzymogenes
produced a large amount of an unknown metabolite that was barely detectable in the untreated culture. Subsequent structural elucidation by nuclear magnetic resonance (NMR) unexpectedly revealed that the metabolite was indole. Indole production was also markedly induced by adrenaline, a known modulator of QseC/QseB. Next, we identified two TCS genes,
L. enzymogenes
qseC
(
Le-qseC
) and
Le-qseB
, in
L. enzymogenes
and found that mutations of
Le-qseC
and
Le-qseB
also led to a dramatic increase in indole production. We then chemically synthesized a fluorescent indole probe that could label the cells. While the
Le-qseB
(cytoplasmic response regulator) mutant was clearly labeled by the probe, the
Le-qseC
(membrane sensor) mutant was not labeled. It was reported previously that indole can enhance antibiotic resistance in bacteria. Therefore, we tested if the dramatic increase in the level of indole production in
L. enzymogenes
upon blocking of
Le-qseC
and
Le-qseB
would lead to enhanced antibiotic resistance. Surprisingly, we found that indole caused the intrinsically multiantibiotic-resistant bacterium
L. enzymogenes
to become susceptible. Point mutations at conserved amino acids in
Le
-QseC also led to antibiotic susceptibility. Because indole is known as an interspecies signal, these findings may have implications.

IMPORTANCE
The environmental bacterium
Lysobacter
is a new source of antibiotic compounds and exhibits intrinsic antibiotic resistance. Here, we found that the inactivation of a two-component regulatory system (TCS) by an inhibitor or by gene deletion led to a remarkable increase in the level of production of a metabolite in
L. enzymogenes
, and this metabolite was identified to be indole. We chemically synthesized a fluorescent indole probe and found that it could label the wild type and a mutant of the TCS cytoplasmic response regulator but not a mutant of the TCS membrane sensor. Indole treatment caused the intrinsically multidrug-resistant bacterium
L. enzymogenes
to be susceptible to antibiotics. Mutations of the TCS sensor also led to antibiotic susceptibility. Because indole is known as an interspecies signal between gut microbiota and mammalian hosts, the observation that indole could render intrinsically resistant
L. enzymogenes
susceptible to common antibiotics may have implications.
Functional Analysis of a Novel β-(1,3)-Glucanase from Corallococcus sp. Strain EGB Containing a Fascin-Like Module
Authors: Zhou Jie , Li Zhoukun , Wu Jiale , Li Lifeng , Li Ding , Ye Xianfeng , Luo Xue , Huang Yan , Cui Zhongli , Cao Hui 
Published: June 2017

ABSTRACT

A novel β-(1,3)-glucanase gene designated
lamC
, cloned from
Corallococcus
sp. strain EGB, contains a fascin-like module and a glycoside hydrolase family 16 (GH16) catalytic module. LamC displays broad hydrolytic activity toward various polysaccharides. Analysis of the hydrolytic products revealed that LamC is an exo-acting enzyme on β-(1,3)(1,3)- and β-(1,6)-linked glucan substrates and an endo-acting enzyme on β-(1,4)-linked glucan and xylan substrates. Site-directed mutagenesis of conserved catalytic Glu residues (E304A and E309A) demonstrated that these activities were derived from the same active site. Excision of the fascin-like module resulted in decreased activity toward β-(1,3)(1,3)-linked glucans. The carbohydrate-binding assay showed that the fascin-like module was a novel β-(1,3)-linked glucan-binding module. The functional characterization of the fascin-like module and catalytic module will help us better understand these enzymes and modules.

IMPORTANCE
In this report of a bacterial β-(1,3)(1,3)-glucanase containing a fascin-like module, we reveal the β-(1,3)(1,3)-glucan-binding function of the fascin-like module present in the N terminus of LamC. LamC displays exo-β-(1,3)/(1,6)-glucanase and endo-β-(1,4)-glucanase/xylanase activities with a single catalytic domain. Thus, LamC was identified as a novel member of the GH16 family.