bluuebunny/crossref_metadata_2025_split huggingface dataset:
Investigation of the Electron Transport Chain to and the Catalytic Activity of the Diheme Cytochrome
c
Peroxidase CcpA of Shewanella oneidensis
Authors: Schütz Björn , Seidel Julian , Sturm Gunnar , Einsle Oliver , Gescher Johannes 
Published: July 2011

ABSTRACT

Bacterial diheme
c
-type cytochrome peroxidases (BCCPs) catalyze the periplasmic reduction of hydrogen peroxide to water. The gammaproteobacterium
Shewanella oneidensis
produces the peroxidase CcpA under a number of anaerobic conditions, including dissimilatory iron-reducing conditions. We wanted to understand the function of this protein in the organism and its putative connection to the electron transport chain to ferric iron. CcpA was isolated and tested for peroxidase activity, and its structural conformation was analyzed by X-ray crystallography. CcpA exhibited
in vitro
peroxidase activity and had a structure typical of diheme peroxidases. It was produced in almost equal amounts under anaerobic and microaerophilic conditions. With 50 mM ferric citrate and 50 μM oxygen in the growth medium, CcpA expression results in a strong selective advantage for the cell, which was detected in competitive growth experiments with wild-type and Δ
ccpA
mutant cells that lack the entire
ccpA
gene due to a markerless deletion. We were unable to reduce CcpA directly with CymA, MtrA, or FccA, which are known key players in the chain of electron transport to ferric iron and fumarate but identified the small monoheme ScyA as a mediator of electron transport between CymA and BCCP. To our knowledge, this is the first detailed description of a complete chain of electron transport to a periplasmic
c
-type cytochrome peroxidase. This study furthermore reports the possibility of establishing a specific electron transport chain using
c
-type cytochromes.
Determining the Extremes of the Cellular NAD(H) Level by Using an Escherichia coli NAD
+
-Auxotrophic Mutant
Authors: Zhou Yongjin , Wang Lei , Yang Fan , Lin Xinping , Zhang Sufang , Zhao Zongbao K. 
Published: July 2011

ABSTRACT

NAD (NAD
+
) and its reduced form (NADH) are omnipresent cofactors in biological systems. However, it is difficult to determine the extremes of the cellular NAD(H) level in live cells because the NAD
+
level is tightly controlled by a biosynthesis regulation mechanism. Here, we developed a strategy to determine the extreme NAD(H) levels in
Escherichia coli
cells that were genetically engineered to be NAD
+
auxotrophic. First, we expressed the
ntt4
gene encoding the NAD(H) transporter in the
E. coli
mutant YJE001, which had a deletion of the
nadC
gene responsible for NAD
+
de novo
biosynthesis, and we showed NTT4 conferred on the mutant strain better growth in the presence of exogenous NAD
+
. We then constructed the NAD
+
-auxotrophic mutant YJE003 by disrupting the essential gene
nadE
, which is responsible for the last step of NAD
+
biosynthesis in cells harboring the
ntt4
gene. The minimal NAD
+
level was determined in M9 medium in proliferating YJE003 cells that were preloaded with NAD
+
, while the maximal NAD(H) level was determined by exposing the cells to high concentrations of exogenous NAD(H). Compared with supplementation of NADH, cells grew faster and had a higher intracellular NAD(H) level when NAD
+
was fed. The intracellular NAD(H) level increased with the increase of exogenous NAD
+
concentration, until it reached a plateau. Thus, a minimal NAD(H) level of 0.039 mM and a maximum of 8.49 mM were determined, which were 0.044× and 9.6× those of wild-type cells, respectively. Finally, the potential application of this strategy in biotechnology is briefly discussed.
Amoeba Host Model for Evaluation of Streptococcus suis Virulence
Authors: Bonifait Laetitia , Charette Steve J. , Filion Geneviève , Gottschalk Marcelo , Grenier Daniel 
Published: July 2011

ABSTRACT

The Gram-positive bacterium
Streptococcus suis
is a major swine pathogen worldwide that causes meningitis, septicemia, and endocarditis. In this study, we demonstrate that the amoeba
Dictyostelium discoideum
can be a relevant alternative system to study the virulence of
S. suis
.
Genome Data Mining and Soil Survey for the Novel Group 5 [NiFe]-Hydrogenase To Explore the Diversity and Ecological Importance of Presumptive High-Affinity H
2
-Oxidizing Bacteria
Authors: Constant Philippe , Chowdhury Soumitra Paul , Hesse Laura , Pratscher Jennifer , Conrad Ralf 
Published: July 2011

ABSTRACT

Streptomyces
soil isolates exhibiting the unique ability to oxidize atmospheric H
2
possess genes specifying a putative high-affinity [NiFe]-hydrogenase. This study was undertaken to explore the taxonomic diversity and the ecological importance of this novel functional group. We propose to designate the genes encoding the small and large subunits of the putative high-affinity hydrogenase
hhyS
and
hhyL
, respectively. Genome data mining revealed that the
hhyL
gene is unevenly distributed in the phyla
Actinobacteria
,
Proteobacteria
,
Chloroflexi
, and
Acidobacteria
. The
hhyL
gene sequences comprised a phylogenetically distinct group, namely, the group 5 [NiFe]-hydrogenase genes. The presumptive high-affinity H
2
-oxidizing bacteria constituting group 5 were shown to possess a hydrogenase gene cluster, including the genes encoding auxiliary and structural components of the enzyme and four additional open reading frames (ORFs) of unknown function. A soil survey confirmed that both high-affinity H
2
oxidation activity and the
hhyL
gene are ubiquitous. A quantitative PCR assay revealed that soil contained 10
6
to 10
8
hhyL
gene copies g (dry weight)
−1
. Assuming one
hhyL
gene copy per genome, the abundance of presumptive high-affinity H
2
-oxidizing bacteria was higher than the maximal population size for which maintenance energy requirements would be fully supplied through the H
2
oxidation activity measured in soil. Our data indicate that the abundance of the
hhyL
gene should not be taken as a reliable proxy for the uptake of atmospheric H
2
by soil, because high-affinity H
2
oxidation is a facultatively mixotrophic metabolism, and microorganisms harboring a nonfunctional group 5 [NiFe]-hydrogenase may occur.
Ecosystem Screening Approach for Pathogen-Associated Microorganisms Affecting Host Disease
Authors: Galiana Eric , Marais Antoine , Mura Catherine , Industri Benoît , Arbiol Gilles , Ponchet Michel 
Published: July 2011

ABSTRACT

The microbial community in which a pathogen evolves is fundamental to disease outcome. Species interacting with a pathogen on the host surface shape the distribution, density, and genetic diversity of the inoculum, but the role of these species is rarely determined. The screening method developed here can be used to characterize pathogen-associated species affecting disease. This strategy involves three steps: (i) constitution of the microbial community, using the pathogen as a trap; (ii) community selection, using extracts from the pathogen as the sole nutrient source; and (iii) molecular identification and the screening of isolates focusing on their effects on the growth of the pathogen
in vitro
and host disease. This approach was applied to a soilborne plant pathogen,
Phytophthora parasitica
, structured in a biofilm, for screening the microbial community from the rhizosphere of
Nicotiana tabacum
(the host). Two of the characterized eukaryotes interfered with the oomycete cycle and may affect the host disease. A
Vorticella
species acted through a mutualistic interaction with
P. parasitica
, disseminating pathogenic material by leaving the biofilm. A
Phoma
species established an amensal interaction with
P. parasitica
, strongly suppressing disease by inhibiting
P. parasitica
germination. This screening method is appropriate for all nonobligate pathogens. It allows the definition of microbial species as promoters or suppressors of a disease for a given biotope. It should also help to identify important microbial relationships for ecology and evolution of pathogens.
Identification of a Toluene-Degrading Bacterium from a Soil Sample through H
2
18
O DNA Stable Isotope Probing
Authors: Woods Angela , Watwood Maribeth , Schwartz Egbert 
Published: July 2011

ABSTRACT

DNA stable isotope probing (DNA-SIP) with H
2
18
O was used to identify a toluene-degrading bacterium in soil amended with 48 ppm toluene. After quantification of toluene degradation rates in soil, DNA was extracted from soil incubated with H
2
18
O, H
2
16
O, H
2
16
O and 48 ppm toluene, or H
2
18
O and 48 ppm toluene. A single DNA band formed along a cesium chloride gradient after isopycnic centrifugation of extracts from soils incubated with H
2
16
O. With extracts from soils to which only H
2
18
O was added, two distinct DNA bands formed, while three bands formed when DNA extracted from soil incubated with both H
2
18
O and toluene was analyzed. We suggest that this third band formed because toluene does not contain any oxygen atoms and toluene-degrading organisms had to transfer oxygen atoms from H
2
18
O into metabolic intermediates to form nucleic acids
de novo
. We extracted the third DNA band and amplified a large fraction of the bacterial 16S rRNA gene. Direct sequencing of the PCR product obtained from the labeled DNA, as well as cloned 16S rRNA amplicons, identified a known toluene degrader,
Rhodococcus jostii
RHA1. A toluene-degrading bacterial strain was subsequently isolated from soil and shown to be
Rhodococcus jostii
RHA1. Finally, quantitative real-time PCR analysis showed that the abundance of the 16S rRNA gene of
Rhodococcus jostii
RHA1 increased in soil after toluene exposure but not in soils from which toluene was withheld. This study indicates that H
2
18
O DNA-SIP can be a useful method for identifying pollutant-degrading bacteria in soil.
Poly(A)-Specific Ribonuclease (PARN-1) Function in Stage-Specific mRNA Turnover in Trypanosoma brucei
Authors: Utter Christopher J. , Garcia Stacey A. , Milone Joseph , Bellofatto Vivian 
Published: July 2011

ABSTRACT

Deadenylation is often the rate-limiting event in regulating the turnover of cellular mRNAs in eukaryotes. Removal of the poly(A) tail initiates mRNA degradation by one of several decay pathways, including deadenylation-dependent decapping, followed by 5′ to 3′ exonuclease decay or 3′ to 5′ exosome-mediated decay. In trypanosomatids, mRNA degradation is important in controlling the expression of differentially expressed genes. Genomic annotation studies have revealed several potential deadenylases. Poly(A)-specific RNase (PARN) is a key deadenylase involved in regulating gene expression in mammals,
Xenopus
oocytes, and higher plants. Trypanosomatids possess three different
PARN
genes,
PARN-1
, -
2
, and -
3
, each of which is expressed at the mRNA level in two life-cycle stages of the human parasite
Trypanosoma brucei
. Here we show that
T. brucei
PARN-1 is an active deadenylase. To determine the role of PARN-1 on mRNA stability
in vivo
, we overexpressed this protein and analyzed perturbations in mRNA steady-state levels as well as mRNA half-life. Interestingly, a subset of mRNAs was affected, including a family of mRNAs that encode stage-specific coat proteins. These data suggest that PARN-1 functions in stage-specific protein production.
YuaB Functions Synergistically with the Exopolysaccharide and TasA Amyloid Fibers To Allow Biofilm Formation by Bacillus subtilis
Authors: Ostrowski Adam , Mehert Angela , Prescott Alan , Kiley Taryn B. , Stanley-Wall Nicola R. 
Published: July 2011

ABSTRACT

During biofilm formation by
Bacillus subtilis
, two extracellular matrix components are synthesized, namely, the TasA amyloid fibers and an exopolysaccharide. In addition, a small protein called YuaB has been shown to allow the biofilm to form. The regulatory protein DegU is known to initiate biofilm formation. In this report we show that the main role of DegU during biofilm formation is to indirectly drive the activation of transcription from the
yuaB
promoter. The N terminus of YuaB constitutes a signal peptide for the Sec transport system. Here we show that the presence of the signal peptide is required for YuaB function. In addition we demonstrate that upon export of YuaB from the cytoplasm, it localizes to the cell wall. We continue with evidence that increased production of TasA and the exopolysaccharide is not sufficient to overcome the effects of a mutation in
yuaB
, demonstrating the unique involvement of YuaB in forming a biofilm. In line with this, YuaB is not involved in correct synthesis, export, or polymerization of either the TasA amyloid fibers or the exopolysaccharide. Taken together, these findings identify YuaB as a protein that plays a novel role during biofilm formation. We hypothesize that YuaB functions synergistically with the known components of the biofilm matrix to facilitate the assembly of the biofilm matrix.
TrbB from Conjugative Plasmid F Is a Structurally Distinct Disulfide Isomerase That Requires DsbD for Redox State Maintenance
Authors: Hemmis Casey W. , Berkmen Mehmet , Eser Markus , Schildbach Joel F. 
Published: July 2011

ABSTRACTTrbB, a periplasmic protein encoded by the conjugative plasmid F, has a predicted thioredoxin-like fold and possesses a C-X-X-C redox active site motif. TrbB may function in the conjugative process by serving as a disulfide bond isomerase, facilitating proper folding of a subset of F-plasmid-encoded proteins in the periplasm. Previous studies have demonstrated that a ΔtrbBF plasmid inEscherichia colilacking DsbCE.coli, its native disulfide bond isomerase, experiences a 10-fold decrease in mating efficiency but have not provided direct evidence for disulfide bond isomerase activity. Here we demonstrate thattrbBcan partially restore transfer of a variant of the distantly related R27 plasmid when both chromosomal and plasmid genes encoding disulfide bond isomerases have been disrupted. In addition, we show that TrbB displays both disulfide bond isomerase and reductase activities on substrates not involved in the conjugative process. Unlike canonical members of the disulfide bond isomerase family, secondary structure predictions suggest that TrbB lacks both an N-terminal dimerization domain and an α-helical domain found in other disulfide bond isomerases. Phylogenetic analyses support the conclusion that TrbB belongs to a unique family of plasmid-based disulfide isomerases. Interestingly, although TrbB diverges structurally from other disulfide bond isomerases, we show that like those isomerases, TrbB relies on DsbD fromE. colifor maintenance of its C-X-X-C redox active site motif.
Genome Sequences of Alicycliphilus denitrificans Strains BC and K601
T
Authors: Oosterkamp Margreet J. , Veuskens Teun , Plugge Caroline M. , Langenhoff Alette A. M. , Gerritse Jan , van Berkel Willem J. H. , Pieper Dietmar H. , Junca Howard , Goodwin Lynne A. , Daligault Hajnalka E. , Bruce David C. , Detter John C. , Tapia Roxanne , Han Cliff S. , Land Miriam L. , Hauser Loren J. , Smidt Hauke , Stams Alfons J. M. 
Published: July 2011

ABSTRACT

Alicycliphilus denitrificans
strain BC and
A. denitrificans
strain K601
T
degrade cyclic hydrocarbons. These strains have been isolated from a mixture of wastewater treatment plant material and benzene-polluted soil and from a wastewater treatment plant, respectively, suggesting their role in bioremediation of soil and water. Although the strains are phylogenetically closely related, there are some clear physiological differences. The hydrocarbon cyclohexanol, for example, can be degraded by strain K601
T
but not by strain BC. Furthermore, both strains can use nitrate and oxygen as an electron acceptor, but only strain BC can use chlorate as electron acceptor. To better understand the nitrate and chlorate reduction mechanisms coupled to the oxidation of cyclic compounds, the genomes of
A. denitrificans
strains BC and K601
T
were sequenced. Here, we report the complete genome sequences of
A. denitrificans
strains BC and K601
T
.
RNase III-Dependent Expression of the
rpsO-pnp
Operon of Streptomyces coelicolor
Authors: Gatewood Marcha L. , Bralley Patricia , Jones George H. 
Published: July 2011

ABSTRACT

We have examined the expression of the
rpsO-pnp
operon in an RNase III (
rnc
) mutant of
Streptomyces coelicolor
. Western blotting demonstrated that polynucleotide phosphorylase (PNPase) levels increased in the
rnc
mutant, JSE1880, compared with the parental strain, M145, and this observation was confirmed by polymerization assays. It was observed that
rpsO-pnp
mRNA levels increased in the
rnc
mutant by 1.6- to 4-fold compared with M145. This increase was observed in exponential, transition, and stationary phases, and the levels of the readthrough transcript, initiated upstream of
rpsO
in the
rpsO-pnp
operon; the
pnp
transcript, initiated in the
rpsO-pnp
intergenic region; and the
rpsO
transcript all increased. The increased levels of these transcripts in JSE1880 reflected increased chemical half-lives for each of the three. We demonstrated further that overexpression of the
rpsO
-
pnp
operon led to significantly higher levels of PNPase activity in JSE1880 compared to M145, reflecting the likelihood that PNPase expression is autoregulated in an RNase III-dependent manner in
S. coelicolor
. To explore further the increase in the level of the
pnp
transcript initiated in the intergenic region in JSE1880, we utilized that transcript as a substrate in assays employing purified
S. coelicolor
RNase III. These assays revealed the presence of hitherto-undiscovered sites of RNase III cleavage of the
pnp
transcript. The position of those sites was determined by primer extension, and they were shown to be situated in the loops of a stem-loop structure.
Legionella pneumophilaRequires Polyamines for Optimal Intracellular Growth
Authors: Nasrallah Gheyath K. , Riveroll Angela L. , Chong Audrey , Murray Lois E. , Lewis P. Jeffrey , Garduño Rafael A. 
Published: July 2011

ABSTRACT

The Gram-negative intracellular pathogen
Legionella pneumophila
replicates in a membrane-bound compartment known as the
Legionella
-containing vacuole (LCV), into which it abundantly releases its chaperonin, HtpB. To determine whether HtpB remains within the LCV or reaches the host cell cytoplasm, we infected U937 human macrophages and CHO cells with
L. pneumophila
expressing a translocation reporter consisting of the
Bordetella pertussis
adenylate cyclase fused to HtpB. These infections led to increased cyclic AMP levels, suggesting that HtpB reaches the host cell cytoplasm. To identify potential functions of cytoplasmic HtpB, we expressed it in the yeast
Saccharomyces cerevisiae
, where HtpB induced pseudohyphal growth. A yeast-two-hybrid screen showed that HtpB interacted with
S
-adenosylmethionine decarboxylase (SAMDC), an essential yeast enzyme (encoded by
SPE2
) that is required for polyamine biosynthesis. Increasing the copy number of
SPE2
induced pseudohyphal growth in
S. cerevisiae
; thus, we speculated that (i) HtpB induces pseudohyphal growth by activating polyamine synthesis and (ii)
L. pneumophila
may require exogenous polyamines for growth. A pharmacological inhibitor of SAMDC significantly reduced
L. pneumophila
replication in L929 mouse cells and U937 macrophages, whereas exogenously added polyamines moderately favored intracellular growth, confirming that polyamines and host SAMDC activity promote
L. pneumophila
proliferation. Bioinformatic analysis revealed that most known enzymes required for polyamine biosynthesis in bacteria (including SAMDC) are absent in
L. pneumophila
, further suggesting a need for exogenous polyamines. We hypothesize that HtpB may function to ensure a supply of polyamines in host cells, which are required for the optimal intracellular growth of
L. pneumophila
.
Overexpression of LolCDE Allows Deletion of the Escherichia coli Gene Encoding Apolipoprotein
N
-Acyltransferase
Authors: Narita Shin-ichiro , Tokuda Hajime 
Published: July 2011

ABSTRACT

Bacterial lipoproteins represent a subset of membrane-associated proteins that are covalently modified with lipids at the N-terminal cysteine. The final step of lipoprotein modification, N-acylation of apolipoproteins, is mediated by apolipoprotein
N
-acyltransferase (Lnt). Examinations with reconstituted proteoliposomes and a conditional mutant previously indicated that N-acylation of lipoproteins is required for their efficient release from the inner membrane catalyzed by LolA and LolCDE, the lipoprotein-specific chaperone and ABC transporter, respectively. Because Lnt is essential for
Escherichia coli
, a mutant lacking Lnt activity has not been isolated. However, we report here that
lnt
-null strains can be constructed when LolCDE is overproduced in strains lacking either the major outer membrane lipoprotein Lpp or transpeptidases that cross-link Lpp with peptidoglycan. Lipoproteins purified from the
lnt
-null strain exhibited increased mobility on SDS-PAGE compared to those from wild-type cells and could be sequenced by Edman degradation, indicating that lipoproteins in this mutant exist as apolipoproteins that lack N-acylation. Overexpression of Lpp in the
lnt
-null strain resulted in the accumulation of apoLpp in the inner membrane and caused growth arrest. In contrast to the release of mature Lpp in the presence of LolA and LolCDE, that of apoLpp from the inner membrane was significantly retarded. Furthermore, the amount of lipoproteins copurified with LolCDE was significantly reduced in the
lnt
-null strain. These results indicate that the affinity of LolCDE for apolipoprotein is very low, and therefore, overexpression of LolCDE is required for its release and sorting to the outer membrane.
Transcriptional Profiling of the Iron Starvation Response in Bordetella pertussis Provides New Insights into Siderophore Utilization and Virulence Gene Expression
Authors: Brickman Timothy J. , Cummings Craig A. , Liew Sin-Yee , Relman David A. , Armstrong Sandra K. 
Published: July 2011

ABSTRACT

Serological studies of patients with pertussis and the identification of antigenic
Bordetella pertussis
proteins support the hypothesis that
B. pertussis
perceives an iron starvation cue and expresses multiple iron source utilization systems in its natural human host environment. Furthermore, previous studies using a murine respiratory tract infection model showed that several of these
B. pertussis
iron systems are required for colonization and persistence and are differentially expressed over the course of infection. The present study examined genome-wide changes in
B. pertussis
gene transcript abundance in response to iron starvation
in vitro
. In addition to known iron source utilization genes, we identified a previously uncharacterized iron-repressed cytoplasmic membrane transporter system,
fbpABC
, that is required for the utilization of multiple structurally distinct siderophores including alcaligin, enterobactin, ferrichrome, and desferrioxamine B. Expression of type III secretion system genes was also found to be upregulated during iron starvation in both
B. pertussis
strain Tohama I and
Bordetella bronchiseptica
strain RB50. In a survey of type III secretion system protein production by an assortment of
B. pertussis
laboratory-adapted and low-passage clinical isolate strains, iron limitation increased the production and secretion of the type III secretion system-specific translocation apparatus tip protein Bsp22 in all Bvg-proficient strains. These results indicate that iron starvation in the infected host is an important environmental cue influencing not only
Bordetella
iron transport gene expression but also the expression of other important virulence-associated genes.