bluuebunny/crossref_metadata_2025_split huggingface dataset:
Functional Analysis of the Single Est1/Ebs1 Homologue in Kluyveromyces lactis Reveals Roles in both Telomere Maintenance and Rapamycin Resistance
Authors: Hsu Min , Yu Eun Young , Sprušanský Ondrej , McEachern Michael J. , Lue Neal F. 
Published: April 2012

ABSTRACT

Est1 and Ebs1 in
Saccharomyces cerevisiae
are paralogous proteins that arose through whole-genome duplication and that serve distinct functions in telomere maintenance and translational regulation. Here we present our functional analysis of the sole Est1/Ebs1 homologue in the related budding yeast
Kluyveromyces lactis
(named
Kl
Est1). We show that similar to other Est1s,
Kl
Est1 is required for normal telomere maintenance
in vivo
and full telomerase primer extension activity
in vitro
.
Kl
Est1 also associates with telomerase RNA (Ter1) and an active telomerase complex in cell extracts. Both the telomere maintenance and the Ter1 association functions of
Kl
Est1 require its N-terminal domain but not its C terminus. Analysis of clusters of point mutations revealed residues in both the N-terminal TPR subdomain and the downstream helical subdomain (DSH) that are important for telomere maintenance and Ter1 association. A UV cross-linking assay was used to establish a direct physical interaction between
Kl
Est1 and a putative stem-loop in Ter1, which also requires both the TPR and DSH subdomains. Moreover, similar to
S. cerevisiae
Ebs1 (
Sc
Ebs1) (but not
Sc
Est1),
Kl
Est1 confers rapamycin sensitivity and may be involved in nonsense-mediated decay. Interestingly, unlike telomere regulation, this apparently separate function of
Kl
Est1 requires its C-terminal domain. Our findings provide insights on the mechanisms and evolution of Est1/Ebs1 homologues in budding yeast and present an attractive model system for analyzing members of this multifunctional protein family.
P65 Truncation Impacts P30 Dynamics during Mycoplasma pneumoniae Gliding
Authors: Hasselbring Benjamin M. , Sheppard Edward S. , Krause Duncan C. 
Published: April 2012

ABSTRACT

The cell wall-less prokaryote
Mycoplasma pneumoniae
is a major cause of community-acquired bronchitis and pneumonia in humans. Colonization is mediated largely by a differentiated terminal organelle, which is also the leading end in gliding motility. Cytadherence-associated proteins P30 and P65 appear to traffic concurrently to the distal end of developing terminal organelles. Here, truncation of P65 due to transposon insertion in the corresponding gene resulted in lower gliding velocity, reduced cytadherence, and decreased steady-state levels of several terminal organelle proteins, including P30. Utilizing fluorescent protein fusions, we followed terminal organelle development over time. New P30 foci appeared at nascent terminal organelles in P65 mutants, as in the wild type. However, with forward cell motility, P30 in the P65 mutants appeared to drag toward the trailing cell pole, where it was released, yielding a fluorescent trail to which truncated P65 colocalized. In contrast, P30 was only rarely observed at the trailing end of gliding wild-type cells. Complementation with the recombinant wild-type P65 allele by transposon delivery restored P65 levels and stabilized P30 localization to the terminal organelle.
The Divergent AmoC
3
Subunit of Ammonia Monooxygenase Functions as Part of a Stress Response System in Nitrosomonas europaea
Authors: Berube Paul M. , Stahl David A. 
Published: April 2012

ABSTRACT

The ammonia monooxygenase of chemolithotrophic ammonia-oxidizing bacteria (AOB) catalyzes the first step in ammonia oxidation by converting ammonia to hydroxylamine. The monooxygenase of
Nitrosomonas europaea
is encoded by two nearly identical operon copies (

amoCAB
1

,
2

). Several AOB, including
N. europaea
, also possess a divergent monocistronic copy of
amoC
(

amoC
3

) of unknown function. Previous work suggested a possible functional role for

amoC
3

as part of the σ
E
stress response regulon during the recovery of
N. europaea
from extended ammonia starvation, thus indicating its importance during the exit of cells from starvation. We here used global transcription analysis to show that expression of

amoC
3

is part of a general poststarvation cellular response system in
N. europaea
. We also found that

amoC
3

is required for an efficient response to some stress conditions, as deleting this gene impaired growth at elevated temperatures and recovery following starvation under high oxygen tensions. Deletion of the σ
32
global stress response regulator demonstrated that the heat shock regulon plays a significant role in mediating the recovery of
N. europaea
from starvation. These findings provide the first described phenotype associated with the divergent AmoC
3
subunit which appears to function as a stress-responsive subunit capable of maintaining ammonia oxidation activity under stress conditions. While this study was limited to starvation and heat shock, it is possible that the AmoC
3
subunit may be responsive to other membrane stressors (e.g., solvent or osmotic shocks) that are prevalent in the environments of AOB.
Clostridium difficile MazF Toxin Exhibits Selective, Not Global, mRNA Cleavage
Authors: Rothenbacher Francesca P. , Suzuki Motoo , Hurley Jennifer M. , Montville Thomas J. , Kirn Thomas J. , Ouyang Ming , Woychik Nancy A. 
Published: April 2012

ABSTRACT

Clostridium difficile
is an important, emerging nosocomial pathogen. The transition from harmless colonization to disease is typically preceded by antimicrobial therapy, which alters the balance of the intestinal flora, enabling
C. difficile
to proliferate in the colon. One of the most perplexing aspects of the
C. difficile
infectious cycle is its ability to survive antimicrobial therapy and transition from inert colonization to active infection. Toxin-antitoxin (TA) systems have been implicated in facilitating persistence after antibiotic treatment. We identified only one TA system in
C. difficile
strain 630 (epidemic type X), designated MazE-cd and MazF-cd, a counterpart of the well-characterized
Escherichia coli
MazEF TA system. This
E. coli
MazF toxin cleaves mRNA at ACA sequences, leading to global mRNA degradation, growth arrest, and death. Likewise, MazF-cd expression in
E. coli
or
Clostridium perfringens
resulted in growth arrest. Primer extension analysis revealed that MazF-cd cleaved RNA at the five-base consensus sequence UACAU, suggesting that the mRNAs susceptible to cleavage comprise a subset of total mRNAs. In agreement, we observed differential cleavage of several mRNAs by MazF-cd
in vivo
, revealing a direct correlation between the number of cleavage recognition sites within a given transcript and its susceptibility to degradation by MazF-cd. Interestingly, upon detailed statistical analyses of the
C. difficile
transcriptome, the major
C. difficile
virulence factor toxin B (TcdB) and CwpV, a cell wall protein involved in aggregation, were predicted to be significantly resistant to MazF-cd cleavage.
Effects of the ERES Pathogenicity Region Regulator Ralp3 on Streptococcus pyogenes Serotype M49 Virulence Factor Expression
Authors: Siemens Nikolai , Fiedler Tomas , Normann Jana , Klein Johannes , Münch Richard , Patenge Nadja , Kreikemeyer Bernd 
Published: April 2012

ABSTRACT

Streptococcus pyogenes
(group A streptococcus [GAS]) is a highly virulent Gram-positive bacterium. For successful infection, GAS expresses many virulence factors, which are clustered together with transcriptional regulators in distinct genomic regions. Ralp3 is a central regulator of the ERES region. In this study, we investigated the role of Ralp3 in GAS M49 pathogenesis. The inactivation of Ralp3 resulted in reduced attachment to and internalization into human keratinocytes. The Δ
ralp3
mutant failed to survive in human blood and serum, and the hyaluronic acid capsule was slightly decreased. In addition, the mutant showed a lower binding capacity to human plasminogen, and the SpeB activity was significantly decreased. Complementation of the Δ
ralp3
mutant restored the wild-type phenotype. The transcriptome and quantitative reverse transcription-PCR analysis of the serotype M49 GAS strain and its isogenic Δ
ralp3
mutant identified 16 genes as upregulated, and 43 genes were found to be downregulated. Among the downregulated genes, there were open reading frames encoding proteins involved in metabolism (e.g., both
lac
operons and the
fru
operon), genes encoding lantibiotics (e.g., the putative salivaricin operon), and ORFs encoding virulence factors (such as the whole Mga core regulon and further genes under Mga control). In summary, the ERES region regulator Ralp3 is an important serotype-specific transcriptional regulator for virulence and metabolic control.
EbfC (YbaB) Is a New Type of Bacterial Nucleoid-Associated Protein and a Global Regulator of Gene Expression in the Lyme Disease Spirochete
Authors: Jutras Brandon L. , Bowman Amy , Brissette Catherine A. , Adams Claire A. , Verma Ashutosh , Chenail Alicia M. , Stevenson Brian 
Published: April 2012

ABSTRACTNearly every known species ofEubacteriaencodes a homolog of theBorrelia burgdorferiEbfC DNA-binding protein. We now demonstrate that fluorescently tagged EbfC associates withB. burgdorferinucleoidsin vivoand that chromatin immunoprecipitation (ChIP) of wild-type EbfC showed it to bindin vivoto sites throughout the genome, two hallmarks of nucleoid-associated proteins. Comparative RNA sequencing (RNA-Seq) of a mutantB. burgdorferistrain that overexpresses EbfC indicated that approximately 4.5% of borrelial genes are significantly impacted by EbfC. TheebfCgene was highly expressed in rapidly growing bacteria, butebfCmRNA was undetectable in stationary phase. Combined with previous data showing that EbfC induces bends in DNA, these results demonstrate that EbfC is a nucleoid-associated protein and lead to the hypothesis thatB. burgdorferiutilizes cellular fluctuations in EbfC levels to globally control transcription of numerous genes. The ubiquity of EbfC proteins inEubacteriasuggests that these results apply to a wide range of pathogens and other bacteria.
Rrp2, a Prokaryotic Enhancer-Like Binding Protein, Is Essential for Viability of Borrelia burgdorferi
Authors: Groshong Ashley M. , Gibbons Nora E. , Yang X. Frank , Blevins Jon S. 
Published: April 2012

ABSTRACT

The Lyme disease spirochete,
Borrelia burgdorferi
, exists in two diverse niches (i.e., an arthropod tick vector and mammalian host) during its enzootic life cycle. To effectively adapt to these unique environments, the bacterium alters the expression of numerous genes, including several major outer surface (lipo)proteins that are required for infection and transmission. An enhancer-binding protein (EBP), known as Rrp2, is one identified activator of the RpoN/RpoS alternative sigma factor cascade. Because initial efforts to generate an
rrp2
deletion strain were unsuccessful, the role of Rrp2 in the activation of the RpoN/RpoS pathway was first defined using a strain of
B. burgdorferi
carrying an
rrp2
point mutant that was defective in its ability to activate RpoN-dependent transcription. The fact that subsequent attempts to disrupt
rrp2
have also been unsuccessful has led investigators to hypothesize that Rrp2 has other undefined functions which are essential for
B. burgdorferi
survival and independent of its EBP function. We used a
lac
-based inducible expression system to generate a conditional
rrp2
mutant in virulent
B. burgdorferi
. In this strain, an isopropyl-β-
d
-thiogalactopyranoside-inducible copy of the
rrp2
gene is expressed in
trans
from a borrelial shuttle vector. We found that the chromosomal copy of
rrp2
could be inactivated only when
rrp2
was induced, and the maintenance of
rrp2
expression was required for the growth of the mutants. In addition, the overexpression of
rrp2
is detrimental to
B. burgdorferi
growth in a manner that is independent of the RpoN/RpoS pathway. These studies provide the first direct evidence that
rrp2
is an essential gene in
B. burgdorferi
.
In Vivo
and
In Vitro
Analyses of Regulation of the Pheromone-Responsive
prgQ
Promoter by the PrgX Pheromone Receptor Protein
Authors: Caserta Enrico , Haemig Heather A. H. , Manias Dawn A. , Tomsic Jerneja , Grundy Frank J. , Henkin Tina M. , Dunny Gary M. 
Published: April 2012

ABSTRACT

Expression of conjugative transfer and virulence functions of the
Enterococcus faecalis
antibiotic resistance plasmid pCF10 is regulated by the interaction of the pheromone receptor protein PrgX with two DNA binding operator sites (XBS1 and XBS2) upstream from the transcription start site of the
prgQ
operon (encoding the pCF10 transfer machinery) and by posttranscriptional mechanisms. Occupancy of both binding sites by PrgX dimers results in repression of the
prgQ
promoter. Structural and genetic studies suggest that the peptide pheromone cCF10 functions by binding to PrgX and altering its oligomerization state, resulting in reduced occupancy of XBSs and increased
prgQ
transcription. The DNA binding activity of PrgX has additional indirect regulatory effects on
prgQ
transcript levels related to the position of the convergently transcribed
prgX
operon. This has complicated interpretation of previous analyses of the control of
prgQ
expression by PrgX. We report here the results of
in vivo
and
in vitro
experiments examining the direct effects of PrgX on transcription from the
prgQ
promoter, as well as quantitative correlation between the concentrations of XBSs, PrgX protein, and
prgQ
promoter activity
in vivo
. The results of electrophoretic mobility shift assays and quantitative analysis of
prgQ
transcription
in vitro
and
in vivo
support the predicted roles of the PrgX DNA binding sites in
prgQ
transcription regulation. The results also suggest the existence of other factors that impede PrgX repression or enhance its antagonism by cCF10
in vivo
.
Escherichia coli DNA Polymerase IV (Pol IV), but Not Pol II, Dynamically Switches with a Stalled Pol III* Replicase
Authors: Heltzel Justin M. H. , Maul Robert W. , Wolff David W. , Sutton Mark D. 
Published: April 2012

ABSTRACT

The
dnaN159
allele encodes a temperature-sensitive mutant form of the β sliding clamp (β159). SOS-induced levels of DNA polymerase IV (Pol IV) confer UV sensitivity upon the
dnaN159
strain, while levels of Pol IV ∼4-fold higher than those induced by the SOS response severely impede its growth. Here, we used mutations in Pol IV that disrupted specific interactions with the β clamp to test our hypothesis that these phenotypes were the result of Pol IV gaining inappropriate access to the replication fork via a Pol III*-Pol IV switch relying on both the rim and cleft of the clamp. Our results clearly demonstrate that Pol IV relied on both the clamp rim and cleft interactions for these phenotypes. In contrast to the case for Pol IV, elevated levels of the other Pols, including Pol II, which was expressed at levels ∼8-fold higher than the normal SOS-induced levels, failed to impede growth of the
dnaN159
strain. These findings suggest that the mechanism used by Pol IV to switch with Pol III* is distinct from those used by the other Pols. Results of experiments utilizing purified components to reconstitute the Pol III*-Pol II switch
in vitro
indicated that Pol II switched equally well with both a stalled and an actively replicating Pol III* in a manner that was independent of the rim contact required by Pol IV. These results provide compelling support for the Pol III*-Pol IV two-step switch model and demonstrate important mechanistic differences in how Pol IV and Pol II switch with Pol III*.
The Entner-Doudoroff Pathway Is Obligatory for Gluconate Utilization and Contributes to the Pathogenicity of Vibrio cholerae
Authors: Patra Tapas , Koley Hemanta , Ramamurthy Thandavarayan , Ghose Asoke C. , Nandy Ranjan K. 
Published: April 2012

ABSTRACT

The Entner-Doudoroff (ED) pathway has recently been shown to play an important role in sugar catabolism for many organisms although very little information is available on the functionality of this pathway in
Vibrio cholerae
, the causative agent of cholera. In this study, activation of the genes
edd
and
eda
, encoding 6-phosphogluconate dehydratase and 2-keto-3-deoxy-6-phosphogluconate aldolase, was used as a marker of a functional ED pathway in
V. cholerae
. Transcriptional activation analyses and gene silencing experiments with cells grown in sugar-supplemented M9 medium demonstrated that the ED pathway is functional in
V. cholerae
and is obligatory for gluconate catabolism. Importantly, selective activation of the ED pathway led to concurrent elevation of transcripts of prime virulence genes (
ctxA
and
tcpA
) and their regulator (
toxT
). Further, lowering of these transcript levels and cholera toxin production
in vitro
by an ED pathway-defective mutant (strain N16961 with a Δ
edd
mutation [Δ
edd
N16961
strain]) suggested the importance of this pathway in regulating
V. cholerae
virulence. The
in vivo
relevance of these data was established as the mutant failed to colonize in suckling mice intestine or to induce fluid accumulation in ligated rabbit ileal loops. Activation of the ED pathway in
V. cholerae
was shown to inhibit biofilm formation
in vitro
that could be reversed in the mutant. As further support for these results, comparative transcriptome analysis with cells grown in the presence of glucose or gluconate revealed that a functional ED pathway led to activation of a subset of previously reported
in vivo
expressed genes. All of these results suggest the importance of the ED pathway in
V. cholerae
pathogenesis.
MexT Functions as a Redox-Responsive Regulator Modulating Disulfide Stress Resistance in Pseudomonas aeruginosa
Authors: Fargier Emilie , Mac Aogáin Micheál , Mooij Marlies J. , Woods David F. , Morrissey John P. , Dobson Alan D. W. , Adams Claire , O'Gara Fergal 
Published: April 2012

ABSTRACT

MexT is a global LysR transcriptional regulator known to modulate antibiotic resistance and virulence in
Pseudomonas aeruginosa
. In this study, a novel role for MexT in mediating intrinsic disulfide stress resistance was demonstrated, representing the first identified phenotype associated with inactivation of this regulator in wild-type cells. Disruption of
mexT
resulted in increased susceptibility to the disulfide stress elicitor diamide [diazenedicarboxylic acid bis(
N
,
N
,-di-methylamide)]. This compound is known to elicit a specific stress response via depletion of reduced glutathione and alteration of the cellular redox environment, implicating MexT in redox control. In support of this, MexT-regulated targets, including the MexEF-OprN multidrug efflux system, were induced by subinhibitory concentrations of diamide. A
mexF
insertion mutant also exhibited increased diamide susceptibility, implicating the MexEF-OprN efflux system in MexT-associated disulfide stress resistance. Purified MexT protein was observed to form an oligomeric complex in the presence of oxidized glutathione, with a calculated redox potential of −189 mV. This value far exceeds the thiol-disulfide redox potential of the bacterial cytoplasm, ensuring that MexT remains reduced under normal physiological conditions. MexT is activated by mutational disruption of the predicted quinone oxidoreductase encoded by
mexS
. Alterations in the cellular redox state were observed in a
mexS
mutant (PA14
nfxC
), supporting a model whereby the perception of MexS-associated redox signals by MexT leads to the induction of the MexEF-OprN efflux system, which, in turn, may mediate disulfide stress resistance via efflux of electrophilic compounds.
Genetic, Biochemical, and Molecular Characterization of the Polypeptide Transport-Associated Domain of Escherichia coli BamA
Authors: Workman Patricia , Heide Kristina , Giuliano Nicolas , Lee Nanhee , Mar James , Vuong Phu , Bennion Drew , Misra Rajeev 
Published: April 2012

ABSTRACT

The BamA protein of
Escherichia coli
plays a central role in the assembly of β-barrel outer membrane proteins (OMPs). The C-terminal domain of BamA folds into an integral outer membrane β-barrel, and the N terminus forms a periplasmic polypeptide transport-associated (POTRA) domain for OMP reception and assembly. We show here that BamA misfolding, caused by the deletion of the R44 residue from the α2 helix of the POTRA 1 domain (ΔR44), can be overcome by the insertion of alanine 2 residues upstream or downstream from the ΔR44 site. This highlights the importance of the side chain orientation of the α2 helix residues for normal POTRA 1 activity. The ΔR44-mediated POTRA folding defect and its correction by the insertion of alanine were further demonstrated by using a construct expressing just the soluble POTRA domain. Besides misfolding, the expression of BamA
ΔR44
from a low-copy-number plasmid confers a severe drug hypersensitivity phenotype. A spontaneous drug-resistant revertant of BamA
ΔR44
was found to carry an A18S substitution in the α1 helix of POTRA 1. In the BamA
ΔR44, A18S
background, OMP biogenesis improved dramatically, and this correlated with improved BamA folding, BamA-SurA interactions, and LptD (lipopolysaccharide transporter) biogenesis. The presence of the A18S substitution in the wild-type BamA protein did not affect the activity of BamA. The discovery of the A18S substitution in the α1 helix of the POTRA 1 domain as a suppressor of the folding defect caused by ΔR44 underscores the importance of the helix 1 and 2 regions in BamA folding.
Mucin Promotes Rapid Surface Motility in Pseudomonas aeruginosa
Authors: Yeung Amy T. Y. , Parayno Alicia , Hancock Robert E. W. 
Published: May 2012

ABSTRACT

An important environmental factor that determines the mode of motility adopted by
Pseudomonas aeruginosa
is the viscosity of the medium, often provided by adjusting agar concentrations
in vitro
. However, the viscous gel-like property of the mucus layer that overlays epithelial surfaces is largely due to the glycoprotein mucin.
P. aeruginosa
is known to swim within 0.3% (wt/vol) agar and swarm on the surface at 0.5% (wt/vol) agar with amino acids as a weak nitrogen source. When physiological concentrations or as little as 0.05% (wt/vol) mucin was added to the swimming agar, in addition to swimming,
P. aeruginosa
was observed to undergo highly accelerated motility on the surface of the agar. The surface motility colonies in the presence of mucin appeared to be circular, with a bright green center surrounded by a thicker white edge. While intact flagella were required for the surface motility in the presence of mucin, type IV pili and rhamnolipid production were not. Replacement of mucin with other wetting agents indicated that the lubricant properties of mucin might contribute to the surface motility. Based on studies with mutants, the quorum-sensing systems (
las
and
rhl
) and the orphan autoinducer receptor QscR played important roles in this form of surface motility. Transcriptional analysis of cells taken from the motility zone revealed the upregulation of genes involved in virulence and resistance. Based on these results, we suggest that mucin may be promoting a new or highly modified form of surface motility, which we propose should be termed “surfing.”

IMPORTANCE
An important factor that dictates the mode of motility adopted by
P. aeruginosa
is the viscosity of the medium, often provided by adjusting agar concentrations
in vitro
. However, the gel-like properties of the mucous layers that overlay epithelial surfaces, such as those of the lung, a major site of
Pseudomonas
infection, are contributed mostly by the production of the glycoprotein mucin. In this study, we added mucin to swimming media and found that it promoted the ability of
P. aeruginosa
to exhibit rapid surface motility. These motility colonies appeared in a circular form, with a bright green center surrounded by a thicker white edge. Interestingly, bacterial cells at the thick edge appeared piled up and lacked flagella, while cells at the motility center had flagella. Our data from various genetic and phenotypic studies suggest that mucin may be promoting a modified form of swarming or a novel form of surface motility in
P. aeruginosa
.