bluuebunny/crossref_metadata_2025_split huggingface dataset:
Comparison of Sterol Import under Aerobic and Anaerobic Conditions in Three Fungal Species, Candida albicans, Candida glabrata, and Saccharomyces cerevisiae
Authors: Zavrel Martin , Hoot Sam J. , White Theodore C. 
Published: March 2013

ABSTRACT

Sterol import has been characterized under various conditions in three distinct fungal species, the model organism
Saccharomyces cerevisiae
and two human fungal pathogens
Candida glabrata
and
Candida albicans
, employing cholesterol, the sterol of higher eukaryotes, as well as its fungal equivalent, ergosterol. Import was confirmed by the detection of esterified cholesterol within the cells. Comparing the three fungal species, we observe sterol import under three different conditions. First, as previously well characterized, we observe sterol import under low oxygen levels in
S. cerevisiae
and
C. glabrata
, which is dependent on the transcription factor Upc2 and/or its orthologs or paralogs. Second, we observe sterol import under aerobic conditions exclusively in the two pathogenic fungi
C. glabrata
and
C. albicans
. Uptake emerges during post-exponential-growth phases, is independent of the characterized Upc2-pathway and is slower compared to the anaerobic uptake in
S. cerevisiae
and
C. glabrata
. Third, we observe under normoxic conditions in
C. glabrata
that Upc2-dependent sterol import can be induced in the presence of fetal bovine serum together with fluconazole. In summary,
C. glabrata
imports sterols both in aerobic and anaerobic conditions, and the limited aerobic uptake can be further stimulated by the presence of serum together with fluconazole.
S. cerevisiae
imports sterols only in anaerobic conditions, demonstrating aerobic sterol exclusion. Finally,
C. albicans
imports sterols exclusively aerobically in post-exponential-growth phases, independent of Upc2. For the first time, we provide direct evidence of sterol import into the human fungal pathogen
C. albicans
, which until now was believed to be incapable of active sterol import.
Agl16, a Thermophilic Glycosyltransferase Mediating the Last Step of
N
-Glycan Biosynthesis in the Thermoacidophilic Crenarchaeon Sulfolobus acidocaldarius
Authors: Meyer Benjamin H. , Peyfoon Elham , Dietrich Carsten , Hitchen Paul , Panico Maria , Morris Howard R. , Dell Anne , Albers Sonja-Verena 
Published: March 2013

ABSTRACT

Recently, the S-layer protein of
Sulfolobus acidocaldarius
was shown to be N-linked with a tribranched hexasaccharide, composed of Man
2
Glc
1
GlcNAc
2
and a sulfated sugar called sulfoquinovose. To identify genes involved in the biosynthesis and attachment of this glycan, markerless in-frame deletions of genes coding for predicted glycosyltransferases were created. The successful deletion of
agl16
, coding for a glycosyltransferase, resulted in the S-layer protein and archaellins having reduced molecular weights, as visualized by Coomassie staining or immunoblotting. This analysis indicated a change in the
N
-glycan composition. Nano-liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses confirmed that the glycan of the S-layer protein from the
agl16
deletion mutant was a pentasaccharide, which was missing a terminal hexose residue. High-performance liquid chromatography (HPLC) analyses of the hydrolyzed
N
-glycan indicated that the missing hexose is a glucose residue. A physiological characterization of the
agl16
deletion mutant revealed a significant effect on the growth at elevated salt concentrations. At 300 mM NaCl, the doubling time of the Δ
agl16
mutant was increased 2-fold compared to that of the background strain. Furthermore, the incomplete glycan structure of the Δ
agl16
deletion strain affected the assembly and function of the archaellum, as exemplified by semisolid Gelrite plate analysis, in which the motility is decreased according to the
N
-glycan size.
The Vitamin B
6
Biosynthesis Pathway in Streptococcus pneumoniae Is Controlled by Pyridoxal 5′-Phosphate and the Transcription Factor PdxR and Has an Impact on Ear Infection
Authors: El Qaidi Samir , Yang Jun , Zhang Jing-Ren , Metzger Dennis W. , Bai Guangchun 
Published: March 2013

ABSTRACT

Vitamin B
6
is an essential cofactor for a large number of enzymes in both prokaryotes and eukaryotes. In this study, we characterized the pyridoxal 5′-phosphate (PLP) biosynthesis pathway in
Streptococcus pneumoniae
. Our results revealed that
S. pneumoniae
possesses a
de novo
vitamin B
6
biosynthesis pathway encoded by the
pdxST
genes. Purified PdxS functionally displayed as PLP synthase, whereas PdxT exhibited glutaminase activity
in vitro
. Deletion of
pdxS
, but not
pdxT
, resulted in a vitamin B
6
auxotrophic mutant. The defective growth of the Δ
pdxS
mutant in a vitamin B
6
-depleted medium could be chemically restored in the presence of the B
6
vitamers at optimal concentrations. By analyzing PdxS expression levels, we demonstrated that the expression of
pdxS
was repressed by PLP and activated by a transcription factor, PdxR. A pneumococcal Δ
pdxR
mutant also exhibited as a vitamin B
6
auxotroph. In addition, we found that disruption of the vitamin B
6
biosynthesis pathway in
S. pneumoniae
caused a significant attenuation in a chinchilla middle ear infection model and a minor attenuation in a mouse pneumonia model, indicating that the impact of vitamin B
6
synthesis on virulence depends upon the bacterial infection niche.
The Nucleotide Excision Repair System of Borrelia burgdorferi Is the Sole Pathway Involved in Repair of DNA Damage by UV Light
Authors: Hardy Pierre-Olivier , Chaconas George 
Published: March 2013

ABSTRACTTo survive and avoid accumulation of mutations caused by DNA damage, the genomes of prokaryotes encode a variety of DNA repair pathways most well characterized inEscherichia coli. Some of these are required for the infectivity of various pathogens. In this study, the importance of 25 DNA repair/recombination genes forBorrelia burgdorferisurvival to UV-induced DNA damage was assessed. In contrast toE. coli, where 15 of these genes have an effect on survival of UV irradiation, disruption of recombinational repair, transcription-coupled repair, methyl-directed mismatch correction, and repair of arrested replication fork pathways did not decrease survival ofB. burgdorferiexposed to UV light. However, the disruption of theB. burgdorferinucleotide excision repair (NER) pathway (uvrA,uvrB,uvrC, anduvrD) resulted in a 10- to 1,000-fold increase in sensitivity to UV light. A functional NER pathway was also shown to be required forB. burgdorferiresistance to nitrosative damage. Finally, disruption ofuvrA,uvrC, anduvrDhad only a minor effect upon murine infection by increasing the time required for dissemination.
SsaA, a Member of a Novel Class of Transcriptional Regulators, Controls Sansanmycin Production in Streptomyces sp. Strain SS through a Feedback Mechanism
Authors: Li Qinglian , Wang Lifei , Xie Yunying , Wang Songmei , Chen Ruxian , Hong Bin 
Published: March 2013

ABSTRACT

Sansanmycins, produced by
Streptomyces
sp. strain SS, are uridyl peptide antibiotics with activities against
Pseudomonas aeruginosa
and multidrug-resistant
Mycobacterium tuberculosis
. In this work, the biosynthetic gene cluster of sansanmycins, comprised of 25 open reading frames (ORFs) showing considerable amino acid sequence identity to those of the pacidamycin and napsamycin gene cluster, was identified. SsaA, the archetype of a novel class of transcriptional regulators, was characterized in the sansanmycin gene cluster, with an N-terminal fork head-associated (FHA) domain and a C-terminal LuxR-type helix-turn-helix (HTH) motif. The disruption of
ssaA
abolished sansanmycin production, as well as the expression of the structural genes for sansanmycin biosynthesis, indicating that SsaA is a pivotal activator for sansanmycin biosynthesis. SsaA was proved to directly bind several putative promoter regions of biosynthetic genes, and comparison of sequences of the binding sites allowed the identification of a consensus SsaA binding sequence, GTMCTGACAN
2
TGTCAGKAC. The DNA binding activity of SsaA was inhibited by sansanmycins A and H in a concentration-dependent manner. Furthermore, sansanmycins A and H were found to directly interact with SsaA. These results indicated that SsaA strictly controls the production of sansanmycins at the transcriptional level in a feedback regulatory mechanism by sensing the accumulation of the end products. As the first characterized regulator of uridyl peptide antibiotic biosynthesis, the understanding of this autoregulatory process involved in sansanmycin biosynthesis will likely provide an effective strategy for rational improvements in the yields of these uridyl peptide antibiotics.
Insights into Denitrification in Methylotenera mobilis from Denitrification Pathway and Methanol Metabolism Mutants
Authors: Mustakhimov Ildar , Kalyuzhnaya Marina G. , Lidstrom Mary E. , Chistoserdova Ludmila 
Published: March 2013

ABSTRACT

We investigated phenotypes of mutants of
Methylotenera mobilis
JLW8 with lesions in genes predicted to encode functions of the denitrification pathway, as well as mutants with mutations in methanol dehydrogenase-like structural genes
xoxF1
and
xoxF2
, in order to obtain insights into denitrification and methanol metabolism by this bacterium. By monitoring the accumulation of nitrous oxide, we demonstrate that a periplasmic nitrate reductase, NAD(P)-linked and copper-containing nitrite reductases, and a nitric oxide reductase are involved in the denitrification pathway and that the pathway must be operational in aerobic conditions. However, only the assimilatory branch of the denitrification pathway was essential for growth on methanol in nitrate-supplemented medium. Mutants with mutations in each of the two
xoxF
genes maintained their ability to grow on methanol, but not the double XoxF mutant, suggesting that XoxF proteins act as methanol dehydrogenase enzymes in
M. mobilis
JLW8. Reduced levels of nitrous oxide accumulated by the XoxF mutants compared to the wild type suggest that these enzymes must be capable of donating electrons for denitrification.
Dynamic Localization of a Transcription Factor in Bacillus subtilis: the LicT Antiterminator Relocalizes in Response to Inducer Availability
Authors: Rothe Fabian M. , Wrede Christoph , Lehnik-Habrink Martin , Görke Boris , Stülke Jörg 
Published: March 2013

ABSTRACT

Bacillus subtilis
transports β-glucosides such as salicin by a dedicated phosphotransferase system (PTS). The expression of the β-glucoside permease BglP is induced in the presence of the substrate salicin, and this induction requires the binding of the antiterminator protein LicT to a specific RNA target in the 5′ region of the
bglP
mRNA to prevent the formation of a transcription terminator. LicT is composed of an N-terminal RNA-binding domain and two consecutive PTS regulation domains, PRD1 and PRD2. In the absence of salicin, LicT is phosphorylated on PRD1 by BglP and thereby inactivated. In the presence of the inducer, the phosphate group from PRD1 is transferred back to BglP and consequently to the incoming substrate, resulting in the activation of LicT. In this study, we have investigated the intracellular localization of LicT. While the protein was evenly distributed in the cell in the absence of the inducer, we observed a subpolar localization of LicT if salicin was present in the medium. Upon addition or removal of the inducer, LicT rapidly relocalized in the cells. This dynamic relocalization did not depend on the binding of LicT to its RNA target sites, since the localization pattern was not affected by deletion of all LicT binding sites. In contrast, experiments with mutants affected in the PTS components as well as mutations of the LicT phosphorylation sites revealed that phosphorylation of LicT by the PTS components plays a major role in the control of the subcellular localization of this RNA-binding transcription factor.
Characterization of the
phd-doc
and
ccd
Toxin-Antitoxin Cassettes from Vibrio Superintegrons
Authors: Guérout Anne-Marie , Iqbal Naeem , Mine Natacha , Ducos-Galand Magaly , Van Melderen Laurence , Mazel Didier 
Published: March 2013

ABSTRACT

Toxin-antitoxin (TA) systems have been reported in the genomes of most bacterial species, and their role when located on the chromosome is still debated. TA systems are particularly abundant in the massive cassette arrays associated with chromosomal superintegrons (SI). Here, we describe the characterization of two superintegron cassettes encoding putative TA systems. The first is the

phd-doc
SI

system identified in
Vibrio cholerae
N16961. We determined its distribution in 36
V. cholerae
strains and among five
V. metschnikovii
strains. We show that this cassette, which is in position 72 of the
V. cholerae
N16961 cassette array, is functional, carries its own promoter, and is expressed from this location. Interestingly, the

phd-doc
SI

system is unable to control its own expression, most likely due to the absence of any DNA-binding domain on the antitoxin. In addition, this SI system is able to cross talk with the canonical P1 phage system. The second cassette that we characterized is the

ccd
Vfi

cassette found in the
V. fischeri
superintegron. We demonstrate that CcdB
Vfi
targets DNA-gyrase, as the canonical CcB
F
toxin, and that

ccd
Vfi

regulates its expression in a fashion similar to the
ccd
F
operon of the conjugative plasmid F. We also establish that this cassette is functional and expressed in its chromosomal context in
V. fischeri
CIP 103206T. We tested its functional interactions with the

ccdAB
F

system and found that CcdA
Vfi
is specific for its associated CcdB
Vfi
and cannot prevent CcdB
F
toxicity. Based on these results, we discuss the possible biological functions of these TA systems in superintegrons.
Influence of Copper Resistance Determinants on Gold Transformation by Cupriavidus metallidurans Strain CH34
Authors: Wiesemann Nicole , Mohr Juliane , Grosse Cornelia , Herzberg Martin , Hause Gerd , Reith Frank , Nies Dietrich H. 
Published: March 2013

ABSTRACTCupriavidus metalliduransis associated with gold grains and may be involved in their formation. Gold(III) complexes influence the transcriptome ofC. metallidurans(F. Reith et al., Proc. Natl. Acad. Sci. U. S. A. 106:17757–17762, 2009), leading to the upregulation of genes involved in the detoxification of reactive oxygen species and metal ions. In a systematic study, the involvement of these systems in gold transformation was investigated. Treatment ofC. metalliduranscells with Au(I) complexes, which occur in this organism's natural environment, led to the upregulation of genes similar to those observed for treatment with Au(III) complexes. The two indigenous plasmids ofC. metallidurans, which harbor several transition metal resistance determinants, were not involved in resistance to Au(I/III) complexes nor in their transformation to metallic nanoparticles. Upregulation of acupA-lacZfusion by the MerR-type regulator CupR with increasing Au(III) concentrations indicated the presence of gold ions in the cytoplasm. A hypothesis stating that the Gig system detoxifies gold complexes by the uptake and reduction of Au(III) to Au(I) or Au(0) reminiscent to detoxification of Hg(II) was disproven. ZupT and other secondary uptake systems for transition metal cations influenced Au(III) resistance but not the upregulation of thecupA-lacZfusion. The two copper-exporting P-type ATPases CupA and CopF were also not essential for gold resistance. ThecopABCDdeterminant on chromosome 2, which encodes periplasmic proteins involved in copper resistance, was required for full gold resistance inC. metallidurans. In conclusion, biomineralization of gold particles via the reduction of mobile Au(I/III) complexes inC. metalliduransappears to primarily occur in the periplasmic space via copper-handling systems.
A Mutant with Aberrant Extracellular LcrV-YscF Interactions Fails To Form Pores and Translocate Yop Effector Proteins but Retains the Ability To Trigger Yop Secretion in Response to Host Cell Contact
Authors: Harmon Dana E. , Murphy Julia L. , Davis Alison J. , Mecsas Joan 
Published: March 2013

ABSTRACT

The plasmid-encoded type three secretion system (TTSS) of
Yersinia
spp. is responsible for the delivery of effector proteins into cells of the innate immune system, where these effectors disrupt the target cells' activity. Successful translocation of effectors into mammalian cells requires
Yersinia
to both insert a translocon into the host cell membrane and sense contact with host cells. To probe the events necessary for translocation, we investigated protein-protein interactions among TTSS components of the needle-translocon complex using a chemical cross-linking-based approach. We detected extracellular protein complexes containing YscF, LcrV, and YopD that were dependent upon needle formation. The formation of these complexes was evaluated in a secretion-competent but translocation-defective mutant, the YscFD28AD46A strain (expressing YscF with the mutations D28A and D46A). We found that one of the YscF and most of the LcrV and YopD cross-linked complexes were nearly absent in this mutant. Furthermore, the YscFD28AD46A strain did not support YopB insertion into mammalian membranes, supporting the idea that the LcrV tip complex is required for YopB insertion and translocon formation. However, the YscFD28AD46A strain did secrete Yops in the presence of host cells, indicating that a translocation-competent tip complex is not required to sense contact with host cells to trigger Yop secretion. In conclusion, in the absence of cross-linkable LcrV-YscF interactions, translocon insertion is abolished, but
Yersinia
still retains the ability to sense cell contact.
Cyclic Di-GMP Modulates the Disease Progression of Erwinia amylovora
Authors: Edmunds Adam C. , Castiblanco Luisa F. , Sundin George W. , Waters Christopher M. 
Published: March 2013

ABSTRACT

The second messenger cyclic di-GMP (c-di-GMP) is a nearly ubiquitous intracellular signal molecule known to regulate various cellular processes, including biofilm formation, motility, and virulence. The intracellular concentration of c-di-GMP is inversely governed by diguanylate cyclase (DGC) enzymes and phosphodiesterase (PDE) enzymes, which synthesize and degrade c-di-GMP, respectively. The role of c-di-GMP in the plant pathogen and causal agent of fire blight disease
Erwinia amylovora
has not been studied previously. Here we demonstrate that three of the five predicted DGC genes in
E. amylovora
(
edc
genes, for

E
rwinia

d
iguanylate
c
yclase),
edcA
,
edcC
, and
edcE
, are active diguanylate cyclases. We show that c-di-GMP positively regulates the secretion of the main exopolysaccharide in
E. amylovora
, amylovoran, leading to increased biofilm formation, and negatively regulates flagellar swimming motility. Although amylovoran secretion and biofilm formation are important for the colonization of plant xylem tissues and the development of systemic infections, deletion of the two biofilm-promoting DGCs increased tissue necrosis in an immature-pear infection assay and an apple shoot infection model, suggesting that c-di-GMP negatively regulates virulence. In addition, c-di-GMP inhibited the expression of
hrpA
, a gene encoding the major structural component of the type III secretion pilus. Our results are the first to describe a role for c-di-GMP in
E. amylovora
and suggest that downregulation of motility and type III secretion by c-di-GMP during infection plays a key role in the coordination of pathogenesis.
SMC Condensation Centers in Bacillus subtilis Are Dynamic Structures
Authors: Kleine Borgmann Luise A. K. , Hummel Hanna , Ulbrich Maximilian H. , Graumann Peter L. 
Published: March 2013

ABSTRACT

SMC and MukB complexes consist of a central SMC dimer and two essential binding partners, ScpA and ScpB (MukE and MukF), and are crucial for correct chromosome compaction and segregation. The complexes form two bipolar assemblies on the chromosome, one in each cell half. Using fluorescence recovery after photobleaching (FRAP), we provide evidence that the SMC complex has high exchange rates. This depends to a considerable degree on
de novo
protein synthesis, revealing that the bacterial SMC complex has high on and off rates for binding to the chromosome. A mutation in SMC that affects ATPase activity and results in exaggerated DNA binding
in vitro
causes a strong segregation defect
in vivo
and affects the localization of the entire SMC complex, which localizes to many more sites in the cell than under normal conditions. These data indicate that ATP turnover is important for the function of
Bacillus subtilis
SMC. In contrast, the centromere protein Spo0J and DNA gyrase showed much less exchange between distinct binding sites on the chromosome than that seen with SMC. Binding of Spo0J to the origin regions was rather static and remained partially conserved until the next cell cycle. Our experiments reveal that the SMC complex has a high, condensin-like turnover rate and that an alteration of the ATPase cycle affects SMC function
in vivo
, while several nucleoid-associated proteins feature limited or slow exchange between different sites on the nucleoid, which may be the basis for epigenetic-like phenomena observed in bacteria.
PelN Is a New Pectate Lyase of Dickeya dadantii with Unusual Characteristics
Authors: Hassan Susan , Shevchik Vladimir E. , Robert Xavier , Hugouvieux-Cotte-Pattat Nicole 
Published: March 2013

ABSTRACT

The plant-pathogenic bacterium
Dickeya dadantii
produces several pectinolytic enzymes that play a major role in the soft-rot disease. Eight characterized endopectate lyases are secreted in the extracellular medium by the type II secretion system, Out. They cleave internal glycosidic bonds of pectin, leading to plant tissue maceration. The
D. dadantii
pectate lyases belong to different families, namely, PL1, PL2, PL3, and PL9. Analysis of the
D. dadantii
3937 genome revealed a gene encoding a new protein of the PL9 family, which already includes the secreted endopectate lyase PelL and the periplasmic exopectate lyase PelX. We demonstrated that PelN is an additional extracellular protein secreted by the Out system. However, PelN has some unusual characteristics. Although most pectate lyases require a very alkaline pH and Ca
2+
for their activity, the PelN activity is optimal at pH 7.4 and in the presence of Fe
2+
as a cofactor. PelN is only weakly affected by the degree of pectin methyl esterification. The PelN structural model, constructed on the basis of the PelL structure, suggests that the PelL global topology and its catalytic amino acids are conserved in PelN. Notable differences concern the presence of additional loops at the PelN surface, and the replacement of PelL charged residues, involved in substrate binding, by aromatic residues in PelN. The
pelN
expression is affected by different environmental conditions, such as pH, osmolarity, and temperature. It is controlled by the repressors KdgR and PecS and by the activator GacA, three regulators of
D. dadantii
pectinase genes. Since a
pelN
mutant had reduced virulence on chicory leaves, the PelN enzyme plays a role in plant infection, despite its low specific activity and its unusual cofactor requirement.