bluuebunny/crossref_metadata_2025_split huggingface dataset:
Production of Pyomelanin, a Second Type of Melanin, via the Tyrosine Degradation Pathway in
Aspergillus fumigatus
Authors: Schmaler-Ripcke Jeannette , Sugareva Venelina , Gebhardt Peter , Winkler Robert , Kniemeyer Olaf , Heinekamp Thorsten , Brakhage Axel A. 
Published: November 2008

ABSTRACT

Aspergillus fumigatus
is the most important airborne fungal pathogen of immunosuppressed humans.
A. fumigatus
is able to produce dihydroxynaphthalene melanin, which is predominantly present in the conidia. Its biosynthesis is an important virulence determinant. Here, we show that
A. fumigatus
is able to produce an alternative melanin, i.e., pyomelanin, by a different pathway, starting from
l
-tyrosine. Proteome analysis indicated that the
l
-tyrosine degradation enzymes are synthesized when the fungus is grown with
l
-tyrosine in the medium. To investigate the pathway in detail, we deleted the genes encoding essential enzymes for pigment production, homogentisate dioxygenase (
hmgA
) and 4-hydroxyphenylpyruvate dioxygenase (
hppD
). Comparative Fourier transform infrared spectroscopy of synthetic pyomelanin and pigment extracted from
A. fumigatus
cultures confirmed the identity of the observed pigment as pyomelanin. In the
hmgA
deletion strain, HmgA activity was abolished and the accumulation of homogentisic acid provoked an increased pigment formation. In contrast, homogentisic acid and pyomelanin were not observed with an
hppD
deletion mutant. Germlings of the
hppD
deletion mutant showed an increased sensitivity to reactive oxygen intermediates. The transcription of both studied genes was induced by
l
-tyrosine. These results confirmed the function of the deleted genes and the predicted pathway in
A. fumigatus
. Homogentisic acid is the major intermediate, and the
l
-tyrosine degradation pathway leading to pyomelanin is similar to that in humans leading to alkaptomelanin.
Regulatory and DNA Repair Genes Contribute to the Desiccation Resistance of
Sinorhizobium meliloti
Rm1021
Authors: Humann Jodi L. , Ziemkiewicz Hope T. , Yurgel Svetlana N. , Kahn Michael L. 
Published: November 2008

ABSTRACT

Sinorhizobium meliloti
can form a nitrogen-fixing symbiotic relationship with alfalfa after bacteria in the soil infect emerging root hairs of the growing plant. To be successful at this, the bacteria must be able to survive in the soil between periods of active plant growth, including when conditions are dry. The ability of
S. meliloti
to withstand desiccation has been known for years, but genes that contribute to this phenotype have not been identified. Transposon mutagenesis was used in combination with novel screening techniques to identify four desiccation-sensitive mutants of
S. meliloti
Rm1021. DNA sequencing of the transposon insertion sites identified three genes with regulatory functions (
relA
,
rpoE2
, and
hpr
) and a DNA repair gene (
uvrC
). Various phenotypes of the mutants were determined, including their behavior on several indicator media and in symbiosis. All of the mutants formed an effective symbiosis with alfalfa. To test the hypothesis that UvrC-related excision repair was important in desiccation resistance,
uvrA
,
uvrB
, and
uvrC
deletion mutants were also constructed. These strains were sensitive to DNA damage induced by UV light and 4-NQO and were also desiccation sensitive. These data indicate that
uvr
gene-mediated DNA repair and the regulation of stress-induced pathways are important for desiccation resistance.
GzSNF1Is Required for Normal Sexual and Asexual Development in the AscomyceteGibberella zeae
Authors: Lee Seung-Ho , Lee Jungkwan , Lee Seunghoon , Park Eun-Hee , Kim Ki-Woo , Kim Myoung-Dong , Yun Sung-Hwan , Lee Yin-Won 
Published: November 2008

ABSTRACTThe sucrose nonfermenting 1 (SNF1) protein kinase of yeast plays a central role in the transcription of glucose-repressible genes in response to glucose starvation. In this study, we deleted an ortholog ofSNF1fromGibberella zeaeto characterize its functions by using a gene replacement strategy. The mycelial growth of deletion mutants (ΔGzSNF1) was reduced by 21 to 74% on diverse carbon sources. The virulence of ΔGzSNF1mutants on barley decreased, and the expression of genes encoding cell-wall-degrading enzymes was reduced. The most distinct phenotypic changes were in sexual and asexual development. ΔGzSNF1mutants produced 30% fewer perithecia, which matured more slowly, and asci that contained one to eight abnormally shaped ascospores. Mutants in which only the GzSNF1catalytic domain was deleted had the same phenotype changes as the ΔGzSNF1strains, but the phenotype was less extreme in the mutants with the regulatory domain deleted. In outcrosses between the ΔGzSNF1mutants, each perithecium contained ∼70% of the abnormal ascospores, and ∼50% of the asci showed unexpected segregation patterns in a single locus tested. The asexual spores of the ΔGzSNF1mutants were shorter and had fewer septa than those of the wild-type strain. The germination and nucleation of both ascospores and conidia were delayed in ΔGzSNF1mutants in comparison with those of the wild-type strain. GzSNF1 expression and localization depended on the developmental stage of the fungus. These results suggest that GzSNF1is critical for normal sexual and asexual development in addition to virulence and the utilization of alternative carbon sources.
Trypanosome Spliced-Leader-Associated RNA (SLA1) Localization and Implications for Spliced-Leader RNA Biogenesis
Authors: Hury Avraham , Goldshmidt Hanoch , Tkacz Itai Dov , Michaeli Shulamit 
Published: November 2008

ABSTRACTSpliced-leader-associated RNA (SLA1) guides the pseudouridylation at position −12 (relative to the 5′ splice site) of the spliced-leader (SL) RNA in all trypanosomatid species. Nevertheless, the exact role of this RNA is currently unknown. Here, we demonstrate that the absence of pseudouridine onLeptomonas collosomaSL RNA has only a minor effect on the ability of this RNA to function intranssplicing in vivo. To investigate the possible role of SLA1 during SL RNA biogenesis, the structure of the SL RNA was examined in permeableTrypanosoma bruceicells depleted for CBF5, the H/ACA pseudouridine synthase, lacking SLA1. Our results suggest that in the absence of SLA1, the SL RNA secondary structure is changed, as was detected by differential sensitivity to oligonucleotide-directed RNase H cleavage, suggesting that the association of SLA1 maintains the SL RNA in a structural form which is distinct from the structure of the SL RNA in the steady state. InT. bruceicells depleted for the SL RNA core protein SmD1, SL RNA first accumulates in large amounts in the nucleus and then is expelled to the cytoplasm. Here, we demonstrate by in vivo aminomethyltrimethyl UV cross-linking studies that under SmD1 depletion, SLA1 remains bound to SL RNA and escorts the SL RNA to the cytoplasm. In situ hybridization with SLA1 and SL RNA demonstrates colocalization between SLA1 and the SL RNA transcription factor tSNAP42, as well as with Sm proteins, suggesting that SLA1 associates with SL RNA early in its biogenesis. These results demonstrate that SLA1 is a unique chaperonic RNA that functions during the early biogenesis of SL RNA to maintain a structure that is most probably suitable for cap 4 modification.
Growth Conditions Regulate the Requirements forCaulobacterChromosome Segregation
Authors: Shebelut Conrad W. , Jensen Rasmus B. , Gitai Zemer 
Published: November 2008

ABSTRACTGrowth environments are important metabolic and developmental regulators. Here we demonstrate a growth environment-dependent effect onCaulobacterchromosome segregation of a small-molecule inhibitor of the MreB bacterial actin cytoskeleton. Our results also implicate ParAB as important segregation determinants, suggesting that multiple distinct mechanisms can mediateCaulobacterchromosome segregation and that their relative contributions can be environmentally regulated.
The Mycobacteriophage D29 Gene
65
Encodes an Early-Expressed Protein That Functions as a Structure-Specific Nuclease
Authors: Giri Nabanita , Bhowmik Priyanka , Bhattacharya Bidisha , Mitra Mahashweta , Das Gupta Sujoy K. 
Published: November 2008

ABSTRACT

The genomes of mycobacteriophages of the L5 family, which includes the lytic phage D29, contain several genes putatively linked to DNA synthesis. One such gene is
65
, which encodes a protein belonging to the RecA/DnaB helicase superfamily. In this study a recombinant version of the mycobacteriophage D29 gp65 was functionally characterized. The results indicated that it is not a helicase as predicted but an exonuclease that removes 3′ arms from forked structures in an ATP-dependent manner. The gp65 exonuclease acts progressively from the 3′ end, until the fork junction is reached. As it goes past, its progress is stalled over a stretch of seven to eight nucleotides immediately downstream of the junction. It efficiently acts on forked structures with single stranded arms. It also acts upon 5′ and 3′ flaps, though with somewhat relaxed specificity, but not on double-stranded forks. Sequence comparison revealed the presence of a KNRXG motif in the C-terminal half of the protein. This is a conserved element found in the RadA/Sms family of DNA repair proteins. A mutation (R203G) in this motif led to complete loss of nuclease activity. This indicated that KNRXG plays an important role in the nuclease function of not only gp65, but possibly other RadA/Sms family proteins as well. This is the first characterization of a bacteriophage-derived RadA/Sms class protein. Given its mode of action, it is very likely that gp65 is involved in processing branched replication intermediates formed during the replication of phage DNA.
The
Pseudomonas aeruginosa pfpI
Gene Plays an Antimutator Role and Provides General Stress Protection
Authors: Rodríguez-Rojas Alexandro , Blázquez Jesús 
Published: November 2008

ABSTRACT

Hypermutator
Pseudomonas aeruginosa
strains, characterized by an increased spontaneous-mutation rate, are found at high frequencies in chronic lung infections. Hypermutability is associated with the loss of antimutator genes related to DNA repair or damage avoidance systems. Only a few antimutator genes have been described in
P. aeruginosa
, although there is some evidence that additional genes may be involved in naturally occurring hypermutability. In order to find new
P. aeruginosa
antimutator genes, we constructed and screened a library of random insertions in the PA14 strain. Some previously described
P. aeruginosa
and/or
Escherichia coli
antimutator genes, such as
mutS
,
mutL
,
uvrD
,
mutT
,
ung
, and
mutY
, were detected, indicating a good coverage of our insertional library. One additional mutant contained an insertion in the
P. aeruginosa
PA14-04650 (
pfpI
) gene, putatively encoding a member of the DJ-1/ThiJ/PfpI superfamily, which includes chaperones, peptidases, and the Parkinson's disease protein DJ-1a. The
pfpI
-defective mutants in both PAO1 and PA14 showed higher spontaneous mutation rates than the wild-type strains, suggesting that PfpI plays a key role in DNA protection under nonstress conditions. Moreover, the inactivation of
pfpI
resulted in a dramatic increase in the H
2
O
2
-induced mutant frequency. Global transcription studies showed the induction of bacteriophage Pf1 genes and the repression of genes related to iron metabolism, suggesting that the increased spontaneous-mutant frequency may be due to reduced protection against the basal level of reactive oxygen species. Finally,
pfpI
mutants are more sensitive to different types of stress and are affected in biofilm formation.
Glycerol Metabolism Is Important for Cytotoxicity of
Mycoplasma pneumoniae
Authors: Hames Claudine , Halbedel Sven , Hoppert Michael , Frey Joachim , Stülke Jörg 
Published: November 2008

ABSTRACT

Glycerol is one of the few carbon sources that can be utilized by
Mycoplasma pneumoniae
. Glycerol metabolism involves uptake by facilitated diffusion, phosphorylation, and the oxidation of glycerol 3-phosphate to dihydroxyacetone phosphate, a glycolytic intermediate. We have analyzed the expression of the genes involved in glycerol metabolism and observed constitutive expression irrespective of the presence of glycerol or preferred carbon sources. Similarly, the enzymatic activity of glycerol kinase is not modulated by HPr-dependent phosphorylation. This lack of regulation is unique among the bacteria for which glycerol metabolism has been studied so far. Two types of enzymes catalyze the oxidation of glycerol 3-phosphate: oxidases and dehydrogenases. Here, we demonstrate that the enzyme encoded by the
M. pneumoniae glpD
gene is a glycerol 3-phosphate oxidase that forms hydrogen peroxide rather than NADH
2
. The formation of hydrogen peroxide by GlpD is crucial for cytotoxic effects of
M. pneumoniae
. A
glpD
mutant exhibited a significantly reduced formation of hydrogen peroxide and a severely reduced cytotoxicity. Attempts to isolate mutants affected in the genes of glycerol metabolism revealed that only the
glpD
gene, encoding the glycerol 3-phosphate oxidase, is dispensable. In contrast, the
glpF
and
glpK
genes, encoding the glycerol facilitator and the glycerol kinase, respectively, are essential in
M. pneumoniae
. Thus, the enzymes of glycerol metabolism are crucial for the pathogenicity of
M. pneumoniae
but also for other essential, yet-to-be-identified functions in the
M. pneumoniae
cell.
Functional Analysis of Three Sulfide:Quinone Oxidoreductase Homologs in
Chlorobaculum tepidum
Authors: Chan Leong-Keat , Morgan-Kiss Rachael M. , Hanson Thomas E. 
Published: November 2008

ABSTRACT

Sulfide:quinone oxidoreductase (SQR) catalyzes sulfide oxidation during sulfide-dependent chemo- and phototrophic growth in bacteria. The green sulfur bacterium
Chlorobaculum tepidum
(formerly
Chlorobium tepidum
) can grow on sulfide as the sole electron donor and sulfur source.
C. tepidum
contains genes encoding three SQR homologs: CT0117, CT0876, and CT1087. This study examined which, if any, of the SQR homologs possess sulfide-dependent ubiquinone reduction activity and are required for growth on sulfide. In contrast to CT0117 and CT0876, transcripts of CT1087 were detected only when cells actively oxidized sulfide. Mutation of CT0117 or CT1087 in
C. tepidum
decreased SQR activity in membrane fractions, and the CT1087 mutant could not grow with ≥6 mM sulfide. Mutation of both CT0117 and CT1087 in
C. tepidum
completely abolished SQR activity, and the double mutant failed to grow with ≥4 mM sulfide. A C-terminal His
6
-tagged CT1087 protein was membrane localized, as was SQR activity. Epitope-tagged CT1087 was detected only when sulfide was actively consumed by cells. Recombinantly produced CT1087 and CT0117 proteins had SQR activity, while CT0876 did not. In summary, we conclude that, under the conditions tested, both CT0117 and CT1087 function as SQR proteins in
C. tepidum
. CT0876 may support the growth of
C. tepidum
at low sulfide concentrations, but no evidence was found for SQR activity associated with this protein.
Identification and Characterization of a Bacterial Transport System for the Uptake of Pyruvate, Propionate, and Acetate in
Corynebacterium glutamicum
Authors: Jolkver Elena , Emer Denise , Ballan Stefan , Krämer Reinhard , Eikmanns Bernhard J. , Marin Kay 
Published: November 2008

ABSTRACT

The metabolism of monocarboxylic acids is of central importance for bacteria in their natural habitat as well as during biotechnological production. Although biosynthesis and degradation are well understood, the transport of such compounds is still a matter of discussion. Here we present the identification and characterization of a new transport system in
Corynebacterium glutamicum
with high affinity for acetate and propionate and with lower affinity for pyruvate. Biochemical analysis of this monocarboxylic acid transporter (MctC) revealed for the first time a quantitative discrimination of passive diffusion and active transport of acetate by bacterial cells. MctC is a secondary transporter and belongs to the class of sodium solute symporters, but it is driven by the electrochemical proton potential. The
mctC
gene is preceded by and cotranscribed with cg0952, a locus encoding a small membrane protein, and the transcription of the cg0952-
mctC
operon is under the control of the transcriptional regulators RamA and RamB. Both of these proteins directly bind to the promoter region of the operon; RamA is essential for expression and RamB exerts a slightly negative control on expression of the cg0952-
mctC
operon.
mctC
expression is induced in the presence of pyruvate and beneficial under substrate-limiting conditions for
C. glutamicum
.
Influence of Operator Site Geometry on Transcriptional Control by the YefM-YoeB Toxin-Antitoxin Complex
Authors: Bailey Simon E. S. , Hayes Finbarr 
Published: November 2008

ABSTRACT

YefM-YoeB is among the most prevalent and well-characterized toxin-antitoxin complexes. YoeB toxin is an endoribonuclease whose activity is inhibited by YefM antitoxin. The regions 5′ of
yefM-yoeB
in diverse bacteria possess conserved sequence motifs that mediate transcriptional autorepression. The
yefM-yoeB
operator site arrangement is exemplified in
Escherichia coli
: a pair of palindromes with core hexamer motifs and a center-to-center distance of 12 bp overlap the
yefM-yoeB
promoter. YefM is an autorepressor that initially recognizes a long palindrome containing the core hexamer, followed by binding to a short repeat. YoeB corepressor greatly enhances the YefM-operator interaction. Scanning mutagenesis demonstrated that the short repeat is crucial for correct interaction of YefM-YoeB with the operator site in vivo and in vitro. Moreover, altering the relative positions of the two palindromes on the DNA helix abrogated YefM-YoeB cooperative interactions with the repeats: complex binding to the long repeat was maintained but was perturbed to the short repeat. Although YefM lacks a canonical DNA binding motif, dual conserved arginine residues embedded in a basic patch of the protein are crucial for operator recognition. Deciphering the molecular basis of toxin-antitoxin transcriptional control will provide key insights into toxin-antitoxin activation and function.
Expression of the
cpdA
Gene, Encoding a 3′,5′-Cyclic AMP (cAMP) Phosphodiesterase, Is Positively Regulated by the cAMP-cAMP Receptor Protein Complex
Authors: Kim Han-Suk , Kim Sung-Min , Lee Hyun-Jung , Park Soon-Jung , Lee Kyu-Ho 
Published: November 2008

ABSTRACT

The intracellular level of cyclic 3′,5′-AMP (cAMP), a signaling molecule that mediates a variety of cellular processes, is finely modulated by the regulation of its synthesis, excretion, and degradation. In this study, cAMP phosphodiesterase (CpdA), an enzyme that catalyzes the conversion of cAMP to AMP, was characterized in a pathogenic bacterium,
Vibrio vulnificus
. The
cpdA
gene exists in an operon composed of
mutT, yqiB, cpdA
, and
yqiA
, the transcription of which was initiated at position −22 upstream of
mutT
. A
cpdA
-null mutant of
V. vulnificus
contained significantly higher levels of cAMP than the wild type but showed no detectable cAMP when a multicopy plasmid of the
cpdA
gene was provided in
trans
, suggesting that CpdA is responsible for cAMP degradation. Cellular contents of the CpdA protein decreased dramatically in both
cya
and
crp
mutants. In addition, levels of expression of the
cpdA
::
luxAB
transcription fusion decreased in
cya
and
crp
mutants. The level of expression of
cpdA
::
luxAB
in the
cya
mutant increased in a concentration-dependent manner upon the exogenous addition of cAMP. The cAMP-cAMP receptor protein (CRP) complex bound directly to the upstream region of
mutT
, which includes a putative CRP-binding sequence centered at position −95.5 relative to the transcription start site. Site-directed mutagenesis or the deletion of this sequence in the
cpdA
::
luxAB
transcription fusion resulted in the loss of regulation by cAMP and CRP. Thus, this study demonstrates that CpdA plays a crucial role in determining the intracellular cAMP level and shows for the first time that the expression of
cpdA
is activated by the cAMP-CRP complex via direct binding to the regulatory region.
The Mevalonate Pathway ofStaphylococcus aureus
Authors: Balibar Carl J. , Shen Xiaoyu , Tao Jianshi 
Published: November 2008

ABSTRACTIsoprenoids are a class of ubiquitous organic molecules synthesized from the five-carbon starter unit isopentenyl pyrophosphate (IPP). Comprising more than 30,000 known natural products, isoprenoids serve various important biological functions in many organisms. In bacteria, undecaprenyl pyrophosphate is absolutely required for the formation of cell wall peptidoglycan and other cell surface structures, while ubiquinones and menaquinones, both containing an essential prenyl moiety, are key electron carriers in respiratory energy generation. There is scant knowledge on the nature and regulation of bacterial isoprenoid pathways. In order to explore the cellular responses to perturbations in the mevalonate pathway, responsible for producing the isoprenoid precursor IPP in many gram-positive bacteria and eukaryotes, we constructed three strains ofStaphylococcus aureusin which each of the mevalonate pathway genes is regulated by an IPTG (isopropyl-β-d-thiogalactopyranoside)-inducible promoter. We used DNA microarrays to profile the transcriptional effects of downregulating the components of the mevalonate pathway inS. aureusand demonstrate that decreased expression of the mevalonate pathway leads to widespread downregulation of primary metabolism genes, an upregulation in virulence factors and cell wall biosynthetic determinants, and surprisingly little compensatory expression in other isoprenoid biosynthetic genes. We subsequently correlate these transcriptional changes with downstream metabolic consequences.
Switching Protein-DNA Recognition Specificity by Single-Amino-Acid Substitutions in the P1parFamily of Plasmid Partition Elements
Authors: Dabrazhynetskaya Alena , Brendler Therese , Ji Xinhua , Austin Stuart 
Published: November 2008

ABSTRACTThe P1, P7, and pMT1parsystems are members of the P1parfamily of plasmid partition elements. Each has a ParA ATPase and a ParB protein that recognizes theparSpartition site of its own plasmid type to promote the active segregation of the plasmid DNA to daughter cells. ParB contacts twoparSmotifs known as BoxA and BoxB, the latter of which determines species specificity. We found that the substitution of a single orthologous amino acid in ParB for that of a different species has major effects on the specificity of recognition. A single change in ParB can cause a complete switch in recognition specificity to that of another species or can abolish specificity. Specificity changes do not necessarily correlate with changes in the gross DNA binding properties of the protein. Molecular modeling suggests that species specificity is determined by the capacity to form a hydrogen bond between ParB residue 288 and the second base in the BoxB sequence. As changes in just one ParB residue and one BoxB base can alter species specificity, plasmids may use such simple changes to evolve new species rapidly.