bluuebunny/crossref_metadata_2025_split huggingface dataset:
A Developmentally Regulated Gene Cluster Involved in Conidial Pigment Biosynthesis in
Aspergillus fumigatus
Authors: Tsai Huei-Fung , Wheeler Michael H. , Chang Yun C. , Kwon-Chung K. J. 
Published: December 2019

ABSTRACT

Aspergillus fumigatus
, a filamentous fungus producing bluish-green conidia, is an important opportunistic pathogen that primarily affects immunocompromised patients. Conidial pigmentation of
A. fumigatus
significantly influences its virulence in a murine model. In the present study, six genes, forming a gene cluster spanning 19 kb, were identified as involved in conidial pigment biosynthesis in
A. fumigatus
. Northern blot analyses showed the six genes to be developmentally regulated and expressed during conidiation. The gene products of
alb1
(for “albino 1”),
arp1
(for “aspergillus reddish-pink 1”), and
arp2
have high similarity to polyketide synthases, scytalone dehydratases, and hydroxynaphthalene reductases, respectively, found in the dihydroxynaphthalene (DHN)-melanin pathway of brown and black fungi. The
abr1
gene (for “aspergillus brown 1”) encodes a putative protein possessing two signatures of multicopper oxidases. The
abr2
gene product has homology to the laccase encoded by the
yA
gene of
Aspergillus nidulans
. The function of
ayg1
(for “aspergillus yellowish-green 1”) remains unknown. Involvement of the six genes in conidial pigmentation was confirmed by the altered conidial color phenotypes that resulted from disruption of each gene in
A. fumigatus
. The presence of a DHN-melanin pathway in
A. fumigatus
was supported by the accumulation of scytalone and flaviolin in the
arp1
deletant, whereas only flaviolin was accumulated in the
arp2
deletants. Scytalone and flaviolin are well-known signature metabolites of the DHN-melanin pathway. Based on DNA sequence similarity, gene disruption results, and biochemical analyses, we conclude that the 19-kb DNA fragment contains a six-gene cluster which is required for conidial pigment biosynthesis in
A. fumigatus
. However, the presence of
abr1
,
abr2
, and
ayg1
in addition to
alb1
,
arp1
, and
arp2
suggests that conidial pigment biosynthesis in
A. fumigatus
is more complex than the known DHN-melanin pathway.
Sulfide-Quinone Reductase from
Rhodobacter capsulatus
: Requirement for Growth, Periplasmic Localization, and Extension of Gene Sequence Analysis
Authors: Schütz Michael , Maldener Iris , Griesbeck Christoph , Hauska Günter 
Published: December 2019

ABSTRACT

The entire sequence of the 3.5-kb fragment of genomic DNA from
Rhodobacter capsulatus
which contains the
sqr
gene and a second complete and two further partial open reading frames has been determined. A correction of the previously published
sqr
gene sequence (M. Schütz, Y. Shahak, E. Padan, and G. Hauska, J. Biol. Chem. 272:9890–9894, 1997) which in the deduced primary structure of the sulfide-quinone reductase changes four positive into four negative charges and the number of amino acids from 425 to 427 was necessary. The correction has no further bearing on the former sequence analysis. Deletion and interruption strains document that sulfide-quinone reductase is essential for photoautotrophic growth on sulfide. The sulfide-oxidizing enzyme is involved in energy conversion, not in detoxification. Studies with an alkaline phosphatase fusion protein reveal a periplasmic localization of the enzyme. Exonuclease treatment of the fusion construct demonstrated that the C-terminal 38 amino acids of sulfide-quinone reductase were required for translocation. An N-terminal signal peptide for translocation was not found in the primary structure of the enzyme. The possibility that the neighboring open reading frame, which contains a double arginine motif, may be involved in translocation has been excluded by gene deletion (rather, the product of this gene functions in an ATP-binding cassette transporter system, together with the product of one of the other open reading frames). The results lead to the conclusion that the sulfide-quinone reductase of
R. capsulatus
functions at the periplasmic surface of the cytoplasmic membrane and that this flavoprotein is translocated by a hitherto-unknown mechanism.
Functional Interactions of a Homolog of Proliferating Cell Nuclear Antigen with DNA Polymerases inArchaea
Authors: Cann Isaac K. O. , Ishino Sonoko , Hayashi Ikuko , Komori Kayoko , Toh Hiroyuki , Morikawa Kosuke , Ishino Yoshizumi 
Published: December 2019

ABSTRACTProliferating cell nuclear antigen (PCNA) is an essential component of the DNA replication and repair machinery in the domainEucarya. We cloned the gene encoding a PCNA homolog (PfuPCNA) from an euryarchaeote,Pyrococcus furiosus, expressed it inEscherichia coli, and characterized the biochemical properties of the gene product. The proteinPfuPCNA stimulated the in vitro primer extension abilities of polymerase (Pol) I and Pol II, which are the two DNA polymerases identified in this organism to date. An immunological experiment showed thatPfuPCNA interacts with both Pol I and Pol II. Pol I is a single polypeptide with a sequence similar to that of family B (α-like) DNA polymerases, while Pol II is a heterodimer.PfuPCNA interacted with DP2, the catalytic subunit of the heterodimeric complex. These results strongly support the idea that the PCNA homolog works as a sliding clamp of DNA polymerases inP. furiosus, and the basic mechanism for the processive DNA synthesis is conserved in the domainsBacteria,Eucarya, andArchaea. The stimulatory effect ofPfuPCNA on the DNA synthesis was observed by using a circular DNA template without the clamp loader (replication factor C [RFC]) in both Pol I and Pol II reactions in contrast to the case of eukaryotic organisms, which are known to require the RFC to open the ring structure of PCNA prior to loading onto a circular DNA. Because RFC homologs have been found in the archaeal genomes, they may permit more efficient stimulation of DNA synthesis by archaeal DNA polymerases in the presence of PCNA. This is the first stage in elucidating the archaeal DNA replication mechanism.
Metabolic Flux Ratio Analysis of Genetic and Environmental Modulations of
Escherichia coli
Central Carbon Metabolism
Authors: Sauer Uwe , Lasko Daniel R. , Fiaux Jocelyne , Hochuli Michel , Glaser Ralf , Szyperski Thomas , Wüthrich Kurt , Bailey James E. 
Published: December 2019

ABSTRACT

The response of
Escherichia coli
central carbon metabolism to genetic and environmental manipulation has been studied by use of a recently developed methodology for metabolic flux ratio (METAFoR) analysis; this methodology can also directly reveal active metabolic pathways. Generation of fluxome data arrays by use of the METAFoR approach is based on two-dimensional
13
C-
1
H correlation nuclear magnetic resonance spectroscopy with fractionally labeled biomass and, in contrast to metabolic flux analysis, does not require measurements of extracellular substrate and metabolite concentrations. METAFoR analyses of
E. coli
strains that moderately overexpress phosphofructokinase, pyruvate kinase, pyruvate decarboxylase, or alcohol dehydrogenase revealed that only a few flux ratios change in concert with the overexpression of these enzymes. Disruption of both pyruvate kinase isoenzymes resulted in altered flux ratios for reactions connecting the phosphoenolpyruvate (PEP) and pyruvate pools but did not significantly alter central metabolism. These data indicate remarkable robustness and rigidity in central carbon metabolism in the presence of genetic variation. More significant physiological changes and flux ratio differences were seen in response to altered environmental conditions. For example, in ammonia-limited chemostat cultures, compared to glucose-limited chemostat cultures, a reduced fraction of PEP molecules was derived through at least one transketolase reaction, and there was a higher relative contribution of anaplerotic PEP carboxylation than of the tricarboxylic acid (TCA) cycle for oxaloacetate synthesis. These two parameters also showed significant variation between aerobic and anaerobic batch cultures. Finally, two reactions catalyzed by PEP carboxykinase and malic enzyme were identified by METAFoR analysis; these had previously been considered absent in
E. coli
cells grown in glucose-containing media. Backward flux from the TCA cycle to glycolysis, as indicated by significant activity of PEP carboxykinase, was found only in glucose-limited chemostat culture, demonstrating that control of this futile cycle activity is relaxed under severe glucose limitation.
Cloning and Sequencing of a Novel
meta
-Cleavage Dioxygenase Gene Whose Product Is Involved in Degradation of γ-Hexachlorocyclohexane in
Sphingomonas paucimobilis
Authors: Miyauchi Keisuke , Adachi Yugo , Nagata Yuji , Takagi Masamichi 
Published: December 2019

ABSTRACT

Sphingomonas
(formerly
Pseudomonas
)
paucimobilis
UT26 utilizes γ-hexachlorocyclohexane (γ-HCH), a halogenated organic insecticide, as a sole source of carbon and energy. In a previous study, we showed that γ-HCH is degraded to chlorohydroquinone (CHQ) and then to hydroquinone (HQ), although the rate of reaction from CHQ to HQ was slow (K. Miyauchi, S. K. Suh, Y. Nagata, and M. Takagi, J. Bacteriol. 180:1354–1359, 1998). In this study, we cloned and characterized a gene, designated
linE
, which is located upstream of
linD
and is directly involved in the degradation of CHQ. The LinE protein consists of 321 amino acids, and all of the amino acids which are reported to be essential for the activity of
meta
-cleavage dioxygenases are conserved in LinE.
Escherichia coli
overproducing LinE could convert both CHQ and HQ, producing γ-hydroxymuconic semialdehyde and maleylacetate, respectively, with consumption of O
2
but could not convert catechol, which is one of the major substrates for
meta
-cleavage dioxygenases. LinE seems to be resistant to the acylchloride, which is the ring cleavage product of CHQ and which seems to react with water to be converted to maleylacetate. These results indicated that LinE is a novel type of
meta
-cleavage dioxygenase, designated (chloro)hydroquinone 1,2-dioxygenase, which cleaves aromatic rings with two hydroxyl groups at
para
positions preferably. This study represents a direct demonstration of a new type of ring cleavage pathway for aromatic compounds, the hydroquinone pathway.
Role of Crotonyl Coenzyme A Reductase in Determining the Ratio of Polyketides Monensin A and Monensin B Produced by
Streptomyces cinnamonensis
Authors: Liu Haibin , Reynolds Kevin A. 
Published: December 2019

ABSTRACT

The
ccr
gene, encoding crotonyl coenzyme A (CoA) reductase (CCR), was cloned from
Streptomyces cinnamonensis
C730.1 and shown to encode a protein with 90% amino acid sequence identity to the CCRs of
Streptomyces collinus
and
Streptomyces coelicolor
. A
ccr
-disrupted mutant,
S. cinnamonensis
L1, was constructed by inserting the
hyg
resistance gene into a unique
Bgl
II site within the
ccr
coding region. By use of the
ermE
* promoter, the
S. collinus ccr
gene was expressed from plasmids in
S. cinnamonensis
C730.1/pHL18 and L1/pHL18. CCR activity in mutant L1 was shown to decrease by more than 90% in both yeast extract-malt extract (YEME) medium and a complex fermentation medium, compared to that in wild-type C730.1. Compared to C730.1, mutants C730.1/pHL18 and L1/pHL18 exhibited a huge increase in CCR activity (14- and 13-fold, respectively) in YEME medium and a moderate increase (3.7- and 2.7-fold, respectively) in the complex fermentation medium. In the complex fermentation medium,
S. cinnamonensis
L1 produced monensins A and B in a ratio of 12:88, dramatically lower than the 50:50 ratio observed for both C730.1 and C730.1/pHL18. Plasmid (pHL18)-based expression of the
S. collinus ccr
gene in mutant L1 increased the monensin A/monensin B ratio to 42:58. Labeling experiments with [1,2-
13
C
2
]acetate demonstrated the same levels of intact incorporation of this material into the butyrate-derived portion of monensin A in both C730.1 and mutant C730.1/pLH18 but a markedly decreased level of such incorporation in mutant L1. The addition of crotonic acid at 15 mM led to significant increases in the monensin A/monensin B ratio in C730.1 and C730.1/pHL18 but had no effect in
S. cinnamonensis
L1. These results demonstrate that CCR plays a significant role in providing butyryl-CoA for monensin A biosynthesis and is present in wild-type
S. cinnamonensis
C730.1 at a level sufficient that the availability of the appropriate substrate (crotonyl-CoA) is limiting.
Random Transposition by Tn
916
in
Desulfitobacterium dehalogenans
Allows for Isolation and Characterization of Halorespiration-Deficient Mutants
Authors: Smidt Hauke , Song Donglin , van der Oost John , de Vos Willem M. 
Published: December 2019

ABSTRACT

To allow for the molecular analysis of halorespiration by the strictly anaerobic gram-positive bacterium
Desulfitobacterium dehalogenans
, halorespiration-deficient mutants were selected and characterized following insertional mutagenesis by the conjugative transposon Tn
916
. To facilitate rapid screening of transconjugants, a highly efficient method for the growth of single colonies on solidified medium has been developed. A streptomycin-resistant mutant of
D. dehalogenans
was isolated and mated with
Enterococcus faecalis
JH2-2 carrying Tn
916
. Insertion of one or two copies of Tn
916
into the chromosome of
D. dehalogenans
was observed. From a total of 2,500 transconjugants, 24 halorespiration-deficient mutants were selected based upon their inability to use 3-chloro-4-hydroxyphenylacetic acid as an electron acceptor. Physiological characterization led to the definition of three phenotypic classes of mutants that differed in their ability to use the additional terminal electron acceptors nitrate and fumarate. The activities of hydrogenase and formate dehydrogenase were determined, and the transposon insertion sites in selected mutants representing the different classes were analyzed on the sequence level following amplification by inverse PCR. The results of the molecular characterization as well as the pleiotropic phenotypes of most mutants indicate that genes coding for common elements shared by the different respiratory chains present in the versatile
D. dehalogenans
have been disrupted.
d
-Allose Catabolism of
Escherichia coli
: Involvement of
alsI
and Regulation of
als
Regulon Expression by Allose and Ribose
Authors: Poulsen Tim S. , Chang Ying-Ying , Hove-Jensen Bjarne 
Published: December 2019

ABSTRACT

Genes involved in allose utilization of
Escherichia coli
K-12 are organized in at least two operons,
alsRBACE
and
alsI
, located next to each other on the chromosome but divergently transcribed. Mutants defective in
alsI
(allose 6-phosphate isomerase gene) and
alsE
(allulose 6-phosphate epimerase gene) were Als
−
. Transcription of the two allose operons, measured as β-galactosidase activity specified by
alsI-lacZ
+
or
alsE-lacZ
+
operon fusions, was induced by allose. Ribose also caused derepression of expression of the regulon under conditions in which ribose phosphate catabolism was impaired.
Providencia stuartii
Genes Activated by Cell-to-Cell Signaling and Identification of a Gene Required for Production or Activity of an Extracellular Factor
Authors: Rather Philip N. , Ding Xuedong , Baca-DeLancey Rita R. , Siddiqui Soofia 
Published: December 2019

ABSTRACT

By utilizing reporter transposons, five
Providencia stuartii
genes that are activated by the accumulation of self-produced extracellular signals have been identified. These genes have been designated
cma
for conditioned medium activated. The presence of conditioned medium from stationary-phase cultures grown in rich media resulted in the premature activation of each gene in cells at early log phase, with activation values ranging from 6- to 26-fold. Preparation of conditioned medium from an M9 salts medium and fractionation by gel filtration chromatography resulted in fractions within the included volume which activated three of the
cma
fusions. In addition, depending on the reporter fusion, peak activity was found in different fractions. The partially purified factors activated in a dose-dependent manner. Characterization of the factors activating the
cma
fusions indicated that they were stable to heat, alkali, and acid. Furthermore, for each
cma
fusion, factor activity was not reproduced by the addition of homoserine lactone, homocysteine thiolactone, pyruvate, Casamino Acids, or α-ketoglutarate. The identities of three
cma
genes have been determined and revealed physiological roles in amino acid biosynthesis and nutrient import. To begin to address the pathways for production of or response to the extracellular factors, we have identified a locus,
aarA
, that is required for the activation of four
cma
fusions. The AarA product was required for factor activity in extracellular supernatants, indicating a possible role in biosynthesis or export.
Unusual Structure of the
attB
Site of the Site-Specific Recombination System of
Lactobacillus delbrueckii
Bacteriophage mv4
Authors: Auvray Frédéric , Coddeville Michèle , Ordonez Romy Catoira , Ritzenthaler Paul 
Published: December 2019

ABSTRACT

The temperate phage mv4 integrates its genome into the chromosome of
Lactobacillus delbrueckii
subsp.
bulgaricus
by site-specific recombination within the 3′ end of a tRNA
Ser
gene. Recombination is catalyzed by the phage-encoded integrase and occurs between the phage
attP
site and the bacterial
attB
site. In this study, we show that the mv4 integrase functions in vivo in
Escherichia coli
and we characterize the bacterial
attB
site with a site-specific recombination test involving compatible plasmids carrying the recombination sites. The importance of particular nucleotides within the
attB
sequence was determined by site-directed mutagenesis. The structure of the
attB
site was found to be simple but rather unusual. A 16-bp DNA fragment was sufficient for function. Unlike most genetic elements that integrate their DNA into tRNA genes, none of the dyad symmetry elements of the tRNA
Ser
gene were present within the minimal
attB
site. No inverted repeats were detected within this site either, in contrast to the lambda site-specific recombination model.
Localization of Synthesis of β1,6-Glucan in
Saccharomyces cerevisiae
Authors: Montijn Roy C. , Vink Edwin , Müller Wally H. , Verkleij Arie J. , Van Den Ende Herman , Henrissat Bernard , Klis Frans M. 
Published: December 2019

ABSTRACT

β1,6-Glucan is a key component of the yeast cell wall, interconnecting cell wall proteins, β1,3-glucan, and chitin. It has been postulated that the synthesis of β1,6-glucan begins in the endoplasmic reticulum with the formation of protein-bound primer structures and that these primer structures are extended in the Golgi complex by two putative glucosyltransferases that are functionally redundant, Kre6 and Skn1. This is followed by maturation steps at the cell surface and by coupling to other cell wall macromolecules. We have reinvestigated the role of Kre6 and Skn1 in the biogenesis of β1,6-glucan. Using hydrophobic cluster analysis, we found that Kre6 and Skn1 show significant similarities to family 16 glycoside hydrolases but not to nucleotide diphospho-sugar glycosyltransferases, indicating that they are glucosyl hydrolases or transglucosylases instead of genuine glucosyltransferases. Next, using immunogold labeling, we tried to visualize intracellular β1,6-glucan in cryofixed
sec1-1
cells which had accumulated secretory vesicles at the restrictive temperature. No intracellular labeling was observed, but the cell surface was heavily labeled. Consistent with this, we could detect substantial amounts of β1,6-glucan in isolated plasma membrane-derived microsomes but not in post-Golgi secretory vesicles. Taken together, our data indicate that the synthesis of β1,6-glucan takes place largely at the cell surface. An alternative function for Kre6 and Skn1 is discussed.
The Genetic Switch Regulating Activity of Early Promoters of the Temperate Lactococcal Bacteriophage TP901-1
Authors: Madsen Peter Lynge , Johansen Annette H. , Hammer Karin , Brøndsted Lone 
Published: December 2019

ABSTRACT

A functional analysis of open reading frame 4 (ORF4) and ORF5 from the temperate lactococcal phage TP901-1 was performed by mutant and deletion analysis combined with transcriptional studies of the early phage promoters
p
R
and
p
L
. ORF4 (180 amino acids) was identified as a phage repressor necessary for repression of both promoters. Furthermore, the presence of ORF4 confers immunity of the host strain to TP901-1. ORF5 (72 amino acids) was found to be able to inhibit repression of the lytic promoter
p
L
by ORF4. Upon transformation with a plasmid containing both ORF4 and ORF5 and their cognate promoters, clonal variation is observed: in each transformant, either
p
L
is open and
p
R
is closed or vice versa. The repression is still dependent on ORF4, and the presence of ORF5 is needed for the clonal variation. Induction of a repressed
p
L
fusion containing
orf4
and
orf5
was obtained by addition of mitomycin C, and the induction was also shown to be dependent on the presence of the RecA protein, even though ORF4 does not contain a recognizable autocleavage site. Our results suggest that the relative amounts of the two proteins ORF4 and ORF5 determine the decision between lytic or lysogenic life cycle after phage infection and that a protein complex consisting of ORF4 and ORF5 may constitute a new type of genetic switch in bacteriophages.
A Multifunctional ATP-Binding Cassette Transporter System from
Vibrio cholerae
Transports Vibriobactin and Enterobactin
Authors: Wyckoff Elizabeth E. , Valle Ana-Maria , Smith Stacey L. , Payne Shelley M. 
Published: December 2019

ABSTRACT

Vibrio cholerae
uses the catechol siderophore vibriobactin for iron transport under iron-limiting conditions. We have identified genes for vibriobactin transport and mapped them within the vibriobactin biosynthetic gene cluster. Within this genetic region we have identified four genes,
viuP
,
viuD
,
viuG
and
viuC
, whose protein products have homology to the periplasmic binding protein, the two integral cytoplasmic membrane proteins, and the ATPase component, respectively, of other iron transport systems. The amino-terminal region of ViuP has homology to a lipoprotein signal sequence, and ViuP could be labeled with [
3
H]palmitic acid. This suggests that ViuP is a membrane lipoprotein. The ViuPDGC system transports both vibriobactin and enterobactin in
Escherichia coli
. In the same assay, the
E. coli
enterobactin transport system, FepBDGC, allowed the utilization of enterobactin but not vibriobactin. Although the entire
viuPDGC
system could complement mutations in
fepB
,
fepD
,
fepG
, or
fepC
, only
viuC
was able to independently complement the corresponding
fep
mutation. This indicates that these proteins usually function as a complex.
V. cholerae
strains carrying a mutation in
viuP
or in
viuG
were constructed by marker exchange. These mutations reduced, but did not completely eliminate, vibriobactin utilization. This suggests that
V. cholerae
contains genes in addition to
viuPDGC
that function in the transport of catechol siderophores.
Promiscuous Origin of a Chimeric Sequence in the
Escherichia coli
O157:H7 Genome
Authors: LeClerc J. Eugene , Li Baoguang , Payne William L. , Cebula Thomas A. 
Published: December 2019

ABSTRACT

A novel sequence of 2.9 kb in the intergenic region between the
mutS
and
rpoS
genes of
Escherichia coli
O157:H7 and closely related strains replaces a sequence of 6.1 kb in
E. coli
K-12 strains. At the same locus in
Shigella dysenteriae
type 1, a sequence identical to that in O157:H7 is bounded by the IS
1
insertion sequence element. Extensive polymorphism in the
mutS-rpoS
chromosomal region is indicative of horizontal transfer events.