bluuebunny/crossref_metadata_2025_split huggingface dataset:
Aspergillus alabamensis
, a New Clinically Relevant Species in the Section
Terrei
Authors: Balajee S. Arunmozhi , Baddley John W. , Peterson Stephen W. , Nickle David , Varga János , Boey Angeline , Lass-Flörl Cornelia , Frisvad Jens C. , Samson Robert A. 
Published: March 2009

ABSTRACT

Phylogenetic analyses of sequences generated from portions of three genes coding for the proteins enolase (
enoA
), β-tubulin (
benA
), and calmodulin (
calM
) of a large number of isolates within the section
Terrei
, genus
Aspergillus
, revealed the presence of a new cryptic species within this section,
Aspergillus alabamensis.
Most members of this new cryptic species were recovered as colonizing isolates from immunocompetent patient populations, had decreased in vitro susceptibilities to the antifungal drug amphotericin B, and were morphologically similar to but genetically distinct from
Aspergillus terreus
isolates.
Silencing of Host CellCYBBGene Expression by the Nuclear Effector AnkA of the Intracellular PathogenAnaplasma phagocytophilum
Authors: Garcia-Garcia Jose C. , Rennoll-Bankert Kristen E. , Pelly Shaaretha , Milstone Aaron M. , Dumler J. Stephen 
Published: March 2009

ABSTRACTCoevolution of intracellular bacterial pathogens and their host cells resulted in the appearance of effector molecules that when translocated into the host cell modulate its function, facilitating bacterial survival within the hostile host environment. Some of these effectors interact with host chromatin and other nuclear components. In this report, we show that the AnkA protein ofAnaplasma phagocytophilum, which is translocated into the host cell nucleus, interacts with gene regulatory regions of host chromatin and is involved in downregulating expression ofCYBB(gp91phox) and other key host defense genes. AnkA effector protein rapidly accumulated in nuclei of infected cells coincident with changes inCYBBtranscription. AnkA interacted with transcriptional regulatory regions of theCYBBlocus at sites where transcriptional regulators bind. AnkA binding to DNA occurred at regions with high AT contents. Mutation of AT stretches at these sites abrogated AnkA binding. Histone H3 acetylation decreased dramatically at theCYBBlocus duringA. phagocytophiluminfection, particularly around AnkA binding sites. Transcription ofCYBBand other defense genes was significantly decreased in AnkA-transfected HL-60 cells. These data suggest a mechanism by which intracellular pathogens directly regulate host cell gene expression mediated by nuclear effectors and changes in host chromatin structure.
The Biosynthetic Pathway for Myxol-2′ Fucoside (Myxoxanthophyll) in the Cyanobacterium
Synechococcus
sp. Strain PCC 7002
Authors: Graham Joel E. , Bryant Donald A. 
Published: March 2009

ABSTRACT

Synechococcus
sp. strain PCC 7002 produces a variety of carotenoids, which comprise predominantly dicylic β-carotene and two dicyclic xanthophylls, zeaxanthin and synechoxanthin. However, this cyanobacterium also produces a monocyclic myxoxanthophyll, which was identified as myxol-2′ fucoside. Compared to the carotenoid glycosides produced by diverse microorganisms, cyanobacterial myxoxanthophyll and closely related compounds are unusual because they are glycosylated on the 2′-OH rather than on the 1′-OH position of the ψ end of the molecule. In this study, the genes encoding two enzymes that modify the ψ end of myxoxanthophyll in
Synechococcus
sp. strain PCC 7002 were identified. Mutational and biochemical studies showed that open reading frame SynPCC7002_A2032, renamed
cruF
, encodes a 1′-hydroxylase and that open reading frame SynPCC7002_A2031, renamed
cruG
, encodes a 2′-O-glycosyltransferase. The enzymatic activity of CruF was verified by chemical characterization of the carotenoid products synthesized when
cruF
was expressed in a lycopene-producing strain of
Escherichia coli
. Database searches showed that homologs of
cruF
and
cruG
occur in the genomes of all sequenced cyanobacterial strains that are known to produce myxol or the acylic xanthophyll oscillaxanthin. The genomes of many other bacteria that produce hydroxylated carotenoids but do not contain
crtC
homologs also contain
cruF
orthologs. Based upon observable intermediates, a complete biosynthetic pathway for myxoxanthophyll is proposed. This study expands the suite of enzymes available for metabolic engineering of carotenoid biosynthetic pathways for biotechnological applications.
Redundancy of Enzymes for Formaldehyde Detoxification in
Pseudomonas putida
Authors: Roca Amalia , Rodríguez-Herva Jose J. , Ramos Juan L. 
Published: March 2009

ABSTRACT

Pseudomonas putida
KT2440 exhibits redundant formaldehyde dehydrogenases and formate dehydrogenases that contribute to the detoxification of formaldehyde, a highly toxic compound. Physical and transcriptional analyses showed that the open reading frame (ORF) PP0328, encoding one of the formaldehyde dehydrogenases, is self-sufficient, whereas the other functional formaldehyde dehydrogenase gene (ORF PP3970) forms an operon with another gene of unknown function. Two formate dehydrogenase gene clusters (PP0489 to PP0492 and PP2183 to PP2186) were identified, and genes in these clusters were found to form operons. All four transcriptional promoters were mapped by primer extension and revealed the presence of noncanonical promoters expressed at basal level in the exponential growth phase and at a higher level in the stationary phase regardless of the presence of extracellular formaldehyde or formate. These promoters were characterized by a 5′-AG-CCA-C/A-CT-3′ conserved region between −7 and −16. To determine the contribution of the different gene products to formaldehyde and formate mineralization, mutants with single and double mutations of formaldehyde dehydrogenases were generated, and the effect of the mutations on formaldehyde catabolism was tested by measuring
14
CO
2
evolution from
14
C-labeled formaldehyde. The results showed that both enzymes contributed to formaldehyde catabolism. A double mutant lacking these two enzymes still evolved CO
2
from formaldehyde, suggesting the presence of one or more still-unidentified formaldehyde dehydrogenases. Mutants with single and double mutations in the clusters for formate dehydrogenases were also generated, and all of them were able to metabolize [
14
C]formate to
14
CO
2
, suggesting a redundancy of functions that was not limited to only the annotated genes. Single and double mutants deficient in formaldehyde dehydrogenases and formate dehydrogenases exhibited longer lag phases than did the parental strain when confronted with concentrations of formaldehyde close to the MICs. This suggests a role for the detoxification system in tolerance to sublethal concentrations of formaldehyde.
Sialic Acid (
N
-Acetyl Neuraminic Acid) Utilization by
Bacteroides fragilis
Requires a Novel
N
-Acetyl Mannosamine Epimerase
Authors: Brigham Christopher , Caughlan Ruth , Gallegos Rene , Dallas Mary Beth , Godoy Veronica G. , Malamy Michael H. 
Published: March 2009

ABSTRACT

We characterized the
nanLET
operon in
Bacteroides fragilis
, whose products are required for the utilization of the sialic acid
N-
acetyl neuraminic acid (NANA) as a carbon and energy source. The first gene of the operon is
nanL
, which codes for an aldolase that cleaves NANA into
N
-acetyl mannosamine (manNAc) and pyruvate. The next gene,
nanE
, codes for a manNAc/
N
-acetylglucosamine (NAG) epimerase, which, intriguingly, possesses more similarity to eukaryotic renin binding proteins than to other bacterial NanE epimerase proteins. Unphosphorylated manNAc is the substrate of NanE, while ATP is a cofactor in the epimerase reaction. The third gene of the operon is
nanT
, which shows similarity to the major transporter facilitator superfamily and is most likely to be a NANA transporter. Deletion of any of these genes eliminates the ability of
B. fragilis
to grow on NANA. Although
B. fragilis
does not normally grow with manNAc as the sole carbon source, we isolated a
B. fragilis
mutant strain that can grow on this substrate, likely due to a mutation in a NAG transporter; both manNAc transport and NAG transport are affected in this strain. Deletion of the
nanE
epimerase gene or the
rokA
hexokinase gene, whose product phosphorylates NAG, in the manNAc-enabled strain abolishes growth on manNAc. Thus,
B. fragilis
possesses a new pathway of NANA utilization, which we show is also found in other
Bacteroides
species.
Characterization of a Novel Thermostable Carboxylesterase fromGeobacillus kaustophilusHTA426 Shows the Existence of a New Carboxylesterase Family
Authors: Montoro-García Silvia , Martínez-Martínez Irene , Navarro-Fernández José , Takami Hideto , García-Carmona Francisco , Sánchez-Ferrer Álvaro 
Published: March 2009

ABSTRACTThe gene GK3045 (741 bp) fromGeobacillus kaustophilusHTA426 was cloned, sequenced, and overexpressed intoEscherichia coliRosetta (DE3). The deduced protein was a 30-kDa monomeric esterase with high homology to carboxylesterases fromGeobacillus thermoleovoransNY (99% identity) andGeobacillus stearothermophilus(97% identity). This protein suffered a proteolytic cut inE. coli, and the problem was overcome by introducing a mutation in the gene (K212R) without affecting the activity. The resulting Est30 showed remarkable thermostability at 65°C, above the optimum growth temperature ofG. kaustophilusHTA426. The optimum pH of the enzyme was 8.0. In addition, the purified enzyme exhibited stability against denaturing agents, like organic solvents, detergents, and urea. The protein catalyzed the hydrolysis ofp-nitrophenyl esters of different acyl chain lengths, confirming the esterase activity. The sequence analysis showed that the protein contains a catalytic triad formed by Ser93, Asp192, and His222, and the Ser of the active site is located in the conserved motif Gly91-X-Ser93-X-Gly95 included in most esterases and lipases. However, this carboxylesterase showed no more than 17% sequence identity with the closest members in the eight families of microbial carboxylesterases. The three-dimensional structure was modeled by sequence alignment and compared with others carboxylesterases. The topological differences suggested the classification of this enzyme and otherGeobacillus-related carboxylesterases in a new α/β hydrolase family different from IV and VI.
Transsexuality in the Rhizosphere: Quorum Sensing Reversibly Converts
Agrobacterium tumefaciens
from Phenotypically Female to Male
Authors: Cho Hongbaek , Pinto Uelinton M. , Winans Stephen C. 
Published: March 2009

ABSTRACT

Conjugative plasmids generally encode proteins that block the conjugative entry of identical or similar plasmids into the host cell, a phenomenon known as entry exclusion. Here, we demonstrate that two Ti plasmids of
Agrobacterium tumefaciens
encode robust entry exclusion functions. Two proteins, TrbJ and TrbK, can each mediate entry exclusion and act synergistically. The
trbJ
and
trbK
genes are included within the
trb
operon, which is tightly regulated by the quorum-sensing regulator TraR and the cognate acylhomoserine lactone. In the absence of quorum-sensing signals, these proteins are not significantly expressed, and cells lacking TrbJ and TrbK are efficient Ti plasmid recipients. In the presence of these signals, these strains block the entry of Ti plasmids and instead become efficient conjugal donors.
An Orthologue of
Bacteroides fragilis
NanH Is the Principal Sialidase in
Tannerella forsythia
Authors: Thompson Hayley , Homer Karen A. , Rao Susmitha , Booth Veronica , Hosie Arthur H. F. 
Published: March 2009

ABSTRACT

Sialidase activity is a putative virulence factor of the anaerobic periodontal pathogen
Tannerella forsythia
, but it is uncertain which genes encode this activity. Characterization of a putative sialidase, SiaHI, by others, indicated that this protein alone may not be responsible for all of the sialidase activity. We describe a second sialidase in
T. forsythia
(TF0035), an orthologue of
Bacteroides fragilis
NanH, and its expression in
Escherichia coli
. Sialidase activity of the expressed NanH was confirmed by using 2′-(4-methylumbelliferyl)-α-
d
-
N
-acetylneuraminic acid as a substrate. Biochemical characterization of the recombinant
T. forsythia
NanH indicated that it was active over a broad pH range, with optimum activity at pH 5.5. This enzyme has high affinity for 2′-(4-methylumbelliferyl)-α-
d
-
N
-acetylneuraminic acid (

K
m

of 32.9 ± 10.3 μM) and rapidly releases 4-methylumbelliferone (
V
max
of 170.8 ± 11.8 nmol of 4-methylumbelliferone min
−1
mg of protein
−1
).
E. coli
lysates containing recombinant
T. forsythia
NanH cleave sialic acid from a range of substrates, with a preference for α2-3 glycosidic linkages. The genes adjacent to
nanH
encode proteins apparently involved in the metabolism of sialic acid, indicating that the NanH sialidase is likely to be involved in nutrient acquisition.
Biochemical Analysis of a β-
d
-Xylosidase and a Bifunctional Xylanase-Ferulic Acid Esterase from a Xylanolytic Gene Cluster in
Prevotella ruminicola
23
Authors: Dodd Dylan , Kocherginskaya Svetlana A. , Spies M. Ashley , Beery Kyle E. , Abbas Charles A. , Mackie Roderick I. , Cann Isaac K. O. 
Published: March 2009

ABSTRACT

Prevotella ruminicola
23 is an obligate anaerobic bacterium in the phylum
Bacteroidetes
that contributes to hemicellulose utilization within the bovine rumen. To gain insight into the cellular machinery that this organism elaborates to degrade the hemicellulosic polymer xylan, we identified and cloned a gene predicted to encode a bifunctional xylanase-ferulic acid esterase (
xyn10D-fae1A
) and expressed the recombinant protein in
Escherichia coli
. Biochemical analysis of purified Xyn10D-Fae1A revealed that this protein possesses both endo-β-1,4-xylanase and ferulic acid esterase activities. A putative glycoside hydrolase (GH) family 3 β-
d
-glucosidase gene, with a novel PA14-like insertion sequence, was identified two genes downstream of
xyn10D-fae1A
. Biochemical analyses of the purified recombinant protein revealed that the putative β-
d
-glucosidase has activity for
p
NP-β-
d
-xylopyranoside,
p
NP-α-
l
-arabinofuranoside, and xylo-oligosaccharides; thus, the gene was designated
xyl3A
. When incubated in combination with Xyn10D-Fae1A, Xyl3A improved the release of xylose monomers from a hemicellulosic xylan substrate, suggesting that these two enzymes function synergistically to depolymerize xylan. Directed mutagenesis studies of Xyn10D-Fae1A mapped the catalytic sites for the two enzymatic functionalities to distinct regions within the polypeptide sequence. When a mutation was introduced into the putative catalytic site for the xylanase domain (E280S), the ferulic acid esterase activity increased threefold, which suggests that the two catalytic domains for Xyn10D-Fae1A are functionally coupled. Directed mutagenesis of conserved residues for Xyl3A resulted in attenuation of activity, which supports the assignment of Xyl3A as a GH family 3 β-
d
-xylosidase.
Genetic Mapping of Secretion and Functional Determinants of the
Vibrio cholerae
TcpF Colonization Factor
Authors: Krebs Shelly J. , Kirn Thomas J. , Taylor Ronald K. 
Published: March 2009

ABSTRACT

Colonization of the human small intestine by
Vibrio cholerae
requires the type IV toxin-coregulated pilus (TCP). TcpF, which is encoded within the
tcp
operon, is secreted from the bacterial cell by the TCP apparatus and is also essential for colonization. Bacteria lacking
tcpF
are deficient in colonization, and anti-TcpF antibodies are protective in the infant mouse cholera model. In order to elucidate the regions of the protein that are required for secretion through the TCP apparatus and for its function in colonization, random mutagenesis of
tcpF
was performed. Analysis of these mutants suggests that multiple regions throughout the protein influence extracellular secretion and that determinants near the C terminus are important for the function of TcpF in colonization. The TcpF proteins of certain environmental
V. cholerae
isolates with 31% to 66% identity to pathogenic
V. cholerae
TcpF showed higher similarity in regions identified as secretion determinants but diverged in regions found to be important for colonization. These environmental TcpF proteins are secreted from the pathogenic strain; however, they do not mediate colonization in the infant mouse model. Here we provide genetic evidence pointing toward regions of TcpF that influence secretion, as well as regions that play an important role in in vivo colonization.
Identification of a Polymyxin Synthetase Gene Cluster of
Paenibacillus polymyxa
and Heterologous Expression of the Gene in
Bacillus subtilis
Authors: Choi Soo-Keun , Park Soo-Young , Kim Rumi , Kim Seong-Bin , Lee Choong-Hwan , Kim Jihyun F. , Park Seung-Hwan 
Published: March 2009

ABSTRACT

Polymyxin, a long-known peptide antibiotic, has recently been reintroduced in clinical practice because it is sometimes the only available antibiotic for the treatment of multidrug-resistant gram-negative pathogenic bacteria. Lack of information on the biosynthetic genes of polymyxin, however, has limited the study of structure-function relationships and the development of improved polymyxins. During whole genome sequencing of
Paenibacillus polymyxa
E681, a plant growth-promoting rhizobacterium, we identified a gene cluster encoding polymyxin synthetase. Here, we report the complete sequence of the gene cluster and its function in polymyxin biosynthesis. The gene cluster spanning the 40.6-kb region consists of five open reading frames, designated
pmxA
,
pmxB
,
pmxC
,
pmxD
, and
pmxE
. The
pmxC
and
pmxD
genes are similar to genes that encode transport proteins, while
pmxA
,
pmxB
, and
pmxE
encode polymyxin synthetases. The insertional disruption of
pmxE
led to a loss of the ability to produce polymyxin. Introduction of the
pmx
gene cluster into the
amyE
locus of the
Bacillus subtilis
chromosome resulted in the production of polymyxin in the presence of extracellularly added
l
-2,4-diaminobutyric acid. Taken together, our findings demonstrate that the
pmx
gene cluster is responsible for polymyxin biosynthesis.
Surface Location of Individual Residues of SlpA Provides Insight into the
Lactobacillus brevis
S-Layer
Authors: Vilen Heikki , Hynönen Ulla , Badelt-Lichtblau Helga , Ilk Nicola , Jääskeläinen Pentti , Torkkeli Mika , Palva Airi 
Published: March 2009

ABSTRACT

Bacterial surface layer (S-layer) proteins are excellent candidates for in vivo and in vitro nanobiotechnological applications because of their ability to self-assemble into two-dimensional lattices that form the outermost layer of many
Eubacteria
and most
Archaea
species. Despite this potential, knowledge about their molecular architecture is limited. In this study, we investigated SlpA, the S-layer protein of the potentially probiotic bacterium
Lactobacillus brevis
ATCC 8287 by cysteine-scanning mutagenesis and chemical modification. We generated a series of 46 mutant proteins by replacing single amino acids with cysteine, which is not present in the wild-type protein. Most of the replaced amino acids were located in the self-assembly domain (residues 179 to 435) that likely faces the outer surface of the lattice. As revealed by electron microscopy, all the mutant proteins were able to form self-assembly products identical to that of the wild type, proving that this replacement does not dramatically alter the protein conformation. The surface accessibility of the sulfhydryl groups introduced was studied with two maleimide-containing marker molecules, TMM(PEG)
12
(molecular weight [MW], 2,360) and AlexaFluor488-maleimide (MW = 720), using both monomeric proteins in solution and proteins allowed to self-assemble on cell wall fragments. Using the acquired data and available domain information, we assigned the mutated residues into four groups according to their location in the protein monomer and lattice structure: outer surface of the lattice (9 residues), inner surface of the lattice (9), protein interior (12), and protein-protein interface/pore regions (16). This information is essential, e.g., in the development of therapeutic and other health-related applications of
Lactobacillus
S-layers.
The Glutathione/Glutaredoxin System Is Essential for Arsenate Reduction in
Synechocystis
sp. Strain PCC 6803
Authors: López-Maury Luis , Sánchez-Riego Ana María , Reyes José Carlos , Florencio Francisco J. 
Published: March 2009

ABSTRACT

Arsenic resistance in
Synechocystis
sp. strain PCC 6803 is mediated by an operon of three genes in which
arsC
codes for an arsenate reductase with unique characteristics. Here we describe the identification of two additional and nearly identical genes coding for arsenate reductases in
Synechocystis
sp. strain PCC 6803, which we have designed
arsI1
and
arsI2
, and the biochemical characterization of both ArsC (arsenate reductase) and ArsI. Functional analysis of single, double, and triple mutants shows that both ArsI enzymes are active arsenate reductases but that their roles in arsenate resistance are essential only in the absence of ArsC. Based on its biochemical properties, ArsC belongs to a family that, though related to thioredoxin-dependent arsenate reductases, uses the glutathione/glutaredoxin system for reduction, whereas ArsI belongs to the previously known glutaredoxin-dependent family. We have also analyzed the role in arsenate resistance of the three glutaredoxins present in
Synechocystis
sp. strain PCC 6803 both in vitro and in vivo. Only the dithiolic glutaredoxins, GrxA (glutaredoxin A) and GrxB (glutaredoxin B), are able to donate electrons to both types of reductases in vitro, while GrxC (glutaredoxin C), a monothiolic glutaredoxin, is unable to donate electrons to either type. Analysis of glutaredoxin mutant strains revealed that only those lacking the
grxA
gene have impaired arsenic resistance.
Searching for Protein-Protein Interactions within the
Bacillus subtilis
Spore Coat
Authors: Krajčíková Daniela , Lukáčová Magda , Müllerová Denisa , Cutting Simon M. , Barák Imrich 
Published: March 2009

ABSTRACT

The capability of endospores of
Bacillus subtilis
to withstand extreme environmental conditions is secured by several attributes. One of them, the protein shell that encases the spore and is known as the coat, provides the spore with its characteristic resistance to toxic chemicals, lytic enzymes, and predation by unicellular and multicellular eukaryotes. Despite most of the components of the spore coat having been identified, we have only a vague understanding of how such a complex structure is assembled. Using the yeast two-hybrid system, we attempted to identify direct contacts among the proteins allocated to the insoluble fraction of the spore coat: CotV, CotW, CotX, CotY, and CotZ. We also examined whether they could interact with CotE, one of the most crucial morphogenetic proteins governing outer coat formation and also present in the insoluble fraction. Out of all 21 possible interactions we tested, 4 were found to be positive. Among these interactions, we confirmed the previous observation that CotE forms homo-oligomers. In addition, we observed homotypic interactions of CotY, strong interactions between CotZ and CotY, and relatively weak, yet significant, interactions between CotV and CotW. The results of this yeast two-hybrid analysis were confirmed by size exclusion chromatography of recombinant coat proteins and a pull-down assay.