bluuebunny/crossref_metadata_2025_split huggingface dataset:
Two Novel, Putatively Cell Wall-Associated and Glycosylphosphatidylinositol-Anchored α-Glucanotransferase Enzymes ofAspergillus niger
Authors: van der Kaaij R. M. , Yuan X.-L. , Franken A. , Ram A. F. J. , Punt P. J. , van der Maarel M. J. E. C. , Dijkhuizen L. 
Published: May 2007

ABSTRACTIn the genome sequence ofAspergillus nigerCBS 513.88, three genes were identified with high similarity to fungal α-amylases. The protein sequences derived from these genes were different in two ways from all described fungal α-amylases: they were predicted to be glycosylphosphatidylinositol anchored, and some highly conserved amino acids of enzymes in the α-amylase family were absent. We expressed two of these enzymes in a suitableA. nigerstrain and characterized the purified proteins. Both enzymes showed transglycosylation activity on donor substrates with α-(1,4)-glycosidic bonds and at least five anhydroglucose units. The enzymes, designated AgtA and AgtB, produced new α-(1,4)-glycosidic bonds and therefore belong to the group of the 4-α-glucanotransferases (EC 2.4.1.25). Their reaction products reached a degree of polymerization of at least 30. Maltose and larger maltooligosaccharides were the most efficient acceptor substrates, although AgtA also used small nigerooligosaccharides containing α-(1,3)-glycosidic bonds as acceptor substrate. AnagtAknockout ofA. nigershowed an increased susceptibility towards the cell wall-disrupting compound calcofluor white, indicating a cell wall integrity defect in this strain. Homologues of AgtA and AgtB are present in other fungal species with α-glucans in their cell walls, but not in yeast species lacking cell wall α-glucan. Possible roles for these enzymes in the synthesis and/or maintenance of the fungal cell wall are discussed.
Development and Application of a Green Fluorescent Protein Sentinel System for Identification of RNA Interference in
Blastomyces dermatitidis
Illuminates the Role of Septin in Morphogenesis and Sporulation
Authors: Krajaejun T. , Gauthier G. M. , Rappleye C. A. , Sullivan T. D. , Klein B. S. 
Published: May 2007

ABSTRACT

A high-throughput strategy for testing gene function would accelerate progress in our understanding of disease pathogenesis for the dimorphic fungus
Blastomyces dermatitidis
, whose genome is being completed. We developed a green fluorescent protein (GFP) sentinel system of gene silencing to rapidly study genes of unknown function. Using Gateway technology to efficiently generate RNA interference plasmids, we cloned a target gene, “X,” next to
GFP
to create one hairpin to knock down the expression of both genes so that diminished GFP reports target gene expression. To test this approach in
B. dermatitidis
, we first used
LACZ
and the virulence gene
BAD1
as targets. The level of GFP reliably reported interference of their expression, leading to rapid detection of gene-silenced transformants. We next investigated a previously unstudied gene encoding septin and explored its possible role in morphogenesis and sporulation. A
CDC11
septin homolog in
B. dermatitidis
localized to the neck of budding yeast cells.
CDC11
-silenced transformants identified with the sentinel system grew slowly as flat or rough colonies on agar. Microscopically, they formed ballooned, distorted yeast cells that failed to bud, and they sporulated poorly as mold. Hence, this GFP sentinel system enables rapid detection of gene silencing and has revealed a pronounced role for septin in morphogenesis, budding, and sporulation of
B. dermatitidis
.
Temperature-Hypersensitive Sites of the Flagellar Switch Component FliG in
Salmonella enterica
Serovar Typhimurium
Authors: Mashimo Takuji , Hashimoto Manami , Yamaguchi Shigeru , Aizawa Shin-Ichi 
Published: May 2007

ABSTRACT

Three flagellar proteins, FliG, FliM, and FliN (FliGMN), are the components of the C ring of the flagellar motor. The genes encoding these proteins are multifunctional; they show three different phenotypes (Fla
−
, Mot
−
, and Che
−
), depending on the sites and types of mutations. Some of the Mot
−
mutants previously characterized are found to be motile. Reexamination of all Mot
−
mutants in
fliGMN
genes so far studied revealed that many of them are actually temperature sensitive (TS); that is, they are motile at 20°C but nonmotile at 37°C. There were two types of TS mutants: one caused a loss of function that was not reversed by a return to the permissive temperature (rigid TS), and the other caused a loss that was reversed (hyper-TS). The rigid TS mutants showed an all-or-none phenotype; that is, once a structure was formed, the structure and function were stable against temperature shifts. All of
fliM
and
fliN
and most of the
fliG
TS mutants belong to this group. On the other hand, the hyper-TS mutants (three of the
fliG
mutants) showed a temporal swimming/stop phenotype, responding to temporal temperature shifts when the structure was formed at a permissive temperature. Those hyper-TS mutation sites are localized in the C-terminal domain of the FliG molecules at sites that are different from the previously proposed functional sites. We discuss a role for this new region of FliG in the torque generation of the flagellar motor.
DNA Polymerases BI and D from the Hyperthermophilic Archaeon
Pyrococcus furiosus
Both Bind to Proliferating Cell Nuclear Antigen with Their C-Terminal PIP-Box Motifs
Authors: Tori Kazuo , Kimizu Megumi , Ishino Sonoko , Ishino Yoshizumi 
Published: May 2007

ABSTRACT

Proliferating cell nuclear antigen (PCNA) is the sliding clamp that is essential for the high processivity of DNA synthesis during DNA replication.
Pyrococcus furiosus
, a hyperthermophilic archaeon, has at least two DNA polymerases, polymerase BI (PolBI) and PolD. Both of the two DNA polymerases interact with the archaeal
P. furiosus
PCNA (PfuPCNA) and perform processive DNA synthesis in vitro. This phenomenon, in addition to the fact that both enzymes display 3′-5′ exonuclease activity, suggests that both DNA polymerases work in replication fork progression. We demonstrated here that both PolBI and PolD functionally interact with PfuPCNA at their C-terminal PIP boxes. The mutant PolBI and PolD enzymes lacking the PIP-box sequence do not respond to the PfuPCNA at all in an in vitro primer extension reaction. This is the first experimental evidence that the PIP-box motif, located at the C termini of the archaeal DNA polymerases, is actually critical for PCNA binding to form a processive DNA-synthesizing complex.
Horizontal Gene Transfer and Homologous Recombination Drive the Evolution of the Nitrogen-Fixing Symbionts ofMedicagoSpecies
Authors: Bailly Xavier , Olivieri Isabelle , Brunel Brigitte , Cleyet-Marel Jean-Claude , Béna Gilles 
Published: May 2007

ABSTRACTUsing nitrogen-fixingSinorhizobiumspecies that interact withMedicagoplants as a model system, we aimed at clarifying how sex has shaped the diversity of bacteria associated with the genusMedicagoon the interspecific and intraspecific scales. To gain insights into the diversification of these symbionts, we inferred a topology that includes the different specificity groups which interact withMedicagospecies, based on sequences of the nodulation gene cluster. Furthermore, 126 bacterial isolates were obtained from two soil samples, usingMedicago truncatulaandMedicago laciniataas host plants, to study the differentiation between populations ofSinorhizobium medicae,Sinorhizobium melilotibv. meliloti, andS. melilotibv. medicaginis. The former two can be associated withM. truncatula(among other species ofMedicago), whereas the last organism is the specific symbiont ofM. laciniata. These bacteria were characterized using a multilocus sequence analysis of four loci, located on the chromosome and on the two megaplasmids ofS. meliloti.The phylogenetic results reveal that several interspecific horizontal gene transfers occurred during the diversification ofMedicagosymbionts. WithinS. meliloti, the analyses show thatnodgenes specific to different host plants have spread to different genetic backgrounds through homologous recombination, preventing further divergence of the different ecotypes. Thus, specialization to different host plant species does not prevent the occurrence of gene flow among host-specific biovars ofS. meliloti, whereas reproductive isolation betweenS. melilotibv. meliloti andS. medicaeis maintained even though these bacteria can cooccur in sympatry on the same individual host plants.
Target Genes and DNA-Binding Sites of the Response Regulator PhoR fromCorynebacterium glutamicum
Authors: Schaaf Sarah , Bott Michael 
Published: May 2007

ABSTRACTThe two-component signal transduction system PhoRS ofCorynebacterium glutamicumis involved in the phosphate (Pi) starvation response. To analyze the binding of unphosphorylated and phosphorylated PhoR to the promoters of phosphate starvation-inducible (psi) genes, this response regulator and the kinase domain of its cognate sensor, PhoS (MBP-PhoSΔ1-246), were overproduced and purified. MBP-PhoSΔ1-246 showed constitutive autophosphorylation activity, and a rapid phosphoryl group transfer from phosphorylated MBP-PhoSΔ1-246 to PhoR was observed. Gel mobility shift assays revealed that phosphorylation increases the DNA-binding affinity of PhoR. The affinity of PhoR∼P to different promoters varied and decreased in the orderpstSCAB>phoRS>phoC>ushA>porB>ugpA>pitA>nucHandphoH1>glpQ1. The binding sites in front ofpstSCABandphoRSwere localized at positions −194 to −176 and −61 to −43 upstream of the transcriptional start sites, respectively. Alignment of these two 19-bp binding sites revealed a high identity in the 5′-terminal part, but not in the 3′-terminal part. As many OmpR-type response regulators bind to direct repeats, the 19-bp sequence might be interpreted as a loosely conserved 8-bp direct repeat separated by 3 bp. This idea was supported by the fact that the highest binding affinity was observed with a perfect 8-bp direct repeat of the sequence CCTGTGAAaatCCTGTGAA. Inspection of the other target promoters revealed sequences with some similarity to this binding motif, which might represent PhoR binding sites. The in vivo relevance of the PhoR-binding site within thephoRSpromoter was supported by reporter gene studies.
CfaD-Dependent Expression of a Novel Extracytoplasmic Protein from EnterotoxigenicEscherichia coli
Authors: Pilonieta M. Carolina , Bodero Maria D. , Munson George P. 
Published: May 2007

ABSTRACTH10407 is a strain of enterotoxigenicEscherichia coli(ETEC) that utilizes CFA/I pili to adhere to surfaces of the small intestine, where it elaborates toxins that cause profuse watery diarrhea in humans. Expression of the CFA/I pilus is positively regulated at the level of transcription by CfaD, a member of the AraC/XylS family. DNase I footprinting revealed that the activator has two binding sites upstream of the pilus promotercfaAp. One site extends from positions −23 to −56, and the other extends from positions −73 to −103 (numbering relative to the transcription start site ofcfaAp). Additional CfaD binding sites were predicted within the genome of H10407 by computational analysis. Two of these sites lie upstream of a previously uncharacterized gene,cexE. In vitro DNase I footprinting confirmed that both sites are genuine binding sites, andcexEp::lacZreporters demonstrated that CfaD is required for the expression ofcexEin vivo. The amino terminus of CexE contains a secretory signal peptide that is removed during translocation across the cytoplasmic membrane through the general secretory pathway. These studies suggest that CexE may be a novel ETEC virulence factor because its expression is controlled by the virulence regulator CfaD, and its distribution is restricted to ETEC.
Quorum-Sensing Regulation of the Biofilm Matrix Genes (pel) ofPseudomonas aeruginosa
Authors: Sakuragi Yumiko , Kolter Roberto 
Published: May 2007

ABSTRACTQuorum sensing (QS) has been previously shown to play an important role in the development ofPseudomonas aeruginosabiofilms (D. G. Davies et al., Science 280:295-298, 1998). Although QS regulation of swarming and DNA release has been shown to play important roles in biofilm development, regulation of genes directly involved in biosynthesis of biofilm matrix has not been described. Here, transcription of thepeloperon, essential for the production of a glucose-rich matrix exopolysaccharide, is shown to be greatly reduced inlasIandrhlImutants. Chemical complementation of thelasImutant with 3-oxo-dodecanoyl homoserine lactone restorespeltranscription to the wild-type level and biofilm formation ability. These findings thus connect QS signaling and transcription of genes responsible for biofilm matrix biosynthesis.
The
Haemophilus influenzae
hFbpABC Fe
3+
Transporter: Analysis of the Membrane Permease and Development of a Gallium-Based Screen for Mutants
Authors: Anderson Damon S. , Adhikari Pratima , Weaver Katherine D. , Crumbliss Alvin L. , Mietzner Timothy A. 
Published: May 2007

ABSTRACT

The obligate human pathogen
Haemophilus influenzae
utilizes a siderophore-independent (free) Fe
3+
transport system to obtain this essential element from the host iron-binding protein transferrin. The hFbpABC transporter is a binding protein-dependent ABC transporter that functions to shuttle (free) Fe
3+
through the periplasm and across the inner membrane of
H. influenzae
. This investigation focuses on the structure and function of the hFbpB membrane permease component of the transporter, a protein that has eluded prior characterization. Based on multiple-sequence alignments between permease orthologs, a series of site-directed mutations targeted at residues within the two conserved permease motifs were generated. The hFbpABC transporter was expressed in a siderophore-deficient
Escherichia coli
background, and effects of mutations were analyzed using growth rescue and radiolabeled
55
Fe
3+
transport assays. Results demonstrate that mutation of the invariant glycine (G418A) within motif 2 led to attenuated transport activity, while mutation of the invariant glycine (G155A/V/E) within motif 1 had no discernible effect on activity. Individual mutations of well-conserved leucines (L154D and L417D) led to attenuated and null transport activities, respectively. As a complement to site-directed methods, a mutant screen based on resistance to the toxic iron analog gallium, an hFbpABC inhibitor, was devised. The screen led to the identification of several significant hFbpB mutations; V497I, I174F, and S475I led to null transport activities, while S146Y resulted in attenuated activity. Significant residues were mapped to a topological model of the hFbpB permease, and the implications of mutations are discussed in light of structural and functional data from related ABC transporters.
Identification of Two
Mycobacterium smegmatis
Lipoproteins Exported by a SecA2-Dependent Pathway
Authors: Gibbons Henry S. , Wolschendorf Frank , Abshire Michelle , Niederweis Michael , Braunstein Miriam 
Published: May 2007

ABSTRACT

The SecA2 protein is part of a specialized protein export system of mycobacteria. We set out to identify proteins exported to the bacterial cell envelope by the mycobacterial SecA2 system. By comparing the protein profiles of cell wall and membrane fractions from wild-type and Δ
secA2
mutant
Mycobacterium smegmatis
, we identified the Msmeg1712 and Msmeg1704 proteins as SecA2-dependent cell envelope proteins. These are the first endogenous
M. smegmatis
proteins identified as dependent on SecA2 for export. Both proteins are homologous to periplasmic sugar-binding proteins of other bacteria, and both contain functional amino-terminal signal sequences with lipobox motifs. These two proteins appeared to be genuine lipoproteins as shown by Triton X-114 fractionation and sensitivity to globomycin, an inhibitor of lipoprotein signal peptidase. The role of SecA2 in the export of these proteins was specific; not all mycobacterial lipoproteins required SecA2 for efficient localization or processing. Finally, Msmeg1704 was recognized by the SecA2 pathway of
Mycobacterium tuberculosis
, as indicated by the appearance of an export intermediate when the protein was expressed in a Δ
secA2
mutant of
M. tuberculosis
. Taken together, these results indicate that a select subset of envelope proteins containing amino-terminal signal sequences can be substrates of the mycobacterial SecA2 pathway and that some determinants for SecA2-dependent export are conserved between
M. smegmatis
and
M. tuberculosis.
Evidence for Modified Mechanisms of Chloroethene Oxidation in
Pseudomonas butanovora
Mutants Containing Single Amino Acid Substitutions in the Hydroxylase α-Subunit of Butane Monooxygenase
Authors: Halsey Kimberly H. , Doughty David M. , Sayavedra-Soto Luis A. , Bottomley Peter J. , Arp Daniel J. 
Published: May 2007

ABSTRACT

The properties of oxidation of dichloroethene (DCE) and trichloroethylene (TCE) by three mutant strains of
Pseudomonas butanovora
containing single amino acid substitutions in the α-subunit of butane monooxygenase hydroxylase (BMOH-α) were compared to the properties of the wild-type strain (Rev WT). The rates of oxidation of three chloroethenes (CEs) were reduced in mutant strain G113N and corresponded with a lower maximum rate of butane oxidation. The rate of TCE degradation was reduced by one-half in mutant strain L279F, whereas the rates of DCE oxidation were the same as those in Rev WT. Evidence was obtained that the composition of products of CE oxidation differed between Rev WT and some of the mutant strains. For example, while Rev WT released nearly all available chlorine stoichiometrically during CE oxidation, strain F321Y released about 40% of the chlorine during 1,2-
cis
-DCE and TCE oxidation, and strain G113N released between 14 and 25% of the available chlorine during oxidation of DCE and 56% of the available chlorine during oxidation of TCE. Whereas Rev WT, strain L279F, and strain F321Y formed stoichiometric amounts of 1,2-
cis
-DCE epoxide during oxidation of 1,2-
cis
-DCE, only about 50% of the 1,2-
cis
-DCE oxidized by strain G113N was detected as the epoxide. Evidence was obtained that 1,2-
cis
-DCE epoxide was a substrate for butane monooxygenase (BMO) that was oxidized after the parent compound was consumed. Yet all of the mutant strains released less than 40% of the available 1,2-
cis
-DCE chlorine, suggesting that they have altered activity towards the epoxide. In addition, strain G113N was unable to degrade the epoxide. TCE epoxide was detected during exposure of Rev WT and strain F321Y to TCE but was not detected with strains L279F and G113N. Lactate-dependent O
2
uptake rates were differentially affected by DCE degradation in the mutant strains, providing evidence that some products released by the altered BMOs reduced the impact of CE on cellular toxicity. The use of CEs as substrates in combination with
P. butanovora
BMOH-α mutants might allow insights into the catalytic mechanism of BMO to be obtained.
A Functional Homing Endonuclease in the
Bacillus anthracis nrdE
Group I Intron
Authors: Nord David , Torrents Eduard , Sjöberg Britt-Marie 
Published: May 2007

ABSTRACT

The essential
Bacillus anthracis nrdE
gene carries a self-splicing group I intron with a putative homing endonuclease belonging to the GIY-YIG family. Here, we show that the
nrdE
pre-mRNA is spliced and that the homing endonuclease cleaves an intronless
nrdE
gene 5 nucleotides (nt) upstream of the intron insertion site, producing 2-nt 3′ extensions. We also show that the sequence required for efficient cleavage spans at least 4 bp upstream and 31 bp downstream of the cleaved coding strand. The position of the recognition sequence in relation to the cleavage position is as expected for a GIY-YIG homing endonuclease. Interestingly,
nrdE
genes from several other
Bacillaceae
were also susceptible to cleavage, with those of
Bacillus cereus
,
Staphylococcus epidermidis
(
nrdE1
),
B. anthracis
, and
Bacillus thuringiensis
serovar konkukian being better substrates than those of
Bacillus subtilis
,
Bacillus lichenformis
, and
S. epidermidis
(
nrdE2
). On the other hand,
nrdE
genes from
Lactococcus lactis
,
Escherichia coli
,
Salmonella enterica
serovar Typhimurium, and
Corynebacterium ammoniagenes
were not cleaved. Intervening sequences (IVSs) residing in protein-coding genes are often found in enzymes involved in DNA metabolism, and the ribonucleotide reductase
nrdE
gene is a frequent target for self-splicing IVSs. A comparison of
nrdE
genes from seven gram-positive low-G+C bacteria, two bacteriophages, and
Nocardia farcinica
showed five different insertion sites for self-splicing IVSs within the coding region of the
nrdE
gene.
Only One of Four Oligopeptide Transport Systems Mediates Nitrogen Nutrition inStaphylococcus aureus
Authors: Hiron Aurelia , Borezée-Durant Elise , Piard Jean-Christophe , Juillard Vincent 
Published: May 2007

ABSTRACTOligopeptides internalized by oligopeptide permease (Opp) transporters play key roles in bacterial nutrition, signaling, and virulence. To date, twooppoperons,opp-1andopp-2, have been identified inStaphylococcus aureus. Systematic in silico analysis of 11 differentS. aureusgenomes revealed the existence of two newoppoperons,opp-3andopp-4, plus anopp-5Agene encoding a putative peptide-binding protein. With the exception ofopp-4, theoppoperons were present in allS. aureusstrains. Within a single strain, the differentoppoperons displayed little sequence similarity and distinct genetic organization. Transcriptional studies showed thatopp-1,opp-2,opp-3, andopp-4operons were polycistronic and thatopp-5Ais monocistronic. We designed a minimal chemically defined medium forS. aureusRN6390 and showed that alloppgenes were expressed but at different levels. Where tested, OppA protein production paralleled transcriptional profiles.opp-3, which encodes proteins most similar to known peptide transport proteins, displayed the highest expression level and was the only transporter to be regulated by specific amino acids, tyrosine and phenylalanine. Defined deletion mutants in one or several peptide permeases were constructed and tested for their capacity to grow in peptide-containing medium. Among the four putative Opp systems, Opp-3 was the only system able to provide oligopeptides for growth, ranging in length from 3 to 8 amino acids. Dipeptides were imported exclusively by DtpT, a proton-driven di- and tripeptide permease. These data provide a first complete inventory of the peptide transport systemsoppanddtpTofS. aureus. Among them, the newly identified Opp-3 appears to be the main Opp system supplying the cell with peptides as nutritional sources.
VapC-1 of Nontypeable
Haemophilus influenzae
Is a Ribonuclease
Authors: Daines Dayle A. , Wu Mack H. , Yuan Sarah Y. 
Published: May 2007

ABSTRACT

Nontypeable
Haemophilus influenzae
(NTHi) organisms are obligate parasites of the human upper respiratory tract that can exist as commensals or pathogens. Toxin-antitoxin (TA) loci are highly conserved gene pairs that encode both a toxin and antitoxin moiety. Seven TA gene families have been identified to date, and NTHi carries two alleles of the
vapBC
family. Here, we have characterized the function of one of the NTHi alleles,
vapBC-1
. The gene pair is transcribed as an operon in two NTHi clinical isolates, and promoter fusions display an inverse relationship to culture density. The antitoxin VapB-1 forms homomultimers both in vitro and in vivo. The expression of the toxin VapC-1 conferred growth inhibition to an
Escherichia coli
expression strain and was successfully purified only when cloned in tandem with its cognate antitoxin. Using total RNA isolated from both
E. coli
and NTHi, we show for the first time that VapC-1 is an RNase that is active on free RNA but does not degrade DNA in vitro. Preincubation of the purified toxin and antitoxin together results in the formation of a protein complex that abrogates the activity of the toxin. We conclude that the NTHi
vapBC-1
gene pair functions as a classical TA locus and that the induction of VapC-1 RNase activity leads to growth inhibition via the mechanism of mRNA cleavage.