bluuebunny/crossref_metadata_2025_split huggingface dataset:
Acetylornithine Transcarbamylase: a Novel Enzyme in Arginine Biosynthesis
Authors: Morizono Hiroki , Cabrera-Luque Juan , Shi Dashuang , Gallegos Rene , Yamaguchi Saori , Yu Xiaolin , Allewell Norma M. , Malamy Michael H. , Tuchman Mendel 
Published: April 2006

ABSTRACT

Ornithine transcarbamylase is a highly conserved enzyme in arginine biosynthesis and the urea cycle. In
Xanthomonas campestris
, the protein annotated as ornithine transcarbamylase, and encoded by the
argF
gene, is unable to synthesize citrulline directly from ornithine. We cloned and overexpressed this
X. campestris
gene in
Escherichia coli
and show that it catalyzes the formation of
N
-acetyl-l-citrulline from
N
-acetyl-l-ornithine and carbamyl phosphate. We now designate this enzyme as an acetylornithine transcarbamylase. The

K
m

values for
N
-acetylornithine and carbamyl phosphate were 1.05 mM and 0.01 mM, respectively. Additional putative transcarbamylases that might also be misannotated were found in the genomes of members of other xanthomonads,
Cytophaga
, and
Bacteroidetes
as well as in DNA sequences of bacteria from environmental isolates. It appears that these different paths for arginine biosynthesis arose very early in evolution and that the canonical ornithine transcarbamylase-dependent pathway became the prevalent form. A potent inhibitor,
N
α
-acetyl-
N
δ
-phosphonoacetyl-
l
-ornithine, was synthesized and showed a midpoint of inhibition at approximately 22 nM; this compound may prove to be a useful starting point for designing inhibitors specific to this novel family of transcarbamylases.
Influence of BrpA on Critical Virulence Attributes ofStreptococcus mutans
Authors: Wen Zezhang T. , Baker Henry V. , Burne Robert A. 
Published: April 2006

ABSTRACTStreptococcus mutans, the primary etiological agent of human dental caries, has developed multiple mechanisms to colonize and form biofilms on the tooth surface. ThebrpAgene codes for a predicted surface-associated protein with apparent roles in biofilm formation, autolysis, and cell division. In this study, we used two models to further characterize the biofilm-forming characteristics of a BrpA-deficient mutant, strain TW14. Compared to those of the parent strain, UA159, TW14 formed long chains and sparse microcolonies on hydroxylapatite disks but failed to accumulate and form three-dimensional biofilms when grown on glucose as the carbohydrate source. The biofilm formation defect was also readily apparent by confocal laser scanning microscopy when flow cells were used to grow biofilms. When subjected to acid killing at pH 2.8 for 45 min, the survival rate of strain TW14 was more than 1 log lower than that of the wild-type strain. TW14 was at least 3 logs more susceptible to killing by 0.2% hydrogen peroxide than was UA159. The expression of more than 200 genes was found by microarray analysis to be altered in cells lacking BrpA (P< 0.01). These results suggest that the loss of BrpA can dramatically influence the transcriptome and significantly affects the regulation of acid and oxidative stress tolerance and biofilm formation inS. mutans, which are key virulence attributes of the organism.
Role Of MinD-Membrane Association in Min Protein Interactions
Authors: Taghbalout Aziz , Ma Luyan , Rothfield Lawrence 
Published: April 2006

ABSTRACT

Division site placement in
Escherichia coli
involves interactions of the MinD protein with MinC and MinE and with other MinD molecules to form membrane-associated polymeric structures. In this work, as part of a study of these interactions, we established that heterologous membrane-associated proteins such as MinD can be targeted to the yeast nuclear membrane, dependent only on the presence of a membrane-binding domain and a nuclear targeting sequence. Targeting to the nuclear membrane was equally effective using the intrinsic MinD membrane-targeting domain or the completely unrelated membrane-targeting domain of cytochrome
b
5
. The chimeric proteins differing in their membrane-targeting sequences were then used to establish the roles of membrane association and specificity of the membrane anchor in MinD interactions, using the yeast two-hybrid system. The chimeric proteins were also used to show that the membrane association of MinD and MinE in
E. coli
cells had no specificity for the membrane anchor, whereas formation of MinDE polar zones and MinE rings required the presence of the native MinD membrane-targeting sequence.
Differential and Dynamic Localization of Topoisomerases in
Bacillus subtilis
Authors: Tadesse Serkalem , Graumann Peter L. 
Published: April 2006

ABSTRACT

Visualization of topoisomerases in live
Bacillus subtilis
cells showed that Topo I, Topo IV, and DNA gyrase differentially localize on the nucleoids but are absent at cytosolic spaces surrounding the nucleoids, suggesting that these topoisomerases interact with many regions of the chromosome. While both subunits of Topo IV were uniformly distributed throughout the nucleoids, Topo I and gyrase formed discrete accumulations, or foci, on the nucleoids in a large fraction of the cells, which showed highly dynamic movements. Three-dimensional time lapse microscopy showed that gyrase foci accumulate and dissipate within a 1-min time scale, revealing dynamic assembly and disassembly of subcelluar topoisomerase centers. Gyrase centers frequently colocalized with the central DNA replication machinery, suggesting a major role for gyrase at the replication fork, while Topo I foci were frequently close to or colocalized with the structural maintenance of chromosomes (SMC) chromosome segregation complex. The findings suggest that different areas of supercoiling exist on the
B. subtilis
nucleoids, which are highly dynamic, with a high degree of positive supercoiling attracting gyrase to the replication machinery and areas of negative supercoiling at the bipolar SMC condensation centers recruiting Topo I.
Comparative Genomics of NAD Biosynthesis in Cyanobacteria
Authors: Gerdes Svetlana Y. , Kurnasov Oleg V. , Shatalin Konstantin , Polanuyer Boris , Sloutsky Roman , Vonstein Veronika , Overbeek Ross , Osterman Andrei L. 
Published: April 2006

ABSTRACTBiosynthesis of NAD(P) cofactors is of special importance for cyanobacteria due to their role in photosynthesis and respiration. Despite significant progress in understanding NAD(P) biosynthetic machinery in some model organisms, relatively little is known about its implementation in cyanobacteria. We addressed this problem by a combination of comparative genome analysis with verification experiments in the model system ofSynechocystissp. strain PCC 6803. A detailed reconstruction of the NAD(P) metabolic subsystem using the SEED genomic platform (http://theseed.uchicago.edu/FIG/index.cgi) helped us accurately annotate respective genes in the entire set of 13 cyanobacterial species with completely sequenced genomes available at the time. Comparative analysis of operational variants implemented in this divergent group allowed us to elucidate both conserved (de novo and universal pathways) and variable (recycling and salvage pathways) aspects of this subsystem. Focused genetic and biochemical experiments confirmed several conjectures about the key aspects of this subsystem. (i) The product of the slr1691 gene, a homolog ofEscherichia coligenenadEcontaining an additional nitrilase-like N-terminal domain, is a NAD synthetase capable of utilizing glutamine as an amide donor in vitro. (ii) The product of the sll1916 gene, a homolog ofE. coligenenadD, is a nicotinic acid mononucleotide-preferring adenylyltransferase. This gene is essential for survival and cannot be compensated for by an alternative nicotinamide mononucleotide (NMN)-preferring adenylyltransferase (slr0787 gene). (iii) The product of the slr0788 gene is a nicotinamide-preferring phosphoribosyltransferase involved in the first step of the two-step nondeamidating utilization of nicotinamide (NMN shunt). (iv) The physiological role of this pathway encoded by a conserved gene cluster, slr0787-slr0788, is likely in the recycling of endogenously generated nicotinamide, as supported by the inability of this organism to utilize exogenously provided niacin. Positional clustering and the cooccurrence profile of the respective genes across a diverse collection of cellular organisms provide evidence of horizontal transfer events in the evolutionary history of this pathway.
Functional Domains of theBacillus subtilisTranscription Factor AraR and Identification of Amino Acids Important for Nucleoprotein Complex Assembly and Effector Binding
Authors: Franco Irina Saraiva , Mota Luís Jaime , Soares Cláudio Manuel , de Sá-Nogueira Isabel 
Published: April 2006

ABSTRACTTheBacillus subtilisAraR transcription factor represses at least 13 genes required for the extracellular degradation of arabinose-containing polysaccharides, transport of arabinose, arabinose oligomers, xylose, and galactose, intracellular degradation of arabinose oligomers, and further catabolism of this sugar. AraR exhibits a chimeric organization comprising a small N-terminal DNA-binding domain that contains a winged helix-turn-helix motif similar to that seen with the GntR family and a larger C-terminal domain homologous to that of the LacI/GalR family. Here, a model for AraR was derived based on the known crystal structures of the FadR and PurR regulators fromEscherichia coli. We have used random mutagenesis, deletion, and construction of chimeric LexA-AraR fusion proteins to map the functional domains of AraR required for DNA binding, dimerization, and effector binding. Moreover, predictions for the functional role of specific residues were tested by site-directed mutagenesis. In vivo analysis identified particular amino acids required for dimer assembly, formation of the nucleoprotein complex, and composition of the sugar-binding cleft. This work presents a structural framework for the function of AraR and provides insight into the mechanistic mode of action of this modular repressor.
Detailed Genomic Analysis of the Wβ and γ Phages InfectingBacillus anthracis: Implications for Evolution of Environmental Fitness and Antibiotic Resistance
Authors: Schuch Raymond , Fischetti Vincent A. 
Published: April 2006

ABSTRACTPhage-mediated lysis has been an essential laboratory tool for rapidly identifyingBacillus anthracisfor more than 40 years, relying on the γ phage derivative of aBacillus cereusprophage called W. The complete genomic sequences of the temperate W phage, referred to as Wβ, and its lytic variant γ were determined and found to encode 53 open reading frames each, spanning 40,864 bp and 37,373 bp, respectively. Direct comparison of the genomes showed that γ evolved through mutations at key loci controlling host recognition, lysogenic growth, and possibly host phenotypic modification. Included are a cluster of point mutations at thegp14tail fiber locus of γ, encoding a protein that, when fused to green fluorescent protein, binds specifically toB. anthracis. A large 2,003-bp deletion was also identified at the γ lysogeny module, explaining its shift from a temperate to a lytic lifestyle. Finally, evidence of recombination was observed at a dicistronic Wβ locus, encoding putative bacterial cell surface-modifying proteins, replaced in γ with a locus, likely obtained from aB. anthracisprophage, encoding demonstrable fosfomycin resistance. Reverse transcriptase PCR analysis confirmed strong induction at the dicistronic Wβ locus and at four other phage loci inB. anthracisand/orB. cereuslysogens. In all, this study represents the first genomic and functional description of two historically important phages and is part of a broader investigation into contributions of phage to theB. anthracislife cycle. Initial findings suggest that lysogeny ofB. anthracispromotes ecological adaptation, rather than virulence, as with other gram-positive pathogens.
Structural Alterations of the Cysteine Desulfurase IscS of
Salmonella enterica
Serovar Typhimurium Reveal Substrate Specificity of IscS in tRNA Thiolation
Authors: Lundgren Hans K. , Björk Glenn R. 
Published: April 2006

ABSTRACT

The cysteine desulfurase IscS in
Salmonella enterica
serovar Typhimurium is required for the formation of all four thiolated nucleosides in tRNA, which is thought to occur via two principally different biosynthetic pathways. The synthesis of 4-thiouridine (s
4
U) and 5-methylaminomethyl-2-thiouridine (mnm
5
s
2
U) occurs by a transfer of sulfur from IscS via various proteins to the target nucleoside in the tRNA, and no iron-sulfur cluster protein participates, whereas the synthesis of 2-thiocytidine (s
2
C) and
N
6
-(4-hydroxyisopentenyl)-2-methylthioadenosine (ms
2
io
6
A) is dependent on iron-sulfur cluster proteins, whose formation and maintenance depend on IscS. Accordingly, inactivation of IscS should result in decreased synthesis of all thiolated nucleosides. We selected mutants defective either in the synthesis of a thiolated nucleoside (mnm
5
s
2
U) specific for the iron-sulfur protein-independent pathway or in the synthesis of a thiolated nucleoside (ms
2
io
6
A) specific for the iron-sulfur protein-dependent pathway. Although we found altered forms of IscS that influenced the synthesis of all thiolated nucleosides, consistent with the model, we also found mutants defective in subsets of thiolated nucleosides. Alterations in the C-terminal region of IscS reduced the level of only ms
2
io
6
A, suggesting that the synthesis of this nucleoside is especially sensitive to minor aberrations in iron-sulfur cluster transfer activity. Our results suggest that IscS has an intrinsic substrate specificity in how it mediates sulfur mobilization and/or iron-sulfur cluster formation and maintenance required for thiolation of tRNA.
Upregulated Transcription of Plasmid and Chromosomal Ribulose Monophosphate Pathway Genes Is Critical for Methanol Assimilation Rate and Methanol Tolerance in the Methylotrophic Bacterium
Bacillus methanolicus
Authors: Jakobsen Øyvind M. , Benichou Aline , Flickinger Michael C. , Valla Svein , Ellingsen Trond E. , Brautaset Trygve 
Published: April 2006

ABSTRACT

The natural plasmid pBM19 carries the key
mdh
gene needed for the oxidation of methanol into formaldehyde by
Bacillus methanolicus
. Five more genes,
glpX
,
fba
,
tkt
,
pfk
, and
rpe
, with deduced roles in the cell primary metabolism, are also located on this plasmid. By using real-time PCR, we show that they are transcriptionally upregulated (6- to 40-fold) in cells utilizing methanol; a similar induction was shown for two chromosomal genes,
hps
and
phi
. These seven genes are involved in the fructose bisphosphate aldolase/sedoheptulose bisphosphatase variant of the ribulose monophosphate (RuMP) pathway for formaldehyde assimilation. Curing of pBM19 causes higher methanol tolerance and reduced formaldehyde tolerance, and the methanol tolerance is reversed to wild-type levels by reintroducing
mdh
. Thus, the RuMP pathway is needed to detoxify the formaldehyde produced by the methanol dehydrogenase-mediated conversion of methanol, and the in vivo transcription levels of
mdh
and the RuMP pathway genes reflect the methanol tolerance level of the cells. The transcriptional inducer of
hps
and
phi
genes is formaldehyde, and not methanol, and introduction of multiple copies of these two genes into
B. methanolicus
made the cells more tolerant of growth on high methanol concentrations. The recombinant strain also had a significantly higher specific growth rate on methanol than the wild type. While pBM19 is critical for growth on methanol and important for formaldehyde detoxification, the maintenance of this plasmid represents a burden for
B. methanolicus
when growing on mannitol. Our data contribute to a new and fundamental understanding of the regulation of
B. methanolicus
methylotrophy.
A CsgD-Independent Pathway for Cellulose Production and Biofilm Formation inEscherichia coli
Authors: Da Re Sandra , Ghigo Jean-Marc 
Published: April 2006

ABSTRACTBacterial growth on a surface often involves the production of a polysaccharide-rich extracellular matrix that provides structural support for the formation of biofilm communities. InSalmonella, cellulose is one of the major constituents of the biofilm matrix. Its production is regulated by CsgD and the diguanylate cyclase AdrA that activates cellulose synthesis at a posttranscriptional level. Here, we studied a collection ofEscherichia coliisolates, and we found that the ability to produce cellulose is a common trait shared by more than 50% of the tested strains. We investigated the genetic determinants of cellulose production and its role in biofilm formation in the commensal strainE. coli1094. By contrast with theSalmonellacellulose regulatory cascade, neither CsgD nor AdrA is required inE. coli1094 to regulate cellulose production. In this strain, an alternative cellulose regulatory pathway is used, which involves the GGDEF domain protein, YedQ. Although AdrA1094is functional, it is weakly expressed inE. coli1094 compared to YedQ, which constitutively activates cellulose production under all tested environmental conditions. The study of cellulose regulation in several otherE. coliisolates showed that, besides the CsgD/AdrA regulatory pathway, both CsgD-independent/YedQ-dependent and CsgD-independent/YedQ-independent pathways are found, indicating that alternative cellulose pathways are common inE. coliand possibly in other cellulose-producingEnterobacteriaceae.
Effects of Phosphorelay Perturbations on Architecture, Sporulation, and Spore Resistance in Biofilms ofBacillus subtilis
Authors: Veening Jan-Willem , Kuipers Oscar P. , Brul Stanley , Hellingwerf Klaas J. , Kort Remco 
Published: April 2006

ABSTRACTThe spore-forming bacteriumBacillus subtilisis able to form highly organized multicellular communities called biofilms. This coordinated bacterial behavior is often lost in domesticated or laboratory strains as a result of planktonic growth in rich media for many generations. However, we show here that the laboratory strainB. subtilis168 is still capable of forming spatially organized multicellular communities on minimal medium agar plates, exemplified by colonies with vein-like structures formed by elevated bundles of cells. In line with the current model for biofilm formation, we demonstrate that overproduction of the phosphorelay components KinA and Spo0A stimulates bundle formation, while overproduction of the transition state regulators AbrB and SinR leads to repression of formation of elevated bundles. Time-lapse fluorescence microscopy studies ofB. subtilisgreen fluorescent protein reporter strains show that bundles are preferential sites for spore formation and that flat structures surrounding the bundles contain vegetative cells. The elevated bundle structures are formed prior to sporulation, in agreement with a genetic developmental program in which these processes are sequentially activated. Perturbations of the phosphorelay by disruption and overexpression of genes that lead to an increased tendency to sporulate result in the segregation of sporulation mutations and decreased heat resistance of spores in biofilms. These results stress the importance of a balanced control of the phosphorelay for biofilm and spore development.
EspF of Enteropathogenic
Escherichia coli
Binds Sorting Nexin 9
Authors: Marchès Oliver , Batchelor Miranda , Shaw Robert K. , Patel Amit , Cummings Nicola , Nagai Takeshi , Sasakawa Chihiro , Carlsson Sven R. , Lundmark Richard , Cougoule Celine , Caron Emmanuelle , Knutton Stuart , Connerton Ian , Frankel Gad 
Published: April 2006

ABSTRACT

EspF of enteropathogenic
Escherichia coli
targets mitochondria and subverts a number of cellular functions. EspF consists of six putative Src homology 3 (SH3) domain binding motifs. In this study we identified sorting nexin 9 (SNX9) as a host cell EspF binding partner protein, which binds EspF via its amino-terminal SH3 region. Coimmunoprecipitation and confocal microscopy showed specific EspF-SNX9 interaction and non-mitochondrial protein colocalization in infected epithelial cells.
Mechanism ofhilARepression by 1,2-Propanediol Consists of Two Distinct Pathways, One Dependent on and the Other Independent of Catabolic Production of Propionate, inSalmonella entericaSerovar Typhimurium
Authors: Nakayama Shu-ichi , Watanabe Haruo 
Published: April 2006

ABSTRACTA glycerol dehydrogenase gene was selected as a multicopy suppressor rescuing the reducedhilAexpression in theSalmonella entericaserovar TyphimuriumcpxAmutant. A substrate of the enzyme, 1,2-propanediol, repressedhilAexpression. The 1,2-propanediol-mediated repression at 150 mM, but not that at 300 mM, was abrogated by blocking the catabolism producing propionate from 1,2-propanediol.
Sigma L Is Important for Cold Shock Adaptation of
Bacillus subtilis
Authors: Wiegeshoff Frank , Beckering Carsten L. , Debarbouille Michel , Marahiel Mohamed A. 
Published: April 2006

ABSTRACT

Although sigma factor-dependent transcriptional regulation was shown to be essential for adaptation to different environmental stimuli, no such sigma factor has been related to the regulation of the cold shock response in
Bacillus subtilis
. In this study, we present genetic evidence for participation of σ
L
(σ
54
) and the two σ
L
-dependent transcriptional enhancers BkdR and YplP in the cold shock response of
Bacillus subtilis
JH642. Single-gene deletion of either
sigL
,
bkdR
, or
yplP
resulted in a cold-sensitive phenotype.
Independent Regulation of MucD, an HtrA-Like Protease in
Pseudomonas aeruginosa
, and the Role of Its Proteolytic Motif in Alginate Gene Regulation
Authors: Wood Lynn F. , Ohman Dennis E. 
Published: April 2006

ABSTRACT

Expression of
mucD
, encoding a homologue of the HtrA(DegP) family of endoserine proteases, was investigated in
Pseudomonas aeruginosa
. Expressed from the
algT-mucABCD
operon, MucD was detected in mucoid (FRD1) and nonmucoid (PAO1) parental strains and also when polar insertions were placed upstream in
algT
or
mucB
. A transcriptional start site for a
mucD
promoter (P
mucD
) was mapped within
mucC
. Expression of single-copy
mucD217
, encoding MucD altered in the protease motif (S217A), was defective in temperature resistance and alginate gene regulation.
Two Putative
c
-Type Multiheme Cytochromes Required for the Expression of OmcB, an Outer Membrane Protein Essential for Optimal Fe(III) Reduction in
Geobacter sulfurreducens
Authors: Kim Byoung-Chan , Qian Xinlei , Leang Ching , Coppi Maddalena V. , Lovley Derek R. 
Published: April 2006

ABSTRACT

Deletion of two homologous
Geobacter sulfurreducens c
-type cytochrome genes,
omcG
and
omcH
, decreased the rate of Fe(III) reduction and decreased the level of an outer membrane cytochrome critical for Fe(III) reduction, OmcB, without affecting its transcription. Expression of either gene restored Fe(III) reduction and OmcB expression, suggesting functional similarity.
Characterization of a Gene Encoding an Acetylase Required for Pyoverdine Synthesis in
Pseudomonas aeruginosa
Authors: Lamont Iain L. , Martin Lois W. , Sims Talia , Scott Amy , Wallace Mary 
Published: April 2006

ABSTRACT

Strains of
Pseudomonas aeruginosa
secrete one of three pyoverdine siderophores (types I to III). We have characterized a gene,
pvdY
II
(for the
pvdY
gene present in type II
P. aeruginosa
strains), that is only present in strains that make type II pyoverdine. A mutation in
pvdY
II
prevented pyoverdine synthesis. Bioinformatic, genetic, and biochemical approaches indicate that the PvdYII enzyme catalyzes acetylation of hydroxyornithine. Expression of
pvdY
II
is repressed by the presence of iron and upregulated by the presence of type II pyoverdine. Characterization of
pvdY
II
provides insights into the molecular basis for production of different pyoverdines by different strains of
P. aeruginosa
.