bluuebunny/crossref_metadata_2025_split huggingface dataset:
Genes of De Novo Pyrimidine Biosynthesis from the Hyperthermoacidophilic Crenarchaeote
Sulfolobus acidocaldarius
: Novel Organization in a Bipolar Operon
Authors: Thia-Toong Thia-Lin , Roovers Martine , Durbecq Virginie , Gigot Daniel , Glansdorff Nicolas , Charlier Daniel 
Published: October 2002

ABSTRACT

Sequencing a 8,519-bp segment of the
Sulfolobus acidocaldarius
genome revealed the existence of a tightly packed bipolar pyrimidine gene cluster encoding the enzymes of de novo UMP synthesis. The G+C content of 35.3% is comparable to that of the entire genome, but intergenic regions exhibit a considerably lower percentage of strong base pairs. Coding regions harbor the classical excess of purines on the coding strand, whereas intergenic regions do not show this bias. Reverse transcription-PCR and primer extension experiments demonstrated the existence of two polycistronic messengers,
pyrEF-orf8
and
pyrBI-orf1-pyrCD-orf2-orf3-orf4
, initiated from a pair of divergent and partially overlapping promoters. The gene order and the grouping in two wings of a bipolar operon constitute a novel organization of
pyr
genes that also occurs in the recently determined genome sequences of
Sulfolobus solfataricus
P2 and
Sulfolobus tokodaii
strain 7; the configuration appears therefore characteristic of
Sulfolobus
. The quasi-leaderless
pyrE
and
pyrB
genes do not bear a Shine-Dalgarno sequence, whereas the initiation codon of promoter-distal genes is preceded at an appropriate distance by a sequence complementary to the 3′ end of 16S rRNA. The polycistronic nature of the
pyr
messengers and the existence of numerous overlaps between contiguous open reading frames suggests the existence of translational coupling.
pyrB
transcription was shown to be approximately twofold repressed in the presence of uracil. The mechanism underlying this modulation is as yet unknown, but it appears to be of a type different from the various attenuation-like mechanisms that regulate
pyrB
transcription in bacteria. In contrast, the
pyrE-pyrB
promoter/control region harbors direct repeats and imperfect palindromes reminiscent of target sites for the binding of a hypothetical regulatory protein(s).
ThenptAGene ofVibrio choleraeEncodes a Functional Sodium-Dependent Phosphate Cotransporter Homologous to the Type II Cotransporters of Eukaryotes
Authors: Lebens Michael , Lundquist Patrik , Söderlund Lars , Todorovic Mirjana , Carlin Nils I. A. 
Published: October 2002

ABSTRACTThenptAgene ofVibrio choleraehas significant protein sequence homology with type II sodium-dependent phosphate (Pi) cotransporters found in animals but not previously identified in prokaryotes. The phylogeny of known type II cotransporter sequences indicates thatnptAmay be either an ancestral gene or a gene acquired from a higher eukaryotic source. The gene was cloned into an expression vector under the control of an inducible promoter and expressed inEscherichia coli. The results demonstrate thatnptAencodes a functional protein with activity similar to that of the animal enzyme, catalyzing high-affinity, sodium-dependent Piuptake with comparable affinities for both sodium and phosphate ions. Furthermore, the activity of NptA is influenced by pH, again in a manner similar to that of the NaPi-2a subtype of the animal enzyme, although it lacks the corresponding REK motif thought to be responsible for this phenomenon. Piuptake activity, a component of which appeared to be sodium dependent, was increased inV. choleraeby phosphate starvation. However, it appears from the use of a reporter gene expressed from thenptApromoter that none of this activity is attributable to the induction of expression fromnptA. It is thus proposed that the physiological function of NptA protein may be the rapid uptake of Piin preparation for rapid growth in nutrient-rich environments and that it may therefore play a role in establishing infection.
Proteolytic Cleavage of the FlhB Homologue YscU of
Yersinia pseudotuberculosis
Is Essential for Bacterial Survival but Not for Type III Secretion
Authors: Lavander Moa , Sundberg Lena , Edqvist Petra J. , Lloyd Scott A. , Wolf-Watz Hans , Forsberg Åke 
Published: October 2002

ABSTRACT

Pathogenic
Yersinia
species employ a type III secretion system (TTSS) to target antihost factors, Yop proteins, into eukaryotic cells. The secretion machinery is constituted of ca. 20 Ysc proteins, nine of which show significant homology to components of the flagellar TTSS. A key event in flagellar assembly is the switch from secreting-assembling hook substrates to filament substrates, a switch regulated by FlhB and FliK. The focus of this study is the FlhB homologue YscU, a bacterial inner membrane protein with a large cytoplasmic C-terminal domain. Our results demonstrate that low levels of YscU were required for functional Yop secretion, whereas higher levels of YscU lowered both Yop secretion and expression. Like FlhB, YscU was cleaved into a 30-kDa N-terminal and a 10-kDa C-terminal part. Expression of the latter in a wild-type strain resulted in elevated Yop secretion. The site of cleavage was at a proline residue, within the strictly conserved amino acid sequence NPTH. A YscU protein with an in-frame deletion of NPTH was cleaved at a different position and was nonfunctional with respect to Yop secretion. Variants of YscU with single substitutions in the conserved NPTH sequence—i.e., N263A, P264A, or T265A—were not cleaved but retained function in Yop secretion. Elevated expression of these YscU variants did, however, result in severe growth inhibition. From this we conclude that YscU cleavage is not a prerequisite for Yop secretion but is rather required to maintain a nontoxic fold.
Simultaneous Coexpression ofBorrelia burgdorferiErp Proteins Occurs through a Specific,erpLocus-Directed Regulatory Mechanism
Authors: El-Hage Nazira , Stevenson Brian 
Published: October 2002

ABSTRACTAn individualBorrelia burgdorferibacterium can encode as many as 13 different Erp (OspE/F-related) proteins from mono-and bicistronic loci that are carried on up to 10 separate plasmids. We demonstrate through multilabel immunofluorescence analyses that individual bacteria simultaneously coexpress their entire Erp protein repertoire. While it has been proposed thatB. burgdorfericontrols expression of Erp and other plasmid-encoded proteins through changes in DNA topology, we observed regulated Erp expression in the absence of detectable differences in DNA supercoiling. Likewise, inhibition of DNA gyrase had no detectable effect on Erp expression. Furthermore, expression of loci physically adjacent toerploci was observed to be independently regulated. It is concluded that Erp expression is regulated by a mechanism(s) directed aterploci and not by a global, plasmid-wide mechanism.
From Genetic Footprinting to Antimicrobial Drug Targets: Examples in Cofactor Biosynthetic Pathways
Authors: Gerdes Svetlana Y. , Scholle Michael D. , D'Souza Mark , Bernal Axel , Baev Mark V. , Farrell Michael , Kurnasov Oleg V. , Daugherty Matthew D. , Mseeh Faika , Polanuyer Boris M. , Campbell John W. , Anantha Shubha , Shatalin Konstantin Y. , Chowdhury Shamim A. K. , Fonstein Michael Y. , Osterman Andrei L. 
Published: October 2002

ABSTRACTNovel drug targets are required in order to design new defenses against antibiotic-resistant pathogens. Comparative genomics provides new opportunities for finding optimal targets among previously unexplored cellular functions, based on an understanding of related biological processes in bacterial pathogens and their hosts. We describe an integrated approach to identification and prioritization of broad-spectrum drug targets. Our strategy is based on genetic footprinting inEscherichia colifollowed by metabolic context analysis of essential gene orthologs in various species. Genes required for viability ofE. coliin rich medium were identified on a whole-genome scale using the genetic footprinting technique. Potential target pathways were deduced from these data and compared with a panel of representative bacterial pathogens by using metabolic reconstructions from genomic data. Conserved and indispensable functions revealed by this analysis potentially represent broad-spectrum antibacterial targets. Further target prioritization involves comparison of the corresponding pathways and individual functions between pathogens and the human host. The most promising targets are validated by direct knockouts in model pathogens. The efficacy of this approach is illustrated using examples from metabolism of adenylate cofactors NAD(P), coenzyme A, and flavin adenine dinucleotide. Several drug targets within these pathways, including three distantly related adenylyltransferases (orthologs of theE. coligenesnadD,coaD, andribF), are discussed in detail.
Functional Annotation of Class I Lysyl-tRNA Synthetase Phylogeny Indicates a Limited Role for Gene Transfer
Authors: Ambrogelly Alexandre , Korencic Dragana , Ibba Michael 
Published: October 2002

ABSTRACTFunctional and comparative genomic studies have previously shown that the essential protein lysyl-tRNA synthetase (LysRS) exists in two unrelated forms. Most prokaryotes and all eukaryotes contain a class II LysRS, whereas most archaea and a few bacteria contain a less common class I LysRS. In bacteria the class I LysRS is only found in the α-proteobacteria and a scattering of other groups, including the spirochetes, while the class I protein is by far the most common form of LysRS in archaea. To investigate this unusual distribution we functionally annotated a representative phylogenetic sampling of LysRS proteins. Class I LysRS proteins from a variety of bacteria and archaea were characterized in vitro by their ability to recognizeEscherichia colitRNALysanticodon mutants. Class I LysRS proteins were found to fall into two distinct groups, those that preferentially recognize the third anticodon nucleotide of tRNALys(U36) and those that recognize both the second and third positions (U35 and U36). Strong recognition of U35 and U36 was confined to the pyrococcus-spirochete grouping within the archaeal branch of the class I LysRS phylogenetic tree, while U36 recognition was seen in other archaea and an example from the α-proteobacteria. Together with the corresponding phylogenetic relationships, these results suggest that despite its comparative rarity the distribution of class I LysRS conforms to the canonical archaeal-bacterial division. The only exception, suggested from both functional and phylogenetic data, appears to be the horizontal transfer of class I LysRS from a pyrococcal progenitor to a limited number of bacteria.
Identification of a DtxR-Regulated Operon That Is Essential for Siderophore-Dependent Iron Uptake inCorynebacterium diphtheriae
Authors: Qian Yilei , Lee John H. , Holmes Randall K. 
Published: October 2002

ABSTRACTThe diphtheria toxin repressor (DtxR) uses Fe2+as a corepressor and inhibits transcription from iron-regulated promoters (IRPs) inCorynebacterium diphtheriae. A new IRP, designated IRP6, was cloned fromC. diphtheriaeby a SELEX-like procedure. DtxR bound to IRP6 in vitro only in the presence of appropriate divalent metal ions, and repression of IRP6 by DtxR in anEscherichia colisystem was iron dependent. The open reading frames (ORFs) downstream from IRP6 and previously described promoter IRP1 were found to encode proteins homologous to components of ATP-binding cassette (ABC) transport systems involved in high-affinity iron uptake in other bacteria. IRP1 and IRP6 were repressed under high-iron conditions in wild-typeC. diphtheriaeC7(β), but they were expressed constitutively in C7(β) mutant strains HC1, HC3, HC4, and HC5, which were shown previously to be defective in corynebactin-dependent iron uptake. A clone of the wild-typeirp6operon (pCM6ABC) complemented the constitutive corynebactin production phenotype of HC1, HC4, and HC5 but not of HC3, whereas a clone of the wild-typeirp1operon failed to complement any of these strains. Complementation by subclones of pCM6ABC demonstrated that mutant alleles ofirp6A,irp6C, andirp6Bwere responsible for the phenotypes of HC1, HC4, and HC5, respectively. Theirp6Aallele in HC1 and theirp6Ballele in HC5 encoded single amino acid substitutions in their predicted protein products, and theirp6Callele in HC4 caused premature chain termination of its predicted protein product. Strain HC3 was found to have a chain-terminating mutation indtxRin addition to a missense mutation in itsirp6Ballele. These findings demonstrated that theirp6operon inC. diphtheriaeencodes a putative ABC transporter, that specific mutant alleles ofirp6A,irp6B, andirp6Care associated with defects in corynebactin-dependent iron uptake, and that complementation of these mutant alleles restores repression of corynebactin production under high-iron growth conditions, most likely as a consequence of restoring siderophore-dependent iron uptake mediated by theirp6operon.
A Novel Histidine-Rich CPx-ATPase from the Filamentous Cyanobacterium
Oscillatoria brevis
Related to Multiple-Heavy-Metal Cotolerance
Authors: Tong Liu , Nakashima Susumu , Shibasaka Mineo , Katsuhara Maki , Kasamo Kunihiro 
Published: October 2002

ABSTRACT

A novel gene related to heavy-metal transport was cloned and identified from the filamentous cyanobacterium
Oscillatoria brevis
. Sequence analysis of the gene (the Bxa1 gene) showed that its product possessed high homology with heavy-metal transport CPx-ATPases. The CPC motif, which is proposed to form putative cation transduction channel, was found in the sixth transmembrane helix. However, instead of the CXXC motif that is present in the N termini of most metal transport CPx-ATPases, Bxa1 contains a unique Cys-Cys (CC) sequence element and histidine-rich motifs as a putative metal binding site. Northern blotting and real-time quantitative reverse transcription-PCR showed that expression of Bxa1 mRNA was induced in vivo by both monovalent (Cu
+
and Ag
+
) and divalent (Zn
2+
and Cd
2+
) heavy-metal ions at similar levels. Experiments on heavy-metal tolerance in
Escherichia coli
with recombinant Bxa1 demonstrated that Bxa1 conferred resistance to both monovalent and divalent heavy metals. This is the first report of a CPx-ATPase responsive to both monovalent and divalent heavy metals.
Temperature- and H-NS-Dependent Regulation of a Plasmid-Encoded Virulence Operon Expressing
Escherichia coli
Hemolysin
Authors: Madrid Cristina , Nieto José M. , Paytubi Sònia , Falconi Maurizio , Gualerzi Claudio O. , Juárez Antonio 
Published: October 2002

ABSTRACT

Proteins H-NS and Hha form a nucleoprotein complex that modulates expression of the thermoregulated
hly
operon of
Escherichia coli.
We have been able to identify two H-NS binding sites in the
hly
regulatory region. One of them partially overlaps the promoter region (site II), and the other is located about 2 kbp upstream (site I). In contrast, Hha protein did not show any preference for specific sequences. In vitro, temperature influences the affinity of H-NS for a DNA fragment containing both binding sites and H-NS-mediated repression of
hly
operon transcription. Deletion analysis of the
hly
regulatory region confirms the relevance of site I for thermoregulation of this operon. We present a model to explain the temperature-modulated repression of the
hly
operon, based on the experiments reported here and other, preexisting data.
A LuxR Homolog Controls Production of Symbiotically Active Extracellular Polysaccharide II by
Sinorhizobium meliloti
Authors: Pellock Brett J. , Teplitski Max , Boinay Ryan P. , Bauer W. Dietz , Walker Graham C. 
Published: October 2002

ABSTRACT

Production of complex extracellular polysaccharides (EPSs) by the nitrogen-fixing soil bacterium
Sinorhizobium meliloti
is required for efficient invasion of root nodules on the host plant alfalfa. Any one of three
S. meliloti
polysaccharides, succinoglycan, EPS II, or K antigen, can mediate infection thread initiation and extension (root nodule invasion) on alfalfa. Of these three polysaccharides, the only symbiotically active polysaccharide produced by
S. meliloti
wild-type strain Rm1021 is succinoglycan. The
expR101
mutation is required to turn on production of symbiotically active forms of EPS II in strain Rm1021. In this study, we have determined the nature of the
expR101
mutation in
S. meliloti
. The
expR101
mutation, a spontaneous dominant mutation, results from precise, reading frame-restoring excision of an insertion sequence from the coding region of
expR
, a gene whose predicted protein product is highly homologous to the
Rhizobium leguminosarum
bv. viciae RhiR protein and a number of other homologs of
Vibrio fischeri
LuxR that function as receptors for
N
-acylhomoserine lactones (AHLs) in quorum-sensing regulation of gene expression.
S. meliloti
ExpR activates transcription of genes involved in EPS II production in a density-dependent fashion, and it does so at much lower cell densities than many quorum-sensing systems. High-pressure liquid chromatographic fractionation of
S. meliloti
culture filtrate extracts revealed at least three peaks with AHL activity, one of which activated ExpR-dependent expression of the
expE
operon.
Differential Expression and Localization of Mn and Fe Superoxide Dismutases in the Heterocystous Cyanobacterium
Anabaena
sp. Strain PCC 7120
Authors: Li Tao , Huang Xu , Zhou Ruanbao , Liu Yingfang , Li Bin , Nomura Chris , Zhao Jindong 
Published: October 2002

ABSTRACT

Superoxide dismutases (Sods) play very important roles in preventing oxidative damages in aerobic organisms. The nitrogen-fixing heterocystous cyanobacterium
Anabaena
sp. strain PCC 7120 has two Sod-encoding genes: a
sodB
, encoding a soluble iron-containing Sod (FeSod), and a
sodA
, encoding a manganese-containing Sod (MnSod). The FeSod was purified and characterized. A recombinant FeSod was also obtained by overproduction in
Escherichia coli.
Immunoblot study of the FeSod during induction of heterocyst differentiation showed that the cells produced six- to eightfold more FeSod 8 h after a shift from a nitrogen-replete condition to a nitrogen-depleted condition. However, the amount of FeSod protein in filaments with mature heterocysts was the same as that in filaments grown with combined nitrogen. Superoxide anion-generating chemicals such as methyl viologen did not induce upregulation of the
sodB
gene expression. The predicted preprotein of the
sodA
gene has a leader peptide and a motif for membrane attachment at the N terminus of the mature protein. Activity staining after gel electrophoresis of the purified thylakoid membranes showed that most of the MnSod in
Anabaena
sp. strain PCC 7120 was located on thylakoids toward the lumenal side. Expression of the
sodA
gene in
E. coli
shows that the leader peptide was required for its activity and the membrane localization of the MnSod. Northern hybridization detected one 0.82-kb transcript of
sodA
. The
sodA
gene was upregulated by methyl viologen, whereas its amount was unchanged during heterocyst differentiation. Immunoblotting and activity staining showed that isolated heterocysts contained a lower but still significant amount of FeSod, suggesting that its function is required in heterocysts. No MnSod was observed in isolated heterocysts. These results show that the two different Sod proteins have differentiated roles in
Anabaena
sp. strain PCC 7120.
Utility of Gram Staining for Evaluation of the Quality of Cystic Fibrosis Sputum Samples
Authors: Nair Bindu , Stapp Jenny , Stapp Lynn , Bugni Linda , Van Dalfsen Jill , Burns Jane L. 
Published: October 2002

ABSTRACTThe microscopic examination of Gram-stained sputum specimens is very helpful in the evaluation of patients with community-acquired pneumonia and has also been recommended for use in cystic fibrosis (CF) patients. This study was undertaken to evaluate that recommendation. One hundred one sputum samples from CF patients were cultured for gram-negative bacilli and examined by Gram staining for both sputum adequacy (using the quality [Q] score) and bacterial morphology. Subjective evaluation of adequacy was also performed and categorized. Based on Q score evaluation, 41% of the samples would have been rejected despite a subjective appearance of purulence. Only three of these rejected samples were culture negative for gram-negative CF pathogens. Correlation between culture results and quantitative Gram stain examination was also poor. These data suggest that subjective evaluation combined with comprehensive bacteriology is superior to Gram staining in identifying pathogens in CF sputum.
In Vitro Comparison of Activities of Terbinafine and Itraconazole against
Paracoccidioides brasiliensis
Authors: Hahn R. C. , Fontes C. J. F. , Batista R. D. , Hamdan J. S. 
Published: October 2002

ABSTRACT

In vitro, terbinafine is highly active against a broad spectrum of pathogenic fungi. We evaluated the activities of terbinafine and itraconazole against 31 isolates of
Paracoccidioides brasiliensis.
The tests were conducted by using a broth macrodilution procedure. MICs, in micrograms per milliliter, were as follows: terbinafine, 0.015 to 1.0 (geometric mean, 0.1188); itraconazole, 0.007 to 0.5 (geometric mean, 0.03165). The usual therapy for paracoccidioidomycosis is sulfonamides, amphotericin B, and azole derivatives (ketoconazole, itraconazole, and fluconazole). In comparison to amphotericin B, azole derivatives allow shorter treatment courses, can be administered orally, and are equally effective. Itraconazole has as high efficacy as ketoconazole, but with superior tolerance. It is the current drug of choice for treatment of paracoccidioidomycosis. The data obtained in this study indicate that terbinafine is active against
P. brasiliensis
in vitro and suggest that this allylamine can be considered a new option as drug therapy for paracoccidioidomycosis.
Inactivation of West Nile Virus during Serologic Testing and Transport
Authors: Mayo Donald R. , Beckwith William H. 
Published: October 2002

ABSTRACT
Inactivation of West Nile virus (WNV) in enzyme-linked immunosorbent assay (ELISA) wash buffer at 37°C was studied, as well as inactivation of WNV in cell culture medium over several days at an ambient temperature (28°C). Aliquots of WNV were removed from the 37°C ELISA wash buffer at 5, 15, 30, and 60 min for the former experiment, while daily aliquots of medium were sampled for the latter experiment. No virus was detected in the wash buffer at 30 and 60 min, while virus was readily detected from cell culture medium over this time. In addition, titers of WNV consistently dropped over a 7-day period at 28°C compared to control suspensions of virus held at 4°C. These observations indicate that WNV is readily inactivated in the presence of detergent-containing buffers. Furthermore, the viability loss at ambient temperature suggests that WNV is easily inactivated during routine transportation and testing of human body fluids such as serum and cerebrospinal fluid.
Corneal Ulcer Caused by the New Fungal SpeciesSarcopodium oculorum
Authors: Guarro Josep , Höfling-Lima Ana Luisa , Gené Josepa , De Freitas Denise , Godoy Patricio , Zorat-Yu Maria Luisa , Zaror Luis , Fischman Olga 
Published: October 2002

ABSTRACTWe describe a case of keratitis caused by a new species of the hyphomycetous genusSarcopodium,S. oculorum. The corneal ulcer developed after 5 months of treatment with corticosteroids in a Brazilian boy diagnosed with allergic conjunctivitis. Fungal hyphae and conidia were detected in corneal scrapings, and repeated cultures were positive for this fungus. The infection was resolved with natamycin and ketoconazole. Eleven antifungals were tested against this fungus, and all except flucytosine and fluconazole showed in vitro activity.