bluuebunny/crossref_metadata_2025_split huggingface dataset:
Discordant Phylogenies within the
rrn
Loci of Rhizobia
Authors: van Berkum Peter , Terefework Zewdu , Paulin Lars , Suomalainen Sini , Lindström Kristina , Eardly Bertrand D. 
Published: May 2003

ABSTRACT

It is evident from complete genome sequencing results that lateral gene transfer and recombination are essential components in the evolutionary process of bacterial genomes. Since this has important implications for bacterial systematics, the primary objective of this study was to compare estimated evolutionary relationships among a representative set of α-
Proteobacteria
by sequencing analysis of three loci within their
rrn
operons. Tree topologies generated with 16S rRNA gene sequences were significantly different from corresponding trees assembled with 23S rRNA gene and internally transcribed space region sequences. Besides the incongruence in tree topologies, evidence that distinct segments along the 16S rRNA gene sequences of bacteria currently classified within the genera
Bradyrhizobium
,
Mesorhizobium
and
Sinorhizobium
have a reticulate evolutionary history was also obtained. Our data have important implications for bacterial taxonomy, because currently most taxonomic decisions are based on comparative 16S rRNA gene sequence analysis. Since phylogenetic placement based on 16S rRNA gene sequence divergence perhaps is questionable, we suggest that the proposals of bacterial nomenclature or changes in their taxonomy that have been made may not necessarily be warranted. Accordingly, a more conservative approach should be taken in the future, in which taxonomic decisions are based on the analysis of a wider variety of loci and comparative analytical methods are used to estimate phylogenetic relationships among the genomes under consideration.
Mutational Analysis of the Carboxy-Terminal (YGX)4Repeat Domain of CpsD, an Autophosphorylating Tyrosine Kinase Required for Capsule Biosynthesis inStreptococcus pneumoniae
Authors: Morona Judy K. , Morona Renato , Miller David C. , Paton James C. 
Published: May 2003

ABSTRACTInStreptococcus pneumoniae, CpsB, CpsC, and CpsD are essential for encapsulation, and mutants containing deletions ofcpsB,cpsC, orcpsDexhibit rough colony morphologies. CpsD is an autophosphorylating protein-tyrosine kinase, CpsC is required for CpsD tyrosine phosphorylation, and CpsB is a phosphotyrosine-protein phosphatase. We have previously shown that autophosphorylation of CpsD at tyrosine attenuates its activity and consequently reduces the level of encapsulation and negatively regulates CPS production. In this study, we further investigated the role of the carboxy-terminal (YGX)4repeat domain of CpsD in encapsulation. A CpsD truncation mutant in which the entire (YGX)4repeat domain was removed was indistinguishable from a strain in which the entirecpsDgene had been deleted, indicating that the carboxy-terminal (YGX)4tail is required for CpsD activity in capsular polysaccharide production. Double mutants having a single tyrosine residue at position 2, 3, or 4 in the (YGX)4repeat domain and lacking CpsB exhibited a rough colony morphology, indicating that in the absence of an active protein-tyrosine phosphatase, phosphorylation of just one of the tyrosine residues in the (YGX)4repeat was sufficient to inactivate CpsD. When various mutants in which CpsD had either one or combinations of two or three tyrosine residues in the (YGX)4repeat domain were examined, only those with three tyrosine residues in the (YGX)4repeat domain were indistinguishable from the wild-type strain. The mutants with either one or two tyrosine residues exhibited mucoid colony morphologies. Further analysis of the mucoid strains indicated that the mucoid phenotype was not due to overproduction of capsular polysaccharide, as these strains actually produced less capsular polysaccharide than the wild-type strain. Thus, the tyrosine residues in the (YGX)4repeat domain are essential for normal functioning of CpsD.
S
-Adenosylmethionine Transport in
Rickettsia prowazekii
Authors: Tucker Aimee M. , Winkler Herbert H. , Driskell Lonnie O. , Wood David O. 
Published: May 2003

ABSTRACT

Rickettsia prowazekii
, the causative agent of epidemic typhus, is an obligate, intracellular, parasitic bacterium that grows within the cytoplasm of eucaryotic host cells. Rickettsiae exploit this intracellular environment by using transport systems for the compounds available in the host cell's cytoplasm. Analysis of the
R. prowazekii
Madrid E genome sequence revealed the presence of a mutation in the rickettsial
metK
gene, the gene encoding the enzyme responsible for the synthesis of
S
-adenosylmethionine (AdoMet). Since AdoMet is required for rickettsial processes, the apparent inability of this strain to synthesize AdoMet suggested the presence of a rickettsial AdoMet transporter. We have confirmed the presence of an AdoMet transporter in the rickettsiae which, to our knowledge, is the first bacterial AdoMet transporter identified. The influx of AdoMet into rickettsiae was a saturable process with a

K
T

of 2.3 μM. Transport was inhibited by
S
-adenosylethionine and
S
-adenosylhomocysteine but not by sinfungin or methionine. Transport was also inhibited by 2,4-dinitrophenol, suggesting an energy-linked transport mechanism, and by
N
-ethylmaleimide. AdoMet transporters with similar properties were also identified in the Breinl strain of
R. prowazekii
and in
Rickettsia typhi
. By screening
Escherichia coli
clone banks for AdoMet transport, the
R. prowazekii
gene coding for a transporter, RP076 (
sam
), was identified. AdoMet transport in
E. coli
containing the
R. prowazekii sam
gene exhibited kinetics similar to that seen in rickettsiae. The existence of a rickettsial transporter for AdoMet raises intriguing questions concerning the evolutionary relationship between the synthesis and transport of this essential metabolite.
Regulation of the Cellulosomal
celS
(
cel48A
) Gene of
Clostridium thermocellum
Is Growth Rate Dependent
Authors: Dror Tali W. , Morag Ely , Rolider Adi , Bayer Edward A. , Lamed Raphael , Shoham Yuval 
Published: May 2003

ABSTRACT

Clostridium thermocellum
produces an extracellular multienzyme complex, termed cellulosome, that allows efficient solubilization of crystalline cellulose. One of the major enzymes in this complex is the CelS (Cel48A) exoglucanase. The regulation of CelS at the protein and transcriptional levels was studied using batch and continuous cultures. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analyses indicated that the amount of CelS in the supernatant fluids of cellobiose-grown cultures is lower than that of cellulose-grown cultures. The transcriptional level of
celS
mRNA was determined quantitatively by RNase protection assays with batch and continuous cultures under carbon and nitrogen limitation. The amount of
celS
mRNA transcripts per cell was about 180 for cells grown under carbon limitation at growth rates of 0.04 to 0.21 h
−1
and 80 and 30 transcripts per cell for batch cultures at growth rates of 0.23 and 0.35 h
−1
, respectively. Under nitrogen limitation, the corresponding levels were 110, 40, and 30 transcripts/cell for growth rates of 0.07, 0.11, and 0.14 h
−1
, respectively. Two major transcriptional start sites were detected at positions −140 and −145 bp, upstream of the translational start site of the
celS
gene. The potential promoters exhibited homology to known sigma factors (i.e., σ
A
and σ
B
) of
Bacillus subtilis
. The relative activity of the two promoters remained constant under the conditions studied and was in agreement with the results of the RNase protection assay, in which the observed transcriptional activity was inversely proportional to the growth rate.
Synthesis of Catalytically Active Form III Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase in Archaea
Authors: Finn Michael W. , Tabita F. Robert 
Published: May 2003

ABSTRACT

Ribulose 1,5 bisphosphate carboxylase/oxygenase (RubisCO) catalyzes the biological reduction and assimilation of carbon dioxide gas to organic carbon; it is the key enzyme responsible for the bulk of organic matter found on earth. Until recently it was believed that there are only two forms of RubisCO, form I and form II. However, the recent completion of several genome-sequencing projects uncovered open reading frames resembling RubisCO in the third domain of life, the archaea. Previous work and homology comparisons suggest that these enzymes represent a third form of RubisCO, form III. While earlier work indicated that two structurally distinct recombinant archaeal RubisCO proteins catalyzed bona fide RubisCO reactions, it was not established that the
rbcL
genes of anaerobic archaea can be transcribed and translated to an active enzyme in the native organisms. In this report, it is shown not only that
Methanococcus jannaschii
,
Archaeoglobus fulgidus
,
Methanosarcina acetivorans
, and
Methanosarcina barkeri
possess open reading frames with the residues required for catalysis but also that the RubisCO protein from these archaea accumulates in an active form under normal growth conditions. In addition, the form III RubisCO gene (
rbcL
) from
M. acetivorans
was shown to complement RubisCO deletion strains of
Rhodobacter capsulatus
and
Rhodobacter sphaeroides
under both photoheterotrophic and photoautotrophic growth conditions. These studies thus indicate for the first time that archaeal form III RubisCO functions in a physiologically significant fashion to fix CO
2
. Furthermore, recombinant
M. jannaschii
,
M. acetivorans
, and
A. fulgidus
RubisCO possess unique properties with respect to quaternary structure, temperature optima, and activity in the presence of molecular oxygen compared to the previously described
Thermococcus kodakaraensis
and halophile proteins.
Repair System for Noncanonical Purines in
Escherichia coli
Authors: Burgis Nicholas E. , Brucker Jason J. , Cunningham Richard P. 
Published: May 2003

ABSTRACT

Exposure of
Escherichia coli
strains deficient in molybdopterin biosynthesis (
moa
) to the purine base
N
-6-hydroxylaminopurine (HAP) is mutagenic and toxic. We show that
moa
mutants exposed to HAP also exhibit elevated mutagenesis, a hyperrecombination phenotype, and increased SOS induction. The
E. coli rdgB
gene encodes a protein homologous to a deoxyribonucleotide triphosphate pyrophosphatase from
Methanococcus jannaschii
that shows a preference for purine base analogs.
moa rdgB
mutants are extremely sensitive to killing by HAP and exhibit increased mutagenesis, recombination, and SOS induction upon HAP exposure. Disruption of the endonuclease V gene,
nfi
, rescues the HAP sensitivity displayed by
moa
and
moa rdgB
mutants and reduces the level of recombination and SOS induction, but it increases the level of mutagenesis. Our results suggest that endonuclease V incision of DNA containing HAP leads to increased recombination and SOS induction and even cell death. Double-strand break repair mutants display an increase in HAP sensitivity, which can be reversed by an
nfi
mutation. This suggests that cell killing may result from an increase in double-strand breaks generated when replication forks encounter endonuclease V-nicked DNA. We propose a pathway for the removal of HAP from purine pools, from deoxynucleotide triphosphate pools, and from DNA, and we suggest a general model for excluding purine base analogs from DNA. The system for HAP removal consists of a molybdoenzyme, thought to detoxify HAP, a deoxyribonucleotide triphosphate pyrophosphatase that removes noncanonical deoxyribonucleotide triphosphates from replication precursor pools, and an endonuclease that initiates the removal of HAP from DNA.
CodY Is a Nutritional Repressor of Flagellar Gene Expression inBacillus subtilis
Authors: Bergara F. , Ibarra C. , Iwamasa J. , Patarroyo J. C. , Aguilera R. , Márquez-Magaña L. M. 
Published: May 2003

ABSTRACTExpression of the σD-dependent flagellin gene,hag, is repressed by the CodY protein in nutrient-rich environments. Analysis of acodYmutant bearing ahag-lacZreporter suggests that the availability of amino acids in the environment is the specific signal that triggers this repression. Further,hag-lacZexpression appears to be sensitive to intracellular GTP levels, as demonstrated by increased expression upon addition of decoyinine. This result is consistent with the postulate that the availability of amino acids in the environment effects intracellular GTP levels through the stringent response. However, the levels ofhag-lacZmeasured upon the addition of subsets of amino acids suggest an additional mechanism(s). CodY is a DNA binding protein that could repress flagellin expression directly by binding to thehagpromoter region, or indirectly by binding to thefla/chepromoter region that governs expression of the σDtranscriptional activator required forhaggene expression. Using an electrophoretic mobility shift assay, we have demonstrated that purified CodY protein binds specifically to both thehagandfla/chepromoter fragments. Additionally, CodY acts as a nutritional repressor of transcription from thefla/chepromoter region that contains two functional promoters. CodY binds to both the σD- and σA-dependent promoters in this region, as demonstrated by DNase I footprint analyses. Footprint analyses of thehaggene demonstrated that CodY binds downstream of its σD-dependent promoter. Taken together, these results identify new members of the CodY regulon that encode motility functions inBacillus subtilisand are controlled by the σDalternate sigma factor.
Characterization of NorR Protein, a Multifunctional Regulator of
norA
Expression in
Staphylococcus aureus
Authors: Truong-Bolduc Que Chi , Zhang Xiamei , Hooper David C. 
Published: May 2003

ABSTRACT

We characterized a
Staphylococcus aureus norA
gene expression regulator, NorR, initially identified from its binding to the
norA
promoter. The
norR
gene was 444 bp in length, located ∼7 kb upstream from the
norA
gene, and encoded a predicted 17.6-kDa protein. Overexpression of
norR
in wild-type
S. aureus
strain ISP794 led to a fourfold decrease in sensitivity to quinolones and ethidium bromide and an increase in the level of
norA
transcripts, suggesting that NorR acts as a positive regulator of
norA
expression. Overexpression of
norR
in
sarA
and
agr
mutants did not alter quinolone sensitivity or levels of
norA
transcription, indicating that the presence of these two global regulatory systems is necessary for NorR to affect the expression of
norA
. Insertion and disruption of
norR
in ISP794 increased resistance to quinolones by 4- to 16-fold but had no effect on
norA
transcription, suggesting that NorR acts as a repressor for another unidentified efflux pump or pumps. These mutants also exhibited an exaggerated clumping phenotype in liquid media, which was complemented fully by a plasmid-encoded
norR
gene. Collectively, these results indicate that NorR is a multifunctional regulator, affecting cell surface properties as well as the expression of NorA and likely other multidrug resistance efflux pumps.
Transcription Regulation Coupling of the DivergentargGandmetYPromoters inEscherichia coliK-12
Authors: Krin Evelyne , Laurent-Winter Christine , Bertin Philippe N. , Danchin Antoine , Kolb Annie 
Published: May 2003

ABSTRACTThe cAMP-catabolite activator protein (CAP) complex is a pleiotropic regulator that regulates a vast number ofEscherichia coligenes, including those involved in carbon metabolism. We identified two new targets of this complex:argG, which encodes the arginosuccinate synthase involved in the arginine biosynthetic pathway, andmetY, which encodes one of the two methionine tRNA initiators, tRNAf2Met. The cAMP-CAP complex activatesargGtranscription and inhibitsmetYtranscription from the same DNA position. We also show that ArgR, the specific repressor of the arginine biosynthetic pathway, together with its arginine cofactor, acts on the regulation ofmetYmediated by CAP. The regulation of the two divergent promoters is thus simultaneously controlled not only by the cAMP-CAP complex, a global regulator, but also by a specific regulator of arginine metabolism, suggesting a previously unsuspected link between carbon metabolism and translation initiation.
Early Colonization Events in the Mutualistic Association between
Steinernema carpocapsae
Nematodes and
Xenorhabdus nematophila
Bacteria
Authors: Martens Eric C. , Heungens Kurt , Goodrich-Blair Heidi 
Published: May 2003

ABSTRACT

The bacterium
Xenorhabdus nematophila
is a mutualist of the entomopathogenic nematode
Steinernema carpocapsae
. During its life cycle, the bacterium exists both separately from the nematode and as an intestinal resident of a nonfeeding nematode form, the infective juvenile (IJ). The progression of
X. nematophila
from an ex vivo existence to a specific and persistent colonization of IJs is a model to understand the mechanisms mediating the initiation and maintenance of benign host-microbe interactions. To help characterize this process, we constructed an
X. nematophila
strain that constitutively expresses green fluorescent protein, which allowed its presence to be monitored within IJs. Using this strain, we showed that few bacterial cells initiate colonization of an individual IJ and that these grow inside the lumen of the IJ intestine in a reproducible polyphasic pattern during colonization. In accordance with these two observations, we demonstrated that the final population of bacteria in a nematode is of predominantly monoclonal origin, suggesting that only one or two bacterial clones initiate or persist during colonization of an individual nematode. These data suggest that
X. nematophila
initiates IJ colonization by competing for limited colonization sites or resources within the nematode intestine. This report represents the first description of the biological interactions occurring between
X. nematophila
and
S. carpocapsae
during the early stages of the colonization process, provides insights into the physiology of
X. nematophila
in its host niche, and will facilitate interpretation of future data regarding the molecular events mediating this process.
Characterization of the
Xanthomonas axonopodis
pv. glycines Hrp Pathogenicity Island
Authors: Kim Jung-Gun , Park Byoung Keun , Yoo Chang-Hyuk , Jeon Eunkyung , Oh Jonghee , Hwang Ingyu 
Published: May 2003

ABSTRACT

We sequenced an approximately 29-kb region from
Xanthomonas axonopodis
pv. glycines that contained the Hrp type III secretion system, and we characterized the genes in this region by Tn
3-gus
mutagenesis and gene expression analyses. From the region,
hrp
(hypersensitive response and pathogenicity) and
hrc
(
hrp
and conserved) genes, which encode type III secretion systems, and
hpa
(
hrp
-associated) genes were identified. The characteristics of the region, such as the presence of many virulence genes, low G+C content, and bordering tRNA genes, satisfied the criteria for a pathogenicity island (PAI) in a bacterium. The PAI was composed of nine
hrp
, nine
hrc
, and eight
hpa
genes with seven plant-inducible promoter boxes. The
hrp
and
hrc
mutants failed to elicit hypersensitive responses in pepper plants but induced hypersensitive responses in all tomato plants tested. The Hrp PAI of
X. axonopodis
pv. glycines resembled the Hrp PAIs of other
Xanthomonas
species, and the Hrp PAI core region was highly conserved. However, in contrast to the PAI of
Pseudomonas syringae
, the regions upstream and downstream from the Hrp PAI core region showed variability in the xanthomonads. In addition, we demonstrate that HpaG, which is located in the Hrp PAI region of
X. axonopodis
pv. glycines, is a response elicitor. Purified HpaG elicited hypersensitive responses at a concentration of 1.0 μM in pepper, tobacco, and
Arabidopsis thaliana
ecotype Cvi-0 by acting as a type III secreted effector protein. However, HpaG failed to elicit hypersensitive responses in tomato, Chinese cabbage, and
A. thaliana
ecotypes Col-0 and Ler. This is the first report to show that the harpin-like effector protein of
Xanthomonas
species exhibits elicitor activity.
Energy-Generating Enzymes ofBurkholderia cepaciaand Their Interactions with Macrophages
Authors: Punj Vasu , Sharma Rachna , Zaborina Olga , Chakrabarty A. M. 
Published: May 2003

ABSTRACTWe previously demonstrated that several clinical and environmental isolates ofBurkholderia cepaciasecreted ATP-utilizing enzymes to the medium; the secretion of these enzymes by cystic fibrosis lung isolate strain 38 was shown to be greatly enhanced in the presence of α2-macroglobulin. Fractionation of the growth medium of cystic fibrosis isolate strain 71 belonging to genomovar I demonstrated the presence of two additional proteins, homologues ofPseudomonas aeruginosaazurin and cytochromec551, which are normally involved in electron transfer during denitrification. A Q-Sepharose column flowthrough fraction of the growth medium ofB. cepaciastrain 71 enriched with the azurin and cytochromec551homologues triggered apoptosis in macrophages and mast cells, leading to their death. Incubation of the Q-Sepharose column flowthrough fraction with antiazurin and anti-cytochromec551antibodies greatly reduced cell death. We cloned and hyperexpressed a gene fromB. cepaciastrain 71 that encodes the homologue ofP. aeruginosaazurin. Such azurin homologues were detected in the growth medium of several strains belonging to genomovars I, III, and VI but not in the growth medium of strains belonging to other genomovars. The growth medium of the strains that elaborated the azurin homologue had high cytotoxicity towards macrophages. Purified azurin homologue was shown to induce apoptosis in macrophages in a caspase-dependent manner and was localized in both the cytosol and nucleus when incubated with or microinjected into macrophages. This is an interesting example of the interaction of a bacterial protein normally involved in cellular energetics with macrophages to effect their cell death.
Genes of the GadX-GadW Regulon inEscherichia coli
Authors: Tucker Don L. , Tucker Nancy , Ma Zhuo , Foster John W. , Miranda Regina L. , Cohen Paul S. , Conway Tyrrell 
Published: May 2003

ABSTRACTAcid in the stomach is thought to be a barrier to bacterial colonization of the intestine.Escherichia coli, however, has three systems for acid resistance, which overcome this barrier. The most effective of these systems is dependent on transport and decarboxylation of glutamate. GadX regulates two genes that encode isoforms of glutamate decarboxylase critical to this system, but additional genes associated with the glutamate-dependent acid resistance system remained to be identified. ThegadXgene and a second downstreamaraC-like transcription factor gene,gadW, were mutated separately and in combination, and the gene expression profiles of the mutants were compared to those of the wild-type strain grown in neutral and acidified media under conditions favoring induction of glutamate-dependent acid resistance. Cluster and principal-component analyses identified 15 GadX-regulated, acid-inducible genes. Reverse transcriptase mapping demonstrated that these genes are organized in 10 operons. Analysis of the strain lacking GadX but possessing GadW confirmed that GadX is a transcriptional activator under acidic growth conditions. Analysis of the strain lacking GadW but possessing GadX indicated that GadW exerts negative control over three GadX target genes. The strain lacking both GadX and GadW was defective in acid induction of most but not all GadX target genes, consistent with the roles of GadW as an inhibitor of GadX-dependent activation of some genes and an activator of other genes. Resistance to acid was decreased under certain conditions in agadXmutant and even more so by combined mutation ofgadXandgadW. However, there was no defect in colonization of the streptomycin-treated mouse model by thegadXmutant in competition with the wild type, and thegadX gadWmutant was a better colonizer than the wild type. Thus,E. colicolonization of the mouse does not appear to require glutamate-dependent acid resistance.