bluuebunny/crossref_metadata_2025 huggingface dataset:
Phosphoprotein with Phosphoglycerate Mutase Activity from the Archaeon
Sulfolobus solfataricus
Authors: Potters M. Ben , Solow Barbara T. , Bischoff Kenneth M. , Graham David E. , Lower Brian H. , Helm Richard , Kennelly Peter J.
Published: March 2003

When soluble extracts of the extreme acidothermophilic archaeon
Sulfolobus solfataricus
were incubated with [γ-
32
P]ATP, several proteins were radiolabeled. One of the more prominent of these, which migrated with a mass of ∼46 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), was purified by column chromatography and SDS-PAGE and subjected to amino acid sequence analysis via both the Edman technique and mass spectroscopy. The best match to the partial sequence obtained was the potential polypeptide product of open reading frame
sso0417
, whose DNA-derived amino acid sequence displayed many features reminiscent of the 2,3-diphosphoglycerate-independent phosphoglycerate (PGA) mutases [iPGMs]. Open reading frame
sso0417
was therefore cloned, and its protein product was expressed in
Escherichia coli
. Assays of its catalytic capabilities revealed that the protein was a moderately effective PGA mutase that also exhibited low levels of phosphohydrolase activity. PGA mutase activity was dependent upon the presence of divalent metal ions such as Co
2+
or Mn
2+
. The recombinant protein underwent autophosphorylation when incubated with either [γ-
32
P]ATP or [γ-
32
P]GTP. The site of phosphorylation was identified as Ser
59
, which corresponds to the catalytically essential serine residue in bacterial and eucaryal iPGMs. The phosphoenzyme intermediate behaved in a chemically and kinetically competent manner. Incubation of the
32
P-labeled phosphoenzyme with 3-PGA resulted in the disappearance of radioactive phosphate and the concomitant appearance of
32
P-labeled PGA at rates comparable to those measured in steady-state assays of PGA mutase activity.
Identification of ZipA, a Signal Recognition Particle-Dependent Protein from
Neisseria gonorrhoeae
Authors: Du Ying , Arvidson Cindy Grove
Published: March 2003

A genetic screen designed to identify proteins that utilize the signal recognition particle (SRP) for targeting in
Escherichia coli
was used to screen a
Neisseria gonorrhoeae
plasmid library. Six plasmids were identified in this screen, and each is predicted to encode one or more putative cytoplasmic membrane (CM) proteins. One of these, pSLO7, has three open reading frames (ORFs), two of which have no similarity to known proteins in GenBank other than sequences from the closely related
N. meningitidis
. Further analyses showed that one of these, SLO7ORF3, encodes a protein that is dependent on the SRP for localization. This gene also appears to be essential in
N. gonorrhoeae
since it was not possible to generate null mutations in the gene. Although appearing unique to
Neisseria
at the DNA sequence level, SLO7ORF3 was found to share some features with the cell division gene
zipA
of
E. coli
. These features included similar chromosomal locations (with respect to linked genes) as well as similarities in the predicted protein domain structures. Here, we show that SLO7ORF3 can complement an
E. coli
conditional
zipA
mutant and therefore encodes a functional ZipA homolog in
N. gonorrhoeae
. This observation is significant in that it is the first ZipA homolog identified in a non-rod-shaped organism. Also interesting is that this is the fourth cell division protein (the others are FtsE, FtsX, and FtsQ) shown to utilize the SRP for localization, which may in part explain why the genes encoding the three SRP components are essential in bacteria.
Genomic Diversification among Archival Strains of
Salmonella enterica
Serovar Typhimurium LT7
Authors: Liu Gui-Rong , Edwards Kelly , Eisenstark Abraham , Fu Ying-Mei , Liu Wei-Qiao , Sanderson Kenneth E. , Johnston Randal N. , Liu Shu-Lin
Published: March 2003

To document genomic changes during long periods of storage, we analyzed
Salmonella enterica
serovar Typhimurium LT7, a mutator strain that was previously reported to have higher rates of mutations compared to other serovar Typhimurium strains such as LT2. Upon plating directly from sealed agar stabs that had been stocked at room temperature for up to four decades, many auxotrophic mutants derived from LT7 gave rise to colonies of different sizes. Restreaking from single colonies consistently yielded colonies of diverse sizes even when we repeated single-colony isolation nine times. Colonies from the first plating had diverse genomic changes among and even within individual vials, including translocations, inversions, duplications, and point mutations, which were detected by rare-cutting endonuclease analysis with pulsed-field gel electrophoresis. Interestingly, even though the colony size kept diversifying, all descendents of the same single colonies from the first plating had the same sets of detected genomic changes. We did not detect any colony size or genome structure diversification in serovar Typhimurium LT7 stocked at −70°C or in serovar Typhimurium LT2 stocked either at −70°C or at room temperature. These results suggest that, although colony size diversification occurred during rapid growth, all detected genomic changes took place during the storage at room temperature and were carried over to their descendents without further changes during rapid growth in rich medium. We constructed a genomic cleavage map on the LT7 strain that had been stocked at −70°C and located all of the detected genomic changes on the map. We speculated on the significance of mutators for survival and evolution under environmentally stressed conditions.
A Single Gene on the Staphylococcal Multiresistance Plasmid pSK1 Encodes a Novel Partitioning System
Authors: Simpson Alice E. , Skurray Ronald A. , Firth Neville
Published: March 2003

Theorf245gene is located immediately upstream of, and divergently transcribed from, the replication initiation gene,rep, of theStaphylococcus aureusmultiresistance plasmid pSK1, and related genes have been found in association with a range of evolutionarily distinct replication genes on plasmids from various gram-positive genera.orf245has been shown previously to extend the segregational stability of a pSK1 minireplicon. Here we describe an investigation into the basis oforf245-mediated stabilization.orf245was not found to influence transcription of pSK1rep, indicating that it is not directly involved in plasmid replication. This was confirmed by demonstrating thatorf245is able to enhance the segregational stability of heterologous theta- and rolling-circle-replicating replicons, suggesting that it encodes a plasmid maintenance function. Evidence inconsistent with postsegregational killing and multimer resolution mechanisms was obtained; however, the intergenic region upstream oforf245was found to mediateorf245-dependent incompatibility, as would be expected if it encodes acis-acting centromere-like site. Taken together, these findings implicate active partitioning as the probable basis of the activity oforf245, which is therefore redesignatedpar. Since it is unrelated to any gene known to play a role in plasmid segregation, it seems likely that pSK1parpotentially represents the prototype of a novel class of active partitioning systems that are distinguished by their capacity to enhance plasmid segregational stability via a single protein-encoding gene.
Roles of YqjH and YqjW, Homologs of the
Escherichia
coli
UmuC/DinB or Y Superfamily of DNA Polymerases, in Stationary-Phase Mutagenesis and UV-Induced Mutagenesis of
Bacillus
subtilis
Authors: Sung Huang-Mo , Yeamans Gabriel , Ross Christian A. , Yasbin Ronald E.
Published: March 2003

YqjH and YqjW are
Bacillus
subtilis
homologs of the UmuC/DinB or Y superfamily of DNA polymerases that are involved in SOS-induced mutagenesis in
Escherichia
coli
. While the functions of YqjH and YqjW in
B
.
subtilis
are still unclear, the comparisons of protein structures demonstrate that YqjH has 36% identity to
E
.
coli
DNA polymerase IV (DinB protein), and YqjW has 26% identity to
E
.
coli
DNA polymerase V (UmuC protein). In this report, we demonstrate that both YqjH and the products of the
yqjW
operon are involved in UV-induced mutagenesis in this bacterium. Furthermore, resistance to UV-induced damage is significantly reduced in cells lacking a functional YqjH protein. Analysis of stationary-phase mutagenesis indicates that absences of YqjH, but not that of YqjW, decreases the ability of
B
.
subtilis
to generate revertants at the
hisC952
allele via this system. These data suggest a role for YqjH in the generation of at least some types of stationary-phase-induced mutagenesis.
Metabolic Enzymes from Psychrophilic Bacteria: Challenge of Adaptation to Low Temperatures in Ornithine Carbamoyltransferase from
Moritella
abyssi
Authors: Xu Ying , Feller Georges , Gerday Charles , Glansdorff Nicolas
Published: March 2003

The enzyme ornithine carbamoyltransferase (OTCase) of
Moritella
abyssi
(OTCase
Mab
), a new, strictly psychrophilic and piezophilic bacterial species, was purified. OTCase
Mab
displays maximal activity at rather low temperatures (23 to 25°C) compared to other cold-active enzymes and is much less thermoresistant than its homologues from
Escherichia
coli
or thermophilic procaryotes. In vitro the enzyme is in equilibrium between a trimeric state and a dodecameric, more stable state. The melting point and denaturation enthalpy changes for the two forms are considerably lower than the corresponding values for the dodecameric
Pyrococcus
furiosus
OTCase and for a thermolabile trimeric mutant thereof. OTCase
Mab
displays higher

K
m

values for ornithine and carbamoyl phosphate than mesophilic and thermophilic OTCases and is only weakly inhibited by the bisubstrate analogue δ-
N
-phosphonoacetyl-
l
-ornithine (PALO). OTCase
Mab
differs from other, nonpsychrophilic OTCases by substitutions in the most conserved motifs, which probably contribute to the comparatively high

K
m

values and the lower sensitivity to PALO. The

K
m

for ornithine, however, is substantially lower at low temperatures. A survey of the catalytic efficiencies (
k
cat
/

K
m

) of OTCases adapted to different temperatures showed that OTCase
Mab
activity remains suboptimal at low temperature despite the 4.5-fold decrease in the

K
m

value for ornithine observed when the temperature is brought from 20 to 5°C. OTCase
Mab
adaptation to cold indicates a trade-off between affinity and catalytic velocity, suggesting that optimization of key metabolic enzymes at low temperatures may be constrained by natural limits.
Enterococcus faecalisPlasmid pAD1-Encoded Fst Toxin Affects Membrane Permeability and Alters Cellular Responses to Lantibiotics
Authors: Weaver Keith E. , Weaver Dariel M. , Wells Carol L. , Waters Christopher M. , Gardner Marshall E. , Ehli Erik A.
Published: March 2003

Fst is a peptide toxin encoded by thepartoxin-antitoxin stability determinant ofEnterococcus faecalisplasmid pAD1. Intracellular overproduction of Fst resulted in simultaneous inhibition of all cellular macromolecular synthesis concomitant with cell growth inhibition and compromised the integrity of the cell membrane. Cells did not lyse or noticeably leak intracellular contents but had specific defects in chromosome partitioning and cell division. Extracellular addition of synthetic Fst had no effect on cell growth. Spontaneous Fst-resistant mutants had a phenotype consistent with changes in membrane composition. Interestingly, overproduction of Fst sensitized cells to the lantibiotic nisin, and Fst-resistant mutants were cross-resistant to nisin and the pAD1-encoded cytolysin.
Sequences near the Active Site in Chimeric Penicillin Binding Proteins 5 and 6 Affect Uniform Morphology of
Escherichia coli
Authors: Ghosh Anindya S. , Young Kevin D.
Published: March 2003

Penicillin binding protein (PBP) 5, a
dd
-carboxypeptidase that removes the terminal
d
-alanine from peptide side chains of peptidoglycan, plays an important role in creating and maintaining the uniform cell shape of
Escherichia coli
. PBP 6, a highly similar homologue, cannot substitute for PBP 5 in this respect. Previously, we localized the shape-maintaining characteristics of PBP 5 to the globular domain that contains the active site (domain I), where PBPs 5 and 6 share substantial identity. To identify the specific segment of domain I responsible for shape control, we created a set of hybrids and determined which ones complemented the aberrant morphology of a misshapen PBP mutant,
E. coli
CS703-1. Fusion proteins were constructed in which 47, 199 and 228 amino-terminal amino acids of one PBP were fused to the corresponding carboxy-terminal amino acids of the other. The morphological phenotype was reversed only by hybrid proteins containing PBP 5 residues 200 to 228, which are located next to the KTG motif of the active site. Because residues 220 to 228 were identical in these proteins, the morphological effect was determined by alterations in amino acids 200 to 219. To confirm the importance of this segment, we constructed mosaic proteins in which these 20 amino acids were grafted from PBP 5 into PBP 6 and vice versa. The PBP 6/5/6 mosaic complemented the aberrant morphology of CS703-1, whereas PBP 5/6/5 did not. Site-directed mutagenesis demonstrated that the Asp
218
and Lys
219
residues were important for shape maintenance by these mosaic PBPs, but the same mutations in wild-type PBP 5 did not eliminate its shape-promoting activity. Homologous enzymes from five other bacteria also complemented the phenotype of CS703-1. The overall conclusion is that creation of a bacterial cell of regular diameter and uniform contour apparently depends primarily on a slight alteration of the enzymatic activity or substrate accessibility at the active site of
E. coli
PBP 5.
The IntP C-Terminal Segment Is Not Required for Excision of Bacteriophage Mx8 from theMyxococcus xanthusChromosome
Authors: Tojo Nobuki , Komano Teruya
Published: March 2003

During lysogenization of myxophage Mx8, phage DNA can be integrated into theattBsite of theMyxococcus xanthuschromosome through site-specific recombination. We previously demonstrated that the Mx8attPsite is located within the coding sequence of the Mx8intPgene. Hence, the integration of Mx8 into theM. xanthuschromosome results in the conversion of the 112-amino-acid C-terminal segment of the IntP protein into a 13-amino-acid C-terminal segment of a new protein, IntR. To examine whether IntR is active for Mx8 excision, we have constructed a series of plasmids carrying various lengths of theintP-attPorintR-attRregions as well as thelacZgene. The integrated Mx8 was excised at a high frequency, indicating that IntR is active for the excision. For Mx8 excision, a gene designatedxiswas shown to be required in addition tointR.
Architecture of a Fur Binding Site: a Comparative Analysis
Authors: Lavrrar Jennifer L. , McIntosh Mark A.
Published: March 2003

Fur is an iron-binding transcriptional repressor that recognizes a 19-bp consensus site of the sequence 5′-GATAATGATAATCATTATC-3′. This site can be defined as three adjacent hexamers of the sequence 5′-GATAAT-3′, with the third being slightly imperfect (an F-F-F configuration), or as two hexamers in the forward orientation separated by one base pair from a third hexamer in the reverse orientation (an F-F-x-R configuration). Although Fur can bind synthetic DNA sequences containing the F-F-F arrangement, most natural binding sites are variations of the F-F-x-R arrangement. The studies presented here compared the ability of Fur to recognize synthetic DNA sequences containing two to four adjacent hexamers with binding to sequences containing variations of the F-F-x-R arrangement (including natural operator sequences from the
entS
and
fepB
promoter regions of
Escherichia coli
). Gel retardation assays showed that the F-F-x-R architecture was necessary for high-affinity Fur-DNA interactions and that contiguous hexamers were not recognized as effectively. In addition, the stoichiometry of Fur at each binding site was determined, showing that Fur interacted with its minimal 19-bp binding site as two overlapping dimers. These data confirm the proposed overlapping-dimer binding model, where the unit of interaction with a single Fur dimer is two inverted hexamers separated by a C:G base pair, with two overlapping units comprising the 19-bp consensus binding site required for the high-affinity interaction with two Fur dimers.
Phase Variation of Ag43 Is Independent of the Oxidation State of OxyR
Authors: Wallecha Anu , Correnti Jason , Munster Vincent , van der Woude Marjan
Published: March 2003

OxyR is a DNA binding protein that differentially regulates a cell's response to hydrogen peroxide-mediated oxidative stress. We previously reported that the reduced form of OxyR is sufficient for repression of transcription of
agn43
from unmethylated template DNA, which is essential for deoxyadenosine methylase (Dam)- and OxyR-dependent phase variation of
agn43
. Here we provide evidence that the oxidized form of OxyR [OxyR(ox)] also represses
agn43
transcription. In vivo, we found that exogenous addition of hydrogen peroxide, sufficient to oxidize OxyR, did not affect the expression of
agn43.
OxyR(ox) repressed in vitro transcription but only from an unmethylated
agn43
template. The −10 sequence of the promoter and three Dam target sequences were protected in an in vitro DNase I footprint assay by OxyR(ox). Furthermore, OxyR(ox) bound to the
agn43
regulatory region DNA with an affinity similar to that for the regulatory regions of
katG
and
oxyS
, which are activated by OxyR(ox), indicating that binding at
agn43
can occur at biologically relevant concentrations. OxyR-dependent regulation of Ag43 expression is therefore unusual in firstly that OxyR binding at
agn43
is dependent on the methylation state of Dam target sequences in its binding site and secondly that OxyR-dependent repression appears to be independent of hydrogen-peroxide mediated oxidative stress and the oxidation state of OxyR.
Interaction of the Initiator Protein of an IncB Plasmid with Its Origin of DNA Replication
Authors: Betteridge T. , Yang J. , Pittard A. J. , Praszkier J.
Published: March 2003

The replication initiator protein RepA of the IncB plasmid pMU720 was purified and used in DNase I protection assays in vitro. RepA protected a 68-bp region of the origin of replication of pMU720. This region, which lies immediately downstream of the DnaA box, contains four copies of the sequence motif 5′AANCNGCAA3′. Mutational analyses identified this sequence as the binding site specifically recognized by RepA (the RepA box). Binding of RepA to the RepA boxes was ordered and sequential, with the box closest to the DnaA binding site (box 1) occupied first and the most distant boxes (boxes 3 and 4) occupied last. However, only boxes 1, 2, and 4 were essential for origin activity, with box 3 playing a lesser role. Changing the spacing between box 1 and the other three boxes affected binding of RepA in vitro and origin activity in vivo, indicating that the RepA molecules bound to
ori
B
interact with one another.
Involvement of the Multidomain Regulatory Protein XynR in Positive Control of Xylanase Gene Expression in the Ruminal Anaerobe
Prevotella bryantii
B
1
4
Authors: Miyazaki Kohji , Miyamoto Hiroyuki , Mercer Derry K. , Hirase Tatsuaki , Martin Jennifer C. , Kojima Yoichi , Flint Harry J.
Published: March 2003

The xylanase gene cluster from the rumen anaerobe
Prevotella bryantii
B
1
4 was found to include a gene (
xynR
) that encodes a multidomain regulatory protein and is downstream from the xylanase and β-xylosidase genes
xynA
and
xynB.
Additional genes identified upstream of
xynA
and
xynB
include
xynD
, which encodes an integral membrane protein that has homology with Na:solute symporters;
xynE
, which is related to the genes encoding acylhydrolases and arylesterases; and
xynF
, which has homology with the genes encoding α-glucuronidases. XynR includes, in a single 833-amino-acid polypeptide, a putative input domain unrelated to other database sequences, a likely transmembrane domain, histidine kinase motifs, response regulator sequences, and a C-terminal AraC-type helix-turn-helix DNA binding domain. Two transcripts (3.7 and 5.8 kb) were detected with a
xynA
probe, and the start site of the 3.7-kb transcript encoding
xynABD
was mapped to a position upstream of
xynD
. The DNA binding domain of XynR was purified after amplification and overexpression in
Escherichia coli
and was found to bind to a 141-bp DNA fragment from the region immediately upstream of
xynD.
In vitro transcription assays demonstrated that XynR stimulates transcription of the 3.7-kb transcript. We concluded that XynR acts as a positive regulator that activates expression of
xynABD
in
P. bryantii
B
1
4. This is the first regulatory protein that demonstrates significant homology with the two-component regulatory protein superfamily and has been shown to be involved in the regulation of polysaccharidase gene expression.
Negative Control of Quorum Sensing by RpoN (σ54) inPseudomonas aeruginosaPAO1
Authors: Heurlier Karin , Dénervaud Valerie , Pessi Gabriella , Reimmann Cornelia , Haas Dieter
Published: March 2003

InPseudomonas aeruginosaPAO1, the expression of several virulence factors such as elastase, rhamnolipids, and hydrogen cyanide depends on quorum-sensing regulation, which involves thelasRIandrhlRIsystems controlled byN-(3-oxododecanoyl)-l-homoserine lactone andN-butyryl-l-homoserine lactone, respectively, as signal molecules. InrpoNmutants lacking the transcription factor σ54, the expression of thelasRandlasIgenes was elevated at low cell densities, whereas expression of therhlRandrhlIgenes was markedly enhanced throughout growth by comparison with the wild type and the complemented mutant strains. As a consequence, therpoNmutants had elevated levels of both signal molecules and overexpressed the biosynthetic genes for elastase, rhamnolipids, and hydrogen cyanide. The quorum-sensing regulatory protein QscR was not involved in the negative control exerted by RpoN. By contrast, in anrpoNmutant, the expression of thegacAglobal regulatory gene was significantly increased during the entire growth cycle, whereas another global regulatory gene,vfr, was downregulated at high cell densities. In conclusion, it appears that GacA levels play an important role, probably indirectly, in the RpoN-dependent modulation of the quorum-sensing machinery ofP. aeruginosa.
Differential Expression of the
Escherichia
coli
Autoaggregation Factor Antigen 43
Authors: Schembri Mark A. , Hjerrild Louise , Gjermansen Morten , Klemm Per
Published: March 2003

Antigen 43 (Ag43) is a self-recognizing surface adhesin found in most
Escherichia
coli
strains. Due to its excellent cell-to-cell aggregation characteristics, Ag43 expression confers clumping and fluffing of cells and promotes biofilm formation. Ag43 expression is repressed by the cellular redox sensor OxyR. Here we used mutant versions of OxyR that are locked in either the reduced or the oxidized form as well as the addition of a simple redox-changing chemical to show that the redox state of OxyR influences Ag43 expression. Furthermore, the redox state of OxyR influences the biofilm-forming potential of
E
.
coli
. Finally, we demonstrated that Ag43-mediated cell aggregation confers significant protection against hydrogen peroxide killing.
The Ascorbate Transporter of
Escherichia coli
Authors: Zhang Zhongge , Aboulwafa Mohammad , Smith Meghan H. , Saier, Milton H.
Published: March 2003

The
sgaTBA
genes of
Escherichia coli
encode a putative 12-transmembrane α-helical segment (12 TMS) transporter, an enzyme IIB-like protein and an enzyme IIA-like protein of the phosphotransferase system (PTS), respectively. We show that all three proteins as well as the energy-coupling PTS proteins, enzyme I and HPr, are required for the anaerobic utilization and uptake of
l
-ascorbate in vivo and its phosphoenolpyruvate-dependent phosphorylation in vitro. The transporter exhibits an apparent

K
m

for
l
-ascorbate of 9 μM and is highly specific. The
sgaTBA
genes are regulated at the transcriptional level by the
yjfQ
gene product, as well as by Crp and Fnr. The
yjfR
gene product is essential for
l
-ascorbate utilization and probably encodes a cytoplasmic
l
-ascorbate 6-phosphate lactonase. We conclude that SgaT represents a novel prototypical enzyme IIC that functions with SgaA and SgaB to allow phosphoryl transfer from HPr(his-P) to
l
-ascorbate via the phosphoryl transfer pathway: PEP → enzyme I-P → HPr-P → IIA-

\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\stackrel{\mathrm{SgaA}}{\mathrm{P}}\) \end{document}

→ IIB-

\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\stackrel{\mathrm{SgaB}}{\mathrm{P}}\) \end{document}

\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\stackrel{\textstyle{\mathrm{IIC^{SgaT}}}}{{\rightarrow}}\) \end{document}

l
-ascorbate-6-P.
The
src
Homology 3-Like Domain of the Diphtheria Toxin Repressor (DtxR) Modulates Repressor Activation through Interaction with the Ancillary Metal Ion-Binding Site
Authors: Love John F. , vanderSpek Johanna C. , Murphy John R.
Published: March 2003

The diphtheria toxin repressor (DtxR) is a transition metal ion-activated repressor that acts as a global regulatory element in the control of iron-sensitive genes in
Corynebacterium diphtheriae
. We recently described (L. Sun, J. C. vanderSpek, and J. R. Murphy, Proc. Natl. Acad. Sci. USA 95:14985-14990, 1998) the isolation and in vivo characterization of a hyperactive mutant of DtxR, DtxR(E175K), that appeared to be constitutively active. We demonstrate here that while DtxR(E175K) remains active in vivo in the presence of 300 μM 2,2′dipyridyl, the purified repressor is, in fact, dependent upon low levels of transition metal ion to transit from the inactive apo form to the active metal ion-bound form of the repressor. Binding studies using 8-anilino-1-naphthalenesulfonic acid suggest that the E175K mutation stabilizes an intermediate of the molten-globule form of the repressor, increasing exposure of hydrophobic residues to solvent. We demonstrate that the hyperactive DtxR(E175K) phenotype is dependent upon an intact ancillary metal ion-binding site (site 1) of the repressor. These observations support the hypothesis that metal ion binding in the ancillary site facilitates the conversion of the inactive apo-repressor to its active, operator-binding conformation. Furthermore, these results support the hypothesis that the C-terminal
src
homology 3-like domain of DtxR plays an active role in the modulation of repressor activity.
YscP and YscU Regulate Substrate Specificity of theYersiniaType III Secretion System
Authors: Edqvist Petra J. , Olsson Jan , Lavander Moa , Sundberg Lena , Forsberg Åke , Wolf-Watz Hans , Lloyd Scott A.
Published: March 2003

PathogenicYersiniaspecies use a type III secretion system to inhibit phagocytosis by eukaryotic cells. At 37°C, the secretion system is assembled, forming a needle-like structure on the bacterial cell surface. Upon eukaryotic cell contact, six effector proteins, called Yops, are translocated into the eukaryotic cell cytosol. Here, we show that ayscPmutant exports an increased amount of the needle component YscF to the bacterial cell surface but is unable to efficiently secrete effector Yops. Mutations in the cytoplasmic domain of the inner membrane protein YscU suppress theyscPphenotype by reducing the level of YscF secretion and increasing the level of Yop secretion. These results suggest that YscP and YscU coordinately regulate the substrate specificity of theYersiniatype III secretion system. Furthermore, we show that YscP and YscU act upstream of the cell contact sensor YopN as well as the inner gatekeeper LcrG in the pathway of substrate export regulation. These results further strengthen the strong evolutionary link between flagellar biosynthesis and type III synthesis.
Mutations Which Uncouple Transport and Phosphorylation in the
d
-Mannitol Phosphotransferase System of
Escherichia coli
K-12 and
Klebsiella pneumoniae
1033-5P14
Authors: Otte Susanne , Scholle Annette , Turgut Sevket , Lengeler Joseph W.
Published: March 2003

Mutants of
Escherichia coli
K-12 were isolated which lack the normal phosphotransferase system-dependent catabolic pathway for
d
-mannitol (Mtl). In some mutants the
pts
genes for the general proteins enzyme I and histidine protein of the phosphoenolpyruvate-dependent carbohydrate phosphotransferase systems were deleted. Other mutants expressed truncated mannitol-specific enzymes II (II
Mtl
) which lacked the IIA
Mtl
or IIBA
Mtl
domain(s), and the
mtlA
genes originated either from
E. coli
K-12 or from
Klebsiella pneumoniae
1033-5P14. The
dalD
gene from
Klebsiella oxytoca
M5a1 was cloned on single-copy plasmids and transformed into the strains described above. This gene encodes an NAD-dependent
d
-arabinitol dehydrogenase (DalD) which converts
d
-arabinitol into
d
-xylulose and also converts
d
-mannitol into
d
-fructose. The different strains were used to isolate mutations which allow efficient transport of mannitol through the nonphosphorylated II
Mtl
complexes by selecting for growth on this polyhydric alcohol. More than 40 different mutants were analyzed to determine their ability to grow on mannitol, as well as their ability to bind and transport free mannitol and, after restoration of the missing domain(s), their ability to phosphorylate mannitol. Four mutations were identified (E218A, E218V, H256P, and H256Y); all of these mutations are located in the highly conserved loop 5 of the IIC membrane-bound transporter, and two are located in its GIHE motif. These mutations were found to affect the various functions in different ways. Interestingly, in the presence of all II
Mtl
variants, whether they were in the truncated form or in the complete form, in the phosphorylated form or in the nonphosphorylated form, and in the wild-type form or in the mutated form, growth occurred on the low-affinity analogue
d
-arabinitol with good efficiency, while only the uncoupled mutated forms transported mannitol at a high rate.
Characterization of a Novel Thermostable
O
-Acetylserine Sulfhydrylase from
Aeropyrum pernix
K1
Authors: Mino Koshiki , Ishikawa Kazuhiko
Published: March 2003

An
O
-acetylserine sulfhydrylase (OASS) from the hyperthermophilic archaeon
Aeropyrum pernix
K1, which shares the pyridoxal 5′-phosphate binding motif with both OASS and cystathionine β-synthase (CBS), was cloned and expressed by using
Escherichia coli
Rosetta(DE3). The purified protein was a dimer and contained pyridoxal 5′-phosphate. It was shown to be an enzyme with CBS activity as well as OASS activity in vitro. The enzyme retained 90% of its activity after a 6-h incubation at 100°C. In the
O
-acetyl-
l
-serine sulfhydrylation reaction, it had a pH optimum of 6.7, apparent

K
m

values for
O
-acetyl-
l
-serine and sulfide of 28 and below 0.2 mM, respectively, and a rate constant of 202 s
−1
. In the
l
-cystathionine synthetic reaction, it showed a broad pH optimum in the range of 8.1 to 8.8, apparent

K
m

values for
l
-serine and
l
-homocysteine of 8 and 0.51 mM, respectively, and a rate constant of 0.7 s
−1
.
A. pernix
OASS has a high activity in the
l
-cysteine desulfurization reaction, which produces sulfide and
S
-(2,3-hydroxy-4-thiobutyl)-
l
-cysteine from
l
-cysteine and dithiothreitol.
TheMoraxella catarrhalisImmunoglobulin D-Binding Protein MID Has Conserved Sequences and Is Regulated by a Mechanism Corresponding to Phase Variation
Authors: Möllenkvist Andrea , Nordström Therése , Halldén Christer , Christensen Jens Jørgen , Forsgren Arne , Riesbeck Kristian
Published: March 2003

The prevalence of theMoraxella catarrhalisimmunoglobulin D (IgD)-binding outer membrane protein MID and its gene was determined in 91 clinical isolates and in 7 culture collection strains. Eighty-four percent of the clinicalMoraxellastrains expressed MID-dependent IgD binding. Themidgene was detected in all strains as revealed by homology of the signal peptide sequence and a conserved area in the 3′ end of the gene. When MID proteins from five different strains were compared, an identity of 65.3 to 85.0% and a similarity of 71.2 to 89.1% were detected. Gene analyses showed several amino acid repeat motifs in the open reading frames, and MID could be called a putative autotransport protein. Interestingly, homopolymeric {polyguanine [poly(G)]} tracts were detected at the 5′ ends within the open reading frames. By flow cytometry, using human IgD and fluorescein isothiocyanate-conjugated anti-IgD polyclonal antibodies, most strains showed two peaks: one high- and one low-intensity peak. All isolates expressing high levels of MID had 1, 2, or 3 triplets of G's in their poly(G) tracts, while strains not expressing MID had 4, 7, 8, or 10 G’s in their poly(G) tracts or point mutations causing a putative preterminated translation. Northern blot analysis revealed that themidgene was regulated at the transcriptional level. Experiments with nonclumping variants ofM.catarrhalisproved that bacteria lost their MID expression by removing a G in their poly(G) tracts.Moraxellastrains isolated from the nasopharynx or from blood and sputum specimens expressed MID at approximately the same frequency. In addition, no variation was observed between strains of different geographical origins (Australia, Europe, Japan, or the United States). MID and themidgene were found solely inM.catarrhalis, whereas relatedNeisseriaandMoraxellaspecies did not express MID. Taken together, MID appears to be a conserved protein that can be found in essentially allM.catarrhalisstrains. Furthermore, MID is governed by poly(G) tracts when bacteria undergo phase variation.
Evidence for Horizontal Transfer of the
Eco
T38I Restriction- Modification Gene to Chromosomal DNA by the P2 Phage and Diversity of Defective P2 Prophages in
Escherichia
coli
TH38 Strains
Authors: Kita Keiko , Kawakami Hideaki , Tanaka Hiroaki
Published: March 2003

A DNA fragment carrying the genes coding for a novel
Eco
T38I restriction endonuclease (R.
Eco
T38I) and
Eco
T38I methyltransferase (M.
Eco
T38I), which recognize G(A/G)GC(C/T)C, was cloned from the chromosomal DNA of
Escherichia
coli
TH38. The endonuclease and methyltransferase genes were in a head-to-head orientation and were separated by a 330-nucleotide intergenic region. A third gene, the C.
Eco
T38I gene, was found in the intergenic region, partially overlapping the R.
Eco
T38I gene. The gene product, C.
Eco
T38I, acted as both a positive regulator of R.
Eco
T38I gene expression and a negative regulator of M.
Eco
T38I gene expression. M.
Eco
T38I purified from recombinant
E
.
coli
cells was shown to be a monomeric protein and to methylate the inner cytosines in the recognition sequence. R.
Eco
T38I was purified from
E
.
coli
HB101 expressing M.
Eco
T38I and formed a homodimer. The
Eco
T38I restriction (R)-modification (M) system (R-M system) was found to be inserted between the
A
and
Q
genes of defective bacteriophage P2, which was lysogenized in the chromosome at
loc
I, one of the P2 phage attachment sites observed in both
E
.
coli
K-12 MG1655 and TH38 chromosomal DNAs. Ten strains of
E
.
coli
TH38 were examined for the presence of the
Eco
T38I R-M gene on the P2 prophage. Conventional PCR analysis and assaying of R activity demonstrated that all strains carried a single copy of the
Eco
T38I R-M gene and expressed R activity but that diversity of excision in the
ogr
,
D
,
H
,
I
, and
J
genes in the defective P2 prophage had arisen.
Heterocyst-Specific Expression of
patB
, a Gene Required for Nitrogen Fixation in
Anabaena
sp. Strain PCC 7120
Authors: Jones Kathryn M. , Buikema William J. , Haselkorn Robert
Published: March 2003

The
patB
gene product is required for growth and survival of the filamentous cyanobacterium
Anabaena
sp. strain PCC 7120 in the absence of combined nitrogen. A
patB
::
gfp
fusion demonstrated that this gene is expressed exclusively in heterocysts.
patB
mutants have a normal initial pattern of heterocyst spacing along the filament but differentiate excess heterocysts after several days in the absence of combined nitrogen. Expression of
hetR
and
patS
, two critical regulators of the heterocyst development cascade, are normal for
patB
mutants, indicating that
patB
acts downstream of them in the differentiation pathway. A
patB
deletion mutant suffers an almost complete cessation of growth and nitrogen fixation within 24 h of combined nitrogen removal. In contrast, a new PatB mutant that is defective in its N-terminal ferredoxin domain, or a previously described mutant that has a frameshift removing its C-terminal helix-turn-helix domain, grows very slowly and differentiates multiple contiguous heterocysts under nitrogen-deficient conditions.
Identification of a New Gene Essential for GerminationofBacillus subtilisSpores withCa2+-Dipicolinate
Authors: Ragkousi Katerina , Eichenberger Patrick , van Ooij Christiaan , Setlow Peter
Published: March 2003

Bacillus subtilisspores can germinate with a 1:1 chelate of Ca2+and dipicolinic acid (DPA), a compound present at high levels in the spore core. Using a genetic screen to identify genes encoding proteins that are specifically involved in spore germination by Ca2+-DPA, three mutations were identified. One was in the gene encoding the cortex lytic enzyme, CwlJ, that was previously shown to be essential for spore germination by Ca2+-DPA. The other two were mapped to an open reading frame,ywdL, encoding a protein of unknown function. Analysis ofywdLexpression showed that the gene is expressed during sporulation in the mother cell compartment of the sporulating cell and that its transcription is σEdependent. Functional characterization of YwdL demonstrated that it is a new spore coat protein that is essential for the presence of CwlJ in the spore coat. Assembly of YwdL itself into the spore coat is dependent on the coat morphogenetic proteins CotE and SpoIVA. However, other than lacking CwlJ,ywdLspores have no obvious defect in their spore coat. Because of the role for YwdL in a part of the spore germination process, we propose renamingywdLas a spore germination gene,gerQ.
Comparative Genomics of
Salmonella
enterica
Serovar Typhi Strains Ty2 and CT18
Authors: Deng Wen , Liou Shian-Ren , Plunkett Guy , Mayhew George F. , Rose Debra J. , Burland Valerie , Kodoyianni Voula , Schwartz David C. , Blattner Frederick R.
Published: March 2003

We present the 4.8-Mb complete genome sequence of
Salmonella
enterica
serovar Typhi strain Ty2, a human-specific pathogen causing typhoid fever. A comparison with the genome sequence of recently isolated
S
.
enterica
serovar Typhi strain CT18 showed that 29 of the 4,646 predicted genes in Ty2 are unique to this strain, while 84 genes are unique to CT18. Both genomes contain more than 200 pseudogenes; 9 of these genes in CT18 are intact in Ty2, while 11 intact CT18 genes are pseudogenes in Ty2. A half-genome interreplichore inversion in Ty2 relative to CT18 was confirmed. The two strains exhibit differences in prophages, insertion sequences, and island structures. While CT18 carries two plasmids, one conferring multiple drug resistance, Ty2 has no plasmids and is sensitive to antibiotics.
TheStreptomyces coelicolorDevelopmental Transcription Factor σBldNIs Synthesized as a Proprotein
Authors: Bibb Maureen J. , Buttner Mark J.
Published: March 2003

bldNis one of a set of genes required for the formation of specialized, spore-bearing aerial hyphae during differentiation in the mycelial bacteriumStreptomyces coelicolor. Previous analysis (M. J. Bibb et al., J. Bacteriol. 182:4606-4616, 2000) showed thatbldNencodes a member of the extracytoplasmic function subfamily of RNA polymerase σ factors and that translation from the most strongly predicted start codon (GTG1) would give rise to a σ factor having an unusual N-terminal extension of ca. 86 residues. Here, by using a combination of site-directed mutagenesis and immunoblot analysis, we provide evidence that allbldNtranslation arises from initiation at GTG1and that the primary translation product is a proprotein (pro-σBldN) that is proteolytically processed to a mature species (σBldN) by removal of most of the unusual N-terminal extension. A time course taken during differentiation of the wild type on solid medium showed early production of pro-σBldNand the subsequent appearance of mature σBldN, which was concomitant with aerial mycelium formation and the disappearance of pro-σBldN. Two genes encoding members of a family of metalloproteases that are involved in the regulated proteolytic processing of transcription factors in other organisms were identified in theS. coelicolorgenome, but their disruption did not affect differentiation or pro-σBldNprocessing.
Localization of rRNA Synthesis inBacillus subtilis: Characterization of Loci Involved in Transcription Focus Formation
Authors: Davies Karen M. , Lewis Peter J.
Published: March 2003

InBacillus subtilis, RNA polymerase becomes concentrated into regions of the nucleoid called transcription foci. With green fluorescent protein-tagged RNA polymerase, these structures are only observed at higher growth rates and have been shown to represent the sites of rRNA synthesis. There are 10 rRNA (rrn) operons distributed around nearly half of the chromosome. In this study we analyzed therrncomposition of transcription foci with fluorescently tagged loci and showed that they comprise the origin-proximal operonrrnObut not the more dispersedrrnEorrrnD. This suggests that transcription foci comprise only the seven origin-proximal operonsrrnO,rrnA,rrnJ,rrnW,rrnI,rrnH, andrrnG. These results have important implications for our understanding of microbial chromosome structure.
CheZ Phosphatase Localizes to Chemoreceptor Patches via CheA-Short
Authors: Cantwell Brian J. , Draheim Roger R. , Weart Richard B. , Nguyen Cameran , Stewart Richard C. , Manson Michael D.
Published: March 2003

We have investigated the conditions required for polar localization of the CheZ phosphatase by using a CheZ-green fluorescent protein fusion protein that, when expressed from a single gene in the chromosome, restored chemotaxis to a Δ
cheZ
strain. Localization was observed in wild-type, Δ
cheZ
, Δ
cheYZ
, and Δ
cheRB
cells but not in cells with
cheA
,
cheW
, or all chemoreceptor genes except
aer
deleted. Cells making only CheA-short (CheA
S
) or CheA lacking the P2 domain also retained normal localization, whereas cells producing only CheA-long or CheA missing the P1 and P2 domains did not. We conclude that CheZ localization requires the truncated C-terminal portion of the P1 domain present in CheA
S
. Missense mutations targeting residues 83 through 120 of CheZ also abolished localization. Two of these mutations do not disrupt chemotaxis, indicating that they specifically prevent interaction with CheA
S
while leaving other activities of CheZ intact.
Genome Organization and Molecular Analysis of the Temperate Bacteriophage MM1 of
Streptococcus pneumoniae
Authors: Obregón Virginia , García José L. , García Ernesto , López Rubens , García Pedro
Published: March 2003

The genome of MM1 (40,248 bp), a temperate bacteriophage from the Spain
23F
-1 multiresistant epidemic clone of
Streptococcus pneumoniae
, is organized in 53 open reading frames (ORFs) and in at least five functional clusters. Bioinformatic and N-terminal amino acid sequence analyses enabled the assignment of possible functions to 26 ORFs. Analyses comparing the MM1 genome with those of other bacteriophages revealed similarities, mainly with genomes of phages infecting gram-positive bacteria, which suggest recent exchange of genes between species colonizing the same habitat.
Structural and Functional Analysis of a Putative Gene Cluster for Palatinose Transport on the Linear Chromosome of
Agrobacterium tumefaciens
MAFF301001
Authors: De Costa Devika M. , Suzuki Katsunori , Yoshida Kazuo
Published: March 2003

We identified a putative
pal
gene cluster (
palR
,
palE
,
palF
,
palG
,
palK
,
palA
, and
palB
) in the plant-tumorigenic bacterium
Agrobacterium tumefaciens
MAFF301001; by sequencing analyses, this cluster was found to be involved in palatinose transport, and its functional importance was revealed by mutational analyses. The
pal
gene products were highly homologous to those of putative trehalose/maltose ABC-type transport systems but were not essential to bacterial growth on trehalose. Insertion mutations in the
palK
and
palE
genes showed the necessity of these genes for bacterial growth and chemotaxis with palatinose as the carbon source, but no inhibition of tumorigenesis was observed. Growth on trehalose and maltose was not influenced by the mutations.
Biofilm Formation by Hyperpiliated Mutants of
Pseudomonas aeruginosa
Authors: Chiang Poney , Burrows Lori L.
Published: March 2003

Under static growth conditions, hyperpiliated, nontwitching
pilT
and
pilU
mutants of
Pseudomonas aeruginosa
formed dense biofilms, showing that adhesion, not twitching motility, is necessary for biofilm initiation. Under flow conditions, the
pilT
mutant formed mushroom-like structures larger than those of the wild type but the
pilU
mutant was defective in biofilm formation. Therefore, twitching motility affects the development of biofilm structure, possibly through modulation of detachment.
Characterization of the
Bacillus subtilis ywtD
Gene, Whose Product Is Involved in γ-Polyglutamic Acid Degradation
Authors: Suzuki Takao , Tahara Yasutaka
Published: March 2003

The
ywtD
gene, which codes for an enzyme that degrades γ-polyglutamic acid (PGA), was cloned from
Bacillus subtilis
IFO16449. The gene is located immediately downstream of
ywsC
and
ywtABC
, a PGA operon involved in PGA biosynthesis, and it showed partial similarity to genes coding for
dl
-endopeptidase, a peptidoglycan-degrading enzyme. The
ywtD
gene, from which signal sequence is excised, was inserted into pET15b, and the recombinant plasmid was then transformed into
Escherichia coli
. Histidine-tagged YwtD was purified from sonicated cells of the transformant. The purified YwtD degraded PGA to yield two hydrolyzed products, a high-molecular-mass product (490 kDa with nearly 100%
l
-glutamic acid) and an 11-kDa product (with
d
-glutamic acid and
l
-glutamic acid in an 80:20 ratio). This finding and results of enzymatic analysis of the two products with carboxypeptidase G suggest that YwtD is a novel enzyme cleaving the γ-glutamyl bond only between
d
- and
l
-glutamic acids of PGA, and it may be designated γ-
dl-
glutamyl hydrolase.
VnfY Is Required for Full Activity of the Vanadium-Containing Dinitrogenase in
Azotobacter vinelandii
Authors: Rüttimann-Johnson Carmen , Rubio Luis M. , Dean Dennis R. , Ludden Paul W.
Published: March 2003

A gene from
Azotobacter vinelandii
whose product exhibits primary sequence similarity to the NifY, NafY, NifX, and VnfX family of proteins, and which is required for effective V-dependent diazotrophic growth, was identified. Because this gene is located downstream from
vnfK
in an arrangement similar to the relative organization of the
nifK
and
nifY
genes, it was designated
vnfY
. A mutant strain having an insertion mutation in
vnfY
has 10-fold less
vnf
dinitrogenase activity and exhibits a greatly diminished level of
49
V label incorporation into the V-dependent dinitrogenase when compared to the wild type. These results indicate that VnfY has a role in the maturation of the V-dependent dinitrogenase, with a specific role in the formation of the V-containing cofactor and/or its insertion into apodinitrogenase.