bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Multifunctional Bacteriophage P2 Cox Protein Requires Oligomerization for Biological Activity
Authors: Eriksson Jesper M. , Haggård-Ljungquist Elisabeth 
Published: July 2002

ABSTRACT

The Cox protein of bacteriophage P2 is a multifunctional protein of 91 amino acids. It is directly involved in the site-specific recombination event leading to excision of P2 DNA out of the host chromosome. In this context, it functions as an architectural protein in the formation of the excisome. Cox is also a transcriptional repressor of the P2 Pc promoter, thereby ensuring lytic growth. Finally it promotes derepression of prophage P4, a nonrelated defective satellite phage, by activating the P4 P
LL
promoter that controls P4 DNA replication. In this case it binds upstream of the P
LL
promoter, which normally is activated by the P4 Delta protein. In this work we have analyzed the native form of the Cox protein in vivo, using a bacteriophage λ
c
I-based oligomerization assay system, and in vitro, using gel filtration, cross-linking agents, and gel retardation assays. We found that P2 Cox has a strong oligomerization function in vivo as well as in vitro. The in vitro analysis indicates that its native form is a tetramer that can self-associate to octamers. Furthermore we show that oligomerization is necessary for the biological activity by characterizing different
cox
mutants and that oligomerization is mediated by the C-terminal region.
The Metabolic Network of
Lactococcus lactis
: Distribution of
14
C-Labeled Substrates between Catabolic and Anabolic Pathways
Authors: Novák L. , Loubiere P. 
Published: July 2002

ABSTRACT

Lactococcus lactis
NCDO 2118 was grown in a simple synthetic medium containing only six essential amino acids and glucose as carbon substrates to determine qualitatively and quantitatively the carbon fluxes into the metabolic network. The specific rates of substrate consumption, product formation, and biomass synthesis, calculated during the exponential growth phase, represented the carbon fluxes within the catabolic and anabolic pathways. The macromolecular composition of the biomass was measured to distribute the global anabolic flux into the specific anabolic pathways. Finally, the distribution of radiolabeled substrates, both into the excreted fermentation end products and into the different macromolecular fractions of biomass, was monitored. The classical end products of lactic acid metabolism (lactate, formate, and acetate) were labeled with glucose, which did not label other excreted products, and to a lesser extent with serine, which was deaminated to pyruvate and represented approximately 10% of the pyruvate flux. Other minor products, keto and hydroxy acids, were produced from glutamate and branched-chain amino acids via deamination and subsequent decarboxylation and/or reduction. Glucose labeled all biomass fractions and accounted for 66% of the cellular carbon, although this represented only 5% of the consumed glucose.
The Stability Region of the Large Virulence Plasmid of
Shigella flexneri
Encodes an Efficient Postsegregational Killing System
Authors: Sayeed Sameera , Reaves Lucretia , Radnedge Lyndsay , Austin Stuart 
Published: July 2002

ABSTRACT

The large virulence plasmid pMYSH6000 of
Shigella flexneri
contains a determinant that is highly effective in stabilizing otherwise unstable plasmids in
Escherichia coli
. Expression of two small contiguous genes,
mvpA
and
mvpT
(formerly termed STBORF1 and STBORF2), was shown to be sufficient for stability. Mutations in
mvpT
abolished plasmid stability, and plasmids expressing only
mvpT
killed the cells unless
mvpA
was supplied from a separate plasmid or from the host chromosome. When replication of a plasmid carrying the minimal
mvp
region was blocked, growth of the culture stopped after a short lag and virtually all of the surviving cells retained the plasmid. Thus, the
mvp
system stabilizes by a highly efficient postsegregational killing (PSK) mechanism, with
mvpT
encoding a cell toxin and
mvpA
encoding an antidote. The regions that surround the
mvp
genes in their original context have an inhibitory effect that attenuates plasmid stabilization and PSK. The region encompassing the
mvp
genes also appears to contain an additional element that can aid propagation of a pSC101-based plasmid under conditions where replication initiation is marginal. However, this appears to be a relatively nonspecific effect of DNA insertion into the plasmid vector.
Control of the Ferric Citrate Transport System of
Escherichia coli
: Mutations in Region 2.1 of the FecI Extracytoplasmic-Function Sigma Factor Suppress Mutations in the FecR Transmembrane Regulatory Protein
Authors: Stiefel Alfred , Mahren Susanne , Ochs Martina , Schindler Petra T. , Enz Sabine , Braun Volkmar 
Published: July 2002

ABSTRACT

Transcription of the ferric citrate transport genes is initiated by binding of ferric citrate to the FecA protein in the outer membrane of
Escherichia coli
K-12. Bound ferric citrate does not have to be transported but initiates a signal that is transmitted by FecA across the outer membrane and by FecR across the cytoplasmic membrane into the cytoplasm, where the FecI extracytoplasmic-function (ECF) sigma factor becomes active. In this study, we isolated transcription initiation-negative missense mutants in the cytoplasmic region of FecR that were located at four sites, L13Q, W19R, W39R, and W50R, which are highly conserved in FecR-like open reading frames of the
Pseudomonas aeruginosa
,
Pseudomonas putida
,
Bordetella pertussis
,
Bordetella bronchiseptica
, and
Caulobacter crescentus
genomes. The cytoplasmic portion of the FecR mutant proteins, FecR
1–85
, did not interact with wild-type FecI, in contrast to wild-type FecR
1–85
, which induced FecI-mediated
fecB
transport gene transcription. Two missense mutations in region 2.1 of FecI, S15A and H20E, partially restored induction of ferric citrate transport gene induction of the
fecR
mutants by ferric citrate. Region 2.1 of ς
70
is thought to bind RNA polymerase core enzyme; the residual activity of mutated FecI in the absence of FecR, however, was not higher than that of wild-type FecI. In addition, missense mutations in the
fecI
promoter region resulted in a twofold increased transcription in
fecR
wild-type cells and a partial restoration of
fec
transport gene transcription in the
fecR
mutants. The mutations reduced binding of the Fe
2+
Fur repressor and as a consequence enhanced
fecI
transcription. The data reveal properties of the FecI ECF factor distinct from those of ς
70
and further support the novel transcription initiation model in which the cytoplasmic portion of FecR is important for FecI activity.
Effects of Mutations in the
Pseudomonas putida miaA
Gene: Regulation of the
trpE
and
trpGDC
Operons in
P. putida
by Attenuation
Authors: Olekhnovich Igor , Gussin Gary N. 
Published: July 2002

ABSTRACT

Tn
5
insertion mutants defective in regulation of the
Pseudomonas putida trpE
and
trpGDC
operons by tryptophan were found to contain insertions in the
P. putida miaA
gene, whose product (in
Escherichia coli
) modifies tRNA
Trp
and is required for attenuation. Nucleotide sequences upstream of
trpE
and
trpG
encode putative leader peptides similar in sequence to leader peptides found in other bacterial species, and the phenotypes of the mutants strongly suggest that transcription of these operons is regulated solely by attenuation.
Identification, Cloning, Expression, and Characterization of the Extracellular Acarbose-Modifying Glycosyltransferase, AcbD, fromActinoplanessp. Strain SE50
Authors: Hemker Michael , Stratmann Ansgar , Goeke Klaus , Schröder Werner , Lenz Jürgen , Piepersberg Wolfgang , Pape Hermann 
Published: July 2002

ABSTRACTAn extracellular enzyme activity in the culture supernatant of the acarbose producerActinoplanessp. strain SE50 catalyzes the transfer of the acarviosyl moiety of acarbose to malto-oligosaccharides. This acarviosyl transferase (ATase) is encoded by a gene,acbD, in the putative biosynthetic gene cluster for the α-glucosidase inhibitor acarbose. TheacbDgene was cloned and heterologously produced inStreptomyces lividansTK23. The recombinant protein was analyzed by enzyme assays. The AcbD protein (724 amino acids) displays all of the features of extracellular α-glucosidases and/or transglycosylases of the α-amylase family and exhibits the highest similarities to several cyclodextrin glucanotransferases (CGTases). However, AcbD had neither α-amylase nor CGTase activity. The AcbD protein was purified to homogeneity, and it was identified by partial protein sequencing of tryptic peptides. AcbD had an apparent molecular mass of 76 kDa and an isoelectric point of 5.0 and required Ca2+ions for activity. The enzyme displayed maximal activity at 30°C and between pH 6.2 and 6.9. TheKmvalues of the ATase for acarbose (donor substrate) and maltose (acceptor substrate) are 0.65 and 0.96 mM, respectively. A wide range of additional donor and acceptor substrates were determined for the enzyme. Acceptors revealed a structural requirement for glucose-analogous structures conserving only the overall stereochemistry, except for the anomeric C atom, and the hydroxyl groups at positions 2, 3, and 4 ofd-glucose. We discuss here the function of the enzyme in the extracellular formation of the series of acarbose-homologous compounds produced byActinoplanessp. strain SE50.
Genetic Studies ofmrp, a Locus Essential for Cellular Aggregation and Sporulation ofMyxococcus xanthus
Authors: Sun Hong , Shi Wenyuan 
Published: July 2002

ABSTRACTUnder starvation conditions,Myxococcus xanthusundergoes a complex developmental process which includes cellular aggregation and sporulation. A transposon insertion mutant (the Tn5-Ω280 mutant) with defects in both aggregation and sporulation was analyzed in this study. The Tn5-Ω280 mutant was found to have a disrupted NtrC-like response regulator designatedMyxococcusregulatory protein B (mrpB). Further sequencing analyses revealed a histidine kinase homolog (mrpA) immediately upstream ofmrpBand a cyclic AMP receptor protein-like transcriptional regulator (mrpC) downstream ofmrpB. In-frame deletion analyses revealed that both themrpBandmrpCgenes were required for cellular aggregation and sporulation but that onlymrpAwas required for sporulation only. Site-specific mutagenesis of the putative phosphorylation site of MrpB, D58, showed that a D58A mutation caused defects in both aggregation and sporulation but that a D58E mutation resulted in only a sporulation defect. Further genetic and molecular analyses with reporter genes and reverse transcription-PCR indicated thatmrpAandmrpBare cotranscribed but thatmrpCis transcribed independently and that all of these genes are developmentally regulated. In addition, MrpB is essential for transcription ofmrpCand MrpC regulates its own transcription. These data indicate that Mrp proteins are important components required forM. xanthusdevelopment. The complicated interaction between Mrp proteins may play an important role in regulating developmental gene expression inM. xanthus.
Genetic Recombination in
Bacillus subtilis
168: Effect of Δ
helD
on DNA Repair and Homologous Recombination
Authors: Carrasco Begoña , Fernández Silvia , Petit Marie-Agnes , Alonso Juan C. 
Published: July 2002

ABSTRACT

The
B. subtilis
Δ
helD
allele rendered cells proficient in transformational recombination and moderately sensitive to methyl methanesulfonate when present in an otherwise Rec
+
strain. The Δ
helD
allele was introduced into
rec
-deficient strains representative of the α (
recF
strain), β (
addA
addB
), γ (
recH
), ɛ (Δ
recU
), and ζ (Δ
recS
) epistatic groups. The Δ
helD
mutation increased the sensitivity to DNA-damaging agents of
addAB,
Δ
recU,
and Δ
recS
cells, did not affect the survival of
recH
cells, and decreased the sensitivity of
recF
cells. Δ
helD
also partially suppressed the DNA repair phenotype of other mutations classified within the α epistatic group, namely the
recL
, Δ
recO,
and
recR
mutations. The Δ
helD
allele marginally reduced plasmid transformation (three- to sevenfold) of mutations classified within the α, β, and γ epistatic groups. Altogether, these data indicate that the loss of helicase IV might stabilize recombination repair intermediates formed in the absence of
recFLOR
and render
recFLOR
,
addAB
, and
recH
cells impaired in plasmid transformation.
The Genome of Archaeal Prophage ΨM100 Encodes the Lytic Enzyme Responsible for Autolysis of
Methanothermobacter wolfeii
Authors: Luo Yongneng , Pfister Peter , Leisinger Thomas , Wasserfallen Alain 
Published: July 2002

ABSTRACT

Methanothermobacter wolfeii
(formerly
Methanobacterium wolfei
), a thermophilic methanoarchaeon whose cultures lyse upon hydrogen starvation, carries a defective prophage called ΨM100 on its chromosome. The nucleotide sequence of ΨM100 and its flanking regions was established and compared to that of the previously sequenced phage ΨM2 of
Methanothermobacter marburgensis
(formerly
Methanobacterium thermoautotrophicum
Marburg). The ΨM100 genome extends over 28,798 bp, and its borders are defined by flanking 21-bp direct repeats of a pure-AT sequence, which very likely forms the core of the putative attachment site where the crossing over occurred during integration. A large fragment of 2,793 bp, IFa, apparently inserted into ΨM100 but is absent in the genome of ΨM2. The remaining part of the ΨM100 genome showed 70.8% nucleotide sequence identity to the whole genome of ΨM2. Thirty-four open reading frames (ORFs) on the forward strand and one ORF on the reverse strand were identified in the ΨM100 genome. Comparison of ΨM100-encoded ORFs to those encoded by phage ΨM2 and to other known protein sequences permitted the assignment of putative functions to some ORFs. The ORF28 protein of ΨM100 was identified as the previously known autolytic enzyme pseudomurein endoisopeptidase PeiW produced by
M. wolfeii
.
Specificities of Eleven Different DNA Methyltransferases of
Helicobacter pylori
Strain 26695
Authors: Vitkute Jolanta , Stankevicius Kornelijus , Tamulaitiene Giedre , Maneliene Zita , Timinskas Albertas , Berg Douglas E. , Janulaitis Arvydas 
Published: July 2002

ABSTRACT

Methyltransferases (MTases) of procaryotes affect general cellular processes such as mismatch repair, regulation of transcription, replication, and transposition, and in some cases may be essential for viability. As components of restriction-modification systems, they contribute to bacterial genetic diversity. The genome of
Helicobacter pylori
strain 26695 contains 25 open reading frames encoding putative DNA MTases. To assess which MTase genes are active, strain 26695 genomic DNA was tested for cleavage by 147 restriction endonucleases; 24 were found that did not cleave this DNA. The specificities of 11 expressed MTases and the genes encoding them were identified from this restriction data, combined with the known sensitivities of restriction endonucleases to specific DNA modification, homology searches, gene cloning and genomic mapping of the methylated bases m
4
C, m
5
C, and m
6
A.
An N-Terminally Truncated RpoS (σ
S
) Protein in
Escherichia coli
Is Active In Vivo and Exhibits Normal Environmental Regulation Even in the Absence of
rpoS
Transcriptional and Translational Control Signals
Authors: Rajkumari K. , Gowrishankar J. 
Published: July 2002

ABSTRACT

RpoS (σ
S
) in
Escherichia coli
is a stationary-phase-specific primary sigma factor of RNA polymerase which is 330 amino acids long and belongs to the eubacterial σ
70
family of proteins. Conserved domain 1.1 at the N-terminal end of σ
70
has been shown to be essential for RNA polymerase function, and its deletion has been shown to result in a dominant-lethal phenotype. We now report that a σ
S
variant with a deletion of its N-terminal 50 amino acids (σ
S
Δ1-50), when expressed in vivo either from a chromosomal
rpoS
::IS
10
allele (in
rho
mutant strains) or from a plasmid-borne arabinose-inducible promoter, is as proficient as the wild type in directing transcription from the
proU
P1 promoter; at three other σ
S
-dependent promoters that were tested (
osmY
,
katE
, and
csiD
), the truncated protein exhibited a three- to sevenfold reduced range of activities. Catabolite repression at the
csiD
promoter (which requires both σ
S
and cyclic AMP [cAMP]-cAMP receptor protein for its activity) was also preserved in the strain expressing σ
S
Δ1-50. The intracellular content of σ
S
Δ1-50 was regulated by culture variables such as growth phase, osmolarity, and temperature in the same manner as that described earlier for σ
S
, even when the truncated protein was expressed from a template that possessed neither the transcriptional nor the translational control elements of wild-type
rpoS
. Our results indicate that, unlike that in σ
70
, the N-terminal domain in σ
S
may not be essential for the protein to function as a sigma factor in vivo. Furthermore, our results suggest that the induction of σ
S
-specific promoters in stationary phase and during growth under conditions of high osmolarity or low temperature is mediated primarily through the regulation of σ
S
protein degradation.
Streptococcus pneumoniaeCapsule Biosynthesis Protein CpsB Is a Novel Manganese-Dependent Phosphotyrosine-Protein Phosphatase
Authors: Morona Judy K. , Morona Renato , Miller David C. , Paton James C. 
Published: July 2002

ABSTRACTThe first four genes of the capsule locus (cps) ofStreptococcus pneumoniae(cpsAtocpsD) are common to most serotypes. We have previously determined that CpsD is an autophosphorylating protein-tyrosine kinase, demonstrated that CpsC is required for CpsD tyrosine-phosphorylation, and shown that CpsB is required for dephosphorylation of CpsD. In the present study we show that CpsB is a novel manganese-dependent phosphotyrosine-protein phosphatase that belongs to the PHP (polymerase and histidinol phosphatase) family of phosphoesterases. We also show that anS. pneumoniaestrain with point mutations incpsB, affecting one of the conserved motifs of CpsB, is unencapsulated and appears to be morphologically identical to a strain in which thecpsBgene had been deleted.
Identification of Virulence Genes in a Pathogenic Strain ofPseudomonas aeruginosaby Representational Difference Analysis
Authors: Choi Ji Young , Sifri Costi D. , Goumnerov Boyan C. , Rahme Laurence G. , Ausubel Frederick M. , Calderwood Stephen B. 
Published: July 2002

ABSTRACTPseudomonas aeruginosais an opportunistic pathogen that may cause severe infections in humans and other vertebrates. In addition, a human clinical isolate ofP. aeruginosa, strain PA14, also causes disease in a variety of nonvertebrate hosts, including plants,Caenorhabditis elegans, and the greater wax moth,Galleria mellonella. This has led to the development of a multihost pathogenesis system in which plants, nematodes, and insects have been used as adjuncts to animal models for the identification ofP. aeruginosavirulence factors. Another approach to identifying virulence genes in bacteria is to take advantage of the natural differences in pathogenicity between isolates of the same species and to use a subtractive hybridization technique to recover relevant genomic differences. The sequenced strain ofP. aeruginosa, strain PAO1, has substantial differences in virulence from strain PA14 in several of the multihost models of pathogenicity, and we have utilized the technique of representational difference analysis (RDA) to directly identify genomic differences betweenP. aeruginosastrains PA14 and PAO1. We have found that thepilC,pilA, anduvrDgenes in strain PA14 differ substantially from their counterparts in strain PAO1. In addition, we have recovered a gene homologous to theybtQgene fromYersinia, which is specifically present in strain PA14 but absent in strain PAO1. Mutation of theybtQhomolog inP. aeruginosastrain PA14 significantly attenuates the virulence of this strain in bothG. mellonellaand a burned mouse model of sepsis to levels comparable to those seen with PAO1. This suggests that the increased virulence ofP. aeruginosastrain PA14 compared to PAO1 may relate to specific genomic differences identifiable by RDA.
Transposable Element IS
Hp608
of
Helicobacter pylori
: Nonrandom Geographic Distribution, Functional Organization, and Insertion Specificity
Authors: Kersulyte Dangeruta , Velapatiño Billie , Dailide Giedrius , Mukhopadhyay Asish K. , Ito Yoshiyuki , Cahuayme Lizbeth , Parkinson Alan J. , Gilman Robert H. , Berg Douglas E. 
Published: July 2002

ABSTRACT

A new member of the IS
605
transposable element family, designated IS
Hp608
, was found by subtractive hybridization in
Helicobacter pylori
. Like the three other insertion sequences (ISs) known in this gastric pathogen, it contains two open reading frames (
orfA
and
orfB
), each related to putative transposase genes of simpler (one-gene) elements in other prokaryotes;
orfB
is also related to the
Salmonella
virulence gene
gipA
. PCR and hybridization tests showed that IS
Hp608
is nonrandomly distributed geographically: it was found in 21% of 194 European and African strains, 14% of 175 Bengali strains, 43% of 131 strains from native Peruvians and Alaska natives, but just 1% of 223 East Asian strains. IS
Hp608
also seemed more abundant in Peruvian gastric cancer strains than gastritis strains (9 of 14 versus 15 of 45, respectively;
P
= 0.04). Two IS
Hp608
types differing by ∼11% in DNA sequence were identified: one was widely distributed geographically, and the other was found only in Peruvian and Alaskan strains. Isolates of a given type differed by ≤2% in DNA sequence, but several recombinant elements were also found. IS
Hp608
marked with a resistance gene was found to (i) transpose in
Escherichia coli
; (ii) generate simple insertions during transposition, not cointegrates; (iii) insert downstream of the motif 5"-TTAC without duplicating target sequences; and (iv) require
orfA
but not
orfB
for its transposition. IS
Hp608
represents a widespread family of novel chimeric mobile DNA elements whose further analysis should provide new insights into transposition mechanisms and into microbial population genetic structure and genome evolution.
The Histone-Like Protein HU Does Not Obstruct Movement of T7 RNA Polymerase in
Escherichia coli
Cells but Stimulates Its Activity
Authors: Morales Pilar , Rouviere-Yaniv Josette , Dreyfus Marc 
Published: July 2002

ABSTRACT

In vivo, RNA polymerases (RNAPs) do not transcribe naked DNA but do transcribe protein-associated DNA. Studies with the model enzyme T7 RNAP have shown that, in eukaryotic cells or in vitro, nucleosomes can inhibit both transcription initiation and elongation. We examine here whether the presence of HU, one of the major histone-like proteins in
Escherichia coli
cells (the genuine milieu for T7 RNAP) affects its activity. An engineered
lac
operon fused to the T7 late promoter was introduced into the chromosome of T7 RNAP-producing strains that either overexpress HU or lack it. The flows of RNAP that enter and exit this operon were compared with regard to the content of HU. We found that the fraction of T7 RNAP molecules that do not reach the end of the
lac
operon (ca. 15%) is the same whether the host cells overexpressed HU or lacked it: thus, the enzyme either freely displaces HU or transcribes through it. However, in these cells, the transcript yield was increased when HU is overexpressed and decreased in the
hup
mutants, presumably reflecting changes in DNA supercoiling. Thus, in contrast to eukaryotic nucleosomes, HU does not impair T7 RNAP activity but has a stimulatory effect. Finally, our results suggest that HU can also influence mRNA stability in vivo.