bluuebunny/crossref_metadata_2025 huggingface dataset:
Oxidative protein folding in bacteria
Authors: Collet Jean‐Francois , Bardwell James C. A.
Published: March 2003

Ten years ago it was thought that disulphide bond formation in prokaryotes occurred spontaneously. Now two pathways involved in disulphide bond formation have been well characterized, the oxidative pathway, which is responsible for the formation of disulphides, and the isomerization pathway, which shuffles incorrectly formed disulphides. Disulphide bonds are donated directly to unfolded polypeptides by the DsbA protein; DsbA is reoxidized by DsbB. DsbB generates disulphides de novo from oxidized quinones.
These quinones are reoxidized by the electron transport chain, showing that disulphide
bond formation is actually driven by electron transport. Disulphide isomerization
requires that incorrect disulphides be attacked using a reduced catalyst, followed
by the redonation of the disulphide, allowing alternative disulphide pairing. Two
isomerases exist in Escherichia coli, DsbC and DsbG. The membrane protein DsbD maintains these disulphide isomerases in their reduced and thereby active form. DsbD is kept reduced by cytosolic thioredoxin in an NADPH‐dependent reaction.
Two domains of a dual‐function NarK protein are required for nitrate uptake, the first step of denitrification in Paracoccus pantotrophus
Authors: Wood Nicholas J. , Alizadeh Tooba , Richardson David J. , Ferguson Stuart J. , Moir James W. B.
Published: March 2003

Uptake of nitrate into the cytoplasm is the first but least well understood step of denitrification; no gene has previously been identified to be necessary for this process. Upstream from the structural genes of the membrane‐bound nitrate reductase (narGHJI) in Paracoccus pantotrophus there is a fusion of two genes,

each homologous to members of the narK family. The single open reading frame

is predicted to encode 24 transmembrane helices, comprising two domains, NarK1 and

NarK2. Analysis of both the accumulation of intracellular nitrite and electron transport

through the nitrate reductase enzyme in narK mutants reveals that NarK1 and

NarK2 are both involved in nitrate uptake. Maximal rate of nitrate transport via

NarK2 was dependent upon nitrite, indicating that NarK2 encodes a nitrate/nitrite

antiporter. The uncouplers S13 and dinitrophenol showed that NarK2 was not dependent

on the proton motive force for maximal nitrate transport activity. Nitrate transport

via NarK1 was dependent on proton motive force, indicating that it is likely to be

a nitrate/proton symporter. Low expression of membrane‐bound nitrate reductase in

narK mutants was counteracted by azide, which induced nitrate reductase expression only if the transcriptional activator NarR was present.
Export of the siderophore enterobactin in Escherichia coli: involvement of a 43 kDa membrane exporter
Authors: Furrer Jason L. , Sanders Douglas N. , Hook‐Barnard India G. , McIntosh Mark A.
Published: March 2003

The enterobactin system for iron transport in Escherichia coli is well characterized
with the exception of the mechanism of enterobactin secretion to the extracellular
environment. Escherichia coli membrane protein P43, encoded by ybdA
in the chromosomal region of genes involved in enterobactin synthesis, shows strong
homology to the 12‐transmembrane segment major facilitator superfamily of export
pumps. A P43‐null mutation was created and siderophore nutrition assays, performed
with a panel of E. coli strains expressing one or more outer membrane receptors
for enterobactin‐related compounds, demonstrated that the P43 mutant was unable to
secrete enterobactin efficiently. Products released from the mutant strain were identified
with thin‐layer chromatography (TLC) and high‐performance liquid chromatography (HPLC),
revealing that the P43 mutant secretes little, if any, enterobactin, but elevated
levels of enterobactin breakdown products 2,3‐ dihydroxybenzoylserine (DHBS) monomer,
dimer, and trimer. These data establish that P43 is a critical component of the E.
coli enterobactin secretion machinery and provides a rationale for the designation
of the previous genetic locus ybdA as entS to reflect its relevant biological function.
A hypermutator phenotype attenuates the virulence of Listeria monocytogenes in a mouse model
Authors: Mérino Delphine , Réglier‐Poupet Hélène , Berche Patrick ,   The European Listeria Genome Consortium, Charbit Alain
Published: March 2003

The integrity of the genetic material of bacteria is guaranteed by a set of distinct repair mechanisms. The participation of these repair systems in bacterial pathogenicity has been addressed only recently. Here, we study for the first time the participation in virulence of the MutSL mismatch repair system of Listeria monocytogenes. The mutS and mutL genes, which are contiguous in the L. monocytogenes chromosome, were identified after in silico analysis. The deduced MutS shares 62% identity with MutS of Bacillus subtilis and 50% identity with HexA, its homologue in Streptococcus pneumoniae; MutL shares 59% identity with MutL of B. subtilis and 47% identity with HexB of S. pneumoniae. Functional analysis of the mutSL locus was studied by constructing a double knock‐out mutant. We showed that the deletion ΔmutSL induces: (i) a 100‐ to 1000‐fold increase in the spontaneous mutation rate; and (ii) a 10‐ to 15‐fold increase in the frequency of transduction, thus demonstrating the role of mutSL of L. monocytogenes in both mismatch repair and homologous recombination. We found that the deletion ΔmutSL moderately affected bacterial virulence, with a 1‐log increase in the lethal dose 50% (LD50) in the mouse. Strikingly, repeated passages of the mutant strain in mice reduced virulence further. Competition assays between wild‐type and mutant strains showed that the deletion ΔmutSL reduced the capacity of L. monocytogenes to survive and multiply in mice. These results thus demonstrate that, for the intracellular pathogen L. monocytogenes, a hypermutator phenotype is more deleterious than profitable to its virulence.
MntR modulates expression of the PerR regulon and superoxide resistance in Staphylococcus aureus through control of manganese uptake
Authors: Horsburgh Malcolm J. , Wharton Stephen J. , Cox Alan G. , Ingham Eileen , Peacock Sharon , Foster Simon J.
Published: March 2003

The Staphylococcus aureus DtxR‐like protein, MntR, controls expression of
the mntABC and mntH genes, which encode putative manganese transporters.
Mutation of mntABC produced a growth defect in metal‐depleted medium and increased
sensitivity to intracellularly generated superoxide radicals. These phenotypes resulted
from diminished uptake of manganese and were rescued by the addition of excess Mn(II).
Resistance to superoxide was incompletely rescued by Mn(II) for STE035 (mntA mntH),
and the strain had reduced virulence in a murine abscess model of infection. Expression
of mntABC was repressed by Mn(II) in an MntR‐dependent manner, which contrasts
with the expression of mntH that was not repressed in elevated Mn(II) and
was decreased in an mntR mutant. This demonstrates that MntR acts as a negative
and positive regulator of these loci re‐spectively. PerR, the peroxide resistance
regulon repressor, acts with MntR to control the expression of mntABC and
manganese uptake. The expression of the PerR‐regulated genes, katA (catalase),
ftn (ferritin) and fur (ferric uptake regulator), was diminished in
STE031 (mntR) when grown in excess Mn(II). Therefore, the control of Mn(II)‐regulated
members of the PerR regulon and the Fur protein is modulated by MntR through its
control of Mn(II) uptake. The co‐ordinated regulation of metal ion homeostasis and
oxidative stress resistance via the regulators MntR, PerR and Fur of S. aureus is discussed.
Replication fork reversal in DNA polymerase III mutants of Escherichia coli: a role for the β clamp
Authors: Grompone Gianfranco , Seigneur Marie , Ehrlich S. Dusko , Michel Bénédicte
Published: March 2003

Certain replication mutations lead in Escherichia coli to a specific reaction
named replication fork reversal: at blocked forks, annealing of the nascent strands
and pairing of the template strands form a four‐way junction. RuvABC‐catalysed resolution
of this Holliday junction causes chromosome double‐strand breaks (DSBs) in a recBC
context and therefore creates a requirement for the recombination proteins RecBC
for viability. In the present work, two mutants were tested for replication fork
reversal: a dnaEts mutant and a dnaNts mutant, affected in the alpha
(polymerase) and beta (processivity clamp) subunits of DNA polymerase III holoenzyme
respectively. In the dnaEts recB strain, RuvABC‐dependent DSBs caused by the
dnaEts mutation occurred at 37°C or 42°C, indicating
the occurrence of replication fork reversal upon partial or complete inactivation
of the DNA polymerase alpha subunit. DSB formation was independent of RecA, RecQ
and the helicase function of PriA. In the dnaNts recB mutant, RuvABC‐dependent
DSB caused by the dnaNts mutation occurred only at semi‐permissive temperature,
37°C, indicating the occurrence of replication fork reversal in conditions
in which the remaining activity of the beta clamp is sufficient for viability. In
contrast, the dnaNts mutation did not cause chromosome breakage at 42°C,
a temperature at which DnaN is totally inactive and the dnaNts mutant is inviable.
We propose that a residual activity of the DNA polymerase III beta clamp is required
for replication fork reversal in the dnaNts mutant.
Mutual dependence of the expression of the cell differentiation regulatory protein HetR and the global nitrogen regulator NtcA during heterocyst development
Authors: Muro‐Pastor Alicia M. , Valladares Ana , Flores Enrique , Herrero Antonia
Published: March 2003

Heterocyst differentiation in the cyanobacterium Anabaena sp. strain PCC
7120 depends on both the global nitrogen regulator NtcA and the cell differentiation
regulatory protein HetR, and induction of hetR upon nitrogen step‐down depends
on NtcA. The use of two out of the four transcription start points (tsps) described
for the hetR gene (those located at positions –728 and –271)
was found to be dependent on NtcA, and the use of the tsp located at position –271
was also dependent on HetR. Thus, autoregulation of hetR could take place
via the activation of transcription from this tsp. Expression of ntcA in nitrogen‐fixing
cultures was higher than in cells growing in the presence of ammonium or nitrate,
and high expression of ntcA under nitrogen deficiency resulted from an increased
use of tsps located at positions –180 and –49. The induction of the
use of these tsps did not take place in ntcA or hetR mutant strains.
These results indicate a mutual dependency in the induction of the regulatory genes
hetR and ntcA that takes place in response to nitrogen step‐down in
Anabaena cells. Expression of the hetC gene, which is also involved in the early steps of heterocyst differentiation, from its NtcA‐dependent tsp was, however, not dependent on HetR.
A single flavoprotein, AppA, integrates both redox and light signals in Rhodobacter sphaeroides
Authors: Braatsch Stephan , Gomelsky Mark , Kuphal Silke , Klug Gabriele
Published: March 2003

Anoxygenic photosynthetic proteobacteria exhibit various light responses, including changing levels of expression of photosynthesis genes. However, the underlying mechanisms are largely unknown. We show that expression of the puf and puc operons encoding structural proteins of the photosynthetic complexes is strongly repressed by blue light under semi‐aerobic growth in Rhodobacter sphaeroides but not in the related species Rhodobacter capsulatus. At very low oxygen tension, puf and puc expression is independent of blue light in both species. Photosynthetic electron transport does not mediate the blue light repression, implying the existence of specific photoreceptors. Here, we show that the flavoprotein AppA is likely to act as the photoreceptor for blue light‐dependent repression during continuous illumination. The FAD cofactor of AppA is essential for the blue light‐dependent sensory transduction of this response. AppA, which is present in R. sphaeroides but not in R. capsulatus, is known to participate in the redox‐dependent control of photosynthesis gene expression. Thus, AppA is the first example of a protein with dual sensing capabilities that integrates both redox and light signals.
Isolation and characterization of capsule structure mutant strains of Cryptococcus neoformans
Authors: Moyrand Frédérique , Klaproth Birgit , Himmelreich Uwe , Dromer Françoise , Janbon Guilhem
Published: March 2003

The capsule of Cryptococcus neoformans is the most obvious virulence factor of this pathogenic yeast. The main capsule constituents are glucuronoxylomannans (GXM). Although several studies have focused on GXM composition and structure, very little is known about their genetics. To elucidate the relationship between the capsule structure and the pathophysiology of the cryptococcosis, genetic screening for mutant strains producing a structurally modified capsule was set up. Using monoclonal antibodies specific for different capsule sugar epitopes, we isolated strains with different mutated capsule structures (Cas mutants). According to their reactivities with various monoclonal antibodies, the mutants were classified into six groups (Cas1 to Cas6). One Cas2 mutant was used to clone the corresponding gene by complementation. This gene (USX1) encodes the previously identified UDP‐xylose synthase. We demonstrated that it is necessary for both capsule xylosylation and C. neoformans virulence.
Natrialba magadii virus φCh1: first complete nucleotide sequence and functional organization of a virus infecting a haloalkaliphilic archaeon
Authors: Klein R. , Baranyi U. , Rössler N. , Greineder B. , Scholz H. , Witte A.
Published: March 2003

The double‐stranded (ds)DNA virus φCh1 infects the haloalkaliphilic archaeon Natrialba magadii. The complete DNA sequence of 58 498 bp of the temperate virus was established, and the probable functions of 21 of 98 φCh1‐encoded open reading frames (ORFs) have been assigned. This knowledge has been used to propose functional modules each required for specific functions during virus development. The φCh1 DNA is terminally redundant and circularly permuted and therefore appears to be packaged by the so‐called headful mechanism. The presence of ORFs encoding homologues of proteins involved in plasmid replication as well as experimental evidence indicate a plasmid‐mediated replication strategy of the virus. Results from nanosequencing of virion components suggest covalent cross‐linking of monomers of at least one of the structural proteins during virus maturation. A comparison of the φCh1 genome with the partly sequenced genome of Halobacterium salinarum virus φH revealed a close relationship between the two viruses, although their host organisms live in distinct environments with respect to the different pH values required for growth.
Dynamic response of catabolic pathways to autoacidification in Lactococcus lactis: transcript profiling and stability in relation to metabolic and energetic constraints
Authors: Even Sergine , Lindley Nic D. , Loubière Pascal , Cocaign‐Bousquet Muriel
Published: March 2003

Summary The dynamic response of the central metabolic pathways to autoacidification (accumulation of organic acid fermentation products) in Lactococcus lactis was investigated in a global manner by integrating molecular data (cellular transcript concentrations, mRNA turnover) within physiological investigations of metabolic and energetic parameters. The decrease in pH associated with the accumulation of organic acids modified the physiological state of the cell considerably. Cytoplasmic acidification led to inhibition of enzyme activities and, consequently, to a diminished catabolic flux through glycolysis and a decreased rate of biochemical energy synthesis. This decrease in energy production together with the increased energy expenditure to counter cytoplasmic acidification led to energetic limitations for biomass synthesis. In these conditions, the specific growth rate decreased progressively, and growth ultimately stopped, although a diminished catabolic flux was maintained in the absence of growth. The cellular response to this phenomenon was to maintain significant levels of mRNA of catabolic genes, involving both continued transcription of the genes and also, in certain cases, an increase in transcript stability. Thus, translation was maintained, and intracellular concentration of certain enzymes increased, partially compensating for the inhibition of activity provoked by the diminished pH. When catabolic activity ceased after prolonged exposure to stress‐induced stationary phase, endogenous RNA catabolism was observed.
SakA MAP kinase is involved in stress signal transduction, sexual development and spore viability in Aspergillus nidulans
Authors: Kawasaki Laura , Sánchez Olivia , Shiozaki Kazuhiro , Aguirre Jesús
Published: March 2003

In eukaryotic cells, environmental stress signals are transmitted by evolutionarily conserved MAPKs, such as Hog1 in the budding yeast Saccharomyces cerevisiae, Spc1 in the fission yeast Schizosaccharomyces pombe and p38/JNK in mammalian cells. Here, we report the identification of the Aspergillus nidulans sakA gene, which encodes a member of the stress MAPK family. The sakA gene is able to complement the S. pombe spc1− defects in both osmo‐regulation and cell cycle progression. Moreover, SakA MAPK is activated in response to osmotic and oxidative stress in both S. pombe and A. nidulans. However, in contrast to hog1 and spc1 mutants, the sakA null mutant is not sensitive to high osmolarity stress, indicating a different regulation of the osmostress response in this fungus. On the other hand, the ΔsakA mutant shows development and cell‐specific phenotypes. First, it displays premature steA‐dependent sexual development. Second, ΔsakA mutant produces asexual spores that are highly sensitive to oxidative and heat shock stress and lose viability upon storage. Indeed, SakA is transiently activated early after induction of conidiation. Our results indicate that SakA MAPK is involved in stress signal transduction and repression of sexual development, and is required for spore stress resistance and survival.
OhrR, a transcription repressor that senses and responds to changes in organic peroxide levels in Xanthomonas campestris pv. phaseoli
Authors: Panmanee Warunya , Vattanaviboon Paiboon , Eiamphungporn Warawan , Whangsuk Wirongrong , Sallabhan Ratiboot , Mongkolsuk Skorn
Published: March 2003

We report the physiological role of OhrR as an organic peroxide sensor and transcription repressor in Xanthomonas campestris pv. phaseoli. In vivo exposure of X. campestris pv. phaseoli to either tert‐butyl or cumene hydroperoxides efficiently neutralized OhrR repression of expression from the OhrR‐regulated P1 promoter. H2O2 was a weak and non‐physiological inducer of the system while other oxidants and metabolites of organic peroxide metabolism did not induce the expression from the P1. Northern blotting results indicated a correlation between concentrations of tert‐butyl hydroperoxide used in the treatment and the induction of ohr (an OhrR‐regulated gene) expression. In addition, the levels of ohr mRNA in cultures induced by various concentrations of tert‐butyl hydroperoxide were reduced in cells with high levels of an organic peroxide metabolising enzyme (AhpC‐AhpF) but not in cells with high catalase levels suggesting that organic peroxide interacts with OhrR. DNA band shift experiments using purified OhrR and the P1 promoter fragment showed that organic peroxide treatment prevented binding of the protein to the P1 promoter by oxidation of OhrR, as the inhibition of binding to the P1 promoter was reversed by addition of a reducing agent, DTT. The highly conserved cysteine residue C22 of OhrR is required for organic peroxide inducible gene expression. A mutant protein, OhrRC22S can repress the P1 promoter activity but is insensitive to organic peroxide treatment. Thus, OhrR is the first transcription repressor characterized that appeared to evolve to physiologically sense organic peroxides.
MetR and CRP bind to the Vibrio harveyi lux promoters and regulate luminescence
Authors: Chatterjee Jaidip , Miyamoto Carol M. , Zouzoulas Athina , Lang B. Franz , Skouris Nicolas , Meighen Edward A.
Published: March 2003

The induction of luminescence in Vibrio harveyi at the later stages of growth is controlled by a quorum‐sensing mechanism in addition to nutritional signals. However, the mechanism of transmission of these signals directly to the lux promoters is unknown and only one regulatory protein, LuxR, has been shown to bind directly to lux promoter DNA. In this report, we have cloned and sequenced two genes, crp and metR, coding for the nutritional regulators, CRP (cAMP receptor protein) and MetR (a LysR homologue), involved in catabolite repression and methionine biosynthesis respectively. The metR gene was cloned based on a general strategy to detect lux DNA‐binding proteins expressed from a genomic library, whereas the crp gene was cloned based on its complementation of an Escherichia coli crp mutant. Both CRP and MetR were shown to bind to lux promoter DNA, with CRP being dependent on the presence of cAMP. Expression studies indicated that the two regulators had opposite effects on luminescence: CRP was an activator and MetR a repressor. Disruption of crp decreased luminescence by about 1000‐fold showing that CRP is a major activator of luminescence the same as LuxR, whereas disruption of MetR resulted in activation of luminescence over 10‐fold, confirming its function as a repressor. Comparison of the levels of the autoinducers involved in quorum sensing excreted by V. harveyi, and the crp and metR mutants, showed that autoinducer production was not significantly different, thus indicating that the nutritional signals do not affect luminescence by changing the levels of the signals required for quorum sensing. Indeed, the large effects of these nutritional sensors show that luminescence is controlled by multiple signals related to the environment and the cell density which must be integrated at the molecular level to control expression at the lux promoters.
Physiological role of the GlnK signal transduction protein of Escherichia coli: survival of nitrogen starvation
Authors: Blauwkamp Timothy A. , Ninfa Alexander J.
Published: March 2003

Escherichia coli
contains two PII‐like signal trans‐duction proteins, PII and GlnK, involved in nitrogen assimilation. We examined the roles of PII and GlnK in controlling expression of
glnALG
,
glnK
and
nac
during the transition from growth on ammonia to nitrogen starvation and vice versa. The PII protein exclusively controlled
glnALG
expression in cells adapted to growth on ammonia, but was unable to limit
nac
and
glnK
expression under conditions of nitrogen starvation. Conversely, GlnK was unable to limit
glnALG
expression in cells adapted to growth on ammonia, but was required to limit expression of the
glnK
and
nac
promoters during nitrogen starvation. In the absence of GlnK, very high expression of the
glnK
and
nac
promoters occurred in nitrogen‐starved cells, and the cells did not reduce
glnK
and
nac
expression when given ammonia. Thus, one specific role of GlnK is to regulate the expression of Ntr genes during nitrogen starvation. GlnK also had a dramatic effect on the ability of cells to survive nitrogen starvation and resume rapid growth when fed ammonia. After being nitrogen starved for as little as 10 h, cells lacking GlnK were unable to resume rapid growth when given ammonia. In contrast, wild‐type cells that were starved immediately resumed rapid growth when fed ammonia. Cells lacking GlnK also showed faster loss of viability during extended nitrogen starvation relative to wild‐type cells. This complex phenotype resulted partly from the requirement for GlnK to regulate
nac
expression; deletion of
nac
restored wild‐type growth rates after ammonia starvation and refeeding to cells lacking GlnK, but did not improve viability during nitrogen starvation. The specific roles of GlnK during nitrogen starvation were not the result of a distinct function of the protein, as expression of PII from the
glnK promoter in cells lacking GlnK restored the wild‐type phenotypes.
A family of autocrine growth factors in Mycobacterium tuberculosis
Authors: Mukamolova Galina V. , Turapov Obolbek A. , Young Danielle I. , Kaprelyants Arseny S. , Kell Douglas B. , Young Michael
Published: March 2003

Mycobacterium tuberculosis and its close relative, Mycobacterium bovis (BCG) contain five genes whose predicted products resemble Rpf from Micrococcus luteus. Rpf is a secreted growth factor, active at picomolar concentrations, which is required for the growth of vegetative cells in minimal media at very low inoculum densities, as well as the resuscitation of dormant cells. We show here that the five cognate proteins from M. tuberculosis have very similar characteristics and properties to those of Rpf. They too stimulate bacterial growth at picomolar (and in some cases, subpicomolar) concentrations. Several lines of evidence indicate that they exert their activity from an extra‐cytoplasmic location, suggesting that they are also involved in intercellular signalling. The five M. tuberculosis proteins show cross‐species activity against M. luteus, Mycobacterium smegmatis and M. bovis (BCG). Actively growing cells of M. bovis (BCG) do not respond to these proteins, whereas bacteria exposed to a prolonged stationary phase do. Affinity‐purified antibodies inhibit bacterial growth in vitro, suggesting that sequestration of these proteins at the cell surface might provide a means to limit or even prevent bacterial multiplication in vivo. The Rpf family of bacterial growth factors may therefore provide novel opportunities for preventing and controlling mycobacterial infections.
A high‐molecular‐weight outer membrane protein of Xanthomonas oryzae pv. oryzae exhibits similarity to non‐fimbrial adhesins of animal pathogenic bacteria and is required for optimum virulence
Authors: Ray Suvendra K. , Rajeshwari R. , Sharma Yogendra , Sonti Ramesh V.
Published: March 2003

Transposon insertions in a novel 3.798 kb open reading frame (ORF) of the rice pathogen, Xanthomonas oryzae pv. oryzae (Xoo) cause virulence deficiency and altered colony/lawn morphology. This ORF encodes a protein, XadA, of 1265 amino acids that exhibits significant similarity to non‐fimbrial adhesins of animal pathogenic bacteria such as Yersinia YadA and Moraxella UspA1. An interesting feature is that the YadA similarity region is repeated six times within the XadA sequence and encompasses almost the entire length of the protein. Anti‐XadA antibodies identified a 110 kDa outer membrane protein that was sensitive to protease treatment of whole cells. XadA expression is induced in minimal medium. Homology modelling suggests that XadA adopts a β‐helix conformation‐like pertactin, a non‐fimbrial adhesin of Bordetella pertussis. This work is the first characterization of a non‐fimbrial adhesin‐like molecule in a plant pathogenic bacterium. It extends our knowledge about the repertoire of homologous virulence factors that are deployed by animal and plant pathogenic bacteria to include functions potentially involved in adhesion.
Selection of plasmid molecules for conjugative transfer and replacement strand synthesis in the donor
Authors: Parker Christopher , Meyer Richard J.
Published: March 2003

Plasmid selection and strand replacement synthesis in donor cells during conjugative transfer was examined by a procedure involving electroporation of test plasmid DNA, containing a base pair mismatch, into donor cells prior to mating. Multiple copies of the plasmid were transferred from a donor cell that allowed vegetative replication of the plasmid. Under conditions non‐permissive for vegetative replication, there were further rounds of transfer after a lag period. Strand replacement in the donor did not depend solely on the initiation mechanism for vegetative replication, indicating a conjugation‐specific mechanism was also available. The lag period between first and second rounds of transfer argues against the transfer of multiple copies into recipients by the spooling of copies generated on a master molecule by rolling‐circle replication.
Knotting dynamics during DNA replication
Authors: Olavarrieta L. , Martínez‐Robles M. L. , Hernández P. , Krimer D. B. , Schvartzman J. B.
Published: March 2003

The topology of plasmid DNA changes continuously as replication progresses. But the dynamics of the process remains to be fully understood. Knotted bubbles form when topo IV knots the daughter duplexes behind the fork in response to their degree of intertwining. Here, we show that knotted bubbles can form during unimpaired DNA replication, but they become more evident in partially replicated intermediates containing a stalled fork. To learn more about the dynamics of knot formation as replication advances, we used two‐dimensional agarose gel electrophoresis to identify knotted bubbles in partially replicated molecules in which the replication fork stalled at different stages of the process. The number and complexity of knotted bubbles rose as a function of bubble size, suggesting that knotting is affected by both precatenane density and bubble size.
Interaction of EnvZ, a sensory histidine kinase, with phosphorylated OmpR, the cognate response regulator
Authors: Yoshida Takeshi , Cai Sheng jian , Inouye Masayori
Published: March 2003

EnvZ is a sensory histidine kinase in Escherichia coli to regulate the phosphorylation of OmpR, its cognate response regulator, required for the expression of genes for outer membrane porin proteins. Here, we re‐examined the recent paper Mattison and Kenney, in which the authors reported that phosphorylated OmpR (OmpR‐P) is unable to bind to EnvZ, thus casting doubts on the role of the EnvZ phosphatase activity in vivo. Using an identical method, the Kd value for the interaction of the fluorescein‐labelled OmpR (Fl‐OmpR) with EnvZc was determined to be 1.96 ± 0.28 µM. We demonstrated that OmpR‐P as well as OmpR inhibited the interaction of Fl‐OmpR with EnvZc. Their 50% inhibitory concentrations were 1.09 ± 0.25 µM and 0.89 ± 0.14 µM, respectively, under the conditions used. The interaction between His‐10‐OmpR and EnvZc was also inhibited almost equally with OmpR‐P and OmpR. Fluorescein labelling of OmpR was highly heterogeneous as detected by mass spectrometry, even though it slightly affected the OmpR phosphorylation (kinase) and the dephosphorylation of OmpR‐P (phosphatase), indicating that EnvZc is able to interact with Fl‐OmpR or Fl‐OmpR‐P as well as with OmpR or OmpR‐P as a substrate. We demonstrated that OmpR‐P is able to interact with EnvZc with a similar affinity to OmpR and serves as an effective substrate for the EnvZ phosphatase. These findings support the hypothesis that osmotic signals regulate the level of the cellular concentration of OmpR‐P by modulating the ratio of kinase to phosphatase activity of the bifunctional enzymatic activities of EnvZ.
Activation of 6‐phosphofructokinase via phosphorylation by Pkn4, a protein Ser/Thr kinase of Myxococcus xanthus
Authors: Nariya Hirofumi , Inouye Sumiko
Published: March 2003

Myxococcus xanthus is a Gram‐negative bacterium that exhibits a communal lifestyle during vegetative growth and multicellular development, forming fruiting bodies filled with spores. It contains at least 13 eukaryotic‐like protein Ser/Thr kinases (PSTKs from Pkn1 to Pkn13). In the present report, we demonstrate that Pkn4, the gene located 18 bp downstream of the gene for 6‐phosphofructokinase (PFK), is a PSTK for M. xanthus PFK (Mx‐PFK), the key regulatory enzyme in glycolysis. Both Pkn4 and Mx‐PFK were expressed in Escherichia coli and purified. Mx‐PFK was found to be phosphorylated by Pkn4 at Thr‐226, which is presumed to be located in the allosteric effector site of the PFK. The phosphorylation of Mx‐PFK enhanced its activity 2.7‐fold, indicating that Pkn4 plays an important role in glucose metabolism. Although PFKs from other organisms are known to be tetrameric enzymes, Mx‐PFK is composed of an octamer and is dissociated to tetramers in the presence of phosphoenolpyruvate (PEP), an allosteric inhibitor for PFK. Furthermore, phosphorylation of PFK by Pkn4 is almost completely inhibited by PEP. Mx‐PFK is associated with the regulatory domain of Pkn4, and this association is inhibited by PEP. This is the first demonstration that a prokaryotic PFK is regulated by phosphorylation by PSTK in prokaryotes.
Proteome analysis of Escherichia coli K‐12 by two‐dimensional native‐state chromatography and MALDI‐MS
Authors: Champion Matthew M. , Campbell Christopher S. , Siegele Deborah A. , Russell David H. , Hu James C.
Published: March 2003

To identify proteins expressed in Escherichia coli K‐12 MG1655 during exponential growth in defined medium, we separated soluble proteins of E. coli over two dimensions of native‐state high‐performance liquid chromatography, and examined the components of the protein mixtures in each of 380 fractions by peptide mass fingerprinting. To date, we have identified the products of 310 genes covering a wide range of cellular functions. Validation of protein assignments was made by comparing the assignments of proteins to specific first‐dimension fractions to proteins visualized by two‐dimensional gel electrophoresis. Co‐fractionation of proteins suggests the possible identities of components of multiprotein complexes. This approach yields high‐throughput gel‐independent identification of proteins. It can also be used to assign identities to spots visualized by two‐dimensional gels, and should be useful to evaluate differences in expressed proteome content and protein complexes among strains or between different physiological states.
Differential analysis of DNA microarray gene expression data
Authors: Hatfield G. Wesley , Hung She‐pin , Baldi Pierre
Published: March 2003

Here, we review briefly the sources of experimental and biological variance that affect the interpretation of high‐dimensional DNA microarray experiments. We discuss methods using a regularized t‐test based on a Bayesian statistical framework that allow the identification of differentially regulated genes with a higher level of confidence than a simple t‐test when only a few experimental replicates are available. We also describe a computational method for calculating the global false‐positive and false‐negative levels inherent in a DNA microarray data set. This method provides a probability of differential expression for each gene based on experiment‐wide false‐positive and ‐negative levels driven by experimental error and biological variance.
Penicillin‐binding protein PBP2 of Escherichia coli localizes preferentially in the lateral wall and at mid‐cell in comparison with the old cell pole
Authors: Den Blaauwen Tanneke , Aarsman Mirjam E. G. , Vischer Norbert O. E. , Nanninga Nanne
Published: March 2003

The localization of penicillin‐binding protein 2 (PBP2) in Escherichia coli has been studied using a functional green fluorescent protein (GFP)–PBP2 fusion protein. PBP2 localized in the bacterial envelope in a spot‐like pattern and also at mid‐cell during cell division. PBP2 disappeared from mid‐cell just before separation of the two daughter cells. It localized with a preference for the cylindrical part of the bacterium in comparison with the old cell poles, which are known to be inert with respect to peptidoglycan synthesis. In contrast to subunits of the divisome, PBP2 failed to localize at mid‐cell when PBP3 was inhibited by the specific antibiotic aztreonam. Therefore, despite its dependency on active PBP3 for localization at mid‐cell, it seems not to be an integral part of the divisome. Cells grown for approximately half a mass doubling time in the presence of the PBP2 inhibitor mecillinam synthesized nascent cell poles with an increased diameter, indicating that PBP2 is required for the maintenance of the correct diameter of the new cell pole.
A new Escherichia coli metabolic competency: growth on fatty acids by a novel anaerobic β‐oxidation pathway
Authors: Campbell John W. , Morgan‐Kiss Rachael M. , E. Cronan John
Published: March 2003

Escherichia coli
uses fatty acids as a sole carbon and energy source during aerobic growth by means of the enzymes encoded by the
fad
regulon. We report that this bacterium can also grow on fatty acids under anaerobic conditions provided that a terminal respiratory electron acceptor such as nitrate is available. This anaerobic utilization pathway is distinct from the well‐studied aerobic pathway in that (i) it proceeds normally in mutant strains lacking various enzymes of the aerobic pathway; (ii) it functions with fatty acids (octanoate and decanoate) that cannot be used by wild‐type
E. coli
strains under aerobic conditions; and (iii) super‐repressor mutants of the
fadR
regulatory locus that block aerobic growth on fatty acids fail to block the anaerobic pathway. We have identified homologues of the FadA, FadB and FadD proteins required for aerobic fatty acid utilization called YfcY, YfcX and YdiD, respectively, which are involved in anaerobic growth on fatty acids. A strong FadR binding site was detected upstream of the
yfcY
gene consistent with microarray analyses, indicating that
yfcYX
expression is negatively regulated by FadR under aerobic growth conditions. In contrast, transcriptional regulation of
ydiD
appears to be independent of FadR, and anaerobic growth on fatty acids is not under FadR control. These three genes are conserved in the available genome sequences of pathogenic
E. coli
,
Shigella
and
Salmonella strains.
An extended role for the nucleoid structuring protein H‐NS in the virulence gene regulatory cascade of Shigella flexneri
Authors: Beloin Christophe , Dorman Charles J.
Published: March 2003

The H‐NS nucleoid structuring protein has been shown previously to play a negative role in controlling virulence gene expression in Shigella flexneri by repressing transcription of the virF and virB regulatory genes and the VirF‐dependent icsA structural gene under non‐permissive growth conditions. Here, we show that H‐NS also acts at the promoters of the VirB‐dependent structural genes in the regulatory cascade. H‐NS protein binds to the promoter regions in vivo and in vitro. The promoters were shown physically and by in silico analysis to contain regions of DNA curvature, a feature of H‐NS binding sites. H‐NS binding sites were determined by DNase I footprinting at the icsB and the virA promoters. The locations of these sites were consistent with a role for H‐NS as a transcription repressor. The VirB‐dependent structural gene promoters were found to respond directly to the H‐NS repressor, revealing a level of control that is additional to that exerted by the H‐NS‐dependent virB activator gene. Moreover, the promoters were sensitive to the level of VirB protein in the cell, requiring a threshold level of VirB to be reached before becoming active. A model is discussed in which the levels of expression of the structural genes reflect the outcome of competition between the countervailing regulatory activities of the H‐NS and VirB proteins.
Linear plasmid SLP2 of Streptomyces lividans is a composite replicon
Authors: Huang Chih‐Hung , Chen Chung‐Yung , Tsai Hsiu‐Hui , Chen Chi , Lin Yi‐Shing , Chen Carton W.
Published: March 2003

SLP2 is a 50 kb linear plasmid in Streptomyces lividans that contains short (44 bp) terminal inverted repeats and covalently bound terminal proteins. The nucleotide sequence of SLP2 was determined. The  rightmost 15.4 kb sequence is identical to that of the host chromosome, including the Tn4811 sequence at the border, which is interrupted by an insertion sequence (IS) element in SLP2. Examination of the flanking target sequences of Tn4811 suggests a previous recombinational event there. The 43 putative protein coding sequences contained many involved in replication (including two terminal protein homologues), partitioning, conjugal transfer and intramycelial spread. The terminally located helicase‐like gene ttrA was necessary for conjugal transfer. The two telomeres diverge significantly in primary sequence, while preserving similar secondary structures. Mini‐linear plasmids containing these telomeres replicated in S. lividans using the chromosomally encoded terminal protein. In addition, two pseudotelomere sequences are present near the left telomere. The G+C content and GC or AT skew profiles exhibit complex distributions. These, plus the inferred recombination at the right arm, indicate that SLP2 has evolved through rounds of exchanges involving at least three replicons.
Transcriptome analysis of Listeria monocytogenes identifies three groups of genes differently regulated by PrfA
Authors: Milohanic Eliane , Glaser Philippe , Coppée Jean‐Yves , Frangeul Lionel , Vega Yolanda , Vázquez‐Boland José A. , Kunst Frank , Cossart Pascale , Buchrieser Carmen
Published: March 2003

PrfA is the major regulator of Listeria virulence gene expression. This protein is a member of the Crp/Fnr family of transcription regulators. To gain a deeper understanding of the PrfA regulon, we constructed a whole‐genome array based on the complete genome sequence of Listeria monocytogenes strain EGDe and evaluated the expression profiles of the wild‐type EGDe and a prfA‐deleted mutant (EGDe ΔprfA). Both strains were grown at 37°C in brain–heart infusion broth (BHI) and BHI supplemented with either activated charcoal, a compound known to enhance virulence gene expression, or cellobiose, a sugar reported to downregulate virulence gene expression in spite of full expression of PrfA. We identified three groups of genes that are regulated differently. Group I comprises, in addition to the 10 already known genes, two new genes, lmo2219 and lmo0788, both positively regulated and preceded by a putative PrfA box. Group II comprises eight negatively regulated genes: lmo0278 is preceded by a putative PrfA box, and the remaining seven genes (lmo0178–lmo0184) are organized in an operon. Group III comprises 53 genes, of which only two (lmo0596 and lmo2067) are preceded by a putative PrfA box. Charcoal addition induced upregulation of group I genes but abolished regulation by PrfA of most group III genes. In the presence of cellobiose, all the group I genes were downregulated, whereas group III genes remained fully activated. Group II genes were repressed in all conditions tested. A comparison of the expression profiles between a second L. monocytogenes strain (P14), its spontaneous mutant expressing a constitutively active PrfA variant (P14prfA*) and its corresponding prfA‐deleted mutant (P14ΔprfA) and the EGDe strain revealed interesting strain‐specific differences. Sequences strongly similar to a sigma B‐dependent promoter were identified upstream of 22 group III genes. These results suggest that PrfA positively regulates a core set of 12 genes preceded by a PrfA box and probably expressed from a sigma A‐dependent promoter. In contrast, a second set of PrfA‐regulated genes lack a PrfA box and are expressed from a sigma B‐dependent promoter. This study reveals that PrfA can act as an activator or a repressor and suggests that PrfA may directly or indirectly activate different sets of genes in association with different sigma factors.
Presence of putative neurotransmitters in the myenteric plexus of the gastrointestinal tract and in the musculature of the urinary bladder of the ferret
Authors: SANN  , HOPPE  , BALDWIN  , GRUNDY  , SCHEMANN
Published: March 2003

The innervation of the musculature in the ferret stomach, ileum, colon and urinary bladder was investigated using immunohistochemistry in noncolchicin‐treated tissues. In the gastrointestinal tract two main subpopulations of myenteric neurones were found: cholinergic neurones expressing choline acetyltransferase (ChAT), which made up 68, 67 and 67% of the neurones in the stomach, ileum and colon, respectively, and nitrergic neurones containing nitric oxide synthase and NADPH‐diaphorase (stomach: 23%, ileum: 21%, colon: 26%). In the stomach, cholinergic neurones expressed substance P (SP, 2% of all neurones), dopamine‐β‐hydroxylase (DBH, 19%) but not tyrosine hydroxylase (TH) or vasoactive intestinal polypeptide (VIP), while nitrergic neurones contained VIP and neuropeptide Y (NPY). TH‐ but not DBH‐immunoreactivity was observed in 4% of gastric neurones. Intense immunoreactivity in the musculature suggests that part of ChAT/SP‐ and NOS/NPY/VIP‐positive neurones function as motorneurones. In the ileum, a high number (32%) of DBH‐positive neurones was demonstrated. About half of the SP‐positive neurones in the ileum also contained calcitonin gene‐related peptide (CGRP). In the urinary bladder, only few intramural ganglia were observed. The smooth muscle was densely innervated by ChAT, NPY and DBH immunoreactive fibres. The data showed that the innervation of the ferret viscera exhibited similarities but also differences as compared with other mammalian species. Some of the chemical coding of myenteric neurones is remarkably similar to that observed in other mammals.
Isopower maps of the electrogastrogram (EGG) after total gastrectomy or total colectomy
Authors: Homma  , Hasegawa  , Maruta  , Watanabe  , Matsuo  , Tamiya  , Nishimaki  , Suzuki  , Muto  , Hatakeyama
Published: March 2003

Isopower or topographic electrogastrograms (EGG) correspond to topographic electroencephalograms. Both project the topographic localizations of the spectral frequencies on the abdominal surface or scalp. This paper compares the pre‐operative control isopower EGG maps with those of total gastrectomy or total colectomy.

EGGs were recorded simultaneously at 27 locations on the epigastro‐abdominal surface. Spectral analysis by the maximal entropy method (MEM) was performed and the ensemble means of pre‐prandial and post‐prandial spectra were calculated. The spectral frequencies were arbitrarily classified into five groups, 1 cycle per minute (cpm) (0–2.4 cpm), 3 cpm (2.5–4.9 cpm), 6 cpm (5.0–7.4 cpm), 8 cpm (7.5–9.9 cpm) and 10 cpm (10.0–12.9 cpm). Maximal power peaks in each spectral group, and electrode locations which were expressed by x–y coordinates were the indicators for making the isopower EGG maps by using a contour map program. Thereafter, the maximal power spots or foci in each spectral group were determined. The pre‐operative maximal power foci of the 1, 8 and 10 cpm groups were distributed rather evenly on the epigastro‐abdominal surface. Those of the 3 and 6 cpm groups, mainly concentrated in the epigastric region, were absent in almost all patients who had undergone total gastrectomy. The infra‐umbilical foci of the 3 and 6 cpm groups completely disappeared after total colectomy. The infra‐umbilical foci of the 3 and 6 cpm groups (2.5–7.4) may reflect the colonic activities and the epigastric 3 cpm foci, the gastric actvities. The pre‐operative maximal power of the 3 cpm foci decreased significantly after total or sub‐total gastrectomy.
Twenty‐four hour ambulatory antroduodenal manometry in normal subjects (co‐operative study)
Authors: Bortolotti M. , Annese V. , Coccia G.
Published: March 2003

Circadian antroduodenal motor activity was studied in 40 normal subjects by means of a portable recording system consisting of a computerized data logger and a probe with microtransducers. The quantitative and qualitative characteristics of contraction events during the interdigestive and digestive periods, as well as during the awake and asleep periods, were analysed. The composition and timing of meals and night recumbence were standardized. In spite of the high interindividual variability in motor parameters, significant differences in the characteristics of interdigestive and digestive periods between waking and sleep states were found. This paper confirms the existence of a circadian variation in antroduodenal motor activity and provides reference values from a large series of normal subjects that can be used for statistical comparisons with those obtained from patients recorded with the same method.