bluuebunny/crossref_metadata_2025 huggingface dataset:
H‐NS‐dependent activation of pectate lyases synthesis in the phytopathogenic bacterium Erwinia chrysanthemi is mediated by the PecT repressor
Authors: Nasser William , Reverchon Sylvie
Published: March 2003

Production of the main virulence determinant pectate lyases (Pels) of the phytopathogenic bacterium Erwinia chrysanthemi is modulated by a complex regulatory network involving the repressor proteins KdgR, PecS and PecT and the activator systems Pir, ExpI–ExpR and CRP. Of these regulators, CRP and PecT are particularly important since the absence of CRP or a slight overproduction of PecT leads to a drastic reduction in synthesis of Pel species. Recently, it has been shown that production of Pel species is strongly reduced in an E. chrysanthemi hns mutant, suggesting an activator function of the nucleoid‐associated protein H‐NS in the expression of the pel genes. Here, we report that the reduced synthesis of Pel species in the hns mutant results from a negative control, exerted by H‐NS, on the transcription of the regulatory gene pecT. This H‐NS/PecT cascade regulation is one of the first elucidations of a positive effect of H‐NS on target gene expression. Moreover, we found that H‐NS also represses the expression of expI, expR and pel genes. H‐NS control is the result of H‐NS binding to extended regions within the pecT, expI, expR and pel genes. Investigation of the simultaneous binding of CRP, RNA polymerase (RNAP) and H‐NS on the pelD gene revealed that these three proteins form a nucleoprotein com‐plex. Together, these data indicate that, by exerting a negative control at multiple levels, H‐NS plays a crucial role in the E. chrysanthemi pel regulatory network.
The alternative sigma factor σE controls antioxidant defences required for Salmonella virulence and stationary‐phase survival
Authors: Testerman Traci L. , Vazquez‐Torres Andrés , Xu Yisheng , Jones‐Carson Jessica , Libby Stephen J. , Fang Ferric C.
Published: March 2003

Bacteria must contend with conditions of nutrient limitation in all natural environments. Complex programmes of gene expression, controlled in part by the alternative sigma factors σS (σ38, RpoS) and σH (σ32, RpoH), allow a number of bacterial species to survive conditions of partial or complete starvation. We show here that the alternative sigma factor σE (σ24, RpoE) also facilitates the survival of Salmonella typhimurium under conditions of nutrient deprivation. Expression of the σE regulon is strongly induced upon entry of Salmonella into stationary phase. A Salmonella mutant lacking σE has reduced survival during stationary phase as well as increased susceptibility to oxidative stress. A Salmonella strain lacking both σE and σS is non‐viable after just 24 h in stationary phase, but survival of these mutants is completely preserved under anaerobic stationary‐phase conditions, suggesting that oxidative injury is one of the major mechanisms of reduced microbial viability during periods of nutrient deprivation. Moreover, the attenuated virulence of σE‐deficient Salmonella for mice can be largely restored by genetic abrogation of the host phagocyte respiratory burst, suggesting that the σE regulon plays an important antioxidant role during Salmonella infection of mammalian hosts.
Role of thioredoxins in the response of Saccharomyces cerevisiae to oxidative stress induced by hydroperoxides
Authors: Garrido Ester Ocón , Grant Chris M.
Published: March 2003

Glutaredoxins and thioredoxins are highly conserved, small, heat‐stable oxidoreductases. The yeast Saccharomyces cerevisiae contains two gene pairs encoding cytoplasmic

glutaredoxins (GRX1, GRX2) and thioredoxins (TRX1, TRX2),

and we have used multiple mutants to determine their roles in medi‐ating resistance

to oxidative stress caused by hydroperoxides. Our data indicate that TRX2

plays the predominant role, as mutants lacking TRX2 are hypersensitive, and

mutants containing TRX2 are resistant to these oxidants. However, the requirement

for TRX2 is only apparent during stationary phase growth, and we present three

lines of evidence that the thioredoxin isoenzymes actually have redundant activities

as antioxidants. First, the trx1 and trx2 mutants show wild‐type resistance

to hydroperoxide during exponential phase growth; secondly, overexpression of either

TRX1 or TRX2 leads to increased resistance to hydroperoxides; and,

thirdly, both Trx1 and Trx2 are equally able to act as cofactors for the thioredoxin

peroxidase, Tsa1. The antioxidant activity of thioredoxins is required for both the

survival of yeast cells as well as protection against oxidative stress during stationary

phase growth, and correlates with an increase in the expression of both TRX1

and TRX2. We show that the requirement for thioredoxins during this growth phase is dependent on their activity as cofactors for the antioxidant enzyme Tsa1, and for regulation of the redox state and protein‐bound levels of the low‐molecular‐weight antioxidant glutathione.
Behaviour of topological marker proteins targeted to the Tat protein transport pathway
Authors: Stanley Nicola R. , Sargent Frank , Buchanan Grant , Shi Jiarong , Stewart Valley , Palmer Tracy , Berks Ben C.
Published: March 2003

The Escherichia coli Tat system mediates Sec‐independent export of protein
precursors bearing twin arginine signal peptides. Formate dehydrogenase‐N is a three‐subunit
membrane‐bound enzyme, in which localization of the FdnG subunit to the membrane
is Tat dependent. FdnG was found in the periplasmic fraction of a mutant lacking
the membrane anchor subunit FdnI, confirming that FdnG is located at the periplasmic
face of the cytoplasmic membrane. However, the phenotypes of gene fusions between
fdnG and the subcellular reporter genes phoA (encoding alkaline phosphatase)
or lacZ (encoding β‐galactosidase) were the opposite of those expected for analogous fusions targeted to the Sec translocase. PhoA fusion experiments have previously been used to argue that the peripheral membrane DmsAB subunits of the Tat‐dependent enzyme dimethyl sulphoxide reductase are located at the cytoplasmic face of the inner membrane. Biochemical data are presented that instead show DmsAB to be at the periplasmic side of the membrane. The behaviour of reporter proteins targeted to the Tat system was analysed in more detail. These data suggest that the Tat and Sec pathways differ in their ability to transport heterologous passenger proteins. They also suggest that caution should be observed when using subcellular reporter fusions to determine the topological organization of Tat‐dependent membrane protein complexes.
Antitermination by GlpP, catabolite repression via CcpA and inducer exclusion triggered by P~GlpK dephosphorylation control Bacillus subtilis glpFK expression
Authors: Darbon Emmanuelle , Servant Pascale , Poncet Sandrine , Deutscher Josef
Published: March 2003

The Bacillus subtilis glpFK operon encoding the glycerol transport facilitator (GlpF) and glycerol kinase (GlpK) is induced by glycerol‐3‐P and repressed by rapidly metabolizable sugars. Carbon catabolite repression (CCR) of glpFK is partly mediated via a catabolite response element cre preceding glpFK. This operator site is recognized by the catabolite control protein A (CcpA) in complex with one of its co‐repressors, P‐Ser‐HPr or P‐Ser‐Crh. HPr is a component of the phosphoenolpyruvate:sugar phos‐photransferase system (PTS), and Crh is an HPr homologue. The hprK‐encoded HPr kinase phosphorylates HPr and Crh at Ser‐46. But in neither ccpA nor hprK mutants was expression of a glpF′–lacZ fusion relieved from CCR, as a second, CcpA‐independent CCR mechanism implying the terminator tglpFK, whose formation is prevented by the glycerol‐3‐P‐activated antiterminator GlpP, is operative. Deletion of tglpFK led to elevated expression of the glpF′–lacZ fusion and to partial relief from CCR. CCR completely disappeared in ΔtglpFK mutants carrying a disruption of ccpA or hprK. The tglpFK‐requiring CCR mechanism seems to be based on insufficient synthesis of glycerol‐3‐P, as CCR of glpFK was absent in ccpA mutants growing on glycerol‐3‐P or synthesizing H230R mutant GlpK. In cells growing on glycerol, glucose prevents the phosphorylation of GlpK by P~His‐HPr. P~GlpK is much more active than GlpK, and the absence of P~GlpK formation in ΔptsHI strains prevents glycerol metabolism. As a consequence, only small amounts of glycerol‐3‐P will be formed in glycerol and glucose‐exposed cells (inducer exclusion). The uptake of glycerol‐3‐P via GlpT provides high concentrations of this metabolite in the ccpA mutant and allows the expression of the glpF′–lacZ fusion even when glucose is present. Similarly, despite the presence of glucose, large amounts of glycerol‐3‐P are formed in a glycerol‐exposed strain synthesizing GlpKH230R, as this mutant GlpK is as active as P~GlpK.
Truncation of merozoite surface protein 3 disrupts its trafficking and that of acidic–basic repeat protein to the surface of Plasmodium falciparum merozoites
Authors: Mills Kerry E. , Pearce J. Andrew , Crabb Brendan S. , Cowman Alan F.
Published: March 2003

Merozoite surface protein 3 (MSP3), an important vaccine candidate, is a soluble polymorphic antigen associated with the surface of Plasmodium falciparum merozoites. The MSP3 sequence contains three blocks of heptad repeats that are consistent with the formation of an intramolecular coiled‐coil. MSP3 also contains a glutamic acid‐rich region and a putative leucine zipper sequence at the C‐terminus. We have disrupted the msp3 gene by homologous recombination, resulting in the expression of a truncated form of MSP3 that lacks the putative leucine zipper sequence but retains the glutamic acid‐rich region and the heptad repeats. Here, we show that truncated MSP3, lacking the putative leucine zipper region, does not localize to the parasitophorous vacuole or interact with the merozoite surface. Furthermore, the acidic–basic repeat antigen (ABRA), which is present on the merozoite surface, also was not localized to the merozoite surface in parasites expressing the truncated form of MSP3. The P. falciparum merozoites lacking MSP3 and ABRA on the surface show reduced invasion into erythrocytes. These results suggest that MSP3 is not absolutely essential for blood stage growth and that the putative leucine zipper region is required for the trafficking of both MSP3 and ABRA to the parasitophorous vacuole.
Selective expression of the β‐subunit of nucleoid‐associated protein HU during cold shock in Escherichia coli
Authors: Giangrossi Mara , Giuliodori Anna Maria , Gualerzi Claudio O. , Pon Cynthia L.
Published: March 2003

Expression of Escherichia coli hupA and hupB, the structural genes

encoding the most abundant nucleoid‐associated proteins HUα and HUβ

has been studied during cold shock. This article demonstrates that: (i) transcriptional

expression of hupA is blocked following a sudden temperature downshift (from

37°C to 10°C), whereas transcription of hupB from the

P2 and P3 promoters is maintained at a constitutive level and is activated de

novo from the P4 promoter; (ii) all three hupB mRNAs (transcribed from

the three natural promoters P2, P3 and P4) become much more stable than the single

hupA transcript; and (iii) the hupB transcripts, unlike that of hupA,

are efficiently translated in vivo during cold acclimation and can be actively

translated in vitro at low temperature. Taken together, the results indicate

that during cold shock the expression of the HUβ subunit is preferentially

stimulated and that of HUα repressed, suggesting that an altered HUα

to HUβ expression ratio resulting in an increase of HUα/HUβ

heterodimers and/or (HUβ)2 homodimers may play an important role during cold adaptation.
Yeast Rio1p is the founding member of a novel subfamily of protein serine kinases involved in the control of cell cycle progression
Authors: Angermayr Michaela , Roidl Andreas , Bandlow Wolfhard
Published: March 2003

Rio1p was identified as a protein serine kinase founding a novel subfamily. It is highly conserved from Archaea to man and only distantly related to previously established protein kinase families. Nevertheless, analysis of multiple protein sequence alignments shows that those amino acid residues that are important for either structure or catalytic activity in conventional protein kinases are also conserved in members of the Rio1p family at the respective positions (corresponding to domains I–XI of protein kinases). Recombinant Rio1p from Escherichia coli and tagged Rio1p from yeast has kinase activity in vitro, and mutation of amino acid residues that are conserved and indispensable for catalytic activity (i.e. ATP‐binding motif, catalytic centre) abrogates activity. RIO1 is essential in yeast and plays a role in cell cycle progression. After sporulation of RIO1/rio1 diploids, RIO1‐disrupted progeny cease growth after one to three cell divisions and arrest as either large unbudded or large‐budded cells. Cells deprived of Rio1p are enlarged and arrest either in G1 or in mitosis mainly with the DNA at the bud neck and short spindles (a phenotype also seen in cells carrying a weak allele), suggesting that Rio1p activity is required for at least at two steps during the cell division cycle: for entrance into S phase and for exit from mitosis. The weak RIO1 allele leads to increased plasmid loss.
HF2: a double‐stranded DNA tailed haloarchaeal virus with a mosaic genome
Authors: Tang Sen‐Lin , Nuttall Stewart , Ngui Katrina , Fisher Clare , Lopez Philippe , Dyall‐Smith Mike
Published: March 2003

HF2 is a haloarchaeal virus infecting two Halorubrum species (Family Halobacteriaceae).
It is lytic, has a head‐and‐tail morphology and belongs to the Myoviridae (contractile
tails). The linear double‐stranded DNA genome was sequenced and found to be 77 670
bp in length, with a mol% G+C of 55.8. A total of 121 likely open reading frames (ORFs) were identified, of which 37 overlapped at start and stop codons. The predicted proteins were usually acidic (average pI of 4.8), and less than about 12% of them had homologues in the sequence databases. Four complete tRNA‐like sequences (tRNA‐Arg, ‐Asx, ‐Pro and ‐Tyr) and an incomplete tRNA‐Thr were detected. A transcription map showed that most of the genome was transcribed and that the synthesis of transcripts occurred in a highly organized and reproducible pattern over a 5 h infection cycle. Transcripts often spanned multiple ORFs, suggesting that viral genes were organized into operons. The predicted ORF and observed transcript directions matched well and showed that transcription is mainly directed inwards from the genome termini, meeting at about 45–48 kb, and this was also a turning point in a cumulative GC‐skew plot. The low point in cumulative GC‐skew, near the left end, was a region rich in short repeats and lacking ORFs, which is likely to be an origin of replication. The HF2 genome is a mosaic of components from widely different sources, demonstrating clearly that viruses of haloarchaea, like their bacteriophage counterparts, are vectors for the exchange and transmission of genetic material between wide taxonomic distances, even across domains.
How do membrane proteins sense water stress?
Authors: Poolman Bert , Blount Paul , Folgering Joost H. A. , Friesen Robert H. E. , Moe Paul C. , Heide Tiemen van der
Published: March 2003

Maintenance of cell turgor is a prerequisite for almost any form of life as it provides a mechanical force for the expansion of the cell envelope. As changes in extracellular osmolality will have similar physicochemical effects on cells from all biological kingdoms, the responses to osmotic stress may be alike in all organisms. The primary response of bacteria to osmotic upshifts involves the activation of transporters, to effect the rapid accumulation of osmo‐protectants, and sensor kinases, to increase the transport and/or biosynthetic capacity for these solutes. Upon osmotic downshift, the excess of cytoplasmic solutes is released via mechanosensitive channel proteins. A number of breakthroughs in the last one or two years have led to tremendous advances in our understanding of the molecular mechanisms of osmosensing in bacteria. The possible mechanisms of osmosensing, and the actual evidence for a particular mechanism, are presented for well studied, osmoregulated transport systems, sensor kinases and mechanosensitive channel proteins. The emerging picture is that intracellular ionic solutes (or ionic strength) serve as a signal for the activation of the upshift‐activated transporters and sensor kinases. For at least one system, there is strong evidence that the signal is transduced to the protein complex via alterations in the protein–lipid interactions rather than direct sensing of ion concentration or ionic strength by the proteins. The osmotic downshift‐activated mechanosensitive channels, on the other hand, sense tension in the membrane but other factors such as hydration state of the protein may affect the equilibrium between open and closed states of the proteins.
The Tol/Pal system function requires an interaction between the C‐terminal domain of TolA and the N‐terminal domain of TolB
Authors: Walburger Anne , Lazdunski Claude , Corda Yves
Published: March 2003

The Tol/Pal system of Escherichia coli is composed of the YbgC, TolQ, TolA, TolR, TolB, Pal and YbgF proteins. It is involved in maintaining the integrity of the outer membrane, and is required for the uptake of group A colicins and DNA of filamentous bacteriophages. To identify new interactions between the components of the Tol/Pal system and gain insight into the mechanism of colicin import, we performed a yeast two‐hybrid screen using the different components of the Tol/Pal system and colicin A. Using this system, we confirmed the already known interactions and identified several new interactions. TolB dimerizes and the periplasmic domain of TolA interacts with YbgF and TolB. Our results indicate that the central domain of TolA (TolAII) is sufficient to interact with YbgF, that the C‐terminal domain of TolA (TolAIII) is sufficient to interact with TolB, and that the amino terminal domain of TolB (D1) is sufficient to bind TolAIII. The TolA/TolB interaction was confirmed by cross‐linking experiments on purified proteins. Moreover, we show that the interaction between TolA and TolB is required for the uptake of colicin A and for the membrane integrity. These results demonstrate that the TolA/TolB interaction allows the formation of a trans‐envelope complex that brings the inner and outer membranes in close proximity.
Autoinduction of the ompR response regulator by acid shock and control of the Salmonella enterica acid tolerance response
Authors: Bang Iel Soo , Audia Jonathon P. , Park Yong Keun , Foster John W.
Published: March 2003

Salmonella enterica serovar Typhimurium periodically experiences acid stress
in a variety of host and non‐host environments. An encounter with non‐lethal acid
stress (pH > 4) induces an assortment of physiological changes, called
the acid tolerance response (ATR), that helps the cell to tolerate extreme low pH
(pH 3). These physiological changes differ in log phase and stationary phase cells
and are controlled by different regulatory proteins. OmpR is an acid‐induced response
regulator critical to the stationary phase ATR but not to the log phase ATR. As OmpR
also controls the expression of the acid‐induced viru‐lence operon ssrAB,
acid shock induction of ompR was examined to gain insight into how Salmonella
links virulence with survival at extreme acid pH. The results indicate that acid
pH induces ompR from a promoter different from that used for basal expression.
Transcription from this promoter is repressed by the histone‐like protein H‐NS and
requires OmpR‐P for induction. The classic sensor kinase EnvZ and acetyl phosphate
collaborate to produce the optimum level of OmpR‐P needed for autoinduction. Although
OmpR‐P is required for acid‐induced expression of ompR in wild‐type cells,
OmpR is not needed for ompR transcription in the absence of H‐NS. Thus, the
role of OmpR‐P in autoinduction is to help to counteract repression by H‐NS. This
evidence, combined with the finding that relaxing DNA supercoiling with novobiocin
also increased ompR transcription, suggests that acid stress induces ompR by altering local DNA topology, not by changing the phosphorylation status of OmpR.
Expression of the Escherichia coli pcnB gene is translationally limited using an inefficient start codon: a second chromosomal example of translation initiated at AUU
Authors: Binns Nigel , Masters Millicent
Published: March 2003

Expression of the gene pcnB, encoding the dispensable Escherichia coli
poly(A) polymerase (PAPI), which is toxic when overproduced, was investigated. Its
promoter was identified and found to be moderately strong when used to express a
β‐galactosidase reporter. Expression levels were not affected by increasing
or decreasing PcnB concentration. Translation of pcnB was found to initiate
from the non‐canonical initiation codon AUU. The only other coli gene reported
to use AUU as initiation codon is infC, which encodes the initiation factor IF‐3. AUU, in common with other rarely used initiation codons, is discriminated against by IF‐3, resulting in the aborting of most AUU‐promoted initiation events. This enables AUU to form part of an autoregulatory circuit controlling IF‐3 production. We show that InfC discrimination reduces PcnB production fivefold. This is the first instance of this mechanism being used to limit severely the production of a potentially toxic product.
Eclipse period without sequestration in Escherichia coli
Authors: Olsson Jan , Dasgupta Santanu , Berg Otto G. , Nordström Kurt
Published: March 2003

The classical Meselson–Stahl density shift experiment was used to determine the length of the eclipse period in Escherichia coli, the minimum time period during which no new initiation is allowed from a newly replicated origin of chromosome replication, oriC. Populations of bacteria growing exponentially in heavy (15NH4+ and 13C6‐glucose) medium were shifted to light (14NH4+ and 12C6‐glucose) medium. The HH‐, HL‐ and LL‐DNA were separated by CsCl density gradient centrifugation, and their relative amounts were determined using radioactive gene‐specific probes. The eclipse period, estimated from the kinetics of conversion of HH‐DNA to HL‐ and LL‐DNA, turned out to be 0.60 generation times for the wild‐type strain. This was invariable for widely varying doubling times (35, 68 and 112 min) and was independent of the chromosome locus at which the eclipse period was measured. For strains with seqA, dam and damseqA mutants, the length of the eclipse period was 0.16, 0.40 and 0.32 generation times respectively. Thus, initiations from oriC were repressed for a considerable proportion of the generation time even when the sequestration function seemed to be severely compromised. The causal relationship between the length of the eclipse period and the synchrony of initiations from oriC is discussed.
Conjugal transfer of the virulence plasmid of Salmonella enterica is regulated by the leucine‐responsive regulatory protein and DNA adenine methylation
Authors: Camacho Eva M. , Casadesús Josep
Published: March 2003

Host‐encoded functions that regulate the transfer operon (tra) in the virulence

plasmid of Salmonella enterica (pSLT) were identified with a genetic screen.

Mutations that decreased tra operon expression mapped in the lrp gene,

which encodes the leucine‐responsive regulatory protein (Lrp). Reduced tra

operon expression in an Lrp− background is caused by lowered transcription

of the traJ gene, which encodes a transcriptional activator of the tra

operon. Gel retardation assays indicated that Lrp binds a DNA region upstream of

the traJ promoter. Deletion of the Lrp binding site resulted in lowered and

Lrp‐independent traJ transcription. Conjugal transfer of pSLT decreased 50‐fold

in a Lrp− background. When a FinO− derivative

of pSLT was used, conjugal transfer from an Lrp− donor decreased

1000‐fold. Mutations that derepressed tra operon expression mapped in dam,

the gene encoding Dam methyltransferase. Expression of the tra operon and

conjugal transfer remain repressed in an Lrp− Dam−

background. These observations support the model that Lrp acts as a conjugation activator

by promoting traJ transcription, whereas Dam methylation acts as a conjugation repressor by activating FinP RNA synthesis. This dual control of conjugal transfer may also operate in other F‐like plasmids such as F and R100.
Molecular microbiology and pathogenesis of Helicobacter and Campylobacter updated: a meeting report of the 11th conference on Campylobacter, Helicobacter and related organisms
Authors: Bereswill Stefan , Kist Manfred
Published: March 2003

The genome analysis of the gastrointestinal pathogens Helicobacter and Campylobacter has stimulated a wealth of new research activities, which are presented every 2 years at the international conferences on Campylobacter, Helicobacter and Related Organisms (CHRO). Both organisms represent excellent models for the identification of new molecular mechanisms involved in pathogenesis, host response and physiolological adaptation in course of acute and chronic infectious diseases. The investigation of their global distribution, pronounced genetic and antigenic diversity as well as the molecu‐lar mechanisms allowing long‐term persistence in hostile and unusual microbial habitats, is a challenge for scientists of many different disciplines world‐wide. With a focus on the molecular microbiology aspects, this review summarizes recent trends in Helicobacter and Campylobacter research by highlighting selected presentations at the 11th CHRO conference. The topics include the discovery of new virulence factors, functional analysis of protein secretion systems, host signalling pathways, adaptation to stress conditions, global gene regulation, and genetic variability.
The control of the yeast H2O2 response by the Msn2/4 transcription factors
Authors: Hasan Rukhsana , Leroy Christophe , Isnard Anne‐Dominique , Labarre Jean , Boy‐Marcotte Emmanuelle , Toledano Michel B.
Published: March 2003

We have analysed the contribution of the Msn2/4 transcription factors and the Ras‐cAMP‐proteine kinase A (PKA) pathway to the control of the yeast H2O2 response. Strains deleted for MSN2 and MSN4 are hypersensitive to H2O2, although they can still adapt to this oxidant. They are also unable to induce 27 proteins of the H2O2 stimulon as shown by quantitative two‐dimensional gel analysis. This peculiar H2O2 tolerance defect, the nature of the proteins of the Msn2/4 regulon, and the partial overlap of this regulon with the Yap1 H2O2‐response regulon, suggest an independent and distinctive role of these two H2O2 stress response pathways. A strain lacking PDE2, and therefore carrying high intracellular cAMP levels, is also hypersensitive to H2O2. In the presence of exogenous cAMP, this strain does not induce the entire H2O2 Msn2/4 regulon and some other proteins. This, and the normal H2O2 induction of a gene reporter under control of the Yap1 regulator when intracellular cAMP level are high, demonstrate that the Ras‐cAMP pathway negatively affects the H2O2 stress response through Msn2/4. However, the high H2O2 sensitivity of a strain lacking the PKA‐negative regulatory subunit Bcy1, is not only the consequence of the inhibition of Msn2/4 but also of Yap1 through a yet undefined mechanism.
Genes of non‐typeable Haemophilus influenzae expressed during interaction with human epithelial cell lines
Authors: Ulsen Peter van , Schilfgaarde Muriel van , Dankert Jacob , Jansen Henk , Alphen Loek van
Published: March 2003

Non‐typeable Haemophilus influenzae may infect the lower respiratory airways of chronic obstructive pulmonary disease patients. We characterized genes of non‐typeable H. influenzae expressed during interaction with two human respiratory tract‐derived epithelial cell lines. A library of 8000 clones was constructed in H. influenzae Rd (rec1) by cloning chromosomal fragments upstream of a promoterless cat gene. Exposure of this library to NCI‐H292 epithelial cell layers in the presence of chloramphenicol (Cam) resulted in survival of bacteria expressing cat. A total of 52 clones were selected that were resistant to Cam in the presence of epithelial cells of cell line NCI‐H292. These did not (n = 42) or hardly grow (n = 10) on sBHI plates containing Cam and were sensitive to Cam in cell culture medium alone. All clones, moreover, survived Cam in the presence of Hep2 epithelial cell layers. Sequence analysis showed that four clones contained sequences without homology to Rd or any other sequence, and therefore contained promoters and parts of open reading frames (ORFs) of novel genes. The other 48 clones were homologous to Rd, and characterization was based upon this genome. Six different functional classes were distinguished: (i) metabolic processes; (ii) stress response; (iii) gene expression; (iv) cell envelope biosynthesis; (v) DNA‐related processes and cell division; and (vi) ORFs encoding proteins of unknown function. The contribution of identified genes to non‐typeable H. influenzae adaptation to the epithelial cell environment is discussed.
RNA editing: complexity and complications
Authors: Stuart Kenneth , Panigrahi Aswini K.
Published: March 2003

RNA editing in Trypanosomatids creates functional mitochondrial mRNAs by extensive uridylate (U) insertion and deletion as specified by small guide RNAs (gRNAs). Editing is catalysed by the multiprotein editosome. Over 20 of its protein components have been identified and additional proteins are likely to function in editing and its regulation. The functions of only a few editosome proteins have been determined. Surprisingly, there are related pairs or sets of editosome proteins, and insertion and deletion editing appear to be functionally and perhaps spatially separate. A model for the editosome is proposed, which has a catalysis domain with separate sectors for insertion and deletion editing. It also contains domains for anchor duplex and upstream RNA binding, which position the sequence to be edited in the catalysis domain.
Never say never again: protein glycosylation in pathogenic bacteria
Authors: Benz Inga , Schmidt M. Alexander
Published: March 2003

In recent years, accumulating evidence for glycosylated bacterial proteins has overthrown an almost dogmatic belief that prokaryotes are not able to synthesize glycoproteins. Now it is widely accepted that eubacteria express glycoproteins. Although, at present, detailed information about glycosylation and structure–function relationships is available for only few eubacterial proteins, the variety of different components and structures observed already indicates that the variations in bacterial glycoproteins seem to exceed the rather limited display found in eukaryotes. Numerous virulence factors of bacterial pathogens have been found to be covalently modified with carbohydrate residues, thereby identifying these factors as true glycoproteins. In several bacterial species, gene clusters suggested to represent a general pro‐tein glycosylation system have been identified. In other cases, genes encoding highly specific glycosyltransferases have been found to be directly linked with virulence genes. These findings raise interesting questions concerning a potential role of glycosylation in pathogenesis. In this review, we will therefore focus on protein glycosylation in Gram‐negative bacterial pathogens.
YopH prevents monocyte chemoattractant protein 1 expression in macrophages and T‐cell proliferation through inactivation of the phosphatidylinositoI 3‐kinase pathway
Authors: Sauvonnet Nathalie , Lambermont Isabelle , Bruggen Pierre van der , Cornelis Guy R.
Published: March 2003

Phosphatidylinositol 3‐kinase (PI 3‐kinase) and its target protein kinase B (Akt) are involved in various processes including internalization, chemotaxis and proliferation. We analysed the activation of Akt in J774 macrophages infected with virulent (pYV+) or avirulent (pYV−) Yersinia enterocolitica. During the early stage of infection with pYV+ and pYV− bacteria, Akt and its targets, glycogen synthase kinase 3 (GSK‐3) and forkhead transcription factor (FKHRL1), became phosphorylated. This phosphorylation induction was inhibited by wortmannin and thus dependent on PI 3‐kinase. When infection was carried out with pYV+ bacteria but not with pYV− bacteria, Akt and its targets became dephosphorylated at later time points. Using single knock‐out mutants in bacterial effector genes, we have determined that the tyrosine phosphatase YopH was responsible for the inactivation of the PI 3‐kinase cascade. In macrophages, this inactivation correlated with the downregulation of mRNA coding for monocyte chemoattractant protein 1 (MCP‐1), suggesting that YopH inhibits recruitment of macrophages to lymph nodes. We also analysed the effects of Y. enterocolitica infection on the proliferation of T lymphocytes. Consistent with the observation that YopH inactivated the Akt pathway, YopH inhibited PI 3‐kinase‐dependent secretion of interleukin 2 and proliferation. These data reveal a new effect of YopH in Yersinia pathogenesis.
A peroxide‐induced zinc uptake system plays an important role in protection against oxidative stress in Bacillus subtilis
Authors: Gaballa Ahmed , Helmann John D.
Published: March 2003

In Bacillus subtilis, hydrogen peroxide (H2O2) induces expression of the PerR regulon including catalase (KatA), alkyl hydroperoxide reductase and the DNA‐binding protein MrgA. We have identified the P‐type metal‐transporting ATPase ZosA (formerly YkvW) as an additional member of the perR regulon. Expression of zosA is induced by H2O2 and repressed by the PerR metalloregulatory protein, which binds to two Per boxes in the promoter region. Physiological studies implicate ZosA in Zn(II) uptake. ZosA functions together with two Zur‐regulated uptake systems and one known efflux system to maintain Zn(II) homeostasis. ZosA is the major pathway for zinc uptake in cells growing with micromolar levels of Zn(II) that are known to repress the two Zur‐regulated transporters. A perR mutant is sensitive to high levels of zinc, and this sensitivity is partially suppressed by a zosA mutation. ZosA is important for resistance to both H2O2 and the thiol‐oxidizing agent diamide. This suggests that increased intracellular Zn(II) may protect thiols from oxidation. In contrast, catalase is critical for H2O2 resistance but does not contribute significantly to diamide resistance. Growth of cells with elevated zinc significantly increases resistance to high concentrations of H2O2, and this effect requires ZosA. Our results indicate that peroxide stress leads to the upregulation of a dedicated Zn(II) uptake system that plays an important role in H2O2 and disulphide stress resistance.
Characterization of a fungal protein kinase from Cryphonectria parasitica and its transcriptional upregulation by hypovirus
Authors: Kim Myoung‐Ju , Choi Jin‐Won , Park Seung‐Moon , Cha Byeong‐Jin , Yang Moon‐Sik , Kim Dae‐Hyuk
Published: March 2003

The chestnut blight fungus Cryphonectria parasitica and its hypovirus comprise useful model system to study the mechanisms of hypoviral infection. We used degenerate primers based on fungal protein kinases to isolate a gene, cppk1, which encodes a novel Ser/Thr protein kinase of C. parasitica. The gene showed highest homology to ptk1, a Ser/Thr protein kinase from Trichoderma reesei. The encoded protein had a predicted mass of 70.5 kDa and a pI of 7.45. Northern blot analyses revealed that the cppk1 transcript was expressed from the beginning of culture, with a slight increase by 5 days of culture. However, its expression was specifically affected by the presence of virus, and it was transcriptionally upregulated in the fungal strain infected with the hypovirus. A kinase assay using Escherichia coli‐derived CpPK1 revealed CpPK1‐specific phosphorylated proteins with estimated masses of 50 kDa and 44 kDa. In addition, the phosphorylation of both proteins was higher in a cell‐free extract from the hypovirulent strain. The increased expression of cppk1 by the introduction of an additional copy results in a subset of viral symptoms of reduced pigmentation and conidiation in a virus‐free isolate. cppk1 overexpression also causes the downregulation of mating factor genes Mf2/1 and Mf2/2, resulting in female sterility. The present study suggests that the hypovirus disturbs fungal signalling by transcriptional upregulation of cppk1, which results in reduced pigmentation and conidiation and female sterility.
A Rho‐dependent phase‐variable transcription terminator controls expression of the FimE recombinase in Escherichia coli
Authors: Joyce Susan A. , Dorman Charles J.
Published: March 2003

The fim switch is a 314 bp segment of invertible chromosomal DNA that is responsible for phase‐variable expression of type 1 fimbriae in Escherichia coli. The switch harbours the promoter of the fimA gene. This codes for the type 1 fimbrial subunit protein and, when the promoter is directed towards fimA (phase ON), the bacteria are fimbriate and, when it is directed away, the cells are afimbriate. The switch lies immediately downstream from the fimE gene, coding for a tyrosine site‐specific recombinase that catalyses inversion of the switch from the ON to the OFF phase. It has been suggested previously that, because the fim switch lies immediately downstream from the fimE gene, expression of FimE could be subject to control by antisense RNA in phase OFF bacteria in which the promoter harboured within the fim switch is oriented against the direction of transcription of the fimE gene. In this study, no effect of inducible fimE antisense RNA, expressed in cis or in trans, on FimE expression was detected. In phase ON cells, fimE mRNA extends across the switch into fimA, whereas in phase OFF cells, it terminates within the switch. This termination is Rho dependent and is abolished in a rho mutant. The extended fimE found in phase ON cells is more stable and results in an approximately fivefold increase in FimE protein compared with phase OFF bacteria. In the absence of Rho factor, fimE mRNA is equally stable in phase ON and phase OFF cells, and the levels of FimE recombinase are also equal.
A novel Myb‐related protein involved in transcriptional activation of encystation genes in Giardia lamblia
Authors: Sun Chin‐Hung , Palm Daniel , McArthur Andrew G. , Svärd Staffan G. , Gillin Frances D.
Published: March 2003

Giardia lamblia
is an important human intestinal parasite that survives outside of the host by differentiation of trophozoites into infectious cysts. Transcriptional regulation is key for encystation gene expression, but the mechanisms are unknown.
Giardia
genome database searches identified a
myb
‐like gene (
gmyb2
) whose expression increased during encystation. Epitope‐tagged gMyb2 localized to both nuclei. DNA binding and mutation analysis showed that gMyb2 binds specifically to C(T/A)ACAG, a c‐Myb‐like target sequence in the promoters of encystation‐induced genes encoding gMyb2, three cyst wall proteins and G6PI‐B, a key enzyme in cyst wall polysaccharide biosynthesis. gMyb2 binding sites were not found in the upstream regions of 31 other giardial genes. Deletion of the putative gMyb2 binding site greatly reduced encystation‐specific promoter activity of
g6pi‐b
. Fusion of gMyb2 binding sites to the constitutive
ran
promoter or
g6pi‐b promoter deletion lacking the gMyb2 binding site in‐duced encystation‐specific expression. gMyb2 may play an important role in transcriptional regulation of encystation genes, and may help co‐ordinate synthesis of cyst wall proteins and polysaccharide. gMyb2 is the first giardial transcription factor to be functionally identified and the first that is associated with upregulation of encystation genes. This work provides a model for study of differential gene regulation in early diverging eukaryotic organisms.
CesT is a bivalent enteropathogenicEscherichia colichaperone required for translocation of both Tir and Map
Authors: Creasey Elizabeth A. , Delahay Robin M. , Bishop Alexandra A. , Shaw Robert K. , Kenny Brendan , Knutton Stuart , Frankel Gad
Published: March 2003

Map is an enteropathogenicEscherichia coli(EPEC) protein that is translocated into eukaryotic cells by a type III secretion system. Although not required for the induction of attaching and effacing (A/E) lesion formation characteristic of EPEC infection, translocated Map is suggested to disrupt mitochondrial membrane potential, which may impact upon subsequent functions of the organelle such as control of cell death. Before secretion, many effector proteins are maintained in the bacterial cytosol by association with a specific chaperone. In EPEC, chaperones have been identified for the effector proteins translocated intimin receptor (Tir) and EspF, and for the translocator proteins EspB and EspD. In this study, we present evidence that the Tir‐specific chaperone, CesT, also performs a chaperone function for Map. Using a combination of biochemical approaches, we demonstrate specific interaction between CesT and Map. Similar to other chaperone–effector pairings, binding is apparent at the amino‐terminus of Map and is indicated to proceed by a similar mechanism to CesT:Tir interaction. Map secretion from acesTmutant strain (SE884) is shown to be reduced and, importantly, its translocation from this strain after infection of HEp‐2 cells is almost totally abrogated. Although other chaperones are reported to have a bivalent binding specificity, CesT is the first member of its family that chaperones more than one protein for translocation.
Amiodarone induces a caffeine‐inhibited, MID1‐depedent rise in free cytoplasmic calcium in Saccharomyces cerevisiae
Authors: Courchesne William E. , Ozturk Sedide
Published: March 2003

Calcium signalling is involved in myriad cellular processes such as mating morphogenesis. Mating in yeast induces changes in cell morphology with a concomitant increase in calcium uptake that is dependent on the MID1 and CCH1 genes. Mid1p and Cch1p are believed to function in a capacitive calcium entry (CCE)‐like process. Amiodarone alters mammalian calcium channel activity but, despite its clinical importance, its molecular mechanisms are not clearly defined. We have shown previously that amiodarone has fungicidal activity against a broad array of fungi. We show here that amiodarone causes a dramatic increase in cytoplasmic calcium ([Ca2+]cyt) in Saccharomyces cerevisiae. The majority of this increase is dependent on extracellular Ca2+ nonetheless, a significant increase in [Ca2+]cyt is still induced by amiodarone when no uptake of extracellular Ca2+ can occur. The influx of extracellular Ca2+ may be a direct effect of amiodarone on a membrane transporter or may be by a CCE mechanism. Uptake of the extracellular Ca2+ is inhibited by caffeine and reduced in strains deleted for the mid1 gene, but not in cells deleted for cch1. Our data are the first demonstrating control of yeast calcium channels by amiodarone and caffeine.
DNA gyrase requirements distinguish the alternate pathways of Mu transposition
Authors: Sokolsky Tanya D. , Baker Tania A.
Published: March 2003

The MuA transposase mediates transposition of bacteriophage Mu through two distinct mechanisms. The first integration event following infection occurs through a non‐replicative mechanism. In contrast, during lytic growth, multiple rounds of replicative transposition amplify the phage genome. We have examined the influence of gyrase and DNA supercoiling on these two transposition pathways using both a gyrase‐inhibiting drug and several distinct gyrase mutants. These experiments reveal that gyrase activity is not essential for integration; both lysogens and recombination intermediates are detected when gyrase is inhibited during Mu infection. In contrast, gyrase inhibition causes severe defects in replicative transposition. In two of the mutants, as well as in drug‐treated cells, replicative transposition is almost completely blocked. Experiments probing for formation of MuA–DNA complexes in vivo reveal that this block occurs very early, during assembly of the transposase complex required for the catalytic steps of recombination. The findings establish that DNA structure‐based signals are used differently for integrative and replicative transposition. We propose that transposase assembly, the committed step for recombination, has evolved to depend on different DNA /architectural signals to control the reaction outcome during these two distinct phases of the phage life cycle.
Glycolipid depletion in antimicrobial therapy
Authors: Svensson Majlis , Frendeus Björn , Butters Terry , Platt Frances , Dwek Raymond , Svanborg Catharina
Published: March 2003

Mucosal pathogens target sites of infection through specific adherence to host glycoconjugate receptors. As a consequence, depletion of such receptors from the cell surface may be expected to inhibit attachment, impair bacterial colonization and reduce the activation of mucosal inflammation. We have used the glucose analogue and glycosphingolipid (GSL) biosynthesis inhibitor N‐butyldeoxynojirimycin (NB‐DNJ) to deplete human uroepithelial cells and the murine urinary tract mucosa of receptors for P‐fimbriated Escherichia coli. NB‐DNJ blocks the ceramide‐specific glucosyltransferase, which catalyses the formation of glucosyl ceramide (GlcCer), the precursor for GSLs. The inhibitor was shown to decrease the GSL content in a dose‐dependent way, and depletion markedly inhibited P‐fimbriated bacterial attachment in vitro. NB‐DNJ‐fed C3H/HeN mice were depleted of GSLs in vivo and showed reduced susceptibility to experimental urinary tract infection with P‐fimbriated E. coli. The mucosal inflammatory response was impaired, as shown by reduced chemokine secretion and lower neutrophil recruitment, and the bacteria colonized the urinary tract less efficiently than in normal mice. These results confirmed the role of P fimbriae‐mediated adherence for colonization and inflammation and point to an interesting alternative to antibiotic treatment for urinary tract infection.
The dual roles of AlgG in C‐5‐epimerization and secretion of alginate polymers in Pseudomonas aeruginosa
Authors: Jain Sumita , Franklin Michael J. , Ertesvåg Helga , Valla Svein , Ohman Dennis E.
Published: March 2003

Pseudomonas aeruginosa
strains causing chronic pulmonary infections in cystic fibrosis patients produce high levels of alginate, an exopolysaccharide that confers a mucoid phenotype. Alginate is a linear polymer of
d
‐mannuronate (M) and variable amounts of its C‐5‐epimer,
l
‐guluronate (G). AlgG is a periplasmic C‐5‐epimerase that converts poly
d
‐mannuronate to the mixed M+G sequence of alginate. To understand better the role and mechanism of AlgG activity, a mutant was constructed in the mucoid strain FRD1 with a defined non‐polar deletion of
algG
. Instead of producing poly mannuronate, the
algG
deletion mutant secreted dialysable uronic acids, as does a mutant lacking the periplasmic protein AlgK. High levels of unsaturated ends and the nuclear magnetic resonance spectroscopy pattern revealed that the small, secreted uronic acids were the products of extensive polymer digestion by AlgL, a periplasmic alginate lyase co‐expressed with AlgG and AlgK. Thus, AlgG is bifunctional with (i) epimerase activity and (ii) a role in protecting alginate from degradation by AlgL during transport through the periplasm. AlgK appears to share the second role. AlgG and AlgK may be part of a periplasmic protein complex, or scaffold, that guides alginate polymers to the outer membrane secretin (AlgE). To characterize the epimerase activity of AlgG further, the
algG4
allele of poly mannuronate‐producing FRD462 was shown to encode a protein lacking only the epimerase function. The sequence of
algG4 has a Ser‐272 to Asn substitution in a serine–threonine‐rich and conserved region of AlgG, which revealed a critical residue for C‐5‐epimerase activity.