bluuebunny/crossref_metadata_2025_split huggingface dataset:
Over 1000 genes are involved in the DNA damage response of Escherichia coli
Authors: Khil Pavel P. , Camerini‐Otero R. Daniel 
Published: March 2003

SummaryChanges in gene expression after treatment of Escherichia coli cultures
with mitomycin C were assessed using gene array technology. Unexpectedly, a large
number of genes (nearly 30% of all genes) displayed significant changes in
their expression level. Analysis and classification of expression profiles of the
corresponding genes allowed us to assign this large number of genes into one or two
dozen small clusters of genes with similar expression profiles. This assignment allowed
us to describe systematically the changes in the level of gene expression in response
to DNA damage. Among the damage‐induced genes, more than 100 are novel. From those
genes involved in DNA metabolism that have not previously been shown to be induced
by DNA damage, the mutS gene involved in mismatch repair is especially noteworthy.
In addition to the SOS response, we observed the induction of other stress response
pathways, such as those of oxidative stress and osmotic protection. Among the genes
that are downregulated in response to DNA damage are numerous protein biosynthesis
genes. Analysis of the gene expression data highlighted the essential involvement
of σs‐regulated genes and the general stress response network in the response to DNA damage.
An aspartate ring at the TolC tunnel entrance determines ion selectivity and presents a target for blocking by large cations
Authors: Andersen Christian , Koronakis Eva , Hughes Colin , Koronakis Vassilis 
Published: March 2003

SummaryThe TolC protein of Escherichia coli comprises an outer membrane β‐barrel
channel and a contiguous α‐helical tunnel spanning the periplasm, providing
an exit duct for protein export and multidrug efflux. It forms a single transmembrane
pore that is open to the outside of the cell but constricted at the peri‐plasmic
tunnel entrance. This sole constriction is lined by a ring of six aspartate residues,
two in each of the three identical monomers. When these were replaced by alanines,
the resulting TolCDADA protein reconstituted normally in black lipid membranes
but showed altered electrophysiological characteristics. In particular, it had lost
the strong pH dependence of the wild type and had switched ion selectivity from cations
to anions. The function of wild‐type TolC as a membrane pore was severely inhibited
by divalent and trivalent cations entering the channel tunnel from the channel (‘extracellular’)
side. Divalent cations bound reversibly to effect complete blocking of the transmembrane
ion flux. Trivalent cations were more potent. Hexamminecobalt bound at nanomolar
concentrations allowed visualization of single blocking events, whereas the smaller
Cr3+ cation bound irreversibly and could also access the cation
binding site via the tunnel entrance. The inhibitory cations had no effect on the
mutant TolCDADA, supporting the view that the aspartate ring is the cation binding site. The electronegative entrance is widely conserved throughout the TolC family, which is essential for efflux and export by Gram‐negative bacteria, suggesting that it could present a general target for drugs.
Polar localization of the Escherichia coli oriC region is independent of the site of replication initiation
Authors: Gordon G. Scott , Shivers Robert P. , Wright Andrew 
Published: March 2003

SummaryThe location of the origin‐linked region of the Escherichia coli chromosome was analysed in strains lacking the core origin locus, oriC. In these strains, which initiate replication from F factors integrated at different locations around the chromosome, origin‐linked DNA remains localized near the cell poles, as in wild‐type cells. In contrast, minichromosomes containing 7 kb of chromosomal DNA including oriC are generally excluded from the ends of the cell. Thus, we propose that positioning of the wild‐type origins at the poles is not a function of their order of replication but a sequence‐specific phenomenon. It is proposed that there are centromere‐like sequences, bordering the wild‐type origin of replication, which are used by host mechanisms to direct the proper placement of the origin region of the chromosome. This function, combined with other host processes, may assure efficient segregation of the E. coli chromosome.
Identification and characterization of genes encoding sex pheromone cAM373 activity in Enterococcus faecalis and Staphylococcus aureus
Authors: Flannagan Susan E. , Clewell Don B. 
Published: March 2003

SummaryThe sex pheromone cAM373 of Enterococcus faecalis and the related staph‐cAM373 of Staphylococcus aureus were found to correspond to heptapeptides located within the C‐termini of the signal sequences of putative prelipoproteins. The deduced mature forms of the lipoproteins share no detectable homology and presumably serve unrelated functions in the cells. The chromosomally encoded genetic determinants for production of the pheromones have been identified and designated camE (encoding cAM373) and camS (encoding staph‐cAM373). Truncated and full‐length clones of camE were generated in Escherichia coli, in which cAM373 activity was expressed. In E. faecalis, insertional inactivation in the middle of camE had no detectable phenotypic effects on the pheromone system. Establishment of an in frame translation stop codon within the signal sequence resulted in reduction of cAM373 activity to 3% of normal levels. The camS determinant has homologues in Staphylococcus epidermidis, Bacillus subtilis and Listeria monocytogenes; however, corresponding heptapeptides present within those sequences do not resemble staph‐cAM373 closely. The particular significance of staph‐cAM373 as a potential intergeneric inducer of transfer‐proficient genetic elements is discussed.
KTI11 and KTI13, Saccharomyces cerevisiae genes controlling sensitivity to G1 arrest induced by Kluyveromyces lactis zymocin
Authors: Fichtner Lars , Schaffrath Raffael 
Published: March 2003

SummaryThe Kluyveromyces lactis zymocin and its γ‐toxin subunit inhibit

cell cycle progression of Saccharomyces cerevisiae. To identify S. cerevisiae

genes conferring zymocin sensitivity, we complemented the unclassified zymocin‐resistant

kti11 and kti13 mutations using a single‐copy yeast library. Thus,

we identified yeast open reading frames (ORFs) YBL071w‐A and YAL020c/ATS1

as KTI11 and KTI13 respectively. Disruption of KTI11 and KTI13

results in the complex tot phenotype observed for the γ‐toxin target

site mutants, tot1–7, and includes zymocin resistance, thermosensitivity,

hypersensitivity to drugs and slow growth. Both loci, KTI11 and KTI13,

are actively transcribed protein‐encoding genes as determined by reverse transcriptase–polymerase

chain reaction (RT–PCR) and in vivo HA epitope tagging. Kti11p is highly

conserved from yeast to man, and Kti13p/Ats1p is related to yeast Prp20p and mammalian

RCC1, components of the Ran–GTP/GDP cycle. Combining disruptions in KTI11

or KTI13 with a deletion in TOT3/ELP3 coding for the RNA polymerase

II (RNAPII) Elongator histone acetyltransferase (HAT) yielded synthetic effects on

slow growth phenotype expression. This suggests genetic interaction and possibly

links KTI11 and KTI13 to Elongator function.
A signal transduction system in Streptomyces coelicolor that activates the expression of a putative cell wall glycan operon in response to vancomycin and other cell wall‐specific antibiotics
Authors: Hong Hee‐Jeon , Paget Mark S. B. , Buttner Mark J. 
Published: March 2003

SummaryWe have investigated a signal transduction system proposed to allow Streptomyces
coelicolor to sense and respond to changes in the integrity of its cell envelope.
The system consists of four proteins, encoded in an operon: σE,
an RNA polymerase σ factor; CseA (formerly ORF202), a protein of unknown
function; CseB, a response regulator; and CseC, a sensor histidine protein kinase
with two predicted transmembrane helices (Cse stands for control of sigma
E). To develop a sensitive bioassay for in‐ducers of the sigE system,
the promoter of the sigE operon (sigEp) was fused to a reporter gene
conferring resistance to kanamycin. Antibiotics that acted as inducers of the sigE
signal transduction system were all inhibitors of intermediate and late steps in
peptidoglycan biosynthesis, including ramoplanin, moenomycin A, bacitracin, several
glycopeptides and some β‐lactams. The cell wall hydrolytic enzyme lysozyme
also acted as an inducer. These data suggest that the CseB–CseC signal transduction
system may be activated by the accumulation of an intermediate in peptidoglycan biosynthesis
or degradationa. A computer‐based searching method was used to identify a σE
target operon of 12 genes (the cwg operon), predicted to specify the biosynthesis
of a cell wall glycan. In low‐Mg2+ medium, transcription of the
cwg operon was induced by vancomycin in a sigE‐dependent manner but,
in high‐Mg2+ medium, there was substantial cwg transcription
in a sigE null mutant, and this sigE‐independent activity was also
induced by vancomycin. Based on these data, we propose a model for the regulation
and function of the σE signal transduction system.
Nac‐mediated repression of the serA promoter of Escherichia coli
Authors: Blauwkamp Timothy A. , Ninfa Alexander J. 
Published: March 2003

Summary Escherichia coli and related bacteria contain two paralogous PII‐like proteins involved in nitrogen regulation, the glnB product, PII, and the glnK product, GlnK. Previous studies have shown that cells lacking both PII and GlnK have a severe growth defect on minimal media, resulting from elevated expression of the Ntr regulon. Here, we show that this growth defect is caused by activity of the nac product, Nac, a LysR‐type transcription factor that is part of the Ntr regulon. Cells with elevated Ntr expression that also contain a null mutation in nac displayed growth rates on minimal medium similar to the wild type. When expressed from high‐copy plasmids, Nac imparts a growth defect to wild‐type cells in an expression level‐dependent manner. Neither expression of Nac nor lack thereof significantly affected Ntr gene expression, suggesting that the activity of Nac at one or more promoters outside the Ntr regulon was responsible for its effects. The growth defect of cells lacking both PII and GlnK was also eliminated upon supplementation of minimal medium with serine or glycine for solid medium or with serine or glycine and glutamine for liquid medium. These observations suggest that high Nac expression results in a reduction in serine biosynthesis. β‐Galactosidase activity expressed from a Mu d1 insertion in serA was reduced approximately 10‐fold in cells with high Nac expression. We hypothesize that one role of Nac is to limit serine biosynthesis as part of a cellular mechanism to reduce metabolism in a co‐ordinated manner when cells become starved for nitrogen.
Effect of var gene disruption on switching in Plasmodium falciparum
Authors: Horrocks Paul , Pinches Robert , Kyes Sue , Kriek Neline , Lee Sarah , Christodoulou Zóe , Newbold Chris I. 
Published: March 2003

SummaryThe molecular mechanisms underpinning switching of variant antigens on the surface of Plasmodium falciparum‐infected erythrocytes are poorly understood. We tested the hypothesis that insertional disruption of the A4var gene, one of two var genes located within the subtelomeric region of one end of chromosome 13, would result in a preferential switch in transcription to the adjacent R29var gene upon rosette selection. In this way, we aimed to mimic the preferential transcription of R29var in rosetting R29 parasites, a parasite line in which the A4var gene is deleted through a chromosome end truncation. Initial analysis of the knock‐out parasite lines shows that the insertional disruption of the A4var gene prevents A4 PfEMP1 expression, but that switching transcription to other var gene variants is unaffected. Furthermore, analysis of var transcription in the knock‐out parasite line during rosette selection shows that, rather than facilitating a switch to R29var gene transcription, this event was suppressed in the transfectants . These data, and the implications for epigenetic transcriptional control of var genes, are discussed.
Identification of Xenorhabdus nematophila genes required for mutualistic colonization of Steinernema carpocapsae nematodes
Authors: Heungens Kurt , Cowles Charles E. , Goodrich‐Blair Heidi 
Published: March 2003

SummaryOne stage in the symbiotic interaction between the bacterium Xenorhabdus nematophila and its nematode host, Steinernema carpocapsae, involves the species‐specific colonization of the nematode intestinal vesicle by the bacterium. To characterize the bacterial molecular determinants that are essential for vesicle colonization, we adapted and applied a signature‐tagged mutagenesis (STM) screen to this system. We identified 15 out of 3000 transposon mutants of X. nematophila with at least a 15‐fold reduction in average vesicle colonization. These 15 mutants harbour disruptions in nine separate loci. Three of these loci have predicted open reading frames (ORFs) with similarity to genes (rpoS, rpoE, lrp) encoding regulatory proteins; two have predicted ORFs with similarity to genes (aroA, serC) encoding amino acid biosynthetic enzymes; one, designated nilB (nematode intestine localization), has an ORF with similarity to a gene encoding a putative outer membrane protein (OmpU) in Neisseria; and three, nilA, nilC and nilD, have no apparent homologues in the public database. nilA, nilB and nilC are linked on a single 4 kb locus. nilB and nilC are > 104‐fold reduced in their ability to colonize the nematode vesicle and are predicted to encode membrane‐localized proteins. The nilD locus contains an extensive repeat region and several small putative ORFs. Other than reduced colonization, the nilB, nilC and nilD mutants did not display alterations in any other phenotype tested, suggesting a specific role for these genes in allowing X. nematophila to associate with the nematode host.
Mechanism of association of adenylate cyclase toxin with the surface of Bordetella pertussis: a role for toxin–filamentous haemagglutinin interaction
Authors: Zaretzky Franca R. , Gray Mary C. , Hewlett Erik L. 
Published: March 2003

SummaryAdenylate cyclase (AC) toxin from Bordetella per‐tussis is unusual in that, unlike most other members of the repeats‐in‐toxin family that are released into the extracellular milieu, it remains associated with the bacterial surface. In this study, we investigated the nature of the association of this toxin with the surface of B. pertussis. AC toxin was extracted from crude outer membrane preparations of B. pertussis with 8 M urea, but only partially with alkaline sodium carbonate and not at all with octylglucoside, suggesting that denaturation of the toxin is necessary for its removal from the membrane. B. pertussis mutants lacking filamentous haemagglutinin (FHA) released significantly more AC toxin into the medium, and AC toxin association with the bacterial surface was partially restored by expression of FHA from a plasmid, suggesting a role for FHA in surface retention of AC toxin. AC toxin distribution was unaffected by the absence of pertactin, or full‐length lipopolysaccharide, or a defect in secretion of pertussis toxin. Using overlay and immunoprecipitation, we found that a direct physical association can occur between AC toxin and FHA. Combined, these findings suggest that FHA may play a role in AC toxin retention on the surface of B. pertussis and raise the possibility of an involvement of adherence mediated by FHA in delivery of AC toxin from the bacterium to the target cell.
The RHO1‐GAPs SAC7, BEM2 and BAG7 control distinct RHO1 functions in Saccharomyces cerevisiae
Authors: Schmidt Anja , Schmelzle Tobias , Hall Michael N. 
Published: March 2003

SummaryIn Saccharomyces cerevisiae, the small GTPase RHO1 plays an essential role in the control of cell wall synthesis and organization of the actin cytoskeleton. Several regulators for RHO1 are known, including the GTPase‐activating proteins (GAPs) SAC7 and BEM2. Here we show that BAG7, identified as the closest homologue of SAC7, also acts as a GAP for RHO1 in vitro and in vivo. Furthermore, we find that BAG7, SAC7, and BEM2 are functionally different in vivo. Overexpression of BAG7 or SAC7, but not BEM2, suppresses the cold sensitivity of a sac7 mutation and the lethality of RHO1 hyperactivation in response to cell wall damage. In contrast, overexpression of BEM2 or SAC7, but not BAG7, downregulates the RHO1‐controlled PKC1‐MPK1 pathway, and disruption of BEM2 or SAC7, but not BAG7, results in increased MPK1 activation. We conclude that BEM2 and SAC7, but not BAG7, are involved in the control of the RHO1‐mediated activation of MPK1, whereas BAG7 and SAC7, but not BEM2, are involved in the regulation of other RHO1 functions. This suggests that different RHO1 GAPs control different RHO1 effector pathways, thus ensuring their individual regulation at the appropriate place and time.
Characterization of the promoter regions involved in galactose‐ and nisin‐mediated induction of the nisA gene in Lactococcus lactis ATCC 11454
Authors: Chandrapati Sailaja , O'Sullivan Daniel J. 
Published: March 2003

SummaryThe nisA promoter is positively regulated in Lactococcus lactis ATCC 11454 by autoinduction via a two‐component NisRK‐mediated system. However, induction of this promoter can also occur when introduced into the plasmid‐free L. lactis LM0230 during growth in galactose or lactose, independent of the NisRK system. In this study, we also characterized this galactose‐mediated induction by determining the nisA start site during growth in galactose, which was identical to the nisA start site upon nisin induction. The region involved in the galactose‐mediated induction of the nisA promoter was investigated by directed deletion analysis of a 200 bp region upstream of the nisA promoter in the transcription fusion pDOC99. The induction of the deletion derivatives by galactose and nisin was compared phenotypically using β‐galactosidase measurements, and the regions necessary for the induction were determined by sequence analysis. Analysis of these regions revealed two sets of a TCT direct repeat [TCT‐N8‐TCT] present at positions (−107 to −94) and (−39 to −26) relative to the transcription initiation site. Disruption of the upstream repeat abolished galactose induction and significantly reduced the nisin induction capacity, suggesting a potential pivotal role for these repeats in transcription induction of the nisA promoter. It was also observed that the galactose‐mediated induction was abolished when a plasmid containing the phosphotransferase system (PTS), phospho‐β‐galactosidase and tagatose pathway genes was introduced into this strain. As this effectively made the Leloir pathway redundant, it points to some component of this pathway as the specific inducer of the nisA promoter.
A novel assembly process of the multicomponent xenobiotic efflux pump in Pseudomonas aeruginosa
Authors: Maseda Hideaki , Kitao Masataka , Eda Shima , Yoshihara Eisaku , Nakae Taiji 
Published: March 2003

SummaryThe nfxC‐type cells of Pseudomonas aeruginosa show resistance to a wide range of structurally and functionally diverse antibiotics, which is a phenomenon that is mainly attributable to the expression of the MexEF‐OprN xenobiotic transporter. The MexF, MexE and OprN subunits of this transporter are located on the inner membrane, the periplasm and the outer membrane, respectively, and are assumed to function as an energy‐dependent transporter, a bridge connecting the inner and outer membranes and outer membrane channel respectively. The nfxC‐type cells showed a single protein band of MexF and OprN, whereas MexE appeared as three distinct bands in an SDS‐polyacrylamide gel electrophoretogram. The mutant cells lacking MexF produced undetectable OprN and only a full‐size of MexE even though the cells had unimpaired oprN and mexE. Expression of the plasmid‐borne MexF in this mutant fully restored OprN and three MexE bands. Another class of mutants producing a full amount of MexF yielded undetectable OprN and two MexE bands lacking the smallest protein species suggesting that the presence of the smallest MexE subunit is required for stabilization of OprN. To identify which part of MexE was needed for stabilization and assembly of OprN, the carboxyl‐terminal‐truncated MexE tagged with polyhistidine was constructed and protein bands were visualized in the presence of MexF with an antibody raised against polyhistidine or MexE. The results revealed that the proteolytic processing of MexE would occur at carboxyl terminal amino acids between 11 and 16, thereby suggesting that the presence of the C‐terminal truncated MexE is essential for stabilization and the proper assembly of OprN. Nucleotide sequencing of mutant mexFs, which produce a wild‐type level of MexF but are unable to support the production of the smallest MexE, thereby destabilizing OprN, revealed that all the mutations were located within two large periplasmic domains of MexF between transmembrane segments 1–2 and 7–8. Taking these findings together, we concluded that two large periplasmic domains of MexF interact with MexE thereby promoting programmed processing of MexE, and this complex eventually assists the correct assembly and sorting of OprN.
A unique, bifunctional site‐specific DNA recombinase from Mycoplasma pulmonis
Authors: Sitaraman Ramakrishnan , Denison Amy M. , Dybvig Kevin 
Published: March 2003

SummarySite‐specific DNA invertible elements often control the production of bacterial surface proteins that are subject to phase variation (ON/OFF switching). Inversion of the DNA element occurs as a result of the reciprocal exchange of DNA catalysed by a specialized enzyme (recombinase) that acts at specific sites. By continually switching the orientation of the invertible element in the chromosome, and consequently the production of the variable protein(s), the cell population remains continually responsive to environmental change such as immunological challenge. In addition to phase‐variable surface proteins, Mycoplasma pulmonis has a family of phase‐variable restriction‐modification enzymes. We report here that a single recombinase in M. pulmonis, HvsR, catalyses independent DNA inversions at non‐homologous loci, causing variations in surface lipoproteins and in the DNA recognition sequence specificity of restriction enzymes. Thus, HvsR is a site‐specific DNA recombinase with dual substrate specificity.