bluuebunny/crossref_metadata_2025 huggingface dataset:
DNA supercoiling‐dependent transcriptional coupling between the divergently transcribed promoters of the ilvYC operon of Escherichia coli is proportional to promoter strengths and transcript lengths
Authors: Opel Michael L. , Hatfield G. Wesley
Published: March 2003

The twin‐domain model of Liu and Wang suggested that high levels of DNA supercoiling generated in the region between closely spaced divergently transcribed promoters could serve to couple the activities of these promoters transcriptionally. In this report, we use topoisomer sets of defined superhelical densities as DNA templates in a purified in vitro transcription system to demonstrate transcriptional coupling between the divergently transcribed ilvY and ilvC promoters of the ilvYC operon of Escherichia coli. Current evidence for this type of DNA supercoiling‐dependent transcriptional coupling, based largely on the in vivo activities of promoters contained in engineered DNA constructs, suggests that the transcription complex must be physically hindered to generate DNA supercoils and to prevent their diffusion throughout the DNA duplex. However, the in vitro results presented here demonstrate that (i) transcriptional coupling is observed between the divergent promoters of the ilvYC operon in the absence of transcript anchoring; (ii) the magnitude of the negative DNA supercoiling generated in the divergent promoter region is proportional to the sum of the global and transcription‐induced superhelicity; and (iii) the magnitude of this transcription‐induced superhelicity is proportional to promoter strengths and transcript lengths.
Molecular cloning and genetic analysis of an indole‐diterpene gene cluster from Penicillium paxilli
Authors: Young Carolyn , McMillan Lisa , Telfer Emily , Scott Barry
Published: March 2003

The indole‐diterpene paxilline is a potent tremorgenic mammalian mycotoxin and a known inhibitor of maxi‐K ion channels. The gene cluster responsible for paxilline biosynthesis in Penicillium paxilli was identified by mapping four large plasmid‐induced chromosome deletions. The cluster is predicted to lie within a 50 kb region of chromosome Va and to contain 17 genes, including a geranylgeranyl pyrophosphate (GGPP) synthase (paxG), two FAD‐dependent monooxygenases (paxM and N), two cytochrome P450 monooxygenases (paxP and Q), a dimethylallyltryptophan (DMAT) synthase (paxD) and two possible transcription factors (paxR and paxS), which contain a Zn(II)2Cys6 DNA‐binding motif. Targeted replacement of paxG confirmed that it is essential for paxilline biosynthesis but dispensable for growth. The GGPP for primary metabolism is predicted to be provided by a second GGPP synthase (ggs1) that was cloned, sequenced and mapped to chromosome IV. Semi‐quantitative reverse transcriptase–polymerase chain reaction analysis demonstrated that the expression of paxG, paxM and paxP in submerged liquid cultures of P. paxilli increased dramatically with the onset of paxilline biosynthesis. In contrast, the expression of β‐tubulin (tub2) and ggs1 was not induced. This is the first description of the molecular cloning and genetic analysis of an indole‐diterpene gene cluster.
Cell cycle and positional constraints on FtsZ localization and the initiation of cell division in Caulobacter crescentus
Authors: Quardokus Ellen M. , Din Neena , Brun Yves V.
Published: March 2003

Swarmer cells of Caulobacter crescentus are devoid of the cell division initiation protein FtsZ and do not replicate DNA. FtsZ is synthesized during the differentiation of swarmer cells into replicating stalked cells. We show that FtsZ first localizes at the incipient stalked pole in differentiating swarmer cells. FtsZ subsequently localizes at the mid‐cell early in the cell cycle. In an effort to understand whether Z‐ring formation and cell constriction are driven solely by the cell cycle‐regulated increase in FtsZ concentration, FtsZ was artificially expressed in swarmer cells at a level equivalent to that found in predivisional cells. Immunofluorescence microscopy showed that, in these swarmer cells, simply increasing FtsZ concentration was not sufficient for Z‐ring formation; Z‐ring formation took place only in stalked cells. Expression of FtsZ in swarmer cells did not alter the timing of cell constriction initiation during the cell cycle but, instead, caused additional constrictions and a delay in cell separation. These additional constrictions were confined to sites close to the original mid‐cell constriction. These results suggest that the timing and placement of Z‐rings is tightly coupled to an early cell cycle event and that cell constriction is not solely dependent on a threshold level of FtsZ.
Analysis of the replicon region and identification of an rRNA operon on pBM400 of Bacillus megaterium QM B1551
Authors: Kunnimalaiyaan Muthusamy , Stevenson David M. , Zhou Yansheng , Vary Patricia S.
Published: March 2003

An 18 633 bp region containing the replicon from the ≈ 53 kb pBM400 plasmid of Bacillus megaterium QM B1551 has been sequenced and characterized. This region contained a complete rRNA operon plus 10 other potential open reading frames (ORFs). The replicon consisted of an upstream promoter and three contiguous genes (repM400, orfB and orfC) that could encode putative proteins of 428, 251 and 289 amino acids respectively. A 1.6 kb minimal replicon was defined and contained most of repM400. OrfB was shown to be required for stability. Three 12 bp identical tandem repeats were located within the coding region of repM400, and their presence on another plasmid caused incompatibility with their own cognate replicon. Nonsense, frameshift and deletion mutations in repM400 prevented replication, but each mutation could be complemented in trans. RepM400 had no significant similarity to sequences in the GenBank database, whereas five other ORFs had some similarity to gene products from other plasmids and the Bacillus genome. An rRNA operon was located upstream of the replication region and is the first rRNA operon to be sequenced from B. megaterium. Its unusual location on non‐essential plasmid DNA has implications for systematics and evolutionary biology.
Hsf1p and Msn2/4p cooperate in the expression of Saccharomyces cerevisiae genes HSP26 and HSP104 in a gene‐ and stress type‐dependent manner
Authors: Amorós Mara , Estruch Francisco
Published: March 2003

Saccharomyces cerevisiae possesses several transcription factors involved in the transcriptional activation of stress‐induced genes. Among them, the heat shock factor (Hsf1p) and the zinc finger proteins of the general stress response (Msn2p and Msn4p) have been shown to play a major role in stress protection. Some heat shock protein (HSP) genes contain both heat shock elements (HSEs) and stress response elements (STREs), suggesting the involvement of both transcription factors in their regulation. Analysis of the stress‐induced expression of two of these genes, HSP26 and HSP104, reveals that the contribution of Hsf1p and Msn2/4p is different depending on the gene and the stress condition.
An analysis of the factory model for chromosome replication and segregation in bacteria
Authors: Sawitzke James , Austin Stuart
Published: March 2003

Recent advances in microscopy have given us important clues as to the nature of chromosome segregation in bacteria. Most current observations favour the view that the process is co‐replicational: DNA replication forks are anchored at the cell centre, and the newly replicated DNA is moved towards the cell poles. This scheme can account for orderly segregation even at high growth rates where multiple replication cycles overlap. We argue that there are five distinct activities directly involved in co‐replicational segregation dynamics. These we refer to as Push, Direct, Condense, Hold and Clear. We attempt to assign one of these roles to each protein implicated in chromosome segregation. The proposed process is very different from mitosis in eukaryotic cells and perhaps more closely resembles the formation of separate sister chromatids during DNA replication.
A glutamate decarboxylase system protects Listeria monocytogenes in gastric fluid
Authors: Cotter Paul D. , Gahan Cormac G. M. , Hill Colin
Published: March 2003

We observed that glutamate greatly enhances the survival of Listeria monocytogenes in gastric fluid, a phenomenon that is directly linked to glutamate decarboxylase activity (GAD). Glutamate‐mediated acid tolerance has been associated in other intestinal genera with the GAD system, in which glutamate is internalized and converted to γ‐aminobutyrate (consuming an intracellular proton) that is subsequently exchanged for another extracellular glutamate via a membrane‐located antiporter. Molecular analysis of L. monocytogenes LO28 revealed the presence of two glutamate decarboxylase homologues, designated gadA and gadB, that are differentially expressed. The gadB gene is co‐transcribed in tandem with an upstream gene, gadC, which encodes a potential glutamate/γ‐aminobutyrate antiporter. Expression of this transcript is upregulated in response to mild acid stress (pH 5.5). In contrast, expression of the monocistronic gadA message was weaker and was not induced by mild acid treatment. Non‐polar deletion mutations resulted in a dramatic decrease in the level of GAD activity and a concomitant decrease in acid resistance in the order LO28 > ΔgadA > ΔgadB = ΔgadC > ΔgadAB for both stationary and logarithmic phase cultures. The exquisite sensitivity of the ΔgadAB mutant to ex vivo porcine and synthetic human gastric fluid demonstrates a clear role for the GAD system in facilitating survival of the organism in the stomach after ingestion and in other low‐pH environments. Furthermore, variations in levels of GAD activity between different strains of L. monocytogenes correlate significantly with levels of tolerance to gastric fluid. Sensitive strains, which include the sequenced L. monocytogenes EGD, exhibit reduced levels of GAD activity. It is clear from this study that expression of GAD by L. monocytogenes strains is an absolute requirement for survival in the stomach environment.
Mismatch repair and the regulation of phase variation in Neisseria meningitidis
Authors: Richardson A. R. , Stojiljkovic I.
Published: March 2003

Neisseria meningitidis controls the expression of several genes involved in host adaptation by a process known as phase variation. The phase variation frequency of haemoglobin (Hb) receptors among clinical isolates of serogroups A, B and C differed drastically, ranging from ≈ 10−6 to 10−2 cfu−1. Frequencies of phase variation are a genetic trait of a particular strain, as two unlinked Hb receptors, hpuAB and hmbR, phase varied with similar frequencies within a given isolate. Based on these frequencies, six Neisserial clinical isolates could be grouped into three distinct classes; slow, medium and fast. An increase in phase variation frequency was accompanied by high rates of spontaneous mutation to rifampicin and nalidixic acid resistance in one medium and one fast strain. The remaining three medium strains displayed elevated levels of phase variation without increases in overall mutability, as they possessed low rates of spontaneous mutation to drug resistance. The mismatch repair system of N. meningitidis was found to play an important role in determining the overall mutability of the clinical isolates. Inactivation of mismatch repair in any strain, regardless of its original phenotype, increased mutability to a level seen in the fast strain. Insertional inactivation of mutS and mutL in the slow strain led to 500‐ and 250‐fold increases in hmbR switching frequency respectively. Concurrently, the frequency of spontaneous point mutations of mutS and mutL mutants from the slow strain was increased 20‐ to 30‐fold to the level seen in the high strain. The status of Dam methylation did not correlate with either the phase variation frequency of Hb receptors or the general mutability of Neisserial strains. Analysis of an expanded set of isolates identified defects in mismatch repair as the genetic basis for strains displaying both the fast Hb switching and high mutation rate phenotypes. In conclusion, elevated frequencies of phase variation were accompanied by increased overall mutability in some N. meningitidis isolates including strains shown to be mismatch repair defective. Other isolates have evolved mechanisms that seem to affect only the switching frequency of phase‐variable genes without an accompanied increased accumulation of spontaneous mutations.
A Streptococcus pneumoniae pathogenicity island encoding an ABC transporter involved in iron uptake and virulence
Authors: Brown Jeremy S. , Gilliland Sarah M. , Holden David W.
Published: March 2003

Restricted iron availability is a major obstacle to growth and survival of pathogenic bacteria during infection. In contrast to Gram‐negative pathogens, little is known about how Gram‐positive pathogens obtain this essential metal. We have identified two Streptococcus pneumoniae genetic loci, pit1 and pit2, encoding homologues of ABC iron transporters that are required for iron uptake by this organism. S. pneumoniae strains containing disrupted copies of either pit1 or pit2 had decreased sensitivity to the iron‐dependent antibiotic streptonigrin, and a strain containing disrupted copies of both pit1 and pit2 was unable to use haemoglobin as an iron source and had a reduced rate of iron uptake. The pit2− strain was moderately and the pit1−/pit2− strain strongly attenuated in virulence in mouse models of pulmonary and systemic infection, showing that the pit loci play a critical role during in vivo growth of S. pneumoniae. The pit2 locus is contained within a 27 kb region of chromosomal DNA that has several features of Gram‐negative bacterial pathogenicity islands. This probable pathogenicity island (PPI‐1) is the first to be described for S. pneumoniae, and its acquisition is likely to have played a significant role in the evolution of this important human pathogen.
Activation and repression of transcription initiation by a distant DNA structural transition
Authors: Sheridan Steven D. , Opel Michael L. , Hatfield G. Wesley
Published: March 2003

Negative superhelical tension can drive local transitions to alternative DNA structures. Long regions of DNA may contain several sites that are susceptible to forming alternative structures. Their relative propensities to undergo transition are ordered according to the energies required for their formation. These energies have two components – the energy needed to drive the transition and the energy relieved by the partial relaxation of superhelicity that the transition provides. This coupling can cause a complex competition among the possible transitions, in which the formation of one energetically favourable alternative structure may inhibit the formation of another within the same domain. In principle, DNA structural competitions can affect the structural and energetic requirements for the initiation of transcription at distant promoter sites. We have tested this possibility by examining the effects of structural transitions on transcription initiation from promoter sites in the same superhelical domain. Specifically, we describe the effects of the presence of a Z‐DNA‐forming DNA sequence on the basal levels of expression of two supercoiling‐sensitive promoters of Escherichia coli, ilvPG and gyrA. We demonstrate transcriptional repression of the ilvPG promoter and activation of the gyrA promoter. We present evidence that this regulation is effected by the superhelically induced B‐ to Z‐DNA transition in a manner that is both orientation and distance independent. We discuss the mechanism of topological coupling between left‐handed Z‐DNA and the regulation of promoter activity. We also discuss the possibility that the coupling of DNA structural transitions and transcriptional activity might be used as a general regulatory mechanism for gene expression.
Pathogenicity island‐dependent activation of Rho GTPases Rac1 and Cdc42 in Helicobacter pylori infection
Authors: Churin Yuri , Kardalinou Efterpi , Meyer Thomas F. , Naumann Michael
Published: March 2003

Helicobacter pylori has been identified as the major aetiological agent in the development of chronic gastritis and duodenal ulcer, and it plays a role in the development of gastric carcinoma. Attachment of H. pylori to gastric epithelial cells leads to nuclear and cytoskeletal responses in host cells. Here, we show that Rho GTPases Rac1 and Cdc42 were activated during infection of gastric epithelial cells with either the wild‐type H. pylori or the mutant strain cagA. In contrast, no activation of Rho GTPases was observed when H. pylori mutant strains (virB7 and PAI) were used that lack functional type IV secretion apparatus. We demonstrated that H. pylori‐induced activation of Rac1 and Cdc42 led to the activation of p21‐activated kinase 1 (PAK1) mediating nuclear responses, whereas the mutant strain PAI had no effect on PAK1 activity. Activation of Rac1, Cdc42 and PAK1 represented a very early event in colonization of gastric epithelial cells by H. pylori. Rac1 and Cdc42 were recruited to the sites of bacterial attachment and are therefore probably involved in the regulation of local and overall cytoskeleton rearrangement in host cells. Finally, actin rearrangement and epithelial cell motility in H. pylori infection depended on the presence of a functional type IV secretion system encoded by the cag pathogenicity island (PAI).
Identification and characterization of a family of secretion‐related small GTPase‐encoding genes from the filamentous fungus Aspergillus niger : a putative SEC4 homologue is not essential for growth
Authors: Punt Peter J. , Seiboth Bernhard , Weenink Xavier O. , Van Zeijl Cora , Lenders Maja , Konetschny Christian , Ram Arthur F. J. , Montijn Roy , Kubicek Christian P. , Van Den Hondel Cees A. M. J. J.
Published: March 2003

DNA fragments containing genetic information for five secretion‐related small GTPases of Aspergillus niger (srgA–E) were isolated and identified as members of different Rab/Ypt subfamilies. This isolation and the search for similar sequences in fungal genomic and EST databases showed that, in contrast to Saccharomyces cerevisiae, filamentous fungi also possess homologues of mammalian Rab2 GTPases. Multiple transcripts with unusually long 5′ and 3′ untranslated regions were found for all srg genes. Their level of expression was independent of the type of carbon source used for growth. Although the transcripts of srgA and srgB were abundant to the same extent throughout the cultivation, that of the other genes peaked during the early growth phase and then declined. Two genes, srgA and srgB, were characterized further. The protein encoded by srgA exhibited relatively low identity (58%) to its closest S. cerevisiae homologue SEC4, whereas the protein encoded by srgB showed 73% identity with S. cerevisiae YPT1. In contrast to other SEC4 homologues, srgA was unable to complement an S. cerevisiae sec4 mutant, and its disruption was not lethal in A. niger. SrgA mutants displayed a twofold increase in their hyphal diameter, unusual apical branching and strongly reduced protein secretion during growth on glucose.
Convergence of two global transcriptional regulators on nitrogen induction of the stress‐acclimation gene nblA in the cyanobacterium Synechococcus sp. PCC 7942
Authors: Luque Ignacio , Zabulon Gérald , Contreras Asunción , Houmard Jean
Published: March 2003

Cyanobacteria respond to environmental stress conditions by degrading their phycobilisomes, the light harvesting complexes for photosynthesis. The expression of nblA, a key gene in this process, is controlled by the response regulator NblR in Synechococcus sp. PCC 7942. Here we show that, under nitrogen stress, nblA is also regulated by NtcA, the global regulator for nitrogen control. NtcA activation of nblA was found to be nitrogen‐specific and did not take place under sulphur stress. Transcripts from the two major transcription start points (tsp) for the nblA gene were induced in response to nitrogen and sulphur starvation. The most active one (tspII) required both NblR and NtcA to induce full nblA expression under nitrogen starvation. NblR and NtcA bound in vitro to a DNA fragment from the nblA promoter region, suggesting that, under nitrogen stress, both NblR and NtcA activate the main regulated promoter (PnblA‐2) by direct DNA‐binding. The structure of PnblA‐2 differs from that of the canonical NtcA‐activated promoter and it is therefore proposed to represent a novel type of NtcA‐dependent promoter. We analysed expression patterns from ntcA and selected NtcA targets in NtcA–, NblR– and wild‐type strains, and discuss data suggesting further interrelations between phycobilisome degradation and nitrogen assimilation regulatory pathways.
CheC is related to the family of flagellar switch proteins and acts independently from CheD to control chemotaxis inBacillus subtilis
Authors: Kirby John R. , Kristich Christopher J. , Saulmon Michael M. , Zimmer Michael A. , Garrity Liam F. , Zhulin Igor B. , Ordal George W.
Published: March 2003

Chemotaxis byBacillus subtilisrequires the inter‐acting chemotaxis proteins CheC and CheD. In this study, we show that CheD is absolutely required for a behavioural response to proline mediated by McpC but is not required for the response to asparagine mediated by McpB. We also show that CheC is not required for the excitation response to asparagine stimulation but is required for adaptation while asparagine remains complexed with the McpB chemoreceptor. CheC displayed an interaction with the histidine kinase CheA as well as with McpB in the yeast two‐hybrid assay, suggesting that the mechanism by which CheC affects adaptation may result from an interaction with the receptor–CheA complex. Furthermore, CheC was found to be related to the family of flagellar switch proteins comprising FliM and FliY but is not present in many proteobacterial genomes in which CheD homologues exist. The distinct physiological roles for CheC and CheD duringB. subtilischemotaxis and the observation that CheD is present in bacterial genomes that lack CheC indicate that these proteins can function independently and may define unique pathways during chemotactic signal transduction. We speculate that CheC interacts with flagellar switch components and dissociates upon CheY‐P binding and subsequently interacts with the receptor complex to facilitate adaptation.
Regulation of capsule gene expression by group A Streptococcus during pharyngeal colonization and invasive infection
Authors: Gryllos Ioannis , Cywes Colette , Shearer Michael H. , Cary Max , Kennedy Ronald C. , Wessels Michael R.
Published: March 2003

Capsular polysaccharide production by group A Streptococcus (GAS) is controlled by transcription of the has operon that encodes the enzymes uniquely required for synthesis of the hyaluronic acid polysaccharide. To investigate the regulation of capsule gene expression during infection, we developed a reporter strain of GAS in which the has operon promoter directed transcription of green fluorescent protein (GFP). Gfp expression was triggered within minutes after introduction of the reporter strain into the peritoneal cavity of mice, as evidenced by the recovery of highly fluorescent GAS from the peritoneum 1 h after challenge. Capsule gene expression was also stimulated in the bloodstream of infected mice, as intensely fluorescent bacteria were observed in blood samples collected after either intraperitoneal or intravenous challenge. Using a similar approach, we also observed rapid induction of capsule gene expression in bacteria inoculated into the pharynx of baboons. Compared to the inoculum, increased green fluorescence was recorded in bacteria recovered from throat swabs collected 1 h after inoculation in all five animals studied. We conclude that introduction of GAS into the pharynx or into deep tissues results in rapid induction of has operon expression, a critical adaptive response that enhances GAS survival in the infected host.
Emerging views on tmRNA‐mediated protein tagging and ribosome rescue
Authors: Gillet Reynald , Felden Brice
Published: March 2003

Transfer‐messenger RNA (tmRNA), also known as SsrA or 10Sa RNA, is a bacterial ribonucleic acid that recycles 70S ribosomes stalled on problematic messenger RNAs (mRNAs) and also contributes to the degradation of incompletely synthesized peptides. tmRNA acts initially as transfer RNA (tRNA), being aminoacylated at its 3′‐end by alanyl‐tRNA synthetase, to add alanine to the stalled polypeptide chain. Resumption of translation ensues not on the mRNA on which the ribosomes were stalled but at an internal position in tmRNA. Termination soon occurs, tmRNA recruiting the appropriate termination factors allowing the release of the tagged protein that is subsequently recognized and degraded by specific cytoplasmic and periplasmic proteases, and permits ribosome recycling. Recent data suggest that tmRNA tags bacterial proteins in three other instances; when ribosomes stall at internal sites; during ‘readthrough’ of canonical termination codons; and when ribosomes are at the termination codon of intact messages. The importance of bacterial tmRNAs for survival, growth under stress, and pathogenesis is also discussed. Recent in vivo and in vitro studies have identified novel ligands of tmRNA. Based on the available experimental evidences, an updated model of tmRNA mediated protein tagging and ribosome rescue in bacteria is presented.
Replication restart in UV‐irradiated Escherichia coli involving pols II, III, V, PriA, RecA and RecFOR proteins
Authors: Rangarajan Savithri , Woodgate Roger , Goodman Myron F.
Published: March 2003

In Escherichia coli, UV‐irradiated cells resume DNA synthesis after a transient
inhibition by a process called replication restart. To elucidate the role of several
key proteins involved in this process, we have analysed the time dependence of replication
restart in strains carrying a combination of mutations in lexA, recA,
polB (pol II), umuDC (pol V), priA, dnaC, recF,
recO or recR. We find that both pol II and the origin‐independent primosome‐assembling
function of PriA are essential for the immediate recovery of DNA synthesis after
UV irradiation. In their absence, translesion replication or ‘replication
readthrough’ occurs ≈ 50 min after UV and is pol V‐dependent. In
a wild‐type, lexA+ background, mutations in recF,
recO or recR block both pathways. Similar results were obtained with
a lexA(Def) recF strain. However, lexA(Def) recO or lexA(Def)
recR strains, although unable to facilitate PriA‐pol II‐dependent restart,
were able to perform pol V‐dependent readthrough. The defects in restart attributed
to mutations in recF, recO or recR were suppressed in a recA730
lexA(Def) strain expressing constitutively activated RecA (RecA*). Our data suggest
that in a wild‐type background, RecF, O and R are important for the induction of
the SOS response and the formation of RecA*‐dependent recombination intermediates
necessary for PriA/Pol II‐dependent replication restart. In con‐trast, only RecF
is required for the activation of RecA that leads to the formation of pol V (UmuD′2C) and facilitates replication readthrough.
Screening for the target gene of cyanobacterial cAMP receptor protein SYCRP1
Authors: Yoshimura Hidehisa , Yanagisawa Shuichi , Kanehisa Minoru , Ohmori Masayuki
Published: March 2003

The target genes for SYCRP1, a cyanobacterial cAMP receptor protein, were surveyed using a DNA micro‐array method. Total RNAs were extracted from a wild‐type strain and a sycrp1 disruptant of Synechocystis sp. PCC 6803, and the respective
gene expression levels were compared. The expression levels of six genes (slr1667,
slr1668, slr2015, slr2016, slr2017 and slr2018) were clearly decreased by the disruption
of the sycrp1 gene. The data suggest that slr1667 and slr1668 constitute one
operon and the other four genes constitute another operon. Transcription start points
for the first genes of these putative operons, which are slr1667 and slr2015, were
determined by primer extension experiments. Gel mobility shift assays and DNase I
footprint analyses were carried out to explore the binding of SYCRP1 to the putative
promoter regions of slr1667 and slr2015. SYCRP1 bound to the specific site in the
5′ upstream region of slr1667 from positions –170 to –155
relative to the transcription start point, while it did not bind to the 5′ upstream region of slr2015. It was concluded that SYCRP1 regulates the expression of the slr1667 gene directly by binding to a specific site in its promoter.
A distinctive single‐stranded DNA‐binding protein from the Archaeon Sulfolobus solfataricus
Authors: Haseltine Cynthia A. , Kowalczykowski Stephen C.
Published: March 2003

Single‐stranded DNA binding proteins (SSBs) have been identified in all three domains of life. Here, we report the identification of a novel crenarchaeal SSB protein that is distinctly different from its euryar‐chaeal counterparts. Rather than comprising four DNA‐binding domains and a zinc‐finger motif within a single polypeptide of 645 amino acids, as for Methanococcus jannaschii, the Sulfolobus solfataricus

SSB protein (SsoSSB) has a single DNA‐binding domain in a polypeptide of just 148

amino acids with a eubacterial‐like acidic C‐terminus. SsoSSB protein was purified

to homogeneity and found to form tetramers in solution, suggesting a quaternary structure

analogous to that of E. coli SSB protein, despite possessing DNA‐binding domains

more similar to those of eukaryotic Replication Protein A (RPA). We demonstrate distributive

binding of SsoSSB to ssDNA at high temperature with an apparent site size of approximately

five nucleotides (nt) per monomer. Additionally, the protein is functional both in

vitro and in vivo, stimulating RecA protein‐mediated DNA strand‐exchange

and rescuing the ssb‐1 lethal mutation of E. coli respectively. We dis‐cuss possible evolutionary relationships amongst the various members of the SSB/RPA family.
Bacterial secrets of secretion: EuroConference on the biology of type IV secretion processes
Authors: Baron Christian , O´Callaghan David , Lanka Erich
Published: March 2003

Type IV secretion systems (TFSS) mediate secretion or direct cell‐to‐cell transfer of virulence factors (proteins or protein–DNA complexes) from many Gram‐negative animal, human and plant pathogens, such as Agrobacterium tumefaciens, Bartonella
tribocorum, Bordetella pertussis, Brucella suis, Helicobacter
pylori, Legionella pneumophila and Rickettsia prowazekii, into
eukaryotic cells. Bacterial conjugation is also classified as a TFSS‐like process
mediating the spread of broad‐host‐range plasmids between Gram‐negative bacteria
such as RP4 and R388, which carry antibiotic resistance genes. Genetic, biochemical,
cell biological and structural biology experiments led to significant progress in
the understanding of several aspects of TFSS processes. X‐ray crystallography revealed
that homologues of the A. tumefaciens inner membrane‐associated proteins VirB11
and VirD4 from H. pylori and R388, respectively, may form channels for substrate
translocation or assembly of the transmembrane TFSS machinery. Biochemical and cell
biological experiments revealed interactions between components of the periplasmic
core components VirB8, VirB9 and VirB10, which may form the translocation channel.
Analysis of A. tumefaciens virulence proteins VirE2 and VirF suggested that
the periplasmic translocation route of the pertussis toxin from B. pertussis
may be more generally valid than previously anticipated. Secretion and modification
of toxins from H. pylori and L. pneumophila profoundly affect host cell metabolism, thus entering the discipline of cellular microbiology. Finally, results from genome sequencing projects revealed the presence of up to three TFSS in a single organism, and the analysis of their interplay and adaptation to different functions will be a future challenge. TFSS‐carrying plasmids were discovered in different ecosystems, suggesting that genetic exchange may speed up their evolution and adaptation to different cell–cell interactions.
Involvement of caspase‐3‐like protein in rapid cell death of Xanthomonas
Authors: Gautam Satyendra , Sharma Arun
Published: March 2003

Xanthomonas campestris pv. glycines strain AM2 (XcgAM2), the aetiological agent of bacterial pustule disease of soybean, as well as some other strains of Xanthomonas including X. campestris pv. malvacearum NCIM 2310 and X. campestris NCIM 2961, exhibited post‐exponential rapid cell death (RCD) in Luria–Bertani (LB) medium. RCD was not displayed by Xanthomonas strains while growing in starch medium. Addition of starch to LB culture of XcgAM2 at any point of incubation during the exponential growth was found to arrest the onset of RCD. RCD in this organism was found to be associated with the synthesis of an endogenous enzyme similar to human caspase‐3, a known marker of apoptosis in eukaryotes. On sodium dodecyl sulphate polyacrylamide gel elecrophoresis (SDS‐PAGE) the XcgAM2 caspase appeared to run along a 55 kDa protein molecular weight marker. The caspase‐3‐like protein was detected in all Xanthomonas strains tested. RCD was not detected in Escherichia coli cultures in LB medium. The caspase‐3‐like enzyme activity or pro‐tein was also found to be absent in this bacterium. Caspase‐3‐like protein or Xanthomonas caspase was detected only in the cells of XcgAM2 growing in LB medium and not in those growing in starch medium. The Xanthomonas caspase protein appeared in cells at around 4 h of incubation, and peaked at around 24 h, before finally disappearing at around 54 h of incubation. However, caspase enzyme activity was detected only 12–13 h after incubation and peaked around 18–20 h. Addition of starch at the beginning or during the period of exponential growth in LB cultures of XcgAM2 terminated the synthesis of this protein. It is presumed that starch acted as the repressor of biosynthesis of the Xanthomonas caspase, thereby preventing the organism from undergoing RCD. The cells undergoing RCD also displayed the other markers of eukaryotic apoptosis. These included binding of annexin V to plasma membrane of cells undergoing RCD and the presence of nicked DNA in culture supernatant as evidenced by the TUNEL (terminal deoxynucleotidyl transferase dUTP nick‐end labelling) assay. Caspase‐negative mutants of XcgAM2 did not display post‐exponential RCD. The importance of RCD in Xanthomonas life cycle is not yet clear, however the phenomenon appears to have similarities with eukaryotic apoptosis.
The C‐terminus of IcmT is essential for pore formation and for intracellular trafficking of Legionella pneumophila within Acanthamoeba polyphaga
Authors: Molmeret Maëlle , Alli O. A. Terry , Radulic Marina , Susa Milorad , Doric Miljenko , Kwaik Yousef Abu
Published: March 2003

We have shown previously that the five rib (release of intracellular
bacteria) mutants of Legionella pneumophila are competent for intracellular
replication but defective in pore formation‐mediated cytolysis and egress from protozoan
and mammalian cells. The rib phenotype results from a point mutation (deletion)
ΔG544 in icmT that is predicted to result in the expression
of a protein truncated by 32 amino acids from the C‐terminus. In contrast to the
rib mutants that are capable of intracellular replication, an icmT
null mutant was completely defective in intracellular replication within mammalian
and protozoan cells, in addition to its defect in pore formation‐mediated cytolysis.
The icmT wild‐type allele complemented the icmT null mutant for both
defects of intracellular replication and pore formation‐mediated cytolysis and egress
from mammalian cells. In contrast, the icmTΔG544 allele
complemented the icmT null mutant for intracellular growth, but not for the
pore‐forming activity. Consistent with their defect in pore formation‐mediated cytotoxicity
in vitro, both mutants failed to cause pulmonary inflammation in A/J mice.
Interestingly, the rib mutant was severely defective in intracellular growth
within Acanthamoeba polyphaga. Confocal laser scanning and electron microscopy
confirmed that the rib mutant and the icmT null mutant were severely
and completely defective, respectively, in intracellular growth in A. polyphaga,
and the respective defects correlated with fusion of the bacterial phagosomes to
lysosomes. Taken together, the data showed that the C‐terminus domain of IcmT is
essential for the pore‐forming activity and is required for intracellular trafficking
and replication within A. polyphaga, but not within mammalian cells.
Bacterial resistance evolution by recruitment of super‐integron gene cassettes
Authors: Rowe‐Magnus Dean A. , Guerout Anne‐Marie , Mazel Didier
Published: March 2003

The capture and spread of antibiotic resistance determinants by integrons underlies the rapid evolution of multiple antibiotic resistance among diverse Gram‐negative clinical isolates. The association of multiple resistance integrons (MRIs) with mobile DNA elements facilitates their transit across phylogenetic boundaries and augments the potential impact of integrons on bacterial evolution. Recently, ancestral chromosomal versions, the super‐integrons (SIs), were found to be genuine components of the genomes of diverse bacterial species. SIs possess evolutionary characteristics and stockpiles of adaptive functions, including cassettes related to antibiotic resistance determinants previously characterized in clinical isolates, which suggest that MRIs and their resistance genes were originally recruited from SIs and their pool of amassed genes. However, the recombination activity of integrons has never been demonstrated in a bacterium other than Escherichia coli. We introduced a naturally occurring

MRI (TpR, SulR) on a conjugative plasmid into Vibrio cholerae,

a species known to harbour a SI. We show that MRIs can randomly recruit genes directly

from the cache of SI cassettes. By applying a selective constraint for the development

of antibiotic resistance, we demonstrate bacterial resistance evolution through the

recruitment a novel, but phenotypically silent, chloramphenicol acetyltransferase

gene from the V. cholerae SI and its precise insertion into the MRI. The resulting

resistance profile (CmR, TpR, SulR) could then be disseminated by conjugation to other clinically relevant pathogens at high frequency. These results demonstrate that otherwise phenotypically sensitive strains may still be a genetic source for the evolution of resistance to clinically relevant antibiotics through integron‐mediated recombination events.
Over 1000 genes are involved in the DNA damage response of Escherichia coli
Authors: Khil Pavel P. , Camerini‐Otero R. Daniel
Published: March 2003

Changes 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

The 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

The 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

The 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

The 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

We 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.