bluuebunny/crossref_metadata_2025 huggingface dataset:
Evidence for a regulatory function of the histone‐like Escherichia coli protein H‐NS in ribosomal RNA synthesis
Authors: Tippner Detlev , Afflerbach Henning , Bradaczek Christiane , Wagner Rolf
Published: October 2006

We have isolated a small Escherichia coli protein which stably Interacts with ribosomal RNA P1 promoter DNA. We present evidence showing that the protein is identical to the histone‐like E. coli protein, H‐NS (H1). Binding of H‐NS to the P1 promoter region is dependent on the DNA curvature. Mapping the H‐NS‐DNA contact sites by nuclease protection and high‐resolution footprinting techniques reveal three H‐NS‐binding domains, and contacts of the protein in the major groove of the bent DNA. The binding region extends from position ‐18 to ‐89, relative to the P1 transcription start site, and shows an overlap with the known binding sites for Fis, another E. coli protein, which acts as transcriptional activator of P1. The binding of H‐NS does not displace Fis; instead, heterologous complexes are formed. Apparently, H‐NS and Fis bind to separated curved DNA segments, with the planes of the curves pointing into different directions, in vitro transcriptional analyses demonstrate that H‐NS represses rRNA P1 promoter‐directed transcription. Repression is most pronounced in the presence of Fis. Thus, H‐NS seems specifically to antagonize Fis‐dependent activation. No comparable inactivation is observed for the second rRNA promoter P2.
Leucine‐responsive regulatory protein and deoxyadenosine methylase control the phase variation and expression of the sfa and daa pili operons in Escherichia coli
Authors: van der Woude M. W. , Low D. A.
Published: October 2006

The Escherichia coli operons daa and sfa encode F1845 and S pill, respectively. In this paper we show that the expression of these operons is under phase variation control at a transcriptional level. The transcription of both operons is dependent on the global regulator leucine‐responsive regulatory protein (Lrp) and deoxyadenosine methylase (Dam). Lrp is required for methylation protection of two GATC sites located within conserved DNA sequences in the regulatory regions of these operons. These GATC sites are differentially methylated, establishing a methylation pattern which is characteristic of either the phase ON or phase OFF state. We also show that Lrp binds to the daa and sfa regulatory regions and that this binding is modulated by the methylation of the GATC sites. These results indicate that the phase variation of the daa and sfa operons is regulated by a mechanism involving differential binding of Lrp owing to methylation of GATC sites in the regulatory region, which is similar to the mechanism that controls phase variation of the pap operon.
IpaB mediates macrophage apoptosis induced by Shigella flexneri
Authors: Zychlinsky Arturo , Kenny Brendan , Ménard Robert , Prévost Marie‐Christine , Holland I. Barry , Sansonetti Philippe J.
Published: October 2006

Shigella flexneri kills macrophages through apoptosis, involving the induction of host cell DNA fragmentation and characteristic morphological changes. Shigella can only cause damage if it escapes from the phagolysosome into the cytoplasm. The S. flexneri cytotoxic genes have been localized to the ipa operon of shigella's virulence plasmid. ipaB, C and D deletion mutants are not invasive and therefore not cytotoxic. In order to distinguish genes involved in the escape from the phagolysosome as distinct from cytotoxicity, we constructed Shigella strains that secrete low amounts of Escherichia coli haemolysin (hlylow). These strains can escape into the cytoplasm of the macrophage even in the absence of the invasion plasmid as verified by electron microscopy and resistance to chloroquine. Macrophages were infected with different ipa mutants expressing hlylow. Both δipaC hlylow and δipaD hlylow were cytotoxic whilst δipaB hlylow and a hlylow strain cured of shigella's pathogenicity plasmid were not. Furthermore, both δipC ahlylow and δipaD hlylow killed through apoptosis as shown by both changes in ultrastructural morphology and fragmentation of the host ceil DNA. These results demonstrate that ipaB is essential for S. flexneri to induce apoptosis in macrophages.
Mutagenesis of Legionella pneumophila using Jn903 dllIacZ: identification of a growth‐phase‐regulated pigmentation gene
Authors: Wlater Lawrence A. , Sadosky Alesia B. , Shuman Howard A.
Published: October 2006

Study of the molecular basis for Legionella pneumophila pathogenicity would be facilitated with an efficient mutagen that can not only mark genomic mutations, but can also be used to reflect gene expression during macrophage infection. A derivative of Jn903, Tn903dlllacZ, is shown to transpose with high efficiency in L. pneumophila. Tn903dlllacZ encodes resistance to kanamycin (KmR) and carries a 5’truncated lacZ gene that can form translational fusions to L. pneumophila genes upon transposition. The cls‐acting Tn903 transposase is supplied outside Tn903dlllacZ, and hence chromosomally integrated copies are stable. KmR LacZ+ insertion mutants of L. pneumophila were isolated and shown by DNA hybridization to carry a single Tn903dlllacZ inserted within their chromosomes at various locations. One particular KmR LacZ+ mutant, AB1156, does not produce the brown pigment (Pig) characteristic of Legionella species. Tn903dlllacZ is responsible for this phenotype since reintroduction of the transposonlinked mutation into a wild‐type background results in a Pig phenotype. L. pneumophila pigment production is normally observed in stationary‐phase growth of cells in culture, and β‐galactosidase activity measured from the pig::lacZ fusion increased during the logarithmic‐phase growth and peaked at the onset of stationary phase. Interestingly, pig::lacZ expression also increased during macrophage infection. The pigment itself, however, does not appear to be required for L. pneumophila to grow within or kill host macrophages.
Molecular cloning and sequence of comK, a gene required for genetic competence in Bacillus subtilis
Authors: van Sinderen Douwe , ten Berge Annelies , Hayema Bert Jan , Hamoen Leendert , Venema Gerard
Published: October 2006

The transformation‐deficient strain E26, Isolated as a pHV60 insertion mutant, was used to isolate comK, a novel transcription unit required for genetic competence in Bacillus subtilis. Mutational analysis and sequence determination showed that comK contained one open reading frame (ORF), which could encode a protein of 192 amino acid residues with a predicted molecular weight of 22 500. An integrated copy of comK not only complemented the competence deficiency of a comK deletion mutant, but also that of strains E26 and FB93. Expression of comK occurred exclusively in glucose‐based minimal medium during the transition to stationary growth phase. Furthermore, the expression of late competence genes appeared to be dependent on the gene product of comK, the expression of which in turn depended on the presence of a functional comL (or srfA) transcription unit. These epistatic interactions indicate that comK is a competence locus occupying an intermediate position in the competence signal transduction network. Primer extension analysis showed that comK has one major transcription start site, preceded by a sequence resembling the consensus promoter used by the σA form of RNA polymerase.
The binding of Pseudomonas aeruginosa pili to glycosphingolipids is a tip‐associated event involving the C‐terminal region of the structural pilin subunit
Authors: Lee K. K. , Sheth H. B. , Wong W. Y. , Sherburne R. , Paranchych W. , Hodges R. S. , Lingwood C. A. , Krivan H. , Irvin R. T.
Published: October 2006

Pili are one of the adhesins of Pseudomonas aeruginosa that mediate adherence to epithelial cell‐surface receptors. The pili of P. aeruginosa strains PAK and PAO were examined and found to bind gangliotetraosyl ceramide (asialo‐GM1) and, to a lesser extend, ll3N‐acetylneuraminosylgangliotetraosyl ceramide (GM1) in solid‐phase binding assays. Asialo‐GM1, but not GM1, inhibited both PAK and PAK pili binding to immobilized asialo‐GM1 on the microtitre plate. PAO pili competitively inhibited PAK pili binding to asialo‐GM1, suggesting the presence of a structurally similar receptor‐binding domain in both pilus types. The interaction between asialo‐GM1 and pili occurs at the pilus tip as asialo‐GM1 coated colloidal gold only decorates the tip of purified pili. Three sets of evidence suggest that the C‐terminal disulphide‐bonded region of the Pseudomonas pilin is exposed at the tip of the pilus: (i) immunocytochemical studies indicate that P. aeruginosa pili have a basal‐tip structural differentiation where the monoclonal antibody (mAb) PK3B recognizes an antigenic epitope displayed only on the basal ends of pili (produced by shearing) while the mAb PK99H, whose antigenic epitope resides in residues 134–140 (Wong et al., 1992), binds only to the tip of PAK pili; (ii) synthetic peptides, PAK(128–144)ox‐OH and PAO(128–144)ox‐OH, which correspond to the C‐terminal disulphide‐bonded region of Pseudomonas pilin are able to bind to asialo‐GM1 and inhibit the binding of pili to the glycolipid; (iii) PK99H was shown to block PAK pilus binding to asialo‐GM1 Monoclonal antibody PK3B had no effect on PAK pili binding to asialo‐GM1 Thus, the adherence of the Pseudomonas pilus to glycosphingolipid receptors is a tip‐associated phenomenon Involving a tip‐exposed C‐terminal region of the pilin structural subunit.
The pili of Pseudomonas aeruginosa strains PAK and PAO bind specifically to the carbohydrate sequence βGalNAc(1–4)βGal found in glycosphingolipids asialo‐GM1 and asialo‐GM2
Authors: Sheth H. B. , Lee K. K. , Wong W. Y. , Srivastava G. , Hindsgaul O. , Hodges R. S. , Paranchych W. , Irvin R. T.
Published: October 2006

Pseudomonas aeruginosa employs pili to mediate adherence to epithelial cell surfaces. The pilus adhesin of P. aeruginosa strains PAK and PAO has been shown to bind to the glycolipid asialo‐GM1 (Lee et al., 1994 —accompanying article). PAK and PAO pili were examined for their abilities to bind to the synthetic βGalNAc(1–4)βGal (a minimal structural carbohydrate receptor sequence of asialo‐GM1 and asialo‐GM2 proposed by Krivan et al., 1988a) using solid‐phase binding assays. Both pill specifically bound to βGalNAc(1–4)βGal. The binding of βGal‐NAc(1–4)βGal‐Biotin to the Immobilized PAK and PAO pili was inhibited by corresponding free pili. The receptor binding domain of the PAK pilus resides in the C‐terminal disulphide‐looped region (residues 128–144) of the pilin structural subunit (Irvin et al., 1989). Biotinylated synthetic peptides corresponding the C‐terminal residues 128–144 of P. aeruginosa PAK and PAO pilin molecules were shown to bind to the βGalNAc(1–4)βGal‐(bovine serum albumin (BSA)). The binding of biotinylated peptides to βGalNAc‐(1–4)βGal‐BSA was inhibited by PAK pili, Ac‐KCTSDQDEOFIPKGCSK‐OH (AcPAK(128–144)ox‐OH) and Ac‐ACKSTQDPMFTPKGCDN‐OH (AcPAO(128–144)ox‐OH) peptides. (In these peptides Ac denotes Nα ‐acetylation of the N‐terminus, ‐OH means a peptide with a free a‐carboxyl group at the C‐terminus and the‘ox’denotes the oxidation of the sulphhydryl groups of Cys–129 and Cys–142.) Both acetylated peptides were also able to inhibit the binding of βGalNAc(1–4)βGal‐biotin to the corresponding BSA‐Peptide(128–144)ox‐OH conjugates. The βGlcNAc(1–3)βGal(1–4)βGlc‐biotin conjugate was unable to specifically bind to either Immobilized PAK and PAO pili or the respective C‐termlnal peptides. The data above demonstrated that the P. aeruginosa pili recognize asialo‐GM1 receptor analogue and that βGalNAc(1–4)βGal disaccharlde is sufficient for binding. Furthermore, the binding to βGalNAc(1–4)βGal was mediated by residues 128–144 of the pilin subunit.
Identification and cloning of a fur homologue from Neisseria meningitidis
Authors: Thomas Christopher E. , Sparlling P. Frederick
Published: October 2006

The iron response in a number of bacterial systems is mediated by fur (ferric uptake regulation)‐like regulatory systems. We have cloned and characterized a gene from Neisseria meningitidis that was homologous to Escherichia coli fur. This clone was capable of modulating expression from both E. coli and neisserial iron‐regulated promoters in response to iron, and it produced a protein that reacted with anti‐E. coli fur serum. Although the DNA and predicted amino acid sequences were very similar to those of four other published fur homologues, meningococcal fur was the most divergent of the group. Inability to construct a meningococcal fur mutant suggested that fur may be essential in this species.
Molecular analysis of RepHI1 A, a minimal replicon of the IncHI1 plasmid R27
Authors: Newnham P. J. , Taylor D. E.
Published: October 2006

R27, a large conjugative plasmid of the HI incompatibility group, was subjected to a subcloning analysis which revealed the presence of a Poll‐independent replicon and determinants contributing to incompatibility within a 2.7 kb SalI/Xbal fragment. The DNA sequence of the minimal replicon revealed the presence of a large open reading frame (ORF) as well two sets of 19 bp repeated oligonucleotides (iterons), In addition to characteristic Escherichia coli origin elements. The protein encoded by the ORF possesses homology with replication initiator proteins encoded by a number of plasmids from different incompatibility groups. Deletion analysis suggested that the iterons are responsible for incompatibility reactions. Dissection of the replicon confirmed this and defined a minimal origin of 230 bp. The putative replication initiator was expressed in an in vitro transcription‐translation system, and the 5’end of the mRNA encoding its synthesis was identified. Transcriptional fusion of the repA promoter to lacZ demonstrated an auto‐regulatory function of RepA. A series of iterons present downstream of the RepA coding sequence are dispensable but are responsible for copy‐number control. The minimal replicon appears to be partition‐defective.
Regulation of the permeability of the gonococcal cell envelope by the mtr system
Authors: Pan Wubin , Spratt Brian G.
Published: October 2006

The mtrR gene of Neisseria gonorrhoeae controls the level of susceptibility to hydrophobic antibiotics and detergents. The mtrR gene was cloned and shown to encode a putative transcriptional repressor. The mtr region was homologous to the envCD and acrAB regions of Escherichia coli, which are also involved in susceptibility to hydrophobic compounds. A homologous repressor protein was encoded by a previously unrecognized open reading frame within both the envCD and acrAB regions. Deletion of mtrR resulted in increased resistance to antibiotics and detergents: the mtrR mutations in two penicillin‐resistant clinical isolates resulted in a change of His–105 to Tyr. We propose that the mtrR repressor allows gonococci to regulate the permeability of its cell envelope in response to environmental signals, so that they can grow in the presence of toxic faecal lipids in the rectum as well as in the genital tract.
The cold‐shock response — a hot topic
Authors: Jones Pamela G. , Inouye Masayori
Published: October 2006

The cold‐shock response of Escherichia coli describes a specific pattern of gene expression In response to abrupt shifts to tower temperatures. This pattern includes the induction of cold‐shock proteins, synthesis of proteins involved in transcription and translation, and repression of heat‐shock proteins. The identified cold‐shock proteins are involved in various cellular functions from supercoiling of DNA to Initiation of translation. The major cold‐shock protein, CspA, has high sequence similarity with three other E. coli proteins ‐ CspB, CspC, and CspD. Using translational lacZ fusions, cspB was found to be cold‐shock inducible at the level of transcription like cspA, while cspC and cspD were not. The Csp proteins, which share sequence similarity with other prokaryotic proteins and with the‘cold‐shock domain’of eukaryotic Y‐box proteins, may have a function in activating transcription or unwinding or masking RNA molecules. Because the cold‐shock response can also be induced by the addition of certain inhibitors of translation, it has been proposed that the state of the ribosome is the physiological sensor for the induction. In addition to E. coll, cold‐shock proteins have also been found in other prokaryotic and eukaryotic organisms.
Reversible topology of a bifunctional transmembrane protein depends upon the charge balance around its transmembrane domain
Authors: Kim Hyeonsuk , Paul Saptarshi , Jennity Joseph , Inouye Masayori
Published: October 2006

Hybrid genes were constructed to express bifunctional hybrid proteins in which staphyloccal nuclease A with or without an amino‐terminai OmpA signal sequence was fused with TEM β‐lactamase (at the carboxyl terminal side) using the signal peptide of the major outer membrane lipoprotein of Escherichia coli as an internal linker. The hybrid proteins were found to be inserted in the membrane. Orientation of the hybrid protein with the OmpA signal peptide showed that the nuclease was translocated into the periplasm and the β‐lactamase remained in the cytoplasm. This indicates that the cleavable OmpA signal peptide served as a secretory signal for nuclease and the internal lipoprotein signal served as the transmembrane anchor, in the absence of the OmpA signal sequence the topology of the hybrid protein was reversed indicating that the internal lipoprotein signal peptide initially served as the signal peptide for the secretion of the carboxy terminal β‐lactamase domain across the membrane and subsequently as a membrane anchoring signal. The role of charged amino acids in the translocation and transmembrane orientation of membrane proteins was also analysed by introducing charged amino acids to either or both sides of the internal lipoprotein signal sequence in the bifunctional hybrid proteins in the absence of the amino‐terminal signal sequence. Introduction of two lysine residues at the carboxy‐terminal side of the internal signal sequence reversed the topology of the transmembrane protein by translocating the aminoterminal nuclease domain across the membrane, leaving the carboxyl terminal β‐actamase domain in the cytoplasm. When three more lysine residues were added to the amino‐terminal side of the internal signal sequence of the same construct the membrane topology flipped back to the original orientation. A similar reversion of the topology could be obtained by introducing negatively charged residues at the amino‐terminal side of the internal signal sequence. Present results demonstrate for the first time that a bifunctional transmembrane protein can be engineered to assume either of the two opposite orientations and that charge balance around the transmembrane domain is a major factor in controlling the topology of a transmembrane protein.
Family of the major cold‐shock protein, CspA (CS7.4), of Escherichia coli, whose members show a high sequence similarity with the eukaryotic Y‐box binding proteins
Authors: Lee Sang Jun , Xie Aiguo , Jiang Weining , Etchegaray Jean‐Pierre , Jones Pamela G. , Inouye Masayori
Published: October 2006

The cspA is a gene of Escherichia coli, whose expression is specifically induced at low temperatures to a level of 13% of total protein synthesis. The CspA protein consisting of 70 amino add residues has high sequence similarity with eukaryotic Y‐box DNA‐binding proteins. We found two independent clones from the Kohara miniset phage collection, which hybridized with a DNA fragment containing cspA. DNA sequencing of these clones confirmed that the two genes are highly homologous to cspA. One designated cspB is mapped at 35 min on the E. coli chromosome and encodes a 71‐residue protein with 79% identity to CspA, while the other, cspC, is mapped at 40 min and encodes a 69‐residue protein with 70% identity. In addition, a DNA sequence upstream of the clpA gene at 19 mm published elsewhere contains an open reading frame for a 74‐residue protein with 45% identity to CspA. All csp genes were fused in the coding regions with the lacZ gene, and the expression of β‐galactosidase was examined for these hybrid genes upon cold shock. A similar cold‐shock induction to cspA was observed for cspB but not cspC and cspD. These results Indicate that E. coli has a family of the cspA gene, some of which are induced by cold shock.
Two novel families of bacterial membrane proteins concerned with nodulation, cell division and transport
Authors: Saier M. H. , Tam R. , Reizer A. , Reizer J.
Published: October 2006

Homology has been established for members of two families of functionally related bacterial membrane proteins. The first family (the resistance/nodulation/cell division (RND) family) Includes (i) two metal‐resistance efflux pumps in Alcaligenes eutrophus (CzcA and CnrA), (ii) three proteins which function together in nodulation of alfalfa roots by Rhizobium meliloti (NoIGHI), and (iii) a cell division protein in Escherichia coli (EnvD). The second family (the putative membrane fusion protein (MFP) family) includes a nodulation protein (NoIF), a cell division protein (EnvC), and a multidrug resistance transport protein (EmrA). We propose that an MFP functions co‐operatively with an RND protein to transport large or hydrophobic molecules across the two membranes of the Gram‐negative bacterial cell envelope.
The molar ratio of σ73 to core polymerase in the obligate intracellular bacterium, Rickettsia prowazekii
Authors: Ding Han‐Fei , Winkler Herbert H.
Published: October 2006

In the obligate Intracellular parasitic bacterium, Rickettsia prowazekii, the molar ratio of σ73 to core RNA polymerase, that is, the degree of saturation of the core polymerase by the catalytically active sigma factor, was very low. This ratio was determined from the radioactivity in rickettsial RNA polymerase immuno‐precipitated from crude extracts of infected L929 cells in which the parasite was exponentially growing. If we assume that, as Is true for the σ subunit, in R. prowazekii and Escherichia coli the β’and β subunits of the RNA polymerase have similar methionine and cysteine contents (the radiolabelled amino acids), the molar ratio of σ73 to core polymerase in R prowazekii would be 0.1. This is in striking contrast to E. Coli where the ratio is typically 0.4. it remains to be established whether this low sigma saturation results In a limitation of active RNA polymerase in R. prowazekii and contributes to its slow growth.
A louse involved in the regulation of replication in plasmid pSC 101
Authors: Manen Danielle , Xia Guixian , Caro Lucien
Published: October 2006

The origin of replication of plasmid pSC101 contains three directly repeated sequences RS1, RS2, and RS3 separated by 22 bp from two palindromic sequences, IR1 and IR2, which are partially homologous to the direct repeats. These inverted repeat (IR) sequences overlap the promoter of the repA gene which encodes a protein essential for plasmid replication. We have shown that RepA binds to the RS sites as a monomer and to the IR sites as a dimer. The influence of the IR1 site, and of the DNA segment that separates it from RS3, on plasmid copy number control has been studied in detail. We show that the integrity of IR1 is essential for efficient replication and plasmid stability, the critical site extending to the left of IR1 proper. We also show that the presence of IR1 modifies profoundly the binding properties of purified RepA protein to a segment of DNA containing the RS sequences. IR1 is separated from its homologous site on RS3 by approximately four turns of the DNA helix. Replication is abolished if this distance is increased by half a turn of the helix but it is restored if the distance is increased by a whole turn. These results suggest a DNA looping interaction, in the initiation of replication, between the RepA dimer that binds iR1 and the RepA monomers that bind the RS sequences.
Disruption of two genes for chitin synthase in the phytopathogenic fungus Ustilago maydis
Authors: Gold S. E. , Kronstad J. W.
Published: October 2006

The phytopathogenic fungus Ustilago maydis exhibits a dimorphic transition in which non‐pathogenic, yeast‐like cells mate to form a pathogenic, filamentous dikaryon. Northern analysis indicated that two chitin synthase genes, chs1 and chs2, from U. maydis are expressed at similar levels in yeast‐like cells and in cells undergoing the mating reaction leading to the filamentous cell type. A mutation was constructed in each of the chitin synthase genes by targeted gene disruption. Each mutant showed a reduction in the level of trypsin‐activated enzyme activity, compared with a wild‐type strain, but retained the wild‐type morphology, the ability to mate and the ability to form the filamentous pathogenic cell type.
Escherichia coli cad operon functions as a supplier of carbon dioxide
Authors: Takayama Miho , Ohyama Takao , Igarashi Kazuel , Kobayashi Hiroshi
Published: October 2006

We examined the gene expression of the Escherichia coli cad operon, which consisted of the genes cadB and cadA (lysine decarboxylase), using cells possessing cadB–lacZ fusion gene. The cad operon was expressed when O2 was limited, and the expression was optimal at pH6.3. The β‐galactosidase activity was lowered by the addition of sodium carbonate to the medium. The expression of the cad operon was reduced in cells containing the plasmid‐encoding ornithine decarboxylase, which produced carbon dioxide, indicating that the gene expression of the cad operon was regulated by carbon dioxide (or its derivatives). It is known that the Krebs cycle is a major pathway for producing carbon dioxide, and that its activity is repressed when O2 is limited. Thus, our present results suggested that the physiological role of the cad operon is to supply carbon dioxide when its internal level is lowered under O2‐limiting conditions at a low pH.
Coupling between mRNA synthesis and mRNA stability in Escherichia coli
Authors: Chow Janet , Dennis Patrick P.
Published: October 2006

Transiently stable products derived from the endonuclease cleavage of transcripts from the secEnusG and rplKAJLrpoBC operons have been identified. Cleavage sites for RNase III occur in the leader of the secEnusG transcript and in the L12‐β intercistronic space of the rplKAJLrpoBC transcript. A single RNase E cleavage site was located in the L1‐L10 intergenic space. Inactivation of RNase III and RNase E results respectively in a one‐ to twofold and a greater than 10‐fold stabilization of five mRNA sequences from within the secE, nusG, L11‐L1, L10 and β encoding cistrons. The relative amounts of each of these five mRNA sequences were found to be nearly constant when measured either in the presence or absence of cleavage by RNase III or RNase E. This clearly implies that any increases in the stability of these mRNA sequences resulting from the inactivation of processing by RNase III or RNase E are counterbalanced by changes in the mRNA synthesis rates. The mechanism that links mRNA synthesis to mRNA decay is not known.
Repression of tonB transcription during anaerobic growth requires Fur binding at the promoter and a second factor binding upstream
Authors: Young Glenn M. , Postle Kathleen
Published: October 2006

Although iron is an essential nutrient, its toxicity at high levels necessitates regulated transport. In Gram‐negative bacteria a central target for regulation is the TonB protein, an energy transducer that couples the cytoplasmic membrane proton motive force to active transport of (FeIII)‐siderophore complexes across the outer membrane. We have previously demonstrated the threefold repression of tonB transcription by excess iron in the presence of Fur repressor protein under aerobic conditions. In this report, we examine tonB regulation under anaerobic conditions where the solubility of iron is not a limiting factor and, presumably, siderophore‐mediated transport is not required. Under these conditions, tonB transcription is repressed at least 10‐foid by excess iron in the presence of Fur, but can be fully derepressed in the absence of Fur. Based on several lines of evidence, this anaerobic repression is not due to increased negative supercoiling as previously postulated. Our results rule out both supercoiling mediated decreased promoter function and increased Fur binding as mediators of anaerobic repression. Under iron‐limiting anaerobic conditions tonB expression is as high or higher than under iron‐limiting aerobic conditions, suggesting that promoter function has not decreased anaerobically. Furthermore, under anaerobic conditions in tonB+ strains, tonB promoter function is insensitive to the gyrase inhibitor novobiocin and to changes in medium osmolarity and temperature, three conditions known to change levels of supercoiling. We also rule out effects of mutations in arcA or fnr as mediators of anaerobic repression. Results from in vivo dimethyl sulphate protection foot‐printing indicate that Fur binds to an operator site between the ‐10 and ‐35 regions of the promoter but not to a less homologous operator site centered at +26. The binding is, if anything, weaker under anaerobic conditions, indicating that anaerobic repression is not mediated through Fur. Additional changes in the in vivo footprint upstream from the promoter implicate a second factor in tonB anaerobic repression. Together, these results suggest that the mechanism responsible for this regulation (and, by analogy, that of other anaerobically repressed, iron‐regulated genes such as cir, exbB, and fhuA) is a novel one.
Isolation and characterization of hypophosphite‐resistant mutants of Escherichia coli: identification of the FocA protein, encoded by the pfl operon, as a putative formate transporter
Authors: Suppmann Bernhard , Sawers Gary
Published: October 2006

Hypophosphite was used as a toxic analogue to identify genes whose products have a putative function in the transport of formate. Two Tn10‐derived insertion mutants were identified that exhibited increased resistance to high concentrations of hypophosphite in the culture medium. The transposon was located in the identical position in the focA (formate channel; previously termed orf) gene of the pfl operon in both mutants. A defined chromosomal focA nonsense mutant, which showed minimal polarity effects on pfl gene expression, had the same phenotype as the insertion mutants. Results obtained using a hycA‐lacZ fusion to monitor changes in the intracellular formate concentration in a focA mutant indicated that the level of formate inside the cell was elevated compared with the wild type. Moreover, it could be shown that there was a corresponding reduction of approximately 50% in the amount of formate excreted by a focA mutant into the culture medium. Taken together, these results indicate that formate accumulates in anaerobic ceils which do not have a functional focA gene product and that one function of FocA may be to export formate from the cell. A further significant result was that hypophosphite could substitute for formate in activating hycA gene expression. This hypophosphite‐dependent activation of hycA gene expression was reduced 10‐fold in a focA null mutant, suggesting that hypophosphite must first enter the cell before it can act as a signal to activate hycA expression. By analogy, these data suggest that FocA may also be functional in the import of formate into anaerobic Escherichia coli cells.

Site‐specific mutagenesis identified the translation initiation codon of focA as a GUG. Therefore, the FocA polypeptide has a molecular weight of 30958. FocA shows significant similarity at both the primary and secondary structural levels with the NirC protein of E. coli and the FdhC protein of Methanobacterium formicicum. All three proteins are predicted to be integral membrane proteins. A detailed in vivo TnphoA mutagenesis study predicted that FocA has six membrane‐spanning segments.
Rho and RNA: models for recognition and response
Authors: Platt Terry
Published: October 2006

Escherichia coli Rho factor Is required for termination of transcription at certain sites by RNA polymerase. Binding to unstructured cytosine‐containing RNA target sites, subsequent RNA‐dependent ATP hydrolysis, and an RNA‐DNA helicase activity that presumably facilitates termination, are considered essential for Rho function. Yet the RNA recognition elements have remained elusive, the parameters relating RNA binding to ATPase activation have been obscure, and the mechanistic steps that integrate Rho's characteristics with its termination function in vitro and in vivo have been largely undefined. Recent work offers new insights into these interactions with results that are both surprising and satisfying in the context of Rho's emerging structure. These include the requirements for binding and ATPase activation by a variety of RNA substrates, dynamic analyses of Rho tracking, helicase and termination activity, and the participation of a new factor (NusG) that interacts with Rho. Models for Rho function are considered in the light of these recent revelations.
Regulation of the Bacillus subtilis trp operon by an RNA‐binding protein
Authors: Gollnick Paul
Published: October 2006

The Bacillus subtilis tryptophan (trpEDCFBA) operon is regulated by transcription attenuation. Transcription is controlled by two alternative RNA secondary structures, which form in the leader transcript. In the presence of L‐tryptophan, a transcription terminator forms and the operon is not expressed, whereas in the absence of tryptophan, an antiterminator structure forms allowing transcription of the operon. The mechanism of selection between these alternative structures involves a trans‐acting RNA‐binding regulatory protein. This protein is the product of the mtrB gene and is called TRAP for trp attenuation p/rotein. TRAP has been shown to bind specifically to trp leader RNA, and to cause transcription of the trp operon to terminate in the leader region. The model for regulation suggests that in the presence of tryptophan, TRAP binds to the leader RNA and induces formation of the transcription terminator structure, whereas in the absence of tryptophan, the protein does not bind and the antiterminator is formed.
Structural dynamics of translating ribosomes: 16S ribosomai RNA bases that may move twice during translocation
Authors: Laughrea Michael
Published: October 2006

Recent footprinting, sedimentation and neutron‐scattering results obtained In vivo or on pre‐translocation and post‐translocation ribosomal complexes are integrated with cross‐linking and immunoelectron microscopy information, it is proposed that the 30S subunit pulses during translocation and that its preand post‐translocation structures are not necessarily identical. Accordingly, transiocation is characterized by three consecutive conformational states of the 30S and 50S subunits. State 1 (the pre‐translocation state) lasts until the elongation factor EF‐G·GTP complex binds to the ribosome or adopts the GTPase conformation. State 2 (the translocation state, or the peak or plateau of the pulse) follows and lasts until EF‐G adopts a subsequent conformation or is released from the ribosome. State 3 (the post‐translocation state) ensues and lasts until A (aminoacyl) site binding of aminoacyl‐tRNA. In state 2, 16S RNA hairpins 26 and 33‐33A, located in the platform and the head of the 30S subunit, respectively, become kinked or twisted, and residue A1503, near the decoding site, becomes exposed. A platform twist is associated with P (peptide) to E (exit) site tRNA movements and a head twist with pivoting of the peptidyl‐tRNA elbow from the A to the P site, around a (retractable?) S19 domain. These twists result in an unlocking of the platform and the head from the 50S subunit. Exposure of A1503 is tentatively associated with movements of mRNA or tRNA anticodon stem‐loops. These twisted or otherwise‐exposed residues readopt their previous setting upon completion of translocation, i.e. states 1 and 3 of 16S RNA differ more from state 2 than from each other. Yet the ribosome is never fully locked or unlocked at any time during elongation. It is unlocked in one or another respect in the pre‐ and post‐translocation states, and unlocked to the largest extent in state 2.

‘Le plus grand Phénomène de la Nature, le plus Merveilleux, est le Mouvement’ (The greatest, the most wonderful Phenomenon in Nature is Motion).

Pierre Moreau de Maupertuis
Expression of Escherichia coli pyruvate oxidase (PoxB) depends on the sigma factor encoded by the rpoS(katF) gene
Authors: Chang Ying‐Ying , Wang Ai‐Yu , Cronan John E.
Published: October 2006

The activity of Escherichia coli pyruvate oxidase (PoxB) was shown to be growth‐phase dependent; the enzyme activity reaches a maximum at early stationary phase. We report that PoxB activity is dependent on a functional rpoS(katF) gene which encodes a σ factor required to transcribe a number of stationary‐phase‐induced genes. PoxB activity as well as the β‐galactosidase encoded by a poxB::lacZ protein fusion was completely abolished in a strain containing a defective rpoS gene. Northern and primer extension analyses showed that poxB expression was regulated at the transcriptional level and was transcribed from a single promoter; the 5′ end of the mRNA being located 27 bp upstream of the translational initiation codon of poxB. The poxB gene was expressed at decreased levels under anaerobiosis; however, the anaerobic regulatory genes arcA, arcB or fnr were not involved in anaerobic poxB gene expression. Expression of the rpoS(katF) gene has been reported to be affected by acetate, the product of PoxB reaction. However, we found that poxB null mutations had no effect on rpoS(katF) expression. Inactivation of two genes involved In acetate metabolism, ackA and pta, had no effect on either poxB or rpoS(katF) expression.
Starvation‐induced expression of SspA and SspB: the effects of a null mutation in sspA on Escherichia coli protein synthesis and survival during growth and prolonged starvation
Authors: Williams Mark D. , Ouyang Tracy X. , Flickinger Michael C.
Published: October 2006

Maxicell labelling and two‐dimensional gel electro‐phoresis (2‐D PAGE) have identified the proteins encoded by sspA and sspB (SspA, SspB) as proteins D27.1 and A25.8, respectively, in the Escherichia coli gene‐protein database. SspA expression increases with decreasing growth rate and is induced by glucose, nitrogen, phosphate or amino acid starvation. The promoter, Pssp, is similar to gearbox promoters. Inactivation of SspA (sspA::neo) blocks sspB expression. [35S]‐methionine‐labelled proteins synthesized during growth and during stationary phase are different in δsspA strains compared to sspA strains. This difference is enhanced during extended stationary phase (24–72 h). Long‐term (10 d) viability of arginine‐starved isogenic strains shows that sspA cultures remain viable significantly longer than δsspA mutants. 2‐D PAGE of proteins expressed during exponential growth shows that expression of at least 11 proteins is altered in δsspA strains. A functional relA gene is required for sspA to affect protein synthesis.
Characterization of the carbon starvation‐inducible and stationary phase‐inducible gene slp encoding an outer membrane lipoprotein in Escherichia coli
Authors: Alexander Denise M. , St John Ann C.
Published: October 2006

Escherichia coli induces the expression of more than 50 proteins in response to starvation for a carbon source. Strains MC7 (csi7::phoA) and MC19 (csi19::phoA) contain fusions of a signal peptide‐deficient phoA reporter sequence to a csi (carbon starvation‐inducible) gene. PhoA expression increased when these strains were deprived of a carbon source or entered stationary phase but did not when the cells were deprived of a nitrogen source or subjected to osmotic, oxidative or thermal stress. Mapping and sequence analysis of the cloned phoA fusions in strains MC7 and MC19 indicated that they had occurred in different locations within the same previously unidentified gene. The wild‐type allele of this gene was cloned and the encoded protein was found to be a new lipoprotein. Therefore we propose to call this locus slp (starvation lipoprotein). The 22 kDa Slp protein is associated with the outer membrane fraction. The slp gene was located at 78.6 centisomes on the E. coli genetic map. The ‐10 and ‐35 regions upstream of the mRNA start site were characteristic of a σ70 promoter. The major transcript from this promoter was sufficiently large to contain slp sequences but not the downstream open reading frame. Induction of β‐galactosidase activity from a slp::lacZ translational fusion during carbon starvation or stationary phase was independent of cAMP, RpoS (KatF) and DnaK, all of which are known to affect the expression of certain starvation‐inducible or stationary phase‐inducible proteins.
Conjugative transposition of Tn916: preferred targets and evidence for conjugative transfer of a single strand and for a double‐stranded circular intermediate
Authors: Scott June R. , Bringel Françoise , Marra Diana , Van Alstine Gaylene , Rudy Christine K.
Published: October 2006

Transposition of conjugative transposons proceeds by excision and formation of a covalently closed circular Intermediate that includes at its joint the six flanking bases from its previous host (coupling sequences). To elucidate the role of the coupling sequences in this process and to determine the sequence of targets used by Tn916, we studied its insertion into a plasmid following conjugation. The results differ from those previously observed when Tn916 was introduced by transformation. They suggest that only one specific strand of the transposon molecule is transferred during the conjugation event and that complementary strand synthesis produces a double‐stranded transposon circle with no mismatches which serves as the reaction intermediate. Tn916 inserts preferentially at specific sites and the same targets are used when Tn916 comes from donors with different coupling sequences. An analysis of the sequences of preferred targets is presented.
A new component of bacteriophage Mu replicative transposition machinery: the Escherichia coli ClpX protein
Authors: Mhammedi‐Alaoul Amine , Pato Martin , Gama Marie‐Jo , Toussaint Ariane
Published: October 2006

We have shown previously that some particular mutations in bacteriophage Mu repressor, the frameshift vir mutations, made the protein very sensitive to the Escherichia coli ATP‐dependent Clp protease. This enzyme is formed by the association between a protease subunit (ClpP) and an ATPase subunit. ClpA, the best characterized of these ATPases, is not required for the degradation of the mutant Mu repressors. Recently, a new potential ClpP associated ATPase, ClpX, has been described. We show here that this new subunit is required for Mu vir repressor degradation. Moreover, ClpX (but not ClpP) was found to be required for normal Mu replication. Thus ClpX has activities that do not require its association with ClpP. In the pathway of Mu replicative transposition, the block resides beyond the strand transfer reaction, i.e. after the transposition reaction per se is completed, suggesting that ClpX is required for the transition to the formation of the active replication complex at one Mu end. This is a new clear‐cut case of the versatile activity of polypeptides that form multi‐component ATP‐dependent proteases.
Transcriptional regulation of prfA and PrfA‐regulated virulence genes in Listeria monocytogenes
Authors: Bohne Jutta , Sokolovic Zeljka , Goebel Werner
Published: October 2006

The ActA protein, the lecithinase PlcB and listeriolysin are the major PrfA‐dependant proteins synthesized when brain‐heart infusion (BHI)‐cultured Listeria monocytogenes is shifted to minimum essential medium (MEM) In the presence of the transcriptional inhibitor rifampicin. Enhanced synthesis of all three proteins under these conditions depends, however, on a short incubation (about 5 min) of the bacteria in MEM without rifampicin, suggesting that induction of these proteins in MEM requires de novo transcription. The enhanced synthesis of these three proteins is observed in the L. monocytogenes wild‐type strains EGD and NCTC 7973, both of which belong to the serotype 1/2a. A significant induction of the bicistronic mRNA for ActA and PlcB is observed in both strains shortly after shifting the bacteria from BHI to MEM. This mRNA as well as the monocistronic listeriolysin (hly)‐specific mRNA is highly stable in L. monocytogenes NCTC 7973 shifted to MEM. In contrast to the actA‐plcB mRNA, no enhanced transcription in MEM is observed for the regulatory prfA gene or for the PrfA‐controlled virulence genes hlyA and plcA in strain NCTC 7973. However, transcription of these genes is induced in strain EGD. Transcriptional induction of the mpl gene is observed in neither strain NCTC 7973 nor in strain EGD. The life‐time of the prfA, plcA, and mpl transcripts is short. ActA was also found to be the most abundant newly synthesized surface protein when the two wild‐type strains of L. monocytogenes replicated within the phagocytic cell line J774. ActA synthesis seemed to be induced in the cytoplasm since the non‐haemolytic mutant M3 did not induce ActA when taken up by J774 cells.
Lon‐dependent proteolysis of CcdA is the key control for activation of CcdB in plasmid‐free segregant bacteria
Authors: Van Melderen Laurence , Bernard Philippe , Couturier Martine
Published: October 2006

The ccd locus contributes to the stability of plasmid F by post‐segregational killing of plasmid‐free bacteria. The ccdB gene product is a potent cell‐killing protein and its activity is negatively regulated by the CcdA protein, in this paper, we show that the CcdA protein is unstable and that the degradation of CcdA is dependent on the Lon protease. Differences in the stability of the killer CcdB protein and its antidote CcdA are the key to post‐segregational killing. Because the half‐life of active CcdA protein is shorter than that of active CcdB protein, persistence of the CcdB protein leads to the death of plasmid‐free bacterial segregants.
Mechanism of action of tetanus and botulinum neurotoxins
Authors: Montecucco Cesare , Schiavo Glampietro
Published: October 2006

The clostridial neurotoxins responsible for tetanus and botulism are metallo‐proteases that enter nerve cells and block neurotransmitter release via zinc‐dependent cleavage of protein components of the neuroexocytosis apparatus. Tetanus neurotoxin (TeNT) binds to the presynaptic membrane of the neuromuscular Junction and is internalized and transported retroaxonally to the spinal cord. Whilst TeNT causes spastic paralysis by acting on the spinal inhibitory interneurons, the seven serotypes of botullnum neurotoxins (BoNT) induce a flaccid paralysis because they intoxicate the neuromuscular junction. TeNT and BoNT serotypes B, D, F and G specifically cleave VAMP/synaptobrevin, a membrane protein of small synaptic vesicles, at different single peptide bonds. Proteins of the presynaptic membrane are specifically attacked by the other BoNTs: serotypes A and E cleave SNAP‐25 at two different sites located within the carboxyl terminus, whereas the specific target of serotype C is syntaxin.
When replication forks stop
Authors: Bierne Hélène , Michel Bénédicte
Published: October 2006

DNA synthesis is an accurate and very processive phenomenon, yet chromosome replication does not proceed at a constant rate and progression of the replication fork can be impeded. Several structural and functional features of the template can modulate the rate of progress of the replication fork. These include DNA secondary structures, DNA damage and occupied protein‐binding sites. In addition, prokaryotes contain sites where replication is specifically arrested. DNA regions at which the replication machinery is blocked or transiently slowed could be particularly susceptible to genome rearrangements. Illegitimate recombination, a ubiquitous phenomenon which may have dramatic consequences, occurs by a variety of mechanisms. The observation that some rearrangements might be facilitated by a pause in replication could provide a clue in elucidating these processes. In support of this, some homologous and illegitimate recombination events have already been correlated with replication pauses or arrest sites.