bluuebunny/crossref_metadata_2025_split huggingface dataset:
An inner‐membrane‐associated virulence protein essential for T‐DNA transfer from Agrobacterium tumefaciens to plants exhibits ATPase activity and similarities to conjugative transfer genes
Authors: Shirasu Ken , Koukolíková‐Nicola Zdena , Hohn Barbara , Kado Clarence I. 
Published: October 2006

SummaryThe 9.5kb virB operon is the largest of the six major operons in the Ti plasmid vir region. This operon contains eleven genes, the largest of which is virB4. This gene encodes an 84kDa protein whose function has not been identified. Its roles in conferring virulence on Agrobacterium tumefaciens and in the T‐DNA transfer process were determined by generating non‐polar mutants by using the Tn5pvirB transposon in which the virB promoter is transcribed downstream of its position of insertion. Several independent mutants were isolated and each insertion site in virB4 was confirmed by nucleotide sequence analysis. These mutants were tested for T‐DNA transfer ability by agroinfection and for tumorigenicity by inoculation in Brassica and Datura. All mutants were agroinfection‐ and tumorigenicity‐negative. These data strongly suggest that virB4 is essential for both the interkingdom transfer of the T‐DNA and virulence. Furthermore, by using anti‐VirB4 serum, the protein product of virB4 was localized to the inner‐membrane fraction of A. tumefaciens. Purified VirB4 protein hydrolyses ATP and this activity was quenched by the anti‐VirB4 serum. The energy generated by VirB4 ATPase therefore may be used to transfer T‐DNA or to assemble the T‐DNA transfer apparatus on the bacterial membrane. Protein sequence analyses revealed striking similarities between VirB4 protein and the proteins required for conjugative transfer, which include TraC, TrwK, and TrbE of plasmids F, R388, and RP4, repectively. These findings suggest that VirB proteins play a direct role in the assembly of a conjugative transfer apparatus required for the transfer of the T‐DNA from A. tumefaciens to plant cells.
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

SummaryWe 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

SummaryThe 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

Summary 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

SummaryStudy 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

SummaryThe 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

SummaryPili 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

SummaryPseudomonas 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

SummaryThe 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

SummaryR27, 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

SummaryThe 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

SummaryThe 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

SummaryHybrid 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

SummaryThe 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

SummaryHomology 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.