bluuebunny/crossref_metadata_2025_split huggingface dataset:
Questions about gonococcal pilus phase‐ and antigenic variation
Authors: Seifert H. Steven 
Published: October 2006

SummaryPathogenic organisms inhabit one of several defined locations within a host where temperature, pH, and nutrients are relatively constant. While the microorganism must adapt to different environments within the host, the host immune system is the most formidable predator that can limit the growth of a pathogen. Neisseria gonorrhoeae (the gonococcus, Gc) is the causative agent of gonorrhoea, and has evolved several systems for varying the antigenicity of different surface antigens, presumably to help evade the effects of the human immune system. The On/Off/On phase variation of surface structure expression also alters the antigenic characteristics of the bacterial cell surface. Antigenic variation of the major subunit of the pilus, pilin, occurs by unidirectional, homologous recombination between a silent locus and the expression locus. The silent loci lie from 1 to 900 kb from the expression locus in the chromosome yet all can donate their sequences to the expression locus. The genetic composition of the pilin loci of two Gc strains has been elucidated, and the types of changes that lead to altered forms of the pilus have been extensively characterized. However, little is known about the precise molecular mechanisms used to allow high‐frequency, non‐reciprocal, chromosomal recombination between pilin loci or about what regulates the process of maintaining chromosome fidelity.
Genetic analysis of the transcriptional arrangement of Azotobacter vinelandii alginate biosynthetic genes: identification of two independent promoters
Authors: Lloret Lourdes , Barreto Rita , León Renato , Moreno Soledad , MartÍnez‐Salazar Jaime , Espín Guadalupe , Soberón‐Chávez Gloria 
Published: October 2006

SummaryThe study of alginate biosynthesis, the exopolysac charide produced by Azotobacter vinelandii and Pseudomonas aeruginosa, might lead to different bio‐technological applications. Here we report the cloning of A. vinelandii algA, the gene coding for the bifunctional enzyme phosphomannose isomerase‐guano‐sine diphospho‐D‐mannose pyrophosphorylase (PMI‐GMP). This gene was selected by the complementation for xanthan gum production of Xanthomonas campestris pv. campestris xanB mutants, which lack this enzymatic activity. The complementing cosmid clones selected, besides containing algA, presented a gene coding for an alginate lyase activity (algL), and some of them also contained algD which codes for GDP‐mannose dehydrogenase. We present here the characterization of the A. vinelandii chromosomal region comprising algD and its promoter region, algA and algL, showing that, as previously reported for P. aeruginosa, A. vinelandii has a cluster of the biosynthetic alginate genes. We provide evidence for the presence of an algD‐independent promoter in this region which transcribes at least algL and algA, and which is regulated in a manner that differs from that of the algD promoter.
In vivo immobilization of enzymatically active polypeptides on the cell surface of Staphylococcus carnosus
Authors: Strauss Andreas , Götz Friedrich 
Published: October 2006

SummaryMany surface proteins of Gram‐positive bacteria are covalently anchored to the cell wall by a ubiquitous mechanism, involving a specific, C‐terminal sorting signal. To achieve cell‐wall immobilization of a normally secreted enzyme in vivo, we constructed a hybrid protein consisting of Staphylococcus hyicus lipase and the C‐terminal region of Staphylococcus aureus fibronectin binding protein B (FnBPB). This region comprised the authentic cell‐wall‐spanning region and cell‐wall sorting signal of FnBPB. Expression of the hybrid protein in Staphylococcus carnosus resulted in efficient cell‐wall anchoring of enzymatically active lipase. The cell‐wall‐immobilized lipase (approximately 10000 molecules per cell) retained more than 80% of the specific activity, compared to the C‐terminally unmodified S. hyicus lipase secreted by S. carnosus cells. After releasing the hybrid protein from the cell wall by lysostaphin treatment, its specific activity was indistinguishable from that of the unmodified lipase. Thus, the C‐terminal region of FnBPB per se was fully compatible with folding of the lipase to an active conformation. To study the influence of the distance between the cell‐wall sorting signal and the C‐terminus of the lipase on the activity of the immobilized lipase, the length of this spacer region was varied. Reduction of the spacer length gradually reduced the activity of the surface‐immobilized lipase. On the other hand, elongation of this spacer did not stimulate the activity of the immobilized lipase, indicating that the spacer must exceed a critical length of approx. 90 amino acids to allow efficient folding of the enzyme, which probably can only be achieved outside the pep‐tidoglycan web of the cell wall. When the lipase was replaced by another enzyme, the Escherichia coliβ‐lactamase, the resulting hybrid was also efficiently anchored in an active conformation to the cell wall of S, carnosus. These results demonstrate that it is possible to immobilize normally soluble enzymes on the cell wall of S. carnosus ‐ without radically altering their catalytic activity ‐ by fusing them to a cell‐wall‐immobilization unit, consisting of a suitable cellwall‐spanning region and a standard cell‐wall sorting signal.
The role of the Saccharomyces cerevisiae CCC1 gene in the homeostasis of manganese ions
Authors: Lapinskas Paula Jean , Lin Su‐Ju , Culotta Valeria Cizewski 
Published: October 2006

Summary We previously reported that oxidative damage in yeast lacking copper/zinc superoxide dismutase (SOD1) can be alleviated through mutations in PMR1, encoding a calcium P‐type ATPase homologue that also functions in manganese homeostasis. In an attempt to further understand the relationship between manganese ions, PMR1 and SOD1, we conducted a search for manganese homeostasis genes that interact with PMR1. A genomic library was screened for genes that, when overexpressed, suppress the manganese hypersensitivity associated with pmr1 mutations. A single clone was isolated that reduced manganese toxicity in both the pmr1 mutant and PMR1 wild‐type yeast. This gene was identified as CCC1, previously shown to function in calcium metabolism. Our studies indicate that, like PMR1, CCC1 functions in the homeostasis of both calcium and manganese ions. The Ccc1p polypeptide was found to localize to a Golgi‐like organelle in yeast cells. Ccc1 p co‐fractionated with a Golgi marker in subcellular fractionation studies and, with immunofluorescence microscopy, Ccc1 p exhibited a punctate pattern of staining typical of yeast Golgi. Our studies suggest that Ccc1 p may act to sequester manganese ions in this organelle and limit the intracellular availability of the metal. First, overexpression of CCC1 reduced manganese cytotoxicity without lowering total accumulation of the metal. Second, overexpression of CCC1 appeared to limit the intracellular availability of the manganese ions needed to support aerobic growth of SOD1 mutants. We provide a model in which Ccc1p and Pmr1p work together to control the intracellular partitioning of manganese ions.
The use of differential display‐PCR to isolate and characterize a Legionella pneumophila locus induced during the intracellular infection of macrophages
Authors: Kwaik Yousef Abu , Pederson Lisa L. 
Published: October 2006

SummaryThe differential display (DD)‐PCR technique has been modified to identify prokaryotic cDNA fragments that are differentially induced by facultative intracellular bacteria in response to the intracellular environment of eukaryotic cells. Several DD‐PCR fragments identified from the intracellular bacterium Legionella pneumophila were induced at 4 h post‐infection of the U937 macrophage‐like cells. From these, a 700 bp fragment was cloned and sequenced. Neither the DNA sequence nor the predicted protein sequence from the open reading frame has similarity to other sequences in genetic databases. Transcription of the chromosomal locus containing the 700 bp fragment (eml, for early stage macrophage‐induced locus) was induced by intracellular bacteria during the first few hours post‐infection of macrophages but the expression was downregulated by 12 h post‐infection. Transcription of eml was not growth phase‐related in vitro, and was not affected by in vitro stress stimuli. A 3.7 kb EcoRI genomic fragment containing the 700 bp DD‐PCR product was cloned. Six mini‐Tn 10 insertions in the 3.7 kb EcoRI fragment were recombined into the L. pneumophila chromosome. Compared to the wild‐type strain, five of the eml isogenic mutants had a similar phenotype of reduced cytopathicity to the U937 cells, showed a 100‐fold increase in killing by macrophages during the first 5h of the intracellular infection, and showed a 100‐fold increase in killing during the first 24 h of infection of the amoeba Hartmanella vermiformis. The 6th mutant had a phenotype indistinguishable from the wild‐type strain. The cytopathicity defect of the mutants to the U937 cells was restored to wild‐type levels by complementation of the mutants with a plasmid containing the 3.7 kb EcoRI fragment. These data showed that the 3.7 kb fragment containing eml is a novel L. pneumophila locus whose expression is uniquely induced by non‐stress stimuli during early stages of the intracellular infection of phagocytic cells. Expression of this locus is required for survival of L. pneumophila within macrophages and within amoebae during early stages of the infection.
Integration host factor alleviates the H‐NS‐mediated repression of the early promoter of bacteriophage Mu
Authors: van Ulsen Peter , Hillebrand Marcel , Zulianello Laurence , van de Putte Pieter , Goosen Nora 
Published: October 2006

Summary Integration host factor (IHF), which is a histone‐like protein, has been shown to positively regulate transcription in two different ways. It can either help the formation of a complex between a transcription factor and RNA polymerase or it can itself activate RNA polymerase without the involvement of other transcription factors. In this study, we present a third mechanism for IHF‐stimulated gene expression, by counteracting the repression by another histone‐like protein, H‐NS. The early (Pe) promoter of bacteriophage Mu is specifically inhibited by H‐NS, both in vivo and in vitro. For this inhibition, H‐NS binds to a large DNA region overlapping the Pe promoter. Binding of IHF to a binding site just upstream of Pe alleviates the H‐NS‐mediated repression of transcription. This same ihf site is also involved in the direct activation of Pe by IHF. In contrast to the direct activation by IHF, however, the alleviating effect of IHF appears not to be dependent on the relevant position of the ihf site on the DNA helix, and it also does not require the presence of the C‐terminal domain of the alpha subunit of RNA polymerase. Footprint analysis shows that binding of IHF to the ihf site destabilizes the interaction of H‐NS with the DNA, not only in the IHF‐binding region but also in the DNA regions flanking the ihf site. These results suggest that IHF disrupts a higher‐order nucleoprotein complex that is formed by H‐NS and the DNA.
Examination of AsmA and its effect on the assembly of Escherichia coli outer membrane proteins
Authors: Deng Ming , Misra Rajeev 
Published: October 2006

Summary
asmA mutations were isolated as extragenic suppressors of an OmpF assembly mutant, OmpF315. This suppressor locus produced a protein that was present in extremely low levels and could only be visualized by Western blotting in cells where AsmA expression was induced from a plasmid. Detailed fractionation analyses showed that AsmA localized with the inner membrane. Curiously, however, the mutant OmpF assembly step influenced by AsmA occurred in the outer membrane, perhaps indicating an indirect involvement of AsmA in the assembly of outer membrane proteins. Biochemical examination of the outer membrane showed that asmA null mutations reduce lipo‐polysaccharide (LPS) levels, thereby lowering the ratios of glycerolphospholipids to LPS and envelope proteins to LPS in the outer membrane. Despite these quantitative alterations, no apparent structural changes in LPS or major phospholipids were noted. Reduced LPS levels in asmA mutants indicate a possible role of AsmA in LPS biogenesis. Data presented in this study suggest that asmA‐mediated OmpF assembly suppression may have been achieved by altering the outer membrane fluidity, thus making it more amenable for the assembly of mutant proteins.
Differential regulation of multiple overlapping promoters in flagellar class II operons in Escherichia coli
Authors: Liu Xiaoying , Matsumura Philip 
Published: October 2006

Summary The Escherichia coli flagellar operons are divided into three categories: classes I, II and III. Expression of class II depends on expression of class I. One of the class II gene products, the FliA protein, is an alternative ß factor (ß28) required for transcription of the class III operons. In this study, we have characterized, in vitro, a role of ß28 in the regulation of the class II operons. Among the three class II operons examined, the fliA and fliL operons, but not the flhB operon, could be transcribed by both ß70 RNA polymerase holoenzyme with FlhD/C (Eß70‐FlhD/C) and ß28 RNA polymerase holoenzyme (Eß28). The flhB operon could only be transcribed by Eß70‐FlhD/C under the conditions used. Both the fliA and fliL operons contained two overlapping promoters oriented in tandem. The transcription of fliA directed by Eß28 could outcompete that by Eß70‐FlhD/C, indicating a positive autoregulation. However, Eß28 could not displace Eß70‐FlhD/C bound to the fliL promoter. The c28‐mediated positive regulation of the class II operons involved a mechanism in which ß28 competed with ß70 for core RNA polymerase. In addition, recruitment of core RNA polymerase from the ß70 ‐10 site to the ß28 ‐10 was facilitated by formation of Eß70‐FlhD/C pre‐initiation complex. Taken together, the three class II promoters investigated are different in terms of their regulation by ß28. We propose that class II operons may be further divided into different subcategories.
A secreted protein tyrosine phosphatase with modular effector domains in the bacterial pathogen Salmonella typhimurlum
Authors: Kaniga Koné , Uralil Jaimol , Bliska James B. , Galán Jorge E. 
Published: October 2006

Summary A number of bacterial pathogens have evolved sophisticated strategies to subvert host‐cell signal‐transduction pathways for their own benefit. These bacteria produce and export proteins capable of specific interactions with key mammalian cell regulatory molecules in order to derail the normal functions of the cells. In this study, we describe the identification of a modular effector protein secreted by the bacterial pathogen Salmonella typhimurium that is required for its full display of virulence. Sequence analysis revealed that a carboxy‐terminal region of this protein, which we have termed SptP, is homologous to the catalytic domains of protein tyrosine phosphatases. Purified SptP protein efficiently dephosphorylated peptide substrates phosphorylated on tyrosine. An engineered mutant of SptP in which a critical Cys residue in the catalytic domain was changed to Ser was devoid of phosphatase activity, indicating a catalytic mechanism similar to that of other tyrosine phosphatases. In addition, an amino‐terminal region of SptP exhibited sequence similarity to the ribosyltransferase exo‐enzyme S from Pseudomonas aeruginosa and the cytotoxin YopE from Yersinia spp. The modular nature of this effector protein may allow multiple interactions with host‐cell signalling functions.
Cis preference of the IS 903 transposase is mediated by a combination of transposase instability and inefficient translation
Authors: Derbyshire Keith M. , Grindley Nigel D. F. 
Published: October 2006

Summary The transposase protein encoded by the insertion element IS 903 belongs to an unusual class of DNA‐binding proteins, termed cis‐acting proteins, that act preferentially at their site of synthesis. Previous work had led us to propose that instability of the IS 903 transposase was a major determinant of its cis preference. Here we describe the isolation of two classes of mutations within the transposase gene that increased action in trans. One class specifically increased trans action without increasing the level of transposition when the mutant gene was located in cis to the transposon. In particular, a threonine‐to‐proline substitution at amino acid 25 (T25P) reduced cis preference about 60‐fold. The half‐life of this mutant transposase was significantly longer than that of the wild‐type transposase, confirming the critical role of protein instability. The second, larger, class of mutations increased the level of transposition both in trans and in cis. The behaviour and location of these mutations were consistent with an increase in gene expression by improving translational initiation. Several of these mutations exerted a disproportionate effect on the action of transposase in trans, implying that translation efficiency may affect more than just the amount of transposase made. Our results indicate that cis preference of the IS 903 transposase is mediated by a combination of transposase instability and inefficient translation initiation.
Mycoplasma pneumoniae cytadherence: unravelling the tie that binds
Authors: Krause Duncan C. 
Published: October 2006

Summary Mycoplasma pneumoniae is the leading cause of pneumonia in older children and young adults. Mycoplasma adherence to the respiratory epithelium (cytadherence) is required for colonization and pathogenesis. Although considered to be among the smallest and simplest known prokaryotes, this cell‐wall‐less bacterium possesses a highly differentiated terminal structure that is thought to be functional in mycoplasma cell division, gliding motility, and cytadherence. Mutant analysis has identified mycoplasma proteins associated with cytadherence, and revealed novel regulatory features. Ultrastructural and biochemical studies have established the subcellular location and interaction of key components, several of which are phosphorylated by ATP‐dependent kinase(s) in a manner that is responsive to changing nutritional conditions. This review summarizes recent progress in defining the composition, organization and regulation of the attachment organelle. What emerges is a picture of M. pneumoniae cytadherence as a multifactorial process that extends well beyond adhesin‐receptor recognition.
Molecular genetic bases of Salmonella entry into host cells
Authors: Galán Jorge E. 
Published: October 2006

Summary Salmonella spp. can enter into non‐phagocytic cells, a property that is essential for their pathogenicity. Recently, considerable progress has been made in the understanding of the molecular genetic bases of this process. It is now evident that Salmonella entry functions are largely encoded on a 35–40 kb region of the Salmonella chromosome located at centisome 63. The majority of the loci in this region encode components of a type III or contact‐dependent secretion system homologous to those described in a variety of animal and plant‐pathogenic bacteria as well as a number of proteins that require this system for their export to the extracellular environment. A somewhat unexpected finding has been the remarkable homology between the Salmonella and Shigella proteins that mediate the entry of these organisms into cultured epithelial cells.
Organization of the origins of replication of the chromosomes of Mycobacterium smegmatis, Mycobacterium leprae and Mycobacterium tuberculosis and isolation of a functional origin from M. smegmatis
Authors: Salazar Leirla , Fsihi Hafida , de Rossi Edda , Riccardi Giovanna , Rios Carmen , Cole Stewart T. , Takiff Howard E. 
Published: October 2006

SummaryThe genus Mycobacterium is composed of species with widely differing growth rates ranging from approximately three hours in Mycobacterium smegmatis to two weeks in Mycobacterium leprae. As DNA replication is coupled to cell duplication, it may be regulated by common mechanisms. The chromosomal regions surrounding the origins of DNA replication from M. smegmatis, M. tuberculosis, and M. leprae have been sequenced, and show very few differences. The gene order, rnpA‐rpmH‐dnaA‐dnaN‐recF‐orf‐gyrB‐gyrA, is the same as in other Gram‐positive organisms. Although the general organization in M. smegmatis is very similar to that of Streptomyces spp., a closely related genus, M. tuberculosis and M. leprae differ as they lack an open reading frame, between dnaN and recF, which is similar to the gnd gene of Escherichia coli. Within the three mycobacterial species, there is extensive sequence conservation in the intergenic regions flanking dnaA, but more variation from the consensus DnaA box sequence was seen than in other bacteria. By means of subcloning experiments, the putative chromosomal origin of replication of M. smegmatis, containing the dnaA‐dnaN region, was shown to promote autonomous replication in M. smegmatis, unlike the corresponding regions from M. tuberculosis or M. leprae.
DNA supercoiling depends on the phosphorylation potential in Escherichia coli
Authors: van Workum Marielle , van Dooren Silvy J. M. , Oldenburg Nienke , Molenaar Douwe , Jensen Peter R. , Snoep Jacky L. , Westerhoff' Hans V. 
Published: October 2006

SummaryATP/ADP ratios were varied in different ways and the degree of negative supercoiling was determined in Escherichia coli. Independent of whether the ATP/ADP ratio was reduced by a shift to anaerobic conditions, by addition of a protonophore (dinitrophenol) or by potassium cyanide addition, DNA supercoiling decreased similarly with the ATP/ADP ratio. The experiments were performed under well‐defined conditions, where oxidative phosphorylation was the dominant route for ATP synthesis, i.e. using a minimal salts medium with succinate as the sole free‐energy and carbon source, and in the presence or absence of ammonia as the nitrogen source. The results of the different experiments were consistent with a single linear relationship between the log(ATP/ADP) and the change in linking number. The dependence of DNA supercoiling on the ATP/ADP ratio was not influenced by inhibitors of transcription or translation. Because the ATP/ADP ratio was modulated in different ways, the unique relationship suggests coupling between the phosphorylation potential and DNA supercoiling. This was most probably mediated by the DNA gyrase, independent of topoisomerase I or transcription.
Co‐operative binding of two Trp repressor dimers to α‐ or β‐centred trp operators
Authors: Günes Çagatay , Staacke Detlev , von Wilcken‐Bergmann Brigitte , Müller‐Hill Benno 
Published: October 2006

SummaryThe α‐centred trp operator binds one dimer of the Trp repressor, whereas the β‐centred trp operator binds two dimers of the Trp repressor (Carey et al., 1991; Haran et al., 1992). The Trp repressor with a Tyr‐Gly‐7 substitution binds almost as well as the wild‐type Trp repressor to the α‐centred trp operator, but it does not bind to the β‐centred trp operator. This confirms that Tyr‐7 is involved in the interaction between Trp repressor dimers, as seen in the crystal structure (Lawson and Carey, 1993). Further experiments with a‐centred trp operator variants showed that positions 1 of the a‐centred trp operators play a crucial role in tetramerisation. The two innermost base pairs of the α‐centred trp operator are not involved in contacts with the dimer of the Trp repressor binding to it. However, substitutions in these positions (T‐A to G‐T) effectively transform the α‐centred trp operator into a β‐centred trp operator, and thus encourage the binding of two Trp repressor dimers to this operator. Finally, we demonstrate, with suitable heterodimers, that one subunit of each dimer suffices to bind to a β‐centred trp operator.