bluuebunny/crossref_metadata_2025_split huggingface dataset:
The general protein‐export pathway is directly required for extracellular pullulanase secretion in Escherichia coli k12
Authors: Pugsley A. P. , Kornacker M. G. , Poquet I. 
Published: October 2006

SummaryPullulanase is an extracellular, cell surface‐anchored lipoprotein produced by Gram‐negative bacteria belonging to the genus Klebsiella. Its correct localization in recombinant Escherichia coli requires the products of 14 genes that are linked to the enzyme structural gene in the Klebsiella chromosome. In addition, we show here that six sec genes (secA, secB, secD, secE, secF, and secY) are all required for processing of the prepulluianase signal peptide to occur. This implies that pullulanase crosses the cytoplamic membrane via the general export pathway of which the sec gene products are essential components. Removal or drastic alteration of the prepullulanase signal peptide cause the enzyme to remain cytoplasmic. We propose that pullulanase secretion occurs in two steps, the first of which is common to all signal peptide‐bearing precursors of exported and secreted proteins, whereas the second is specifically involved in translocating pullulanase to the cell surface.
Promoter sequence requirements for Fnr‐dependent activation of transcription of the narGHJI operon
Authors: Walker M. S. , DeMoss J. A. 
Published: October 2006

SummaryThe sequence requirements for Fnr‐dependent transcription of the narGHJI operon of Escherichia coli were studied in plasmids carrying a narG::lacZ protein fusion with the 5′ end of the promoter deleted so that expression was controlled exclusively by Fnr. These plasmids were subjected to in vitro mutagenesis, and β‐galactostdase activities were determined in transformed strains after aerobic and anaerobic growth. A single base‐pair change in the Fnr box, a sequence which is highly conserved in all Fnr‐dependent promoters, essentially abolished anaerobic induction of expression. Primer extension analysis located the transcription start site 57 bp upstream from the narG translation start site and placed the Fnr box at a position centred between –41 and –42 bases from the transcription start site. The position of the Fnr box relative to the transcription start site was critical for anaerobic induction of expression. The deletion of 2bp or addition of 4, 6, 10, 14, 20, 22, 28, 30, and 40 bp immediately downstream from the Fnr box abolished anaerobic induction. Sequence changes between the Fnr box and the transcription start site had different effects, depending upon the region mutagenized. Base changes immediately downstream from the Fnr box, including bases –20 to –29, did not lead to any decrease in anaerobic induction of expression, but in most instances resulted in increased expression. Base changes further downstream prevented anaerobic induction of expression and suggested the requirement for a –10 hexamer which is partially homologous to the –10 consensus sequence for σ70‐specific promoters of E. coli. In addition, specific bases in the region immediately 5′ to the –10 hexamer were found to be required for full anaerobic induction. The critical location of the Fnr box and the absence of sequence specificity for the region which separates the Fnr box from the –10 region suggests that the interaction of Fnr with the Fnr box may replace the requirement for a –35 sequence in the activation of transcription from the narGHJI promoter.
Detection of a gene encoding a phosphatidylinositol‐specific phospholipase C that is co‐ordinately expressed with listeriolysin in Listeria monocytogenes
Authors: Leimeister‐Wächter M. , Domann E. , Chakraborty T. 
Published: October 2006

SummaryA phosphatidylinositol‐specific phospholipase C (PI‐PLC) that is unique to the pathogenic Listeria species L. monocytogenes and L. ivanovii has been detected. Deletion analysis performed with Escherichia coli recombinants expressing PI‐PLC activity together with maxicell analysis showed that a 34kDa polypeptide was responsible for this activity. Nucleotide sequencing revealed that the gene encoding this polypeptide comprises 317 amino acid residues with a 22‐amino‐acid signal peptide. This gene, designated pic for phosphatidylInositol‐specific phospholipase C, is located back to back with the listeriolysin gene on the chromosome of L. monocytogenes where these genes are transcribed by divergent non‐overlapping promoters. Expression of the pic gene is dependent on the product of the prfA gene, which also regulates expression of the listeriolysin gene in L. monocytogenes.
Nucleotide sequence of the Escherichia coli thymidine kinase gene provides evidence for conservation of functional domains and quaternary structure
Authors: Black M. E. , Hruby D. E. 
Published: October 2006

SummaryUsing λ bacteriophage clones from the Kohara Escherichia coli library spanning minutes 25.5 to 26.5 on the E. coli chromosome (strain W3110), two overlapping DNA fragments were identified which were able to confer thymidine kinase (TK) enzyme activity to a TK‐ strain of E. coli (KY895). This genetic complementation assay was used in concert with subcloning procedures to identify the minimal region (a 900bp EcoRI‐SalI fragment) which contained the E. coli thymidine kinase gene (tdk.) The nucleotide sequence of the Eco RI–SalI fragment and a small portion of the adjoining downstream fragment was determined. Computer analysis of the derived sequence indicated the presence of a rightward‐reading open reading frame of 615bp which was capable of encoding a 205‐amino‐acid polypeptide with a predicted Mr, of 23458 daltons. The in vivo transcriptional activity of this locus was confirmed by Northern blot hybridization analysis of RNA isolated from E. coli JM101 or KY895 which detected a 650‐nucleotide RNA transcribed from this region. This places the tdk gene at approximately minute 27.35 on the E. coli W3110 chromosome, about 15 kb downstream from the narG locus and approximately 25 kb upstream of the trp operon. Although the predicted Mr of the E. coli TK protein was 23.5 kDa, gel‐filtration analyses suggested that, like eukaryotic thymidine kinases, the active form of this enzyme is a multimeric complex. Furthermore, comparisons between the predicted amino acid sequences of the TK proteins encoded by E. coli, bacteriophage T4, and a mammalian poxvirus (vaccinia virus) revealed several regions of amino acid identity, one of which appears to be the ATP‐binding site, thereby suggesting that the essential functional domains of this enzyme have been well‐conserved throughout evolution.
Functioning of the stable signal peptide of the pCloDF13‐encoded bacteriocin release protein
Authors: Luirink J. , Duim B. , de Gier J. W. L. , Oudega B. 
Published: October 2006

SummaryThe pCloDF13‐encoded bacteriocin release protein (BRP) is a lipoprotein which is synthesized as a precursor with an amino‐terminal signal peptide that appears to be stable after cleavage. The role of the stable signal peptide in the functioning of the BRP was studied with respect to the release of cloacin DF13, ‘lysis’ and leakage of periplasmic proteins. The BRP gene fragment encoding the stable signal peptide was replaced by a fragment encoding the unstable peptide of the murein lipoprotein (Lpp). The resulting hybrid protein was normally acylated and processed by signal peptidase II, leaving no stable signal peptide in the cells. Expression of the hybrid protein did not result in the specific release of cloacin DF13, whereas ‘lysis’ and the release of periplasmic enzymes were unaffected. These results indicated a role for the stable BRP signal peptide in the translocation of cloacin DF13 across the cytoplasmic membrane.
The role of a stress‐response protein in Salmonella typhimurium virulence
Authors: Johnson K. , Charles I. , Dougan G. , Pickard D. , O'Gaora P. , Costa G. , Ali T. , Miller I. , Hormaeche C. 
Published: October 2006

SummaryWe recently described the use of selective transposon mutagenesis to generate a series of a virulent mutants of a pathogenic strain of Salmonella typhimurium. Cloning and sequencing of the insertion sites from two of these mutants reveals that both have identical locations within an open reading frame that is highly homologous to a gene, htrA, encoding a heat‐shock protein in Escherichia coli. DNA sequence analysis of S. typhimurium htrA reveals the presence of a gene capable of encoding a protein with a calculated Mr of 49316 that has 88.7% protein:protein homology with its E. coli counterpart. In E. coli, lesions in this gene, also known as degP, reduce proteolytic degradation of aberrant periplasmic proteins. Characteristics of the S. typhimurium htrA mutants, 046 and 014, in vivo and in vitro suggested that they are avirulent because of impaired ability to survive and/or replicate in host tissues. In vitro, the S. typhimurium htrA mutants 046 and 014 are not temperature‐sensitive but were found to be more susceptible to oxidative stress than the parent, suggesting that they may be less able to withstand oxidative killing within macrophages.
PhoP/PhoQ: macrophage‐specific modulators of Salmonella virulence?
Authors: Miller S. I. 
Published: October 2006

SummaryThe regulation of gene expression by the two‐component regulatory system PhoP/PhoQ is necessary for Salmonella typhimurium survival within macro‐phages, defensin resistance, acid resistance, and murine typhoid fever pathogenesis. Salmonella experience multiple environments during mammalian infection and survival requires tightly regulated gene expression. Alter phagocytosis by macrophages, signal transduction by PhoQ results in the transcription of QhoP‐activated genes (pags) encoding proteins essential to bacterial survival and virulence. One such gene, pagC, encodes an envelope protein with amino acid similarity to an epithelial cell invasion protein of Yersina enterocolitica, Ail, and a bacteriophage λ outer membrane protein, Lom. The PhoP and PhoQ proteins can also repress the synthesis of proteins, encoded by phoP repressed genes (prgs), when pags are maximally expressed. If prgs encode receptors for toxic compounds, prg repression may protect the cell within macrophages when pag expression is most necessary. At least one prg locus, prgH, is required for full S. typhimurium mouse virulence. Within the macrophage, different environments may stimulate a switch from pag to prg expression that is necessary to Salmonella survival, prg expression may also be necessary for surviving non‐macrophage environments. Study of the PhoP regulon should lead to the discovery of new virulence factors, increase knowledge of how gene regulation is essential to bacterial virulence, and perhaps lead to the development of better vaccines for typhoid fever.Salmonella species experience multiple environments within mammalian hosts
Energy requirements for protein translocation across the Escherichia coli inner membrane
Authors: Geller B. L. 
Published: October 2006

SummaryBoth ATP and an electrochemical potential play roles in translocating proteins across the Inner membrane of Escherichia coli. Recent discoveries have dissected the overall transmembrane movement into separate subreactions with different energy requirements, identified a translocation ATPase, and reconstituted both energy‐requiring steps of the reaction from purified components. A more refined understanding of the energetics of this fundamental process is beginning to provide answers about the basic issues of how proteins move across the hydrophobic membrane barrier.
Sequencing, mutational analysis, and transcriptional regulation of the Escherichia coli htrB gene
Authors: Karow M. , Georgopoulos C. 
Published: October 2006

SummaryThe Escherichia coli htrB gene was originally discovered because its insertional inactivation led to an exquisitely temperature‐sensitive phenotype in rich media, i.e. the ability to form colonies at temperatures below 32°C, but not above 33°C. The htrB gene has been sequenced. It can potentially code for two proteins, with Mr values of 35407 Da and 8669 Da, that are encoded by overlapping, divergent open reading frames. Our data are consistent with the 35407 Da protein being HtrB. Northern blot analysis clearly shows that the monocistronic htrB message is not under heat‐shock regulation. We have also sequenced the flanking DNA and have discovered a new gene, designated orf39.9, located immediately adjacent to htrB, but divergently transcribed.
On the evolution of functional secondary metabolites (natural products)
Authors: Stone M. J. , Williams D. H. 
Published: October 2006

SummaryIt is argued that organisms have evolved the ability to biosynthesize secondary metabolites (natural products) because of the selectional advantages they obtain as a result of the functions of the compounds. The clustering together of antibiotic biosynthesis, regulation, and resistance genes implies that these genes have been selected as a group and that the antibiotics function in antagonistic capacities in nature. Pleiotropic switching, the simultaneous expression of sporulation and antibiotic biosynthesis genes, is interpreted in terms of the defence roles of antibiotics. We suggest a general mechanism for the evolution of secondary metabolite biosynthesis pathways, and argue against the hypothesis that modern antibiotics had prebiotic effector functions, on the basis that it does not account for modern bio‐synthetic pathways.
spbA locus ensures the segregational stability of pTH1030, a novel type of Gram‐positive replicon
Authors: Lereclus D. , Arantes O. 
Published: October 2006

SummaryThe replication region of the plasmid pHT1030 of Bacillus thuringiensis was previously mapped to a 2.9 kb DNA fragment. The DNA sequence was analysed and it was shown that the minimal replicon resides within a 1 kb fragment of DNA carrying no potential protein coding sequence. Moreover, no production of single‐stranded DNA intermediates was detected in the plasmid‐containing cells. pHT1030 therefore belongs to a class of replicons not previously described in Gram‐positive bacteria. Examination of the segregational stability of deletion derivatives of pHT1030 in bacilli defined two stability regions. One is located within the minimal replicon of pHT1030, whereas the second (spbA) is not required for replication. spbA encodes a 15 kDa protein and ensures the segregational stability of the plasmid. This effect of spbA is particularly highlighted in sporulation. The absence of the spbA locus gives rise to plasmid‐free spores at high frequency, whereas the spbA+ plasmids are stably maintained. The stability of the plasmids during sporulation seems to be correlated with an unequal division of the cell by the sporulation septum.
Haemolysin‐derived synthetic peptides with pore‐forming and haemolytic activity
Authors: Oropeza‐Wekerle R.‐L. , Muller S. , Briand J.‐P. , Benz R. , Schmid A. , Goebel W. 
Published: October 2006

Summary Escherichia coli haemolysin (Hlya) is a pore‐forming protein which belongs to the family of ‘Repeat‐toxins’ (RTX)(Lo et al., 1989; Lally et al., 1989; Kraig et al., (1990). A model for the pore‐forming structure of HlyA has been proposed (Ludwig et al., 1991) which consists of eight transmembrane segments all present in this hydrophobic region of HlyA. We report here that two synthetic peptides of 10 and 8 amino acids in length (Pep1 and Pep2, respectively), which are derived from transmembrane segment V, are able to form pores in an artificial lipid bilayer. In addition, Pep1 exhibits strong haemolytic activity when tested on human red blood cells (HRBCs). The haemolytic activity of Pep1 and of E. coli haemolysin is completely inhibited by antibodies raised against Pep1.
Membrane‐associated GTPases in bacteria
Authors: March Paul E. 
Published: October 2006

SummaryMembers of the GTPase superfamily are extremely important in regulating membrane signalling pathways in all cells. This review focuses on membrane‐associated GTPases that have been described in prokaryotes. In bacteria, LepA and NodO are very similar to protein synthesis elongation factors but apparently have membrane‐related functions. The amino acid sequences of FtsY and Ffh are clearly related to eukaryotic factors involved in protein secretion. Obg and Era are not closely related to any GTPase subgroup according to amino acid sequence comparisons, but they are essential for viability. In spite of similarities to well‐studied eukaryotic proteins the signalling pathways of these cellular regulators, with the exception of NodQ, have not yet been elucidated.
A system of transposon mutagenesis for bacteriophage T4
Authors: Woodworth Denise L. , Kreuzer Kenneth N. 
Published: October 2006

SummaryWe have developed a system of transposon mutagenesis for bacteriophage T4. The transposon is a plasmid derivative of Tn5 which contains the essential T4 gene 24, permitting a direct selection for transposition events into a gene 24‐deteted phage. The transposition occurred at a frequency of only 10‐7 per progeny phage, even though a dam‐ host was used to increase transposition frequency. Phage strains with a transposon insert were distinguished from most pseudorevertants of the gene 24 deletion by plaque hybridization using a transposon‐specific probe. Mapping analysis showed that the transposon inserts into a large number of sites in the T4 genome, probably with a preference for certain regions. The transposon insertions in four strains were analysed by DNA sequencing using primers that hybridize to each end of the transposon and read out into the T4 genome. In each case, a 9 bp T4 target sequence had been duplicated and the insertions had occurred exactly at the IS50 ends of the transposon, demonstrating that bona fide transposition had occurred. Finally, the transposon insert strains were screened on the TabG Escherichia coli strain, which inhibits the growth of T4 motA mutants, and a motA transposon insert strain was found.
The Aeromonas hydrophila exeE gene, required both for protein secretion and normal outer membrane biogenesis, is a member of a general secretion pathway
Authors: Jiang Bo , Howard S. Peter 
Published: October 2006

SummaryThe Aeromonas hydrophila Tn5–751 insertion mutant L1.97 is unable to secrete extracellular proteins, and is fragile because of defective assembly of its outer membrane. A Kpnl 4.1 kb fragment, which complements this mutant when supplied with an exogenous promoter, was isolated and sequenced. It contains two complete genes, exeE and exeF, plus fragments of two others and may form part of an operon. The exeE and exef open reading frames encode 501‐residue M, 55882 and 388‐residue M, 43431 proteins, respectively. These genes were expressed in vitro and their initiation codons verified by deletion analysis. Tn5–751 had inserted near the centre of the exeE gene in the L1.97 strain. Subclones of the Kpnl 4.1 kb fragment which contained only the exeE gene fully complemented the mutation, indicating that its function is required both for extracellular secretion and outer membrane assembly. ExeE and ExeF are highly similar to other proteins which have been shown to be involved in extracellular secretion, suggesting that an additional export apparatus beyond that required for inner membrane translocation may be part of the physiology of many Gram‐negative bacteria.
Expression of extracellular phospholipase from Serratia liquefaciens is growth‐phase‐dependent, catabolite‐repressed and regulated by anaerobiosis
Authors: Givskov Michael , Molin Søren 
Published: October 2006

SummaryMany members of the genus Serratia synthesize and excrete a number of extracellular hydrolytic enzymes. One of these is the phospholipase A1 from Serratia liquefaciens, the expression of which is growth‐phase‐dependent. Through the use of gene fusions and primer extension analysis we show that the expression of phospholipase is subject to positive transcriptional regulation of a dual promoter system; one promoter positioned approximately 600bp upstream from the phIA gene is responsible for the induction of phospholipase expression under anaerobic conditions, and the other promoter positioned 50bp upstream from the phIA gene is subject to catabolite repression and induced during the transition from exponential to late log‐phase of bacterial growth. On the basis of sequence homology and behaviour in the relevant Escherichia coli mutants, we suggest the distant promoter to be Fnr‐controlled and the proximal phIA promoter to be a member of the FIbB‐controlled flagellar‐chemotaxis regulon.
Mip protein of Legionella pneumophila exhibits peptidyl‐prolyl‐cis/trans isomerase (PPIase) activity
Authors: Fischer Gunter , Bang Holger , Ludwig Birgit , Mann Karlheinz , Hacker Jörg 
Published: October 2006

Summary Legionella pneumophila is an intracellular parasite which is able to survive and multiply in human monocytes and alveolar macrophages. The Mip (macro‐phage infectivity potentiator) protein has been shown to be an essential virulence factor. A search of translated nucleic acid data bases has shown that the Mip protein from strain Wadsworth possesses regions homologous to those found in the FK506‐binding proteins (FKBPs) of several different eukaryotic organisms. FKBPs are able to bind to the immunosuppressant macrolide FK506 and possess peptidyl‐prolyl cis/trans isomerase (PPIase) activity. The gene coding for the Mip protein was cloned from the chromosome of L. pneumophila strain Philadelphia I and sequenced. It was synthesized in Escherichia coli K‐12 and after purification it exhibited PPIase activity catalysing the slow cis/trans isomerization of prolyl peptide bonds in oligopeptides. Mip is inhibited by FK506 and fully resistant to cyclosporin A, as was also found for the recently characterized FKBP‐type PPlases of eukaryotes. However, the N‐terminal extension of Mip and/or the substitutions of the variable amino acids in the C‐terminal FKBP core lead to variations, when compared with eukaryotic FKBPs, in substrate specificity with the oligopeptide substrates of type Suc‐Ala‐Xaa‐Pro‐Phe‐4‐nitroanilide. Nevertheless, the Legionella Mip factor represents a bacterial gene product which shares some characteristics normally found in eukaryotic proteins. In view of the activity of PPIases in protein‐folding reactions, such prokaryotic FKBP analogues may represent a new class of bacterial pathogenicity factors.
MicroReview Control of translation initiation in Saccharomyces cerevisiae
Authors: Yoon Heejeong , Donahue Thomas F. 
Published: October 2006

SummaryThe first observations regarding the control of translation initiation in the yeast Saccharomyces cerevisiae were made by Fred Sherman and his colleagues in 1971. Elegant genetic studies of the CYC1 gene resulted in the formulation of ‘Sherman's Rules’ for translation initiation as follows: (i) AUG is the only initiator codon. (ii) the most proximal AUG from the 5’end of a message will serve as the start site of translation; and (iii) if the upstream AUG codon is mutated then initiation begins at the next available AUG in the message. Hidden within these rules is the mechanism of eukaryotic translation initiation, as these very same rules were later shown to apply to higher eukaryotic organisms and were formulated into the scanning model. However, only in the past five years has yeast been taken seriously as an organism for studying the mechanism of eukaryotic translation initiation. The basis for this is that the yeast genes for at least four mammalian translation initiation factor homologues have been identified and the number is growing. Similar factors suggest similar mechanisms for translation initiation between yeast and mammals. For some translation initiation factors, the genetics of yeast has provided new insights into their function. A mechanism for regulating translation initiation in mammalian cells is now evident in yeast. It seems clear that the molecular genetics of yeast coupled with the available in vitro translation system will provide a wealth of information in the future regarding translational control and regulatory mechanisms. The purpose of this review is to summarize what is known about translational control in S. cerevisiae.