bluuebunny/crossref_metadata_2025_split huggingface dataset:
Microevolution within a clonal population of pathogenic bacteria: recombination, gene duplication and horizontal genetic exchange in the opa gene family of Neisseria meningitidis
Authors: Hobbs Marcia M. , Seiler Andrea , Achtman Mark , Cannon Janne G. 
Published: October 2006

SummaryOpacity (Opa) proteins are a family of antigenically variable outer‐membrane proteins of Neisseria meningitidis. Even among clonally related epidemic meningococcal isolates, there is greater variation of Opa protein expression than can be accounted for by the opa gene repertoire of any individual strain. We characterized the opa genes of eight closely related Isolates of serogroup A N. meningitidis (subgroup IV‐1) from a recent meningitis epidemic in West Africa. DNA sequence analysis and Southern blot experiments indicated that changes occurred in the opa genes of these bacteria as they spread through the human population, over a relatively short period of time. Such changes in one or a few loci within a clonal population are referred to as microevolution. The distribution of sequences present in hypervariable (HV) regions of the opa genes suggests that duplication of all or part of opa genes into other opa loci changed the repertoire of Opa proteins that could be expressed. Additional variability in this gene family appears to have been introduced by horizontal exchange of opa sequences from other meningococcal strains and from Neisseria gonorrhoeae. These results indicate that processes of recombination and genetic exchange contributed to variability in major surface antigens of this clonal population of pathogenic bacteria.
The cis‐effect of a nascent peptide on its translating ribosome: influence of the cat‐86 leader pentapeptide on translation termination at leader codon 6
Authors: Rogers Elizabeth J. , Lovett Paul S. 
Published: October 2006

SummaryInducible cat genes from Gram‐positive bacteria are regulated by translation attenuation. The inducer chloramphenicol stalls a ribosome at a specific site in the leader of cat transcripts; this destabillzes a downstream stem‐loop structure that normally sequesters the ribosome‐binding site for the cat structural gene. The five‐amino‐acid peptide MVKTD that is synthesized when a ribosome has translated to the leader induction site is an inhibitor of peptidyl transferase In vitro. Thus, the peptide may be the in vivo determinant of the site of ribosome stalling. Here we provide evidence that the leader pentapeptide can exert a cis‐effect on its translating ribosome In vivo. Converting leader codon 6 to the ochre codon results in expression of cat‐86 in the absence of Inducer. We term this autoinduction. Autoinduction is abolished by mutations that change the amino‐acid sequence of the leader peptide but have no, or little, effect on the sequence of nucleotides at the leader stall site. In contrast, four nucleotide changes within the leader site occupied by the stalled ribosome that result in synonymous codon replacements do not diminish autoinduction. Our evidence indicates that the cat‐86 leader pentapeptide can alter the function of its translating ribosome.
Thermoregulation in Yersinia enterocolitica is coincident with changes in DNA supercoiling
Authors: Rohde John R. , Fox James M. , Minnich Scott A. 
Published: October 2006

Summary Yersinia enterocolitica is a facultative intracellular parasite, displaying the ability to grow saprophytically or invade and persist intracellularly in the mammalian reticuloendothelial system. The transition between such diverse environments requires the co‐ordinated regulation of specific sets of genes on both the chromosome and virulence plasmid. Temperature has a profound pleiotropic effect on gene expression and phenotypically promotes alterations in cell morphology, outer‐membrane protein synthesis, urease production, lipopolysaccharide synthesis, motility, and synthesis of genes involved in invasion of euKaryotic host cells. By examining thermoregulated flagella biosynthesis, we have determined that motility is repressed at 25° C (permissive temperature) with subinhibitory concentrations of novobiocin. These conditions also induce virulence gene expression suggesting novobiocin addition stimulates, at least partially, a high‐temperature environment. Furthermore, temperature‐shift experiments, using Y. enterocolitica containing pACYC184 as a reporter plasmid, indicate that thermo‐induced alterations of DNA supercoiling coincide with temperature‐induced phenotypic changes. A class of putative DNA gyrase mutant (novobiocin resistant) likewise demonstrates the 37° C phenotype when cultured at 25°C; it is non‐motile, urease negative, calcium growth dependent, and positive for Yop expression. These results support a model implicating DNA topology as a contributing factor of Y. enterocolitica thermoregulation.
Characterization of the autophosphorylation of Era, an essential Escherichia coli GTPase
Authors: Sood Poonam , Lerner Claude G. , Shimamoto Toshi , Lu Qing , Inouye Masayori 
Published: October 2006

SummaryEra is an essential protein in Escherichia coli which binds both GTP and GDP and has an intrinsic GTPase activity. Studies on the role of GTP/GDP binding and GTPase activity in an attempt to understand its function lead to the observation that Era is autophosphorylated. The autophosphorylated reaction is specific for GTP and cannot use ATP as a phosphoryl group donor. The reaction velocity is of first order with respect to protein concentration, suggesting an intramolecular mechanism. Autophosphorylation occurs at serine and threonine residues. The major phosphorylated tryptic peptide isolated after autophosphorylation has been identified as ISITSR, from residue 33 to 38. The peptide contains the site of phosphorylation and two potential sites for serine and threonine phosphorylation. Subsequently, both the threonine residue at position 36 and the serine residue at position 37 were altered to alanine. The double mutant Era, but not individual single mutants, was unable to functionally complement the growth of an E. coli strain which cannot produce wild‐type Era protein at high temperature. This suggests that either threonine 36 or serine 37 has to exist for the function of Era In vivo. phosphorylation of Era was also examined by two‐dimensional gel electrophoresis. Era has been previously assigned two distinct positions having two different X‐Y co‐ordinates: one of the spots (H032.0) was identified as phosphorylated Era, indicating that a substantial portion of Era in the cell is indeed phosphorylated. Therefore, Era autophosphorylation is likely to play an important physiological role in the cell. The sequence encoding the C‐terminus previously published had a missing C between A900 and GgO1. As a resuit of the frameshift, Era consists of 301 residues, 15 fewer than originaiiy reported.
Differential expression of multiple exo‐cellobiohydrolase I‐like genes in the lignin‐degrading fungus Phanerochaete chrysosporium
Authors: Sims Paul F. G. , Soares‐Felipe M. Sueli , Wang Qi , Gent Manda E. , Tempelaars Corinne , Broda Paul 
Published: October 2006

SummaryThe genome of Phanerochaete chrysosporium strain ME446 contains multiple, non‐allelic, cellobiohydrolase I (CSHI)‐like sequences, at least two of which are expressed in a cellulose‐dependent manner. Each of the expressed genes contains two identically positioned introns within its coding region. The lengths and sequences of these introns are different and one is not excised from all transcripts, raising the possibility that subtly different protein products may be expressed from a common gene. Introns are also present upstream of both genes but these differ in number and position, as well as sequence and length. Endoglucanase‐like sequences could not be identified and it is suggested that variant CBHI‐like proteins may provide endoglucanase activity in this fungus.
Peptidase activity of Escherichia coli aminopeptidase A is not required for its role in Xer site‐specific recombination
Authors: McCulloch Richard , Burke Mary E. , Sherratt David J. 
Published: October 2006

SummaryXer site‐specific recombination is required for the stable inheritance of multicopy plasmids and the normal segregation of the bacterial chromosome in Escherichia coli.Two related recombinases and two accessory proteins are essential for Xer‐mediated recombination at cer, a recombination site in the plasmid ColE1 The accessory proteins, ArgR and PepA, function in ensuring that the Xer recombination reaction acts exclusively intramolecularly, converting plasmid dimers into monomers and not vice versa. PepA is an amino‐exopeptidase, but its molecular role in the Xer recombination mechanism is unclear. Here we show that a mutation directed at the presumptive active site of PepA creates a protein with no detectable peptidase activity in vitro or in vivo, but which still functions normality in Xer site‐specific recombination at cer.
Molecular characterization of intact, but cryptic, flagellin genes in the genus Shigella
Authors: Tominaga Akira , Mahmoud Mohamed Abdel‐Hakim , Mukaihara Takafumi , Enomoto Masatoshi 
Published: October 2006

SummaryFlagellin genes (fliC) were detected in two species of the genus Shigella. The fliCSF gene cloned from Shigella flexneri produced normal‐type flagella in an Escherichia coli δ fliC strain while the fliCSS genes from two Shigella sonnei strains produced curly‐type flagella and their expression is repressible by Salmonella FljA repressor. The fliCSF gene (1650bp) shared high similarity with the E. coli fliCE gene not only in the 5′ and 3′ constant sequences but also in the upstream and downstream sequences. The fliCS genes (1572 bp) shared high similarity with the Salmonella typhimurium fliCs gene in the operator and 3’constant sequences and also shared high similarity with the fliCE gene in the downstream sequence, suggesting that the fliC gene has undergone horizontal transfer and recombination. Differences In nucleotide sequences of the central variable regions among the four fliC genes, including fliCE and fliCS, suggest that they started differentiation in each lineage approximately 80 million years ago. Loss of motility in Shigella seems to be evolutionarily a recent event.
Molecular characterization of PulE, a protein required for pullulanase secretion
Authors: Possot Odile , Pugsley Anthony P. 
Published: October 2006

SummarypulE, one of 14 genes specifically required for pullulanase secretion in Klebsiella oxytoca, codes for a putative nucleotide‐binding protein. Subcellular fractioNatlon indicated that the majority of PulE in Escherichia coli cells expressing all 14 secretion genes is mainly associated with the cytoplasmic membrane through both hydrophobic and non‐hydrophobic interactions. Mutational analysis revealed that one of the two regions of PulE that are conserved in many nucteotide‐binding proteins (Walker box A) is essential for pullulanase secretion. Likewise, mutations that removed aspartate residues from each of two regions immediately downstream from the Walker box A also reduced secretion. These aspartate‐rich regions are highly conserved in all 16 known PulE homologues but not in any other nucleotide‐binding proteins. Altogether, these results Indicate that PulE might belong to a new family of nucleotide‐binding proteins. The protein could not be cross‐linked to the photoactivatable ATP analogue azido‐ATP, however. Most pulE point or deletion mutations which prevented pullulanase secretion exhibited transdominance when expressed at high levels in cells producing wild‐type PulE protein. Evidence presented suggests that PulE might be a homodimer.
Arsenate reduction mediated by the plasmid‐encoded ArsC protein is coupled to glutathione
Authors: Oden Kristine L. , Gladysheva Tatiana B. , Rosen Barry P. 
Published: October 2006

SummaryResistance to arsenate conferred on Escherichia coli by the ars operon of plasmid R773 requires both the product of the arsC gene and reduction of arsenate to arsenate. A genetic analysis was performed to identify the source of reducing potential in vivo. in addition to the ars genes, arsenate resistance required the products of the gor gene for glutathione reductase and the gshA and gshB genes for glutathione synthesis. Mutations in the trx and grx genes for thioredoxin and glutaredoxin, respectively, had no effect on arsenate resistance. Although resistance required the arsC gene, the rate of reduction of arsenate to arsenate was nearly the same in cells lacking the ars operon. In strains deficient in glutathione biosynthesis this endogenous reduction was greatly diminished, and cells exhibited increased sensitivity to arsenate. When glutathione was supplied exogenously to such mutants, resistance was restored only to cells expressing the ars operon, and only such cells had detectable arsenate reduction after addition of glutathione. Since ArsC‐catalysed reduction of arsenate provides high level resistance, physical coupling of the ArsC reaction to efflux of the resulting arsenite is hypothesised.
Lytic conversion of Escherichia coli by bacteriophage T5: blocking of the FhuA receptor protein by a lipoprotein expressed early during infection
Authors: Decker Katja , Krauel Verena , Meesmann Anke , Heller Knut J. 
Published: October 2006

SummaryThe nucleotide sequence of the region between the oad gene, encoding the host specificity protein, and the right‐terminal repetition of bacteriophage T5 DNA was determined. Five small open reading frames, the first of which was called IIp, were detected, which apparently formed an operon transcribed from a promoter that overlapped the oad promoter. Both promoters were confirmed by primer extension assays. Using mRNA isolated at different times after T5 infection, the IIp and oad promoters were identified as early and late promoters, respectively. The N‐terminus of the IIp gene product possess a signal sequence and a processing site characteristic of lipoproteins. After subcloning and expression of IIp, its product Lip was identified as a 7.8 kDa polypeptide. Acylation of Lip was confirmed by addition of globomycin, which resulted in the accumulation of the unprocessed precursor form. FhuA4 cells synthesizing Lip were resistant to phage T5. Resistance was caused by inhibition of adsorption of T5 to its FhuA receptor protein. Resistance could be overcome by derepression of fhuA transcription, suggesting a blocking of FhuA by direct interaction with Lip. Since Lip‐mediated T5 resistance has several aspects in common with the phenomenon of lysogenic conversion, we suggest that it should be called lytic conversion.
Why are mammalian alkaline phosphatases much more active than bacterial alkaline phosphatases?
Authors: Murphy Jennifer E. , Kantrowitz Evan R. 
Published: October 2006

SummaryMammalian alkaline phosphatases are 20‐30‐fold more active than the corresponding bacterial enzymes even though their amino acid sequences are 25–30% absolutely conserved. In the active‐site region there are two noticeable differences between the sequences of the bacterial and mammalian enzymes, in the Escherichia coli enzyme positions 153 and 328 are Asp and Lys, respectively, but in the mammalian enzymes His is observed at both of these positions. Site‐specific mutagenesis, genetic and X‐ray crystallographic data, which will be summarized here, suggest that the His substitutions at positions 153 and 328 are primarily responsible for the differences in properties between the bacterial and mammalian alkaline phosphatases.
The ins and outs of protein splicing elements
Authors: Colston M. J. , Davis E. O. 
Published: October 2006

SummaryProtein splicing involves the removal of an internal protein sequence from a precursor molecule and the ligation of the two flanking sequences to produce a mature protein product, in a post‐translational event analogous to the removal of an intron from rRNA. Protein splicing introns, or‘inteins’appear to be a novel type of genetic element capable of mediating gene conversion of an‘intein‐less’allele, and hence promoting their own dissemination. The mechanism by which protein splicing is achieved is probably entirety encoded within the internal protein sequence, or intein, and does not require other accessory molecules. Although the concept of protein splicing inteins as selfish genetic elements of no immediate consequence to the host organism has emerged, this interpretation is questioned by recent evidence that in at least one example there appears to have been selection for protein splicing.
The 24 kDa N‐terminal sub‐domain of the DNA gyrase B protein binds coumarin drugs
Authors: Gilbert E. Jane , Maxwell Anthony 
Published: October 2006

SummaryA number of lines of evidence suggest that the N‐terminal sub‐domain of the DNA gyrase B protein contains the binding site for the coumarin antibiotics. We have engineered a clone which encodes a 24 kDa protein which represents this domain. Bacteria which overproduce this protein show an elevated level of resistance to coumarins, suggestive of binding of the 24 kDa protein to the drugs In vivo. In vitro we find that the 24 kDa protein does not interact with the gyrase A or B proteins or with DNA, and fails to hydrolyse ATP or show significant binding to ATP, ADP or ADPNP. However, we show that the 24 kDa protein binds coumarin drugs as tightly as the Intact B protein. A number of experiments suggest that the Interaction of the coumarins with the protein is predominantly hydrophobic in nature.
Synthesis of ribosomal proteins during growth of Streptomyces coelicolor
Authors: Blanco Gloria , Rodicio M. Rosario , Puglia Anna Maria , Méndez Carmen , Thompson Charles J. , Salas José A. 
Published: October 2006

SummaryChanges in expression of ribosomal protein genes during growth and stationary phase of Streptomyces coelicolor A3(2) in liquid medium were studied. Proteins being synthesized were pulse‐labelled with [35 S]‐methionine, separated by two‐dimensional poly‐acrylamide gel electrophoresis, and quantified using the Bioimage computer software. Most of the ribosomal proteins were synthesized throughout the life cycle. Exceptions were two proteins whose synthesis drastically decreased at the approach of stationary phase. These two proteins were identified in purified ribosomes as homologues of Escherichia coli ribosomal proteins L10 and L7/L12, using antibodies raised against fusion proteins between these ribosomal proteins and Escherichia coliβ‐galactosldase. The genes (rplJ and rplL) encoding the L10 and L7/L12 proteins were contained in a 1.2 kb BamHl fragment that was cloned and sequenced. The linkage and order of the genes coincide with other L10‐L7/L12 operons. However, L11 and L1 genes were not present immediately upstream of the L10 gene, as is the case for E. coli and other bacteria. Instead, two open reading frames of unknown function were found immediately upstream of the L10 gene, in an adjacent 1.9 kb BamHl fragment.
Sequence and function of LuxO, a negative regulator of luminescence in Vibrio harveyi
Authors: Bassler Bonnie L. , Wright Miriam , Silverman Michael R. 
Published: October 2006

SummaryDensity‐dependent expression of luminescence in Vibrio harveyi is regulated by the concentration of extracellular signal molecules (autoinducers) In the culture medium. A recombinant clone that restored function to one class of spontaneous dim mutants was found to encode a function required for the density‐dependent response. Transposon Tn5 Insertions in the recombinant clone were isolated, and the mutations were transferred to the genome of V. harveyi for examination of mutant phenotypes. Expression of luminescence in V. harveyi strains with transposon insertions in one locus, luxO, was independent of the density of the culture and was similar in intensity to the maximal level observed in wild‐type bacteria. Sequence analysis of luxO revealed one open reading frame that encoded a protein, LuxO, similar in amino acid sequence to the response regulator domain of the family of two‐component, signal transduction proteins. The constitutive phenotype of LuxO∼ mutants indicates that LuxO acts negatively to control expression of luminescence, and relief of repression by LuxO in the wild type could result from interactions with other components in the Lux signalling system.
Post‐transcriptional regulation of the groEL1 gene of Streptomyces albus
Authors: Servant Pascale , Thompson Charles J. , Mazodier Philippe 
Published: October 2006

SummaryThermally induced expression of the heat‐shock gene groEL is subject to post‐transcriptional regulation in Streptomyces albus. When S. albus cells were shifted from 30°C to 41°C, synthesis of three GroEL‐like proteins was induced from two genes transcribed from associated promoters P1 and P2. Surprisingly, analyses of transcriptional fusions of these promoters with various reporter genes Indicated constitutive expression independent of heat shock. In contrast, neo expression was thermally inducible as a GroELI‐APH translational fusion protein. Furthermore, expression of the groEL1‐neo gene was heat Inducible even after the groEL1 promoter region was replaced by a heteroiogous non‐heat‐inducible promoter such as the Escherichia coli lac promoter. Finally, synthesis of GroE proteins, as well as the GroEL‐APH fusion protein, was heat inducible when their transcription was inhibited by rifampicin. Post‐transcriptional regulatory signals needed for heat‐induced GroEL1 synthesis were mapped within of the groEL 1 structural gene.
Regulation of transcription at the ndh promoter of Escherichia coli by FNR and novel factors
Authors: Green J. , Guest J. R. 
Published: October 2006

SummaryFNR Is a transcriptional regulator that controls gene expression in response to oxygen limitation in Escherichia coli. The NADH dehydrogenase II gene (ndh) is repressed by FNR under anaerobic conditions. Repression is not simply due to occlusion of the promoter (‐35 and ‐10) region by FNR because adjacent pairs of FNR monomers were found to bind at two sites centred at ‐50.5 and ‐94.S in the ndh promoter region without preventing RNA polymerase binding. However, contact between RNA polymerase and the ‐132 to ‐62 region of the non‐coding strand of ndh DNA, and RNA polymerase‐mediated open complex formation, were prevented by bound FNR. The upstream FNR‐binding site (‐94.5) was needed for efficient FNR‐dependent repression of ndh transcription if. vitro, and also for repression of an ndh‐lacZ fusion in vivo. Anaerobic ndh repression may thus involve the binding of two pairs of FNR monomers upstream of the ‐35 region, which prevents essential RNA polymerase‐DNA contacts in the upstream region as well as inhibiting RNA polymerase function by direct FNR interaction. Expression of the ndh‐lacZ fusion in an fnr deletion strain was enhanced by anaerobic growth in rich medium or minimal medium supplemented with amino acids. Furthermore, two proteins (Mr 12000 and 35000) which interact with and may activate transcription from the ndh promoter under these conditions were detected by gel retardation analysis. These putative amino acid‐responsive activators may thus offset FNR‐mediated repression and maintain a low level of anaerobic ndh expression for regulating the NAD+NADH ratio during growth in rich media.