bluuebunny/crossref_metadata_2025 huggingface dataset:
Effector‐induced self‐association and conformational changes in the enhancer‐binding protein NTRC
Authors: Farez‐Vidal M. Esther , Wilson Timothy J. , Davidson Barrie E. , Howlett Geoffrey J. , Austin Sara , Dixon Ray A.
Published: November 2003

The Klebsiella pneumoniae nitrogen regulatory protein NTRC is a response regulator which activates transcription in response to nitrogen limitation, and is a member of the family of σN‐dependent enhancer‐binding proteins. Using limited trypsin digestion, two domains of NTRC were detected and conformational changes within the protein in response to the binding of ligands were also observed. In the absence of ligands, the major digestion products were 42, 36 and 12.5 kDa bands corresponding to the central plus C‐terminal domain, the central domain, and the N‐terminal domains, respectively. Upon binding of purine but not pyrimidine nucleotides, the 36 kDa band was insensitive to further proteolysis, indicative of a conformational change in the central domain. Analysis of the dependence of this insensitivity on ATPγS concentration suggested an apparent dissociation constant (Kd) for ATPγS of 150 μM. In the presence of DNA, both the central and C‐terminal domains of NTRC were insensitive to proteolytic cleavage, indicative of a further conformational change. NTRC S160F, a mutant form of NTRC that is active in the absence of phosphorylation, was more stable to proteolysis than the wild‐type protein. This mutant protein is apparently locked in a conformation resembling the DNA‐bound form of wild‐type NTRC. The involvement of ligands in self‐association was studied using sedimentation equilibrium analysis. In the absence of ligand, wild‐type NTRC displayed a monomer–dimer equilibrium with a Kd of 6 μM. In the presence of ATPγS the equilibrium was shifted towards the dimer form (Kd = 0.8 μM). A similar dissociation constant for the monomer–dimer interaction was observed with NTRC S160F in the absence of ATPγS (Kd = 0.5 μM). The addition of ATPγS induced a significant association of NTRC S160F to higher‐order states with a dimer–octamer model producing a slightly, but not significantly better fit to the data than a dimer–hexamer model. We propose that ligand‐mediated self‐association provides a common mechanism for activation of this class of transcriptional regulatory proteins.
The GalF protein of Escherichia coli is not a UDP‐glucose pyrophosphorylase but interacts with the GalU protein possibly to regulate cellular levels of UDP‐glucose
Authors: Marolda Cristina L. , Valvano Miguel A.
Published: November 2003

We report the functional characterization of the galF gene of strain VW187 (Escherichia coli O7:K1), which encodes a polypeptide displaying structural features common to bacterial UDP‐glucose pyrophosphorylases, including the E. coli GalU protein. These enzymes catalyse a reversible reaction converting UTP and glucose‐1‐phosphate into UDP‐glucose and PPi. We show that, although the GalF protein is expressed in vivo, GalF‐expressing plasmids cannot complement the phenotype of a galU mutant and extracts from this mutant which only produces GalF are enzymatically inactive. In contrast, the presence of GalU and GalF proteins in the same cell‐free extract caused a significant reduction in the rate of pyrophosphorolysis (conversion of UDP‐glucose into glucose‐1‐phosphate) but no significant effect on the kinetics of synthesis of UDP‐glucose. The presence of GalF also increased the thermal stability of the enzyme in vitro. The effect of GalF in the biochemical properties of the UDP‐glucose pyrophosphorylase required the co‐synthesis of GalF and GalU, suggesting that they could interact as components of the oligomeric enzyme. The physical interaction of GalU and GalF was demonstrated in vivo by the co‐expression of both proteins as fusion products using a yeast two‐hybrid system. Furthermore, using a pair of galF +/galU + and galF/galU + isogenic strains, we demonstrated that the presence of GalF is associated with an increased concentration of intracellular UDP‐glucose as well as with an enhancement of the thermal stability of the UDP‐glucose pyrophosphorylase in vivo. We propose that GalF is a non‐catalytic subunit of the UDP‐glucose pyrophosphorylase modulating the enzyme activity to increase the formation of UDP‐glucose, and this function is important for bacterial adaptation to conditions of stress.
An oligopeptide permease responsible for the import of an extracellular signal governing aerial mycelium formation in Streptomyces coelicolor
Authors: Nodwell Justin R. , McGovern Karen , Losick Richard
Published: November 2003

Morphological differentiation in the filamentous bacterium Streptomyces coelicolor is believed to involve a mechanism of extracellular signalling that culminates with the formation of an aerial mycelium. We have identified a gene cluster designated bldK in which insertional and deletion mutations cause a block in aerial mycelium formation. Extracellular complementation experiments indicate that bldK defines a step in a cascade of extracellular signals; colonies of a bldK‐mutant strain extracellularly complement bld261‐mutant colonies, and are themselves extracellularly complemented by bldA‐and bldH‐mutant colonies. The bldK locus, which is located at 5 o'clock on the genetic map and within Ase I fragment ‘N’ on the physical map, consists of five adjacent open reading frames. These genes specify homologues of the subunits of the oligopeptide‐permease family of ATP‐binding cassette (ABC) membrane‐spanning transporters. Because bldK mutations confer resistance to the toxic tripeptide bialaphos, it is inferred that BldK is an oligopeptide importer. We propose that the BldK transporter is responsible for the import of an extracellular signalling molecule produced under the control of the wild‐type product of the bld261 gene. The BldK‐imported signal, in turn, causes the production of a second extracellular signal molecule that depends on the products of bldA and bldH for its action.
Re‐examination of the role of the periplasmic domain of EnvZ in sensing of osmolarity signals in Escherichia coli
Authors: Leonardo Michael R. , Forst Steven
Published: November 2003

In Escherichia coli, EnvZ senses changes in the osmotic conditions of the growth environment and controls the phosphorylated state of the regulatory protein, OmpR. OmpR‐phosphate regulates the expression of the porin genes, ompF and ompC. To investigate the role of the periplasmic domain of EnvZ in sensing of osmolarity signals, portions of this domain were deleted. Cells containing the EnvZ mutant proteins were able to regulate normally the production of OmpF and OmpC in response to changes in osmolarity. The periplasmic domain of EnvZ was also replaced with the non‐homologous periplasmic domain of the histidine kinase PhoR of Bacillus subtilis. Osmoregulation of OmpF and OmpC production in cells containing the PhoR–EnvZ hybrid protein was indistinguishable from that in cells containing wild‐type EnvZ. Identical results were obtained with an envZ–pta/ack strain, which could not synthesize acetyl phosphate. Thus, acetyl phosphate was not involved in the regulation of ompF and ompC observed in this study. These results indicate that the periplasmic domain of EnvZ is not essential for sensing of osmolarity signals.
Subtype‐specific interactions of type C staphylococcal enterotoxins with the T‐cell receptor
Authors: Deringer James R. , Ely R. James , Stauffacher Cynthia V. , Bohach Gregory A.
Published: November 2003

The goal of this study was to investigate the molecular interaction between superantigens and the T‐cell receptor (TCR). Using a quantitative polymerase chain reaction (PCR) to assess T‐cell proliferation profiles, we found that SEB, SEC1, SEC2 and SEC3 expanded human T cells bearing Vβ3, Vβ12, Vβ13.2, Vβ14, Vβ15, Vβ17 and Vβ20. SEC2 and SEC3 have the additional ability to expand T cells bearing Vβ13.1, and their expansion of Vβ3 was markedly reduced compared to SEB and SEC1. Based on the activity of SEC1 mutants containing single amino acid substitutions, we concluded that the differential abilities of these native toxins to stimulate Vβ3 and Vβ13.1 was determined by the residue in position 26, located in the base of the SEC α3 cavity. The SEC1 mutant, in which Val in position 26 was substituted with the analogous SEC2/SEC3 residue (Tyr), generated a Vβ expansion profile that was indistinguishable from those generated by SEC2 and SEC3. Using these findings, the co‐ordinates of a recently reported murine TCR β‐chain crystal structure, and other documented information, we propose a compatible molecular model for the interaction of SEC3 with the T‐cell receptor. In this model complex, the complementarity‐determining regions (CDRs) 1 and 2 and the hypervariable loop 4 of the Vβ element contact SEC3 predominantly through residues in the α3 cavity of the toxin. CDR3 of the β chain is not involved in any toxin contacts. The proposed model not only includes contacts identified in previous mutagenesis studies, but is also consistent with the ability of tyrosine and valine in position 26 to differentially affect the expansion of Vβs 3 and 13.1 by the SEC superantigens.
Three conserved consensus sequences identify the NAD‐binding site of ADP‐ribosylating enzymes, expressed by eukaryotes, bacteria and T‐even bacteriophages
Authors: Domenighini Mario , Rappuoli Rino
Published: November 2003

It has been previously reported that the three‐dimensional structures of the NAD‐binding and catalytic site of bacterial toxins with ADP‐ribosylating activity are superimposable, and that the key amino acids for the enzymatic activity are conserved. The model includes an NAD‐binding and catalytic site formed by an α‐helix bent over a β‐strand, surrounded by two β‐strands bearing a Glu and a His, or Arg, that are required for catalysis. We show here that the model can be extended to comprise all proteins with ADP‐ribosylating activity known to date, including all eukaryotic mono‐ and poly‐ADP‐ribosyltransferases, the bacterial ADP‐ribosylating enzymes which do not have toxic activity, and the analogous enzymes encoded by T‐even bacteriophages. We show that, in addition to the common Glu and Arg/His amino acids previously identified, the conserved motifs can be extended as follows: (i) the Arg/His motif is usually arom‐His/Arg (where ‘arom’ is an aromatic residue); (ii) in the sequences of the CT group the β‐strand forming part of the ‘scaffold’ of the catalytic cavity has an arom‐ph‐Ser‐Thr‐Ser‐ph consensus (where ‘ph’ represents a hydrophobic residue); and (iii) the motif centred in the key glutamic residue is Glu/Gln‐X‐Glu; while (iv) in the sequences of the DT group the NAD‐binding motif is Tyr‐X10‐Tyr. We believe that the model proposed not only accounts for all ADP‐ribosylating proteins known to date, but it is likely to fit other enzymes (currently being analysed) which possess such an activity.
The C‐terminal domain of the Rhizobium leguminosarum chitin synthase NodC is important for function and determines the orientation of the N‐terminal region in the inner membrane
Authors: Barny Marie‐Anne , Schoonejans Eric , Economou Anastassios , Johnston Andrew W. B. , Downie J. Allan
Published: November 2003

The nod C genes from rhizobia encode an N‐acetylglucosaminyl transferase (chitin synthase) involved in the formation of lipo‐chito‐oligosaccharide Nod factors that initiate root nodule morphogenesis in legume plants. NodC proteins have two hydrophobic domains, one of about 21 residues at the N‐terminus and a longer one, which could consist of two or three transmembrane spans, near the C‐terminus. These two hydrophobic domains flank a large hydrophilic region that shows extensive homology with other β‐glycosyl transferases. The topology of NodC in the inner membrane of Rhizobium leguminosarum biovar viciae was analysed using a series of gene fusions encoding proteins in which NodC was fused to alkaline phosphatase (PhoA) lacking an N‐terminal transit sequence or to β‐galactosidase (LacZ). Our data support a model in which the N‐terminal hydrophobic domain spans the membrane in a Nout–Cin orientation, with the adjacent large hydrophilic domain being exposed to the cytoplasm. This orientation appears to depend upon the presence of the hydrophobic region near the C‐terminus. We propose that this hydrophobic region contains three transmembrane spans, such that the C‐terminus of NodC is located in the periplasm. A short region of about 40 amino acids, encompassing the last transmembrane span, is essential for the function of NodC. Our model for NodC topology suggests that most of NodC, including the region showing most similarity to other β‐glycosyl transferases, is exposed to the cytoplasm, where it is likely that polymerization of N‐acetyl glucoasamine occurs. Such a model is incompatible with previous reports suggesting that NodC spans both inner and outer membranes.
aarD, a Providencia stuartii homologue of cydD: role in 2′‐N‐acetyltransferase expression, cell morphology and growth in the presence of an extracellular factor
Authors: Macinga David R. , Rather Philip N.
Published: November 2003

In a search for genes involved in regulation of the 2′‐N‐acetyltransferase in Providencia stuartii, a mini‐Tn5 Cm insertion has been isolated in a locus designated aarD. The aarD1::mini‐Tn5 Cm mutation resulted in a 4.7‐fold increase in the levels of β‐galactosidase accumulation from an aac(2′)–lacZ transcriptional fusion and a 32‐fold increase in the levels of gentamicin resistance in P. stuartii. The wild‐type aarD locus was cloned on a 5.0 kb Cla I fragment and complemented the aarD1 mutation. Nucleotide sequence analysis of this fragment identified two large open reading frames whose deduced products displayed significant amino acid identity, 64% and 64%, respectively, to the CydD and CydC proteins of Escherichia coli, which are involved in formation of the cytochrome d oxidase complex. Physical mapping indicated the aarD1::mini‐Tn5 Cm insertion was within the open reading homologous to CydD. The strain containing the aarD1 mutation was unable to grow in the presence of toluidine blue or on glycerol minimal media in the presence of zinc, suggesting that aarD is functionally equivalent to cydD. Additional phenotypes resulting from the aarD1 mutation included: altered cell morphology, a reduced growth rate and the inability of cells to grow beyond early log phase. Further examination of this phenomenon revealed that the aarD1 mutant was unable to grow in the presence of a self‐produced extracellular factor(s). This novel phenotype was not limited to P. stuartii as E. coli cydD and ΔcydAB::kan mutants were also sensitive to a self‐produced extracellular factor.
Characterization of pectin methylesterase B, an outer membrane lipoprotein of Erwinia chrysanthemi 3937
Authors: Shevchik Vladimir E. , Condemine Guy , Hugouvieux‐Cotte‐Pattat Nicole , Robert‐Baudouy Janine
Published: November 2003

The secretion of extracellular pectinases, among which there are least six isoenzymes of pectate lyase and one pectin methylesterase, allows the phytopathogenic bacterium Erwinia chrysanthemi to degrade pectin. A gene coding for a novel pectin methylesterase has been cloned from an E. chrysanthemi strain 3937 gene library. This gene, pemB, codes for a 433‐amino‐acid protein. The PemB N‐terminal region has the characteristics of lipoprotein signal sequences. We have shown that the PemB precursor is processed and that palmitate is incorporated into the mature protein. The PemB lipoprotein is not released into the extracellular medium and is localized in the outer membrane. The PemB sequence presents homology with other pectin methylesterases from bacterial and plant origin. pemB‐like proteins were detected in four other E. chrysanthemi strains but not in Erwinia carotovora strains. PemB was overproduced in Escherichia coli and purified to homogeneity. PemB activity is strongly increased by non‐ionic detergents. The enzyme is more active on methylated oligogalacturonides than on pectin, and it is necessary for the growth of the bacteria on oligomeric substrates. PemB is more probably involved in the degradation of methylated oligogalacturonides present in the periplasm of the bacteria, rather than in a direct action on extracellular pectin. pemB expression is inducible in the presence of pectin and is controlled by the negative regulator KdgR.
A cytochrome c biogenesis gene involved in pyoverdine production in Pseudomonas fluorescens ATCC 17400
Authors: Gaballa Ahmed , Koedam Nico , Cornelis Pierre
Published: November 2003

Pseudomonas fluorescens ATCC 17400 produces pyoverdine under iron‐limiting conditions. A Tn5 mutant, 2G11, produced lower amounts of different pyoverdine forms and was unable to grow under iron limitation caused by ethylenediamine‐di(o‐hydroxy‐phenylacetic acid) (EDDHA) or zinc. This mutant was complemented by a 9.6 kb HindIII–BamHI DNA fragment that contained eight contiguous open reading frames (ORFs cytA to cytH ). The proteins possibly encoded by this polycistronic gene cluster were all similar to the products of cytochrome c biogenesis genes from, amongst others, Rhodobacter capsulatus and Bradyrhizobium japonicum, not only in terms of amino acid sequence, but also in the overall hydropathy index of these proteins. By TnphoA mutagenesis and site‐specific gene replacement it was found that the first three ORFs (cytA to cytC ) were essential for cytochrome c production while only the product of cytA was needed for normal pyoverdine production. The presence of a putative haem‐binding site in the CytA protein (WGSWWVWD) was confirmed. From analysis of a constructed phoA fusion, a periplasmic location was found for this motif. The ability of the cytA gene to restore both cytochrome c and pyoverdine production suggests the involvement of this particular gene both in haem and in pyoverdine transport in P. fluorescens.
The afimbrial adhesive sheath encoded by the afa‐3 gene cluster of pathogenic Escherichia coli is composed of two adhesins
Authors: Garcia Marie‐Isabelle , Gounon Pierre , Courcoux Pascale , Labigne Agnès , Le Bouguénec Chantal
Published: November 2003

The afa‐3 gene cluster determines the formation of an afimbrial adhesive sheath that is expressed by uropathogenic as well as diarrhoea‐associated Escherichia coli strains. It contains six genes (afaA–afaF ), among which the afaE3 gene is known to code for the structural AfaE‐III adhesin (previously designated AFA‐III), whereas no role has yet been identified for the afaD gene product. The afa‐3 gene cluster is closely related to the daa operon that codes for an adhesin, the F1845 adhesin, which is highly related to the AfaE‐III adhesin; however, unlike the AfaE‐III adhesin, F1845 is a fimbrial adhesin. Reported in this work is the construction of chimeras between the afa‐3 and daa operons. Analyses of the phenotypes conferred by these afa‐3/daa chimeric clusters allowed us to conclude that the biogenesis of a fimbrial or an afimbrial adhesin is fully determined by the amino acid sequence of the AfaE‐III and F1845 adhesins. Moreover, the role of the AfaD product in the biosynthesis of the afimbrial sheath was assessed by immunogold and immunofluorescence experiments. The AfaD and the AfaE‐III products were purified and used to raise rabbit and mouse antisera. Similar to AfaE‐III, AfaD was found to be a surface‐exposed protein as well as an adhesin; both AfaD and AfaE‐III are concomittantly expressed by the bacterial cell. These results demonstrate, for the first time, that the afimbrial adhesive sheath expressed by pathogenic E. coli is composed of two adhesins.
Heat‐shock and general stress response in Bacillus subtilis
Authors: Hecker Michael , Schumann Wolfgang , Völker Uwe
Published: November 2003

The induction of stress proteins is an important component of the adaptional network of a non‐growing cell of Bacillus subtilis. A diverse range of stresses such as heat shock, salt stress, ethanol, starvation for oxygen or nutrients etc. induce the same set of proteins, called general stress proteins. Although the adaptive functions of these proteins are largely unknown, they are proposed to provide general and rather non‐specific protection of the cell under these adverse conditions. In addition to these non‐specific general stress proteins, all extracellular signals induce a set of specific stress proteins that may confer specific protection against a particular stress factor. In B. subtilis at least three different classes of heat‐inducible genes can be defined by their common regulatory characteristics: Class I genes, as exemplified by the dnaK and groE operons, are most efficiently induced by heat stress. Their expression involves a σA‐dependent promoter, an inverted repeat (called the CIRCE element) highly conserved among eubacteria, and probably a repressor interacting with the CIRCE element. The majority of general stress genes (class II, more than 40) are induced at σB‐dependent promoters by different growth‐inhibiting conditions. The activation of σB by stress or starvation is the crucial event in the induction of this large stress regulon. Only a few genes, including lonclpCclpP, and ftsH, can respond to different stress factors independently of σB or CIRCE (class III). Stress induction of these genes occurs at promoters presumably recognized by σA and probably involves additional regulatory elements which remain to be defined.
Interchange of the active and silent S‐layer protein genes of Lactobacillus acidophilus by inversion of the chromosomal slp segment
Authors: Boot Hein J. , Kolen Carin P. A. M. , Pouwels Peter H.
Published: November 2003

The most‐dominant surface‐exposed protein in many bacterial species is the S‐protein. This protein crystallises into a regular monolayer on the outside surface of the bacteria: the S‐layer. Lactobacillus acidophilus harbours two S‐protein‐encoding genes, slpA and slpB, only one of which (slpA ) is expressed. In this study, we show by polymerase chain reaction (PCR) analysis that slpA and slpB are located on a 6 kb chromosomal segment, in opposite orientations. In a small fraction of the bacterial population, this segment is inverted. The inversion leads to interchanging of the expressed and silent S‐protein‐encoding genes, and places the formerly silent gene behind the S‐promoter which is located outside the inverted segment. A 26 bp sequence showing a high degree of similarity with the consensus sequence recognized by the Din family of invertases is present in the region where recombination occurs. Expression of the slpA gene seems to be favoured under laboratory growth conditions because 99.7% of the chromosomes of an L. acidophilus ATCC 4356 broth culture had the slpA gene present at the slp expression site.
A cytoplasmic gene‐shuffle system in Kluyveromyces lactis: use of epitope tagging to detect a killer plasmid‐encoded gene product
Authors: Schaffrath Raffael , Meacock Peter A.
Published: November 2003

A novel gene shuffle approach has been developed for investigating the functions of genes on the cytoplasmic linear DNA killer plasmids of Kluyveromyces lactis. By transplacing k2ORF5 from the larger plasmid pGKL2(k2) onto pGKL1(k1) we have shown this gene to be essential and functionally interchangeable between plasmids. Once transferred onto k1, k2ORF5 is fully able to complement a k2ORF50 deletion on k2 in trans, giving rise to yeast strains containing only the two recombinant plasmid forms. Additionally, the in vivo product of k2ORF5 has been identified as a 19.5 kDa protein by transplacing an epitope‐tagged k2ORF5 allele from k2 to k1. The ease of detection of the tagged ORF5 product in comparison to TRF1, the gene product of k2ORF10, indicates that Orf5p is one of the most abundant k2 products, implying structural rather than regulatory function.
Cloning of an Aeromonas hydrophila type IV pilus biogenesis gene cluster: complementation of pilus assembly functions and characterization of a type IV leader peptidase/N‐methyltransferase required for extracellular protein secretion
Authors: Pepe Cynthia M. , Eklund Melvin W. , Strom Mark S.
Published: November 2003

Aeromonas hydrophila secretes several extracellular proteins that are associated with virulence including an enterotoxin, a protease, and the hole‐forming toxin, aerolysin. These degradative enzymes and toxins are exported by a conserved pathway found in many Gram‐negative bacteria. In Pseudomonas aeruginosa this export pathway and type IV pilus biogenesis are dependent on the product of the pilD gene. PilD is a bifunctional enzyme that processes components of the extracellular secretory pathway as well as a type IV prepilin. An A. hydrophila genomic library was transferred into a P. aeruginosa pilD mutant that is defective for type IV pilus biogenesis. The A. hydrophila pilD homologue, tapD, was identified by its ability to complement the pilD mutation in P. aeruginosa. Transconjugants containing tapD were sensitive to the type IV pilus‐specific phage, PO4. Sequence data revealed that tapD is part of a cluster of genes (tapABCD) that are homologous to P. aeruginosa type IV pilus biogenesis genes (pilABCD). We showed that TapB and TapC are functionally homologous to P. aeruginosa PilB and PilC, the first such functional complementation of pilus assembly demonstrated between bacteria that express type IV pili. In vitro studies revealed that TapD has both endopeptidase and N‐methyltransferase activities using P. aeruginosa prepilin as substrate. Furthermore, we show that tapD is required for extracellular secretion of aerolysin and protease, indicating that tapD may play an important role in the virulence of A. hydrophila
Analysis of the localization of STE6/CFTR chimeras in a Saccharomyces cerevisiae model for the cystic fibrosis defect CFTRΔF508
Authors: Paddon Chris , Loayza Diego , Vangelista Luca , Solari Roberto , Michaelis Susan
Published: November 2003

The use of yeast as a model system to study mammalian proteins is attractive, because yeast genetic tools can be utilized if a suitable phenotype is created. STE6, the Saccharomyces cerevisiae a‐factor mating pheromone transporter, and CFTR, the mammalian cystic fibrosis transmembrane conductance regulator, are both members of the ATP binding cassette (ABC) superfamily. Teem et al. (1993) described a yeast model for studying a mutant form of the cystic fibrosis protein, CFTRΔF508. The model involved expression of a chimeric molecule in which a portion of yeast STE6 was replaced with the corresponding region from mammalian CFTR. The STE6/CFTR chimera complemented a ste6 mutant strain for mating, indicating that it could export a‐factor. However, mating efficiency was dramatically reduced upon introduction of ΔF508, providing a yeast phenotype for this mutation. In human cells, the ΔF508 mutation results in retention of CFTR in the endoplasmic reticulum (ER), and possibly in reduction of its chloride‐channel activity. Here we examine the basis for the differences in STE6 activity promoted by the wild‐type and mutant STE6/CFTR chimeras. By analysis of protein stability and subcellular localization, we find that the mutant chimera is not ER‐retained in yeast. We conclude that the molecular basis for the reduced mating of the STE6/CFTRΔF508 chimera must reflect a reduction in its capacity to transport a‐factor, rather than mistrafficking. Thus, STE6/CFTRΔF508 in yeast appears to be a good genetic model to probe certain aspects of protein function, but not to study protein localization.
ahpC, a gene involved in isoniazid resistance of the Mycobacterium tuberculosis complex
Authors: Wilson Theresa M. , Collins Desmond M.
Published: November 2003

A gene conferring low‐level isoniazid (INH) resistance on Mycobacterium smegmatis was isolated from a cosmid library of the genome of an INH‐resistant Mycobacterium bovis strain. The gene had good homology with ahpC, the product of which is a subunit of alkyl hydroperoxide reductase, and also with a family of thiol‐specific antioxidant enzymes. A mutation was found in the promoter upon comparison with the equivalent DNA sequence from the INH‐sensitive parent strain. Promoter sequences from other INH‐sensitive and INH‐resistant M. bovis and Mycobacterium tuberculosis strains were sequenced and the mutation was found only in the INH‐resistant strains. An INH‐resistant M. tuberculosis strain also had an additional mutation in the promoter region. The wild‐type promoter and promoters with one and two mutations were ligated into a reporter plasmid containing the lacZ gene. The presence of the first mutation resulted in a sixfold induction of β‐galactosidase activity, and the presence of both mutations caused a 10‐fold induction. Increased expression of AhpC may account for some of the INH resistance of strains of the M. tuberculosis complex.
Lignocellulose degradation by Phanerochaete chrysosporium: gene families and gene expression for a complex process
Authors: Broda Paul , Birch Paul R. J. , Brooks Paul R. , Sims Paul F. G.
Published: November 2003

Phanerochaete chrysosporium completely degrades lignocellulose. The most recalcitrant component, lignin, is oxidized by the radical products of lignin and manganese peroxidases, whereas cellulose and hemicellulose are hydrolysed. Both peroxidases and cellulases exist as complex families at both the DNA and protein levels. The lignin peroxidases may function principally when mycelium‐bound and, therefore, undetectable in culture supernatants. Moreover, methods for the study of P. chrysosporium must be applicable to solid substrate as well as liquid‐culture conditions. For these reasons, detailed studies of gene expression, made possible by the reverse transcriptase–polymerase chain reaction method, are essential. Such studies reveal that gene families are subject to differential expression. The cellulase system has some differences from that of Trichoderma reesei; the distinction made between the activities of exocellobiohydrolases and endoglucanases needs to be re‐appraised in both species. Current studies also seek to reconstruct the systems of degradation of lignocellulose and its individual components by heterologous expression of individual proteins in recombinant systems, and their use in mechanistic studies singly and in combinations.
Plasmid transfer and expression of the transfer (tra) gene product of plasmid pIJ101 are temporally regulated during the Streptomyces lividans life cycle
Authors: Pettis Gregg S. , Cohen Stanley N.
Published: November 2003

We previously have shown that a chromosomally integrated copy of the tra gene of plasmid pIJ101 under the control of the KorA repressor protein, which regulates transcription of tra as well as its own synthesis, can promote the conjugal transfer of both chromosomal and plasmid genes in Streptomyces lividans. Using an antibody generated against a fusion protein containing the C‐terminal portion of Tra, we show here that this essential conjugation protein is present in membrane fractions of both surface‐grown S. lividans, which mate readily, and of cells grown in liquid culture, where mating has not been found. Expression of Tra during the S. lividans life cycle was temporally regulated and was reduced late during vegetative growth so that little or no Tra protein was detected in cells as they began to differentiate morphologically and produce secondary metabolites. Comparison of the membrane concentration of Tra protein with tra mRNA concentration during the S. lividans life cycle indicated that the disappearance of Tra is post‐transcriptionally controlled and thus is not mediated by KorA. The results of ‘interrupted mating’ experiments, together with the time of appearance of Tra in S. lividans membranes, indicate that the intermycelial transfer of pIJ101 in S. lividans is complete by the onset of cellular differentiation.
Regulation of competence for genetic transformation in Streptococcus pneumoniae by an auto‐induced peptide pheromone and a two‐component regulatory system
Authors: Pestova E. V. , Håvarstein L. S. , Morrison D. A.
Published: November 2003

The regulation of competence for genetic transformation in Streptococcus pneumoniae depends on a quorum‐sensing system, but the only molecular elements of the system whose specific role have been identified are an extracellular peptide signal and an ABC‐transporter required for its export. Here we show that transcription of comC, the gene encoding a predicted 41‐residue precursor peptide that is thought to be processed and secreted as the 17‐residue mature competence activator, increased approximately 40‐fold above its basal level of expression in response to exogenous synthetic activator, consistent with earlier experiments indicating that the activator acts autocatalytically. We also describe two new genes, comD and comE, that encode members of histidine protein kinase and response‐regulator families and are linked to comC. Disruption of comE abolished both response to synthetic activator peptide and endogenous competence induction.
Identification of the streptococcal competence‐pheromone receptor
Authors: Håvarstein Leiv Sigve , Gaustad Peter , Nes Ingolf F. , Morrison Donald A.
Published: November 2003

Competence for genetic transformation in certain species of streptococci has been known for many years to be induced by a secreted protease‐sensitive pheromone, referred to as the competence factor or activator, which acts as a quorum‐sensing signal to co‐ordinate expression of late competence genes. We recently reported identification of the pheromone of Streptococcus pneumoniae strain Rx as a small unmodified peptide, which was termed competence‐stimulating peptide (CSP). By identifying the gene (comC) encoding the Rx CSP we were able to show that it is synthesized as a precursor peptide containing an N‐terminal double‐glycine type leader. In the present work, we describe two alleles of the corresponding gene from Streptococcus gordonii strains Challis and NCTC 7865, which are strains with distinct competence pheromones and corresponding specific pheromone reactivities. In addition, the nucleic acid sequences of two genes located downstream of comC were determined; interestingly, these genes encode a two‐component signal transduction system. We therefore speculated that their products, a histidine kinase (ComD) and its cognate response regulator (ComE), act downstream of the CSP in competence regulation. By tracing the CSP specificity of the competence response in these strains to strain‐specific alleles of comD, we obtained evidence demonstrating that the histidine kinase ComD is the competence‐pheromone receptor.
The effects on Escherichia coli of expression of the cloned bacteriophage T4 nucleoid disruption (ndd ) gene
Authors: Bouet Jean‐Yves , Campo Nathalie J. , Krisch Henry M. , Louarn Jean‐Michel
Published: November 2003

Immediately after T4 bacteriophage infection, the Escherichia coli nucleoid undergoes rapid delocalization. The ndd gene of T4 is responsible for this nuclear disruption phenomenon. We have cloned two alleles of this gene and studied the effects of their expression on E. coli cells. We have shown that the Ndd protein (i) is able to reproduce the disruption of the nucleoid characteristic of T4 infection, (ii) is highly toxic and results in a logarithmic decrease in cell viability, and (iii) inhibits genomic DNA replication by blocking progression of replication forks. Induction of Ndd does not result in degradation of genomic DNA and does not significantly alter the general processes of transcription and translation during the entire period of exponential cell death. These results support the notion that the target of Ndd is some aspect of the nucleoid architecture.
A new PrfA‐regulated gene of Listeria monocytogenes encoding a small, secreted protein which belongs to the family of internalins
Authors: Engelbrecht Fredi , Chun Soo‐Kyung , Ochs Christian , Hess Jürgen , Lottspeich Friedrich , Goebel Werner , Sokolovic Zeljka
Published: November 2003

A mutant of Listeria monocytogenes EGD was constructed that carries an extended deletion removing the entire PrfA‐regulated gene cluster from plcA to plcB and a second deletion inactivating the inlA gene. Upon supplementation of this mutant with multiple gene copies of prfA, a protein of 30 kDa was detected in the supernatant of the mutant strain. The gene encoding this protein was obtained by direct and inverse polymerase chain reaction using oligonucleotide primers that were deduced from partial amino acid sequences of the purified 30 kDa protein. The amino acid sequence of the gene product revealed a protein of 297 amino acids that carried eight repeat units with high homology to those of the two known internalin proteins A and B. This secretory protein, termed internalin C, is much smaller than InlA or InlB and its complete sequence is related to the two known internalins. The gene inlC is transcribed into a monocistronic mRNA from a single promoter which shows a typical consensus sequence for PrfA‐binding at the position −40. In contrast to the transcription of the inlAB operon, which is downregulated after shift of an L. monocytogenes EGD culture from brain–heart infusion into minimum essential medium (MEM), transcription of inlC is induced in MEM like most of the other known PrfA‐regulated virulence genes. In addition, inlC is strongly transcribed in the cytoplasm of phagocytic J774 cells whereas inlA is poorly transcribed under these conditions, suggesting that internalin C may play a role in a late stage of L. monocytogenes infection rather than in the uptake of L. monocytogenes by non‐professional phagocytic cells. An inlC deletion mutant shows reduced virulence when tested in an intravenous mouse model, but intracellular replication of the mutant in Caco‐2 and J774 cells appears to be comparable with that of the wild‐type strain.
Internal pH crisis, lysine decarboxylase and the acid tolerance response of Salmonella typhimurium
Authors: Park Yong‐Keun , Bearson Bradley , Bang Seong Ho , Bang Iel Soo , Foster John W.
Published: November 2003

Salmonella typhimurium possesses an adaptive response to acid that increases survival during exposure to extremely low pH values. The acid tolerance response (ATR) includes both log‐phase and stationary‐phase systems. The log‐phase ATR appears to require two components for maximum acid tolerance, namely an inducible pH homeostasis system, and a series of acid‐shock proteins. We have discovered one of what appears to be a series of inducible exigency pH homeostasis systems that contribute to acid tolerance in extreme acid environments. The low pH‐inducible lysine decarboxylase was shown to contribute significantly to pH homeostasis in environments as low as pH 3.0. Under the conditions tested, both lysine decarboxylase and σs‐dependent acid‐shock proteins were required for acid tolerance but only lysine decarboxylase contributed to pH homeostasis. The cadBA operon encoding lysine decarboxylase and a lysine/cadaverine antiporter were cloned from S. typhimurium and were found to be 79% homologous to the cadBA operon from Escherichia coli. The results suggest that S. typhimurium has a variety of means of fulfilling the pH homeostasis requirement of the ATR in the form of inducible amino acid decarboxylases.
Accessory proteins impose site selectivity during ColE1 dimer resolution
Authors: Guhathakurta Anjan , Viney Ian , Summers David
Published: November 2003

The cer–Xer dimer resolution system of plasmid ColE1 is highly selective, acting only at sites on the same molecule and in direct repeat. Recombination requires the XerCD recombinase and accessory proteins ArgR and PepA. The Escherichia coli chromosome dimer resolution site dif and the type II hybrid site use the same recombinase but are independent of ArgR and PepA and show no site selectivity. This has led to the proposal that ArgR and PepA are responsible for the imposition of constraint. We describe here the characterization of a novel class of ‘conditionally constrained’ multimer resolution sites whose properties support this hypothesis. In the presence of ArgR and PepA, plasmids containing conditionally constrained sites are monomeric, but in their absence, extensive multimerisation is seen. A mutant ArgR derivative (ArgR110), which is defective in cer‐mediated dimer resolution, remains able to prevent plasmid multimerisation by a conditionally constrained site. This implies that the accessory factors block recombination in trans rather than ensuring rapid multimer resolution. When the distance between the ArgR and XerCD binding sites in a conditionally constrained site was altered by a non‐integral number of helical turns, the site became unconstrained. Constraint was restored by the insertion of a full helical turn.
In vitro analysis of mRNA processing by RNase E in the pap operon of Escherichia coli
Authors: Naureckiene Saule , Uhlin Bernt Eric
Published: November 2003

Differential gene expression from operons encoding fimbrial adhesins in Escherichia coli involves processing and differential decay of polycistronic transcripts. Previous analyses of mRNA processing in vivo using ribonuclease mutants of E. coli have given different results with the different fimbrial gene systems tested. For the pap operon from uropathogenic E. coli, the results suggested that the mRNA processing is dependent on ribonuclease E (RNase E), whereas in other fimbrial operons with similar genetic organisation, the processing was concluded to be RNase E independent. We have developed an in vitro system allowing us to assess the cleavage of pap mRNA, to study the mRNA processing of a fimbrial operon in more detail, and to define the enzymatic activity and target. The results of this study establish that RNase E does indeed cleave the papBA intercistronic transcript. Analysis of the cleavage products reveals that in vitro RNase E can cleave the mRNA at other positions in addition to the site preferentially cleaved in vivo. The specificity of the cleavage pattern was assessed using transcripts derived from mutants with base substitutions near, or within, the major in vivo cleavage site. Such mutants have alternative cleavage sites. A common feature of the different cleavage sites is a high A/U nucleotide content, similar to other known RNase E cleavage sites. Features of the secondary structure of the papBA intercistronic mRNA were investigated using single‐strand‐specific and double‐strand‐specific nucleases. The secondary structure model derived from stability calculations and our results from the nuclease‐probing experiments indicate that the positions subject to RNase E cleavage are mainly single stranded and flanked by more stable stem–loop structures. The results are consistent with the notion that an mRNA conformation exposing A/U‐rich, non‐paired regions constitutes the target, i.e. a flexible determinant, for processing by RNase E in the pap transcript. The findings are discussed in relation to the existence of a potential recognition site for RNase E and the analysis of RNase E cleavages in other RNA molecules.
Influence of pili, fibrils, and capsule on in vitro adherence by Haemophilus influenzae type b
Authors: St. Geme, III Joseph W. , Cutter David
Published: November 2003

Haemophilus influenzae type b is an encapsulated bacterium that initiates infection by colonizing the upper respiratory epithelium. In vitro studies indicate that H. influenzae type b is capable of expressing two morphologically distinct filamentous adhesive structures, referred to as pili and fibrils, respectively. In this study, we examined adherence to a variety of human epithelial‐cell types and demonstrated that pili and fibrils have separate cellular binding specificities. In addition, we found that capsular material inhibits fibril recognition of the host‐cell surface. This inhibitory effect was reduced when bacteria were grown to stationary phase, reflecting diminished encapsulation. However, when growth medium was supplemented with Mg2+, stationary‐phase organisms were relatively heavily encapsulated and non‐adherent. These observations suggest that encapsulation can be modulated in response to growth phase or environmental signals. It is possible that encapsulation is down‐modulated early in the infectious process in order to avoid interfering with colonization. In contrast, encapsulation may be up‐modulated between hosts and during bacteremia, where it appears to confer a selective advantage. We speculate that this model may also apply to other encapsulated pathogens.
Recombination in mycobacteria
Authors: McFadden Johnjoe
Published: November 2003

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB) is thought to infect a quarter of the world’s population and accounts for 3 million deaths each year. Leprosy, caused by Mycobacterium leprae continues to afflict millions. In many countries, the incidence of TB is increasing due to its association with acquired immune deficiency syndrome (AIDS) and the emergence of multidrug resistance strains of tubercle bacilli. Genes that encode major antigens, enzymes, potential virulence determinants and drug resistance in mycobacteria have been isolated and characterized; however, further genetic analysis of pathogenic mycobacteria has been severely hampered by the difficulty in precisely defining the phenotype of both wild‐type and mutant genes by utilizing homologous recombination to perform allele exchange. Recombination mechanisms have been intensely studied in Escherichia coli but it is unclear how far mechanistic pathways elucidated in this species are applicable to other organisms, such as mycobacteria. The aim of this review is to examine what is currently known about homologous recombination in mycobacteria. A model is proposed to account for both low levels of homologous recombination and high levels of illegitimate recombination found in the tubercle bacillus.
Characterization of a Mycobacterium smegmatis gene that confers resistance to phages L5 and D29 when overexpressed
Authors: Barsom Elizabeth K. , Hatfull Graham F.
Published: November 2003

Bacteriophage infection requires a specific interaction with the outer surface of a bacterial host followed by interaction with the cell membrane and phage DNA injection. Phages of the mycobacteria encounter a cell wall that is rich in unusual lipid‐ and sugar‐containing components which form a formidable barrier that must be passed to gain access to the membrane. We describe here a gene of Mycobacterium smegmatis that confers resistance to mycobacteriophages L5 and D29. The phage‐resistance phenotype results not from mutation but from elevated expression of a wild‐type gene. It appears that the product of this multicopy phage‐resistance (mpr) gene may alter the structure of the host cell wall or membrane, thereby inhibiting productive phage DNA injection.
Definition of a consensus DNA‐binding site for the Escherichia coli pleiotropic regulatory protein, FruR
Authors: Nègre Didier , Bonod‐Bidaud Christelle , Geourjon Christophe , Deléage Gilbert , Cozzone Alain J. , Cortay Jean‐Claude
Published: November 2003

The FruR regulator of Escherichia coli controls the initiation of transcription of several operons encoding a variety of proteins involved in carbon and energy metabolism. The sequence determinants of the FruR‐binding site were analysed by using 6× His‐tagged FruR and a series of double‐stranded randomized oligonucleotides. FruR consensus binding sites were selected and characterized by several consecutive rounds of the polymerase chain reaction‐assisted binding‐site selection method (BSS) using nitrocellulose‐immobilized DNA‐binding protein. FruR was demonstrated to require, for binding, an 8 bp left half‐site motif and a 3 bp conserved right half‐site with the following sequence: 5′‐GNNGAATC/GNT‐3′. In this sequence, the left half‐site AATC/ consensus tetranucleotide is a typical motif of the DNA‐binding site of the regulators of the GalR–LacI family. On the other hand, the high degree of degeneracy found in the right half‐site of this palindrome‐like structure indicated that FruR, which is a tetramer in solution, interacts asymmetrically with the two half‐sites of its operator. However, potentially FruR‐target sites showing a high degree of symmetry were detected in 13 genes/operons. Among these, we have focused our interest on the pfkA gene, encoding phosphofructokinase‐1, which is negatively regulated by FruR.