bluuebunny/crossref_metadata_2025 huggingface dataset:
Glycerol‐3‐phosphate‐mediated repression of malT in Escherichia coli does not require metabolism, depends on enzyme IIAGlc and is mediated by cAMP levels
Authors: Eppler Tanja , Boos Winfried
Published: March 2003

malT encodes the central activator of the maltose system in Escherichia coli, a gene that is typically under positive control of the cAMP/CAP catabolite repression system. When cells were grown in tryptone broth, the addition of glycerol reduced malT expression two‐ to threefold. Phosphorylation of glycerol to glycerol‐3‐phosphate (G3P) was necessary for this repression, but further metabolism to dihydroxyacetone phosphate was not. Mutants lacking adenylate cyclase and harbouring a crp* mutation (synthesizing a cAMP receptor protein that is independent of cAMP) no longer repressed a transcriptional malT–lacZ fusion but still repressed a translational malT–lacZ fusion. Similar results were obtained with a mutant lacking enzyme IIAGlc. For the translational fusion (in a cya crp* genetic background) to be repressed by glycerol, a drop to pH 5 of the growth medium was necessary. Thus, while transcriptional repression by glycerol requires enzyme IIAGlc, cAMP and CAP, pH‐mediated translational repression is cAMP independent. Other sugars that are not transported by the phosphotransferase system, most notably d‐xylose, showed the same effect as glycerol.
Identification and characterization of a Streptococcus pyogenes ABC transporter with multiple specificity for metal cations
Authors: Janulczyk Robert , Pallon Jan , Björck Lars
Published: March 2003

Metal ions are crucial trace elements for bacteria infecting the human host. The LraI (lipoprotein receptor‐associated antigen I) transporter in Streptococcus spp. belongs to the superfamily of ABC transporters. The transporter consists of a lipoprotein, an ATP‐binding protein and a hydrophobic integral membrane protein. Here, we describe a new member of the LraI family in the important human pathogen Streptococcus pyogenes. The system was identified in silico by analysis of the S. pyogenes Genome Sequencing Project. The S. pyogenes operon exhibits an atypical organization compared with equivalents in other Streptococcus spp. The presence and atypical organization of the operon was verified in a number of S. pyogenes strains of different serotypes. Transcriptional analysis of the LraI operon demonstrates a polycistronic transcription attenuated by a stable stem–loop structure, which allows the lipoprotein to be expressed in larger quantities than the other two components. The localization of the native lipoprotein at the bacterial surface was shown by proteolytic digestion of S. pyogenes bacteria and NH2‐terminal sequencing of a released lipoprotein fragment. Recombinant lipoprotein was expressed as a GST fusion protein, and studies of molecular interactions with metal radioisotopes demonstrated that the protein has affinity for Zn(II), Fe(III) and Cu(II). Zn(II) and Cu(II) were found to compete for the same binding site, whereas Fe(III) uses a second site. Also, proton‐induced X‐ray analysis of lipoprotein samples identified iron, copper and zinc. Finally, a mutant strain lacking a functional mtsABC operon was generated and showed reduced uptake of 55Fe and 65Zn compared with the wild‐type strain. The operon encoding this novel ABC transporter with multiple specificity for metal cations is designated mtsABC, for metal transporter of Streptococcus.
Characterization of a candidate Borrelia burgdorferiβ3‐chain integrin ligand identified using a phage display library
Authors: Coburn Jenifer , Chege Wambui , Magoun Loranne , Bodary Sarah C. , Leong John M.
Published: March 2003

The spirochaetal agents of Lyme disease, Borrelia burgdorferi (sensu lato) bind to integrins αIIbβ3, αvβ3 and α5β1 in purified form and on the surfaces of human cells. Using a phage display library of B. burgdorferi (sensu stricto) DNA, a candidate ligand for β3‐chain integrins was identified. The native B. burgdorferi protein, termed p66, is known to be recognized by human Lyme disease patient sera and to be expressed on the surface of the spirochaete. We show here that recombinant p66 binds specifically to β3‐chain integrins and inhibits attachment of intact B. burgdorferi to the same integrins. When expressed on the surface of Escherichia coli, this protein increases the attachment of E. coli to a transfected cell line that expresses αvβ3, but not to the parental cell line, which expresses no β3‐chain integrins. Localization of p66 on the surface of B. burgdorferi, the ability of recombinant forms of the protein to bind to β3‐chain integrins and the fact that p66 and B. burgdorferi bind to β3‐chain integrins in a mutually exclusive manner make p66 an attractive candidate bacterial ligand for integrins αIIbβ3 and αvβ3.
Helicase motifs: the engine that powers DNA unwinding
Authors: Hall Mark C. , Matson Steven W.
Published: March 2003

Helicases play essential roles in nearly all DNA metabolic transactions and have been implicated in a variety of human genetic disorders. A hallmark of these enzymes is the existence of a set of highly conserved amino acid sequences termed the ‘helicase motifs’ that were hypothesized to be critical for helicase function. These motifs are shared by another group of enzymes involved in chromatin remodelling. Numerous structure–function studies, targeting highly conserved residues within the helicase motifs, have been instrumental in uncovering the functional significance of these regions. Recently, the results of these mutational studies were augmented by the solution of the three‐dimensional crystal structure of three different helicases. The structural model for each helicase revealed that the conserved motifs are clustered together, forming a nucleotide‐binding pocket and a portion of the nucleic acid binding site. This result is gratifying, as it is consistent with structure–function studies suggesting that all the conserved motifs are involved in the nucleotide hydrolysis reaction. Here, we review helicase structure–function studies in the light of the recent crystal structure reports. The current data support a model for helicase action in which the conserved motifs define an engine that powers the unwinding of duplex nucleic acids, using energy derived from nucleotide hydrolysis and conformational changes that allow the transduction of energy between the nucleotide and nucleic acid binding sites. In addition, this ATP‐hydrolysing engine is apparently also associated with proteins involved in chromatin remodelling and provides the energy required to alter protein‐DNA structure, rather than duplex DNA or RNA structure.
Initial events in the degradation of the polycistronic puf mRNA in Rhodobacter capsulatus and consequences for further processing steps
Authors: Heck Claudia , Balzer Angelika , Fuhrmann Oliver , Klug Gabriele
Published: March 2003

Individual segments of the polycistronic puf mRNA of Rhodobacter capsulatus exhibit extremely different half‐lives contributing to the stoichiometry of light‐harvesting and reaction centre complexes of this facultative phototrophic bacterium. While earlier investigations shed light on the processes leading to the degradation of the 2.7 kb pufBALMX mRNA and, consequently, to the formation of the highly stable 0.5 kb pufBA mRNA processing product, we have now investigated the initial events in the degradation of the highly unstable 3.2 kb pufQBALMX primary transcript. Sequence modifications of two putative RNase E recognition sites within the pufQ coding region provide strong evidence that RNase E‐mediated cleavage of a sequence at the 3′ end of pufQ is involved in rate‐limiting cleavage of the primary pufQBALMX transcript in vivo. The putative RNase E recognition sequence at the 5′ end of pufQ is cleaved in vitro but does not contribute to rate‐limiting cleavage in vivo. Analysis of the decay of puf mRNA segments transcribed from wild‐type and mutated puf DNA sequences in R. capsulatus and Escherichia coli reveal that RNase E‐mediated cleavage within the pufQ mRNA sequence also affects the stability of the 0.5 kb pufBA processing product. These findings demonstrate that the stability of a certain mRNA segment depends on the pathway of processing of its precursor molecule.
A PP2C phosphatase containing a PAS domain is required to convey signals of energy stress to the σB transcription factor of Bacillus subtilis
Authors: Vijay Kamni , Brody Margaret S. , Fredlund Elisabeth , Price Chester W.
Published: March 2003

The σB transcription factor of the bacterium Bacillus subtilis is activated by growth‐limiting energy or environmental challenge to direct the synthesis of more than 100 general stress proteins. Although the signal transduction pathway that conveys these stress signals to σB is becoming increasingly well understood, how environmental or energy stress signals enter this pathway remains unknown. We show here that two PP2C serine phosphatases — RsbP, which is required for response to energy stress, and RsbU, which is required for response to environmental stress — each converge on the RsbV regulator of σB. According to the current understanding of σB regulation, in unstressed cells the phosphorylated RsbV anti‐anti‐σ is unable to complex the RsbW anti‐σ, which is then free to bind and inactivate σB. We can now advance the model that either PP2C phosphatase, when triggered by its particular class of stress, can remove the phosphate from RsbV and thereby activate σB. The action of the previously described RsbU is known to be controlled by dedicated upstream signalling components that are activated by environmental stress. The action of the RsbP phosphatase described here requires an energy stress, which we suggest is sensed, at least in part, by the PAS domain in the amino‐terminal region of the RsbP phosphatase. In other bacterial signalling proteins, similar PAS domains and their associated chromophores directly sense changes in intracellular redox potential to control the activity of a linked output domain.
Deletion of the unique gene encoding a typical histone H1 has no apparent phenotype in Aspergillus nidulans
Authors: Ramón Ana , Muro‐Pastor María Isabel , Scazzocchio Claudio , Gonzalez Ramon
Published: March 2003

We have cloned the H1 histone gene (hhoA) of Aspergillus nidulans. This single‐copy gene codes for a typical linker histone with one central globular domain. The open reading frame is interrupted by six introns. The position of the first intron is identical to that of introns found in some plant histones. An H1–GFP fusion shows exclusive nuclear localization, whereas chromosomal localization can be observed during condensation at mitosis. Surprisingly, the deletion of hhoA results in no obvious phenotype. The nucleosomal repeat length and susceptibility to micrococcal nuclease digestion of A. nidulans chromatin are unchanged in the deleted strain. The nucleosomal organization of a number of promoters, including in particular the strictly regulated niiA‐niaD bidirectional promoter is not affected.
A genomic analysis of two‐component signal transduction in Streptococcus pneumoniae
Authors: Throup John P. , Koretke Kristin K. , Bryant Alexander P. , Ingraham Karen A. , Chalker Alison F. , Ge Yigong , Marra Andrea , Wallis Nicola G. , Brown James R. , Holmes David J. , Rosenberg Martin , Burnham Martin K. R.
Published: March 2003

A genomics‐based approach was used to identify the entire gene complement of putative two‐component signal transduction systems (TCSTSs) in Streptococcus pneumoniae. A total of 14 open reading frames (ORFs) were identified as putative response regulators, 13 of which were adjacent to genes encoding probable histidine kinases. Both the histidine kinase and response regulator proteins were categorized into subfamilies on the basis of phylogeny. Through a systematic programme of mutagenesis, the importance of each novel TCSTS was determined with respect to viability and pathogenicity. One TCSTS was identified that was essential for the growth of S. pneumoniaeThis locus was highly homologous to the yycFG gene pair encoding the essential response regulator/histidine kinase proteins identified in Bacillus subtilis and Staphylococcus aureus. Separate deletions of eight other loci led in each case to a dramatic attenuation of growth in a mouse respiratory tract infection model, suggesting that these signal transduction systems are important for the in vivo adaptation and pathogenesis of S. pneumoniae. The identification of conserved TCSTSs important for both pathogenicity and viability in a Gram‐positive pathogen highlights the potential of two‐component signal transduction as a multicomponent target for antibacterial drug discovery.
Molecular cloning of the calcium and sodium ATPases in Neurospora crassa
Authors: Benito Begoña , Garciadeblás Blanca , Rodríguez‐Navarro Alonso
Published: March 2003

Using PCR, reverse transcription‐PCR (RT‐PCR) and colony hybridization in a genomic library, we isolated six genes which encode type II P‐type ATPases in Neurospora crassa. The six full‐length cDNAs were cloned in a yeast expression vector and transformed into Saccharomyces cerevisiae null Ca2+‐ or Na+‐ATPase mutants. Three cDNAs suppressed the defect of the Ca2+ mutant and two of these protected from Mn2+ toxicity. One cDNA suppressed the defect of the Na+ mutant and two cDNAs were not functional in S. cerevisiae. The expression of the transcripts of the six genes in the presence of Ca2+, Na+, high pH or supporting an osmotic shock indicated that, with the exception of one of the Ca2+‐ATPases, the main function of the cloned ATPases is the adaptation to stress conditions. The relationship between the cloned fungal Ca2+‐ and Na+‐ATPases and plant type II P‐ATPases is discussed.
Identification of a candidate glycosaminoglycan‐binding adhesin of the Lyme disease spirochete Borrelia burgdorferi
Authors: Parveen Nikhat , Leong John M.
Published: March 2003

Binding of glycosaminoglycans (GAGs) by Borrelia burgdorferi, the Lyme disease spirochete, has the potential to promote the colonization of diverse tissues. GAG binding by B. burgdorferi is associated with haemagglutination and we have identified a 26 kDa protein, which we have termed Bgp (BorreliaGAG‐binding protein), on the basis of its ability to bind to heparin and erythrocytes. Bgp was found in outer membrane fractions of B. burgdorferi and on the surface of intact bacteria, as assayed by labelling with a membrane‐impermeable biotinylating agent or anti‐Bgp antibodies. Purified recombinant Bgp agglutinated erythrocytes, binds to the same spectrum of GAGs as the B. burgdorferi strain from which the cloned bgp sequence was obtained, and inhibited B. burgdorferi binding to purified GAGs and to cultured mammalian cells. Thus, Bgp is a strong candidate for a GAG‐binding adhesin of B. burgdorferi.
Identification of additional genes belonging to the LexA regulon in Escherichia coli
Authors: Fernández de Henestrosa Antonio R. , Ogi Tomoo , Aoyagi Sayura , Chafin David , Hayes Jeffrey J. , Ohmori Haruo , Woodgate Roger
Published: March 2003

Exposure of Escherichia coli to a variety of DNA‐damaging agents results in the induction of the global ‘SOS response’. Expression of many of the genes in the SOS regulon are controlled by the LexA protein. LexA acts as a transcriptional repressor of these unlinked genes by binding to specific sequences (LexA boxes) located within the promoter region of each LexA‐regulated gene. Alignment of 20 LexA binding sites found in the E. coli chromosome reveals a consensus of 5′‐TACTG(TA)5CAGTA‐3′. DNA sequences that exhibit a close match to the consensus are said to have a low heterology index and bind LexA tightly, whereas those that are more diverged have a high heterology index and are not expected to bind LexA. By using this heterology index, together with other search criteria, such as the location of the putative LexA box relative to a gene or to promoter elements, we have performed computational searches of the entire E. coli genome to identify novel LexA‐regulated genes. These searches identified a total of 69 potential LexA‐regulated genes/operons with a heterology index of < 15 and included all previously characterized LexA‐regulated genes. Probes were made to the remaining genes, and these were screened by Northern analysis for damage‐inducible gene expression in a wild‐type lexA+ cell, constitutive expression in a lexA(Def) cell and basal expression in a non‐inducible lexA(Ind−) cell. These experiments have allowed us to identify seven new LexA‐regulated genes, thus bringing the present number of genes in the E. coli LexA regulon to 31. The potential function of each newly identified LexA‐regulated gene is discussed.
A monomeric histidine kinase derived from EnvZ, an Escherichia coli osmosensor
Authors: Qin Ling , Dutta Rinku , Kurokawa Hirofumi , Ikura Mitsuhiko , Inouye Masayori
Published: March 2003

Histidine kinases function as dimers. The kinase domain of the osmosensing histidine kinase EnvZ of Escherichia coli consists of two domains: domain A (67 residues) responsible for histidine phosphotransfer and dimerization, and domain B (161 residues) responsible for the catalytic and ATP‐binding function. The individual structures of these two domains have been recently solved by NMR spectroscopy. Here, we demonstrate that an enzymatically functional monomeric histidine kinase can be constructed by fusing in tandem two domains A and one domain B to produce a single polypeptide (A–A–B). We show that this protein, EnvZc[AAB], is soluble and exists as a stable monomer. The autophosphorylation and OmpR kinase activities of the monomeric EnvZc[AAB] are similar to that of the wild‐type EnvZ, while OmpR‐binding and phosphatase functions are reduced. V8 protease digestion and mutational analyses indicate that His‐243 of only the amino proximal domain A is phosphorylated. Based on these results, molecular models are proposed for the structures of EnvZc[AAB] and the kinase domain of EnvZ. The present results demonstrate for the first time the construction of a functional, monomeric histidine kinase, further structural studies of which may provide important insights into the structure–function relationships of histidine kinases.
Extracellular targeting of choline‐binding proteins in Streptococcus pneumoniae by a zinc metalloprotease
Authors: Novak Rodger , Charpentier Emmanuelle , Braun Johann S. , Park Eun , Murti Surekha , Tuomanen Elaine , Masure Robert
Published: March 2003

A genetic‐based search for surface proteins of Streptococcus pneumoniae involved in adhesion identified a putative zinc metalloprotease (ZmpB). ZmpB shared high amino acid sequence similarities with IgA1 proteases of Gram‐positive bacteria, but ZmpB had neither IgA1 nor IgA2 protease activity. Analysis of a family of surface‐expressed proteins, the choline‐binding proteins (Cbp’s), in a zmpB‐deficient mutant demonstrated a global loss of surface expression of CbpA, CbpE, CbpF and CbpJ. CbpA was detected within the cytoplasm. The zmpB‐deficient mutant also failed to lyse with penicillin, a sign of lack of function of the Cbp LytA. Immunodetection studies revealed that the autolysin (LytA), normally located on the cell wall, was trapped in the cytoplasm colocalized with DNA and the transformation protein CinA. Trafficking of CinA and RecA to the cell membrane during genetic competence was also not observed in the zmpB‐deficient mutant. These results suggest a protease dependent regulatory mechanism governing the translocation of CinA and the Cbp's LytA and CbpA of S. pneumoniae.
The G‐protein FlhF has a role in polar flagellar placement and general stress response induction in Pseudomonas putida
Authors: Pandza S. , Baetens M. , Park C. H. , Au T. , Keyhan M. , Matin A.
Published: March 2003

The flhF gene of Pseudomonas putida, which encodes a GTP‐binding protein, is part of the flagellar–motility–chemotaxis operon. Its disruption leads to a random flagellar arrangement in the mutant (MK107) and loss of directional motility in contrast to the wild type, which has polar flagella. The return of a normal flhF allele restores polar flagella and normal motility to MK107; its overexpression triples the flagellar number but does not restore directional motility. As FlhF is homologous to the receptor protein of the signal recognition particle (SRP) pathway of membrane protein translocation, this pathway may have a role in polar flagellar placement in P. putida. MK107 is also compromised in the development of the starvation‐induced general stress resistance (SGSR) and effective synthesis of several starvation and exponential phase proteins. While somewhat increased protein secretion in MK107 may contribute to its SGSR impairment, the altered protein synthesis pattern also appears to have a role.
Competence regulation by oxygen in Streptococcus pneumoniae: involvement of ciaRH and comCDE
Authors: Echenique José R. , Chapuy‐Regaud Sabine , Trombe Marie‐Claude
Published: March 2003

Anaerobic aerotolerant Streptococcus pneumoniae modulates its genetic transformability and its virulence in response to the oxygen concentration. The activity of a single protein encoded by nox and showing NADH oxidase activity is involved in these adaptive responses to O2. Northern blot analysis of wild‐type cultures grown under aerobic and microaerobic conditions indicated transcriptional control of comCDE by O2. An O2‐independent mutant strain carrying the gain‐of‐function mutation comE38KE was isolated and its analysis showed that ComE is a key point in competence stimulation by O2. Plasmid insertion mutations in ciaRH revealed that this two component signal‐transducing system negatively regulates comCDE transcription. The level of comCDE transcripts appears as a major control point in competence regulation by O2 and also by growth phase and cell density.
The thermostabilizing domain of the modular xylanase XynA ofThermotoga maritimarepresents a novel type of binding domain with affinity for soluble xylan and mixed‐linkage β‐1,3/β‐1,4‐glucan
Authors: Meissner Kirsten , Wassenberg Doris , Liebl Wolfgang
Published: March 2003

Thermotoga maritimaXynA is an extremely thermostable modular enzyme with five domains (A1–A2–B–C1–C2). Its catalytic domain (–B–) is flanked by duplicated non‐catalytic domains. The C‐terminal repeated domains represent cellulose‐binding domains (CBDs). Xylanase domains related to the N‐terminal domains of XynA (A1–A2) are called thermostabilizing domains because their deletion normally leads to increased thermosensitivity of the enzymes. It was found that a glutathione‐S‐transferase (GST) hybrid protein (GST‐A1A2) containing both A‐domains of XynA can interact with various soluble xylan preparations and with mixed‐linkage β‐1,3/β‐1,4‐glucans. GST‐A1A2 showed no affinity for insoluble microcrystalline cellulose, whereas, vice versa, GST‐C2, which contains the C‐terminal CBD of XynA, did not interact with soluble xylan. Another hybrid protein, GST‐A2, displayed the same binding properties as GST‐A1A2, indicating that A2 alone can also promote xylan binding. The dissociation constants for the binding of xylose, xylobiose, xylotriose, xylotetraose and xylopentaose by GST‐A2, as determined at 20°C by fluorescence quench experiments, were 8.1 × 10−3 M, 2.3 × 10−4 M, 2.3 × 10−5 M, 2.5 × 10−6 M and 1.1 × 10−6 M respectively. The A‐domains of XynA, which are designated as xylan binding domains (XBD), are, from the structural as well as the functional point of view, prototypes of a novel class of binding domains. More than 50 related protein segments with hitherto unknown function were detected in about 30 other multidomain β‐glycanases, among them putative plant (Arabidopsis thaliana) xylanases. It is argued that polysaccharide binding and not thermostabilization is the main function of A‐like domains.
Study of chromosome rearrangements associated with the trpE26 mutation of Bacillus subtilis
Authors: Regamey Alexandre , Lazarevic Vladimir , Hauser Philippe , Karamata Dimitri
Published: March 2003

Chromosome rearrangements involved in the formation of merodiploid strains in the Bacillus subtilis 168–166 system were explained by postulating the existence of intrachromosomal homology regions. This working hypothesis was tested by analysing sequences and restriction patterns of the, as yet uncharacterized, junctions between chromosome segments undergoing rearrangements in parent, 168 trpC2 and 166 trpE26, as well as in derived merodiploid strains. Identification, at the Ia/Ib chromosome junction of both parent strains, of a 1.3 kb segment nearly identical to a segment of prophage SPβ established the existence of one of the postulated homology sequences. Inspection of relevant junctions revealed that a set of different homology regions, derived from prophage SPβ, plays a key role in the formation of so‐called trpE30, trpE30+, as well as of new class I merodiploids. Analysis of junctions involved in the transfer of the trpE26 mutation, i.e. simultaneous translocation of chromosome segment C and rotation of the terminal relative to the origin moiety of the chromosome, did not confirm the presence of any sequence suitable for homologous recombination. We propose a model involving simultaneous introduction of four donor DNA molecules, each comprising a different relevant junction, and their pairing with the junction regions of the recipient chromosome. The resolution of this structure, resting on homologous recombination, would confer the donor chromosome structure to the recipient, achieving some kind of ‘transstamping’. In addition, a rather regular pattern of inverse and direct short sequence repeats in regions flanking the breaking points could be correlated with the initial, X‐ray‐induced, rearrangement.
A single glutaredoxin or thioredoxin gene is essential for viability in the yeast Saccharomyces cerevisiae
Authors: Draculic Tamara , Dawes Ian W. , Grant Chris M.
Published: March 2003

Glutaredoxins and thioredoxins are small heat‐stable oxidoreductases that have been conserved throughout evolution. The yeast Saccharomyces cerevisiae contains two gene pairs encoding cytoplasmic glutaredoxins (GRX1, GRX2) and thioredoxins (TRX1, TRX2). We report here that the quadruple trx1 trx2 grx1 grx2 mutant is inviable and that either a single glutaredoxin or a single thioredoxin (i.e. grx1 grx2 trx1, grx1 grx2 trx2, grx1 trx1 trx2, grx2 trx1 trx2) is essential for viability. Loss of both thioredoxins has been reported previously to lead to methionine auxotrophy consistent with thioredoxins being the sole reductants for 3′‐phosphoadenosine 5′‐phosphosulphate reductase (PAPS) in yeast. However, we present evidence for the existence of a novel yeast hydrogen donor for PAPS reductase, as strains lacking both thioredoxins assimilated sulphate under conditions that minimized the generation of reactive oxygen species (low aeration and absence of functional mitochondria). In addition, the assimilation of [35S]‐sulphate was approximately 60‐fold higher in the trx1 trx2 grx1 and trx1 trx2 grx2 mutants compared with the trx1 trx2 mutant. Furthermore, in contrast to the trx1 trx2 mutant, the trx1 trx2 grx2 mutant grew on minimal agar plates, and the trx1 trx2 grx1 mutant grew on minimal agar plates under anaerobic conditions. We propose a model in which the novel reductase activity normally functions in the repair of oxidant‐mediated protein damage but, under conditions that minimize the generation of reactive oxygen species, it can serve as a hydrogen donor for PAPS reductase.
Structure and function of bacterial outer membrane proteins: barrels in a nutshell
Authors: Koebnik Ralf , Locher Kaspar P. , Van Gelder Patrick
Published: March 2003

The outer membrane protects Gram‐negative bacteria against a harsh environment. At the same time, the embedded proteins fulfil a number of tasks that are crucial to the bacterial cell, such as solute and protein translocation, as well as signal transduction. Unlike membrane proteins from all other sources, integral outer membrane proteins do not consist of transmembrane α‐helices, but instead fold into antiparallel β‐barrels. Over recent years, the atomic structures of several outer membrane proteins, belonging to six families, have been determined. They include the OmpA membrane domain, the OmpX protein, phospholipase A, general porins (OmpF, PhoE), substrate‐specific porins (LamB, ScrY) and the TonB‐dependent iron siderophore transporters FhuA and FepA. These crystallographic studies have yielded invaluable insight into and decisively advanced the understanding of the functions of these intriguing proteins. Our review is aimed at discussing their common principles and peculiarities as well as open questions associated with them.
Phase variation of a β‐1,3 galactosyltransferase involved in generation of the ganglioside GM1‐like lipo‐oligosaccharide of Campylobacter jejuni
Authors: Linton Dennis , Gilbert Michel , Hitchen Paul G. , Dell Anne , Morris Howard R. , Wakarchuk Warren W. , Gregson Norman A. , Wren Brendan W.
Published: March 2003

Ganglioside mimicry by Campylobacter jejuni lipo‐oligosaccharide (LOS) is thought to be a critical factor in the triggering of the Guillain–Barré and Miller–Fisher syndrome neuropathies after C. jejuni infection. The combination of a completed genome sequence and a ganglioside GM1‐like LOS structure makes C. jejuni NCTC 11168 a useful model strain for the identification and characterization of the genes involved in the biosynthesis of ganglioside‐mimicking LOS. Genome analysis identified a putative LOS biosynthetic cluster and, from this, we describe a putative gene (ORF Cj1139c), which we have termed wlaN, with a significant level of similarity to a number of bacterial glycosyltransferases. Mutation of this gene in C. jejuni NCTC 11168 resulted in a LOS molecule of increased electrophoretic mobility, which also failed to bind cholera toxin. Comparison of LOS structural data from wild type and the mutant strain indicated lack of a terminal β‐1,3‐linked galactose residue in the latter. The wlaN gene product was demonstrated unambiguously as a β‐1,3 galactosyltransferase responsible for converting GM2‐like LOS structures to GM1‐like by in vitro expression. We also show that the presence of an intragenic homopolymeric tract renders the expression of a functional wlaN gene product phase variable, resulting in distinct C. jejuni NCTC 11168 cell populations with alternate GM1 or GM2 ganglioside‐mimicking LOS structures. The distribution of wlaN among a number of C. jejuni strains with known LOS structure was determined and, for C. jejuni NCTC 12500, similar wlaN gene phase variation was shown to occur, so that this strain has the potential to synthesize a GM1‐like LOS structure as well as the ganglioside GM2‐like LOS structure proposed in the literature.
Divergent structure of the ComQXPA quorum‐sensing components: molecular basis of strain‐specific communication mechanism in Bacillus subtilis
Authors: Tran Lam‐Son Phan , Nagai Toshiro , Itoh Yoshifumi
Published: March 2003

In Bacillus subtilis, the ComQXPA quorum‐sensing system controls cell density‐dependent phenotypes such as the production of degradative enzymes and antibiotics and the development of genetic competence. Bacillus subtilis (natto) NAF12, a mutant defective in poly‐γ‐glutamate (γ‐PGA) production, was derived from B. subtilis (natto) NAF4 by Tn917‐LTV1 insertional mutagenesis. Determination of the mutant DNA sequences flanking the Tn917‐LTV1 insert revealed that the insertion had inactivated comP in this mutant, indicating that γ‐PGA synthesis in B. subtilis (natto) is under the control of the ComP–ComA signal transduction system. A comparison of the amino acid sequences revealed striking variation in the primary structures of ComQ (44% identity), ComX (26%) and the sensor domain of ComP (36%) between B. subtilis (natto) NAF4 and B. subtilis 168. In contrast, the amino acid and nucleotide sequences of the kinase domains of ComP and of the ComA response regulator share 95% and 100% identity respectively. The comP genes of NAF4 and 168 restored the impaired competence of B. subtilis BD1658 (comP::cat) and γ‐PGA production of B. subtilis (natto) NAF12 (comP::Tn917‐LTV1) to only 15% of the level achieved by the respective parent comP genes. However, when introduced together with the cognate comQ and comX genes, the comP genes restored the relevant defect of the heterologous comP mutants nearly to wild‐type levels. Analogous to the comCDE system of Streptococcus strains and the agrBCDE system of Staphylococcus aureus, the concerted variation in the comQXP genes appears to establish specific intercellular communication between B. subtilis strains sharing the same pheromone system.
A protein with similarity to the human retinoblastoma binding protein 2 acts specifically as a repressor for genes regulated by the b mating type locus in Ustilago maydis
Authors: Quadbeck‐Seeger Claudia , Wanner Gerhard , Huber Sieglinde , Kahmann Regine , Kämper Jörg
Published: March 2003

Pathogenic development in the corn smut fungus Ustilago maydis is controlled by a heterodimer of the two homeodomain proteins bE and bW which are encoded by the b mating type locus. The bE/bW heterodimer is thought to achieve its function as a transcriptional regulator of pathogenicity genes, either directly by binding to cis regulatory sequences or indirectly via a b‐dependent regulatory cascade.

In a screen for components of the b‐dependent regulatory cascade we have isolated Rum1 (regulator U. maydis 1), a protein with similarities to the human retinoblastoma binding protein 2. Deletion of rum1 results in expression of several b regulated genes independently from their activation via the bE/bW heterodimer. rum1 mutant strains remain pathogenic, proliferate in planta, but fail to produce spores. The defect leads to an arrest in spore development at a defined stage before the spore wall is generated. Deduced from the highly conserved domain structure of Rum1 that includes a DNA‐binding motif and a region known to facilitate the interaction with histone deacetylases, we propose that Rum1 functions as a transcriptional repressor through the modulation of chromatin structure.
Evidence that Ralstonia eutropha (Alcaligenes eutrophus) contains a functional homologue of the Ralstonia solanacearum Phc cell density sensing system
Authors: Garg Ram P. , Yindeeyoungyeon Wandee , Gilis Anja , Denny Timothy P. , Van Der Lelie Daniel , Schell MarK. A.
Published: March 2003

In the phytopathogen Ralstonia (Pseudomonas) solanacearum, control of many virulence genes is partly mediated by the Phc cell density sensing system. Phc uses a novel self‐produced signal molecule [3‐hydroxypalmitic acid methyl ester (3‐OH PAME)], an atypical two‐component system (PhcS/PhcR), and a LysR‐type activator (PhcA) to regulate a reversible switching between two different physiological states. While Phc is present in most R. solanacearum strains, it is apparently absent from other pseudomonad plant pathogens and prokaryotic genomes that have been sequenced. Here, we report discovery of a phcA orthologue in the non‐pathogenic, facultative chemolithoautotroph Ralstonia eutropha (Alcaligenes eutrophus) that fully complements R. solanacearum phcA mutants. We also demonstrate that some R. eutropha produce an extracellular factor that complements R. solanacearum mutants deficient in production of the 3‐OH PAME signal molecule that controls phcA. Additionally, Southern blot hybridization analysis suggested that R. eutropha harbours other Phc components, such as PhcB (a biosynthetic enzyme for 3‐OH PAME) and PhcS (a 3‐OH PAME‐responsive sensor kinase). Analysis of a phcA‐null mutant of R. eutropha showed that phcA (and probably Phc) positively activates motility, in contrast to R. solanacearum where it represses motility. Similarly, the R. eutropha phcA mutant was unaffected in siderophore production, whereas inactivation of phcA in R. solanacearum increases siderophore production. Although our data strongly suggest that R. eutropha has a functional Phc‐like system and support the phylogeny of Ralstonia, it implies that Phc may have a different physiological and ecological function in R. eutropha.
Evidence of recent lateral gene transfer among hyperthermophilic Archaea
Authors: DiRuggiero Jocelyne , Dunn Diane , Maeder Dennis L. , Holley‐Shanks Rhonda , Chatard Jerome , Horlacher Reinhold , Robb Frank T. , Boos Winfried , Weiss Robert B.
Published: March 2003

A total of 153 nucleotide differences were found over a contiguous 16 kb region between two hyperthermophilic Archaea, Pyrococcus furiosus and Thermococcus litoralis. The 16 kb region in P. furiosus is flanked by insertion sequence (IS) elements with inverted and direct repeats. Both IS elements contain a single open reading frame (ORF) encoding a putative protein of 233 amino acids identified as a transposase. This 16 kb region has the features of a typical bacterial composite transposon and represents a possible mechanism for lateral gene transfer between Archaea or possibly between Archaea and Bacteria. A total of 23 homologous IS elements was found in the genome sequence of P. furiosus, whereas no full‐length IS elements were identified in the genomes of Pyrococcus abyssi and Pyrococcus horikoshii. Only one IS element was found in T. litoralis. In P. furiosus and T. litoralis, the 16 kb region contains an ABC transport system for maltose and trehalose that was characterized biochemically for T. litoralis. Regulation of expression studies showed that the malE gene, located on the transposon, and the encoded trehalose/maltose‐binding protein (TMBP) are induced in the presence of maltose and trehalose in both P. furiosus and T. litoralis. The implications of transposition as a mechanism for lateral gene transfer among Archaea are discussed.
G‐protein signalling mediates differential production of toxic secondary metabolites
Authors: Tag Andrew , Hicks Julie , Garifullina Gulnara , Ake Charles , Phillips T. D. , Beremand Marian , Keller Nancy
Published: March 2003

Filamentous fungi elaborate a complex array of secondary metabolites, including antibiotics and mycotoxins. As many of these compounds pose significant economic and health concerns, elucidation of the underlying cellular mechanisms that control their production is essential. Previous work revealed that synthesis of the carcinogenic mycotoxins sterigmatocystin (ST) and aflatoxin (AF) in Aspergillus species is negatively controlled by FadA, the α‐subunit of a heterotrimeric G‐protein. In sharp contrast, we show here that the dominant activating fadA allele, fadAG42R, stimulates transcription of a gene from the A. nidulans penicillin (PN) gene cluster and elevates penicillin production. Thus, FadA has opposite roles in regulating the biosynthesis of a potent antibiotic (PN) and a lethal mycotoxin (ST) in A. nidulans. Furthermore, expression of fadAG42R in Fusarium sporotrichioides increases trichothecene (TR) mycotoxin production and alters TR gene expression. Our findings reveal that a G‐protein defines an important control point for differential expression of fungal secondary metabolites within and across fungal genera. These data provide critical evidence suggesting that targeting G‐protein signal transduction pathways as a means of controlling or preventing the production of a single mycotoxin could have serious undesirable consequences with regard to the production of other secondary metabolites.
The txtAB genes of the plant pathogen Streptomyces acidiscabies encode a peptide synthetase required for phytotoxin thaxtomin A production and pathogenicity
Authors: Healy Frank G. , Wach Michael , Krasnoff Stuart B. , Gibson Donna M. , Loria Rosemary
Published: March 2003

Four Streptomyces species have been described as the causal agents of scab disease, which affects economically important root and tuber crops worldwide. These species produce a family of cyclic dipeptides, the thaxtomins, which alone mimic disease symptomatology. Structural considerations suggest that thaxtomins are synthesized non‐ribosomally. Degenerate oligonucleotide primers were used to amplify conserved portions of the acyladenylation module of peptide synthetase genes from genomic DNA of representatives of the four species. Pairwise Southern hybridizations identified a peptide synthetase acyladenylation module conserved among three species. The complete nucleotide sequences of two peptide synthetase genes (txtAB) were determined from S. acidiscabies 84.104 cosmid library clones. The organization of the deduced TxtA and TxtB peptide synthetase catalytic domains is consistent with the formation of N‐methylated cyclic dipeptides such as thaxtomins. Based on high‐performance liquid chromatography (HPLC) analysis, thaxtomin A production was abolished in txtA gene disruption mutants. Although the growth and morphological characteristics of the mutants were identical to those of the parent strain, txtA mutants were avirulent on potato tubers. Moreover, introduction of the thaxtomin synthetase cosmid into a txtA mutant restored both pathogenicity and thaxtomin A production, demonstrating a critical role for thaxtomins in pathogenesis.
Critical protective role of bacterial superoxide dismutase in Rhizobium–legume symbiosis
Authors: Santos Renata , Hérouart Didier , Puppo Alain , Touati Danièle
Published: March 2003

In nitrogen‐poor soils, rhizobia elicit nodule formation on legume roots, within which they differentiate into bacteroids that fix atmospheric nitrogen. Protection against reactive oxygen species (ROS) was anticipated to play an important role in Rhizobium–legume symbiosis because nitrogenase is extremely oxygen sensitive. We deleted the sodA gene encoding the sole cytoplasmic superoxide dismutase (SOD) of Sinorhizobium meliloti. The resulting mutant, deficient in superoxide dismutase, grew almost normally and was only moderately sensitive to oxidative stress when free living. In contrast, its symbiotic properties in alfalfa were drastically affected. Nitrogen‐fixing ability was severely impaired. More strikingly, most SOD‐deficient bacteria did not reach the differentiation stage of nitrogen‐fixing bacteroids. The SOD‐deficient mutant nodulated poorly and displayed abnormal infection. After release into plant cells, a large number of bacteria failed to differentiate into bacteroids and rapidly underwent senescence. Thus, bacterial SOD plays a key protective role in the symbiotic process.
Endoribonuclease RNase III is essential in Bacillus subtilis
Authors: Herskovitz Michelle A. , Bechhofer David H.
Published: March 2003

The rncS gene of Bacillus subtilis encodes Bs‐RNase III, a narrow‐specificity endoribonuclease. Previous attempts to disrupt rncS were unsuccessful. Here, a strain was constructed in which Bs‐RNase III expression was dependent upon transcription of rncS from a temperature‐sensitive plasmid. Growth of this strain at the non‐permissive temperature resulted in 90–95% cell death, and virtually all the cells that survived retained the rncS‐expressing plasmid. Thus, we conclude that rncS is essential in B. subtilis. The rncS conditional strain also revealed that Bs‐RNase III participates in the processing of ribosomal RNA, in addition to processing small cytoplasmic RNA, a member of the signal recognition particle RNA family. Most significantly, a rare rncS null strain was isolated that will aid further study of the critical role Bs‐RNase III plays in B. subtilis.
Increased bar minigene mRNA stability during cell growth inhibition
Authors: Valadez J. Gerardo , Hernández‐Sánchez Javier , Magos Marco A. , Ontiveros Carlos , Guarneros Gabriel
Published: March 2003

Bacteriophage lambda is unable to grow vegetatively on Escherichia coli mutants defective in peptidyl‐tRNA hydrolase (Pth) activity. Mutations which allow phage growth on the defective host have been located at regions named bar in the lambda genome. Expression of wild‐type bar regions from plasmid constructs results in inhibition of protein synthesis and lethality to Pth‐defective cells. Two of these wild‐type bar regions, barI+ and barII+, contain minigenes with similar AUG–AUA–stop codon sequences preceded by different Shine–Dalgarno (SD) and spacer regions. The induced expression of barI+ and barII+ regions from plasmid constructs resulted in similar patterns of protein synthesis inhibition and cell growth arrest. Therefore, these deleterious effects may stem from translation of the transcripts containing the minigene two‐codon ‘ORF’ (open reading frame). To test for this possibility, we assayed the effect of point mutations within the barI minigene. The results showed that a base pair substitution within the SD and the two‐codon ‘ORF’ sequences affected protein synthesis and cell growth inhibition. In addition, mRNA stability was altered in each mutant. Higher mRNA stability correlated with the more toxic minigenes. We argue that this effect may be caused by ribosome protection of the mRNA in paused complexes as a result of deficiency of specific tRNA.
Conserved amino acid motifs from the novel Piv/MooV family of transposases and site‐specific recombinases are required for catalysis of DNA inversion by Piv
Authors: Tobiason Deborah M. , Buchner John M. , Thiel William H. , Gernert Kim M. , Karls Anna C. Glasgow
Published: March 2003

Piv, a site‐specific invertase from Moraxella lacunata, exhibits amino acid homology with the transposases of the IS110/IS492 family of insertion elements. The functions of conserved amino acid motifs that define this novel family of both transposases and site‐specific recombinases (Piv/MooV family) were examined by mutagenesis of fully conserved amino acids within each motif in Piv. All Piv mutants altered in conserved residues were defective for in vivo inversion of the M. lacunata invertible DNA segment, but competent for in vivo binding to Piv DNA recognition sequences. Although the primary amino acid sequences of the Piv/MooV recombinases do not contain a conserved DDE motif, which defines the retroviral integrase/transposase (IN/Tnps) family, the predicted secondary structural elements of Piv align well with those of the IN/Tnps for which crystal structures have been determined. Molecular modelling of Piv based on these alignments predicts that E59, conserved as either E or D in the Piv/MooV family, forms a catalytic pocket with the conserved D9 and D101 residues. Analysis of Piv E59G confirms a role for E59 in catalysis of inversion. These results suggest that Piv and the related IS110/IS492 transposases mediate DNA recombination by a common mechanism involving a catalytic DED or DDD motif.
RcoA has pleiotropic effects on Aspergillus nidulans cellular development
Authors: Hicks Julie , Lockington Robin A. , Strauss Joseph , Dieringer Daniel , Kubicek Christian P. , Kelly Joan , Keller Nancy
Published: March 2003

Aspergillus nidulans rcoA encodes a member of the WD repeat family of proteins. The RcoA protein shares sequence similarity with other members of this protein family, including the Saccharomyces cerevisiae Tup1p and Neurospora crassa RCO1. Tup1p is involved in negative regulation of an array of functions including carbon catabolite repression. RCO1 functions in regulating pleiotropic developmental processes, but not carbon catabolite repression. In A. nidulans, deletion of rcoA (ΔrcoA), a recessive mutation, resulted in gross defects in vegetative growth, asexual spore production and sterigmatocystin (ST) biosynthesis. Expression of the asexual and ST pathway‐specific regulatory genes, brlA and aflR, respectively, but not the signal transduction genes (i.e. flbA, fluG or fadA) regulating brlA and aflR expression was delayed (brlA) or eliminated (aflR) in a ΔrcoA strain. Overexpression of aflR in a ΔrcoA strain could not rescue normal expression of downstream targets of AflR. CreA‐dependent carbon catabolite repression of starch and ethanol utilization was only weakly affected in a ΔrcoA strain. The strong role of RcoA in development, vegetative growth and ST production, compared with a relatively weak role in carbon catabolite repression, is similar to the role of RCO1 in N. crassa.