bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of novel VirR/VirS‐regulated genes in Clostridium perfringens
Authors: Banu Sayera , Ohtani Kaori , Yaguchi Harumi , Swe Tint , Cole Stewart T. , Hayashi Hideo , Shimizu Tohru
Published: March 2003

Novel genes that are regulated in Clostridium perfringens by the two‐component regulatory system, VirR/VirS, were identified using a differential display method. A plasmid library was constructed from C. perfringens chromosomal DNA, and the plasmids were hybridized with cDNA probes prepared from total RNA of wild‐type strain 13 and its virR mutant derivative TS133. Three clones were identified that carry newly identified VirR/VirS‐regulated genes, two of which were positively regulated and one of which was negatively regulated. Genes located on the identified clones were deduced by nucleotide sequencing, and the target genes of the VirR/VirS system were identified with a set of Northern hybridizations. A 4.9 kb mRNA transcribing the metB (cystathionine gamma‐synthase), cysK (cysteine synthase) and ygaG (hypothetical protein) genes was negatively regulated, whereas 1.6 and 6.0 kb transcripts encoding ptp (protein tyrosine phosphatase) and cpd (2′,3′‐cyclic nucleotide 2′‐phosphodiesterase) respectively, were shown to be positively regulated by the VirR/VirS system. The other gene, hyp7, whose transcript was positively regulated by the VirR/VirS system, was shown to activate the transcription of the colA (kappa‐toxin) and plc (alpha‐toxin) genes, but not the pfoA (theta‐toxin) gene in C. perfringens. These results suggested that the global regulatory system VirR/VirS could regulate various genes, other than toxin genes, both positively and negatively and that the hyp7 gene might encode a novel regulatory factor for toxin production in C. perfringens.
Interactions among components of the Salmonella flagellar export apparatus and its substrates
Authors: Minamino Tohru , Macnab Robert M.
Published: March 2003

We have examined the cytoplasmic components (FliH, FliI and FliJ) of the type III flagellar protein export apparatus, plus the cytoplasmic domains (FlhAC and FlhBC) of two of its six membrane components. FliH, FlhAC and FliJ, when overproduced, caused inhibition of motility of wild‐type cells and inhibition of the export of substrates such as the hook protein FlgE. Co‐overproduction of FliH and FliI substantially relieved the inhibition caused by FliH, suggesting that it is excess free FliH that is inhibitory and that FliH and FliI form a complex. We purified His‐FLAG‐tagged versions of: (i) export components FliH, FliI, FliJ, FlhAC and FlhBC; (ii) rod/hook‐type export substrates FlgB (rod protein), FlgE (hook protein), FlgD (hook capping protein) and FliE (basal body protein); and (iii) filament‐type export substrates FlgK and FlgL (hook–filament junction proteins) and FliC (flagellin). We tested for protein–protein interactions by affinity blotting. In many cases, a given protein interacted with more than one other component, indicating that there are likely to be multiple dynamic interactions or interactions that involve more than two components. Interactions of FlhBC with rod/hook‐type substrates were strong, whereas those with filament‐type substrates were very weak; this may reflect the role of FlhB in substrate specificity switching. We propose a model for the flagellar export apparatus in which FlhA and FlhB and the other four integral membrane proteins of the apparatus form a complex at the base of the flagellar motor. A soluble complex of at least three proteins (FliH, FliI and FliJ) bind the protein to be exported and then interact with the complex at the motor to deliver the protein, which is then exported in an ATP‐dependent process mediated by FliI.
Escherichia coli DipZ: anatomy of a transmembrane protein disulphide reductase in which three pairs of cysteine residues, one in each of three domains, contribute differentially to function
Authors: Gordon Euan H. J. , Page M. Dudley , Willis Anthony C. , Ferguson Stuart J.
Published: March 2003

DipZ is a bacterial cytoplasmic membrane protein that transfers reducing power from the cytoplasm to the periplasm so as to facilitate the formation of correct disulphide bonds and c‐type cytochromes in the latter compartment. Topological analysis using gene fusions between the Escherichia coli dipZ and either E. coli phoA or lacZ shows that DipZ has a highly hydrophobic central domain comprising eight transmembrane α‐helices plus periplasmic globular N‐terminal and C‐terminal domains. The previously assigned translational start codon for the E. coli DipZ was shown to be incorrect and the protein to be larger than previously thought. The experimentally determined translational start position indicates that an additional α‐helix at the N‐terminus acts as a cleavable signal peptide so that the N‐terminus of the mature protein is located in the periplasm. The newly assigned 5′ end of the dipZ gene was shown to be preceded by a functional ribosome‐binding site. The hydrophobic central domain and both of the periplasmic globular domains each have a pair of highly conserved cysteine residues, and it was shown by site directed mutagenesis that all six conserved cysteine residues contribute to DipZ function.
The extremely halophilic archaeon Haloferax volcanii has two very different dihydrofolate reductases
Authors: Ortenberg Ron , Rozenblatt‐Rosen Orit , Mevarech Moshe
Published: March 2003

The gene encoding dihydrofolate reductase, hdrA, from the extremely halophilic archaeon Haloferax volcanii was previously isolated from a spontaneous trimethoprim‐resistant mutant in a DNA sequence that had undergone amplification. Here, we show that deletion of hdrA did not affect growth in minimal medium and that the strain carrying the deletion remained sensitive to trimethoprim. A spontaneous trimethoprim‐resistant colony was isolated in the hdrA deletion strain and found to possess a new DNA amplification. Sequencing of the amplification revealed a second, substantially different, dihydrofolate reductase gene, hdrB, which was found to be located immediately downstream of the thymidylate synthase gene, hts. The physiological role of hDHFR‐1 and hDHFR‐2 was determined by generating Haloferax volcanii strains in which each gene, hdrA or hdrB, or both genes were deleted. It was found that hdrB alone can support growth of Haloferax volcanii in minimal medium, whereas hdrA alone can support growth of Haloferax volcanii in minimal medium only when the medium is supplemented with thymidine. It was also shown that, in contrast to Escherichia coli, the ΔhdrA, ΔhdrB double deletion mutant is viable in the presence of a functional thymidylate synthase gene. The hdrB gene was overexpressed in Escherichia coli and the enzyme purified to homogeneity. The biochemical properties of the new enzyme (hDHFR‐2) are markedly different from those of hDHFR‐1. The use of the dihydrofolate reductase and thymidylate synthase genes as stable selectable markers is described.
A large‐scale study of Yap1p‐dependent genes in normal aerobic and H2O2‐stress conditions: the role of Yap1p in cell proliferation control in yeast
Authors: Dumond Hélène , Danielou Nolwenn , Pinto Moïse , Bolotin‐Fukuhara Monique
Published: March 2003

Yeast genes regulated by the transcriptional activator Yap1p were screened by two independent methods: (i) use of a LacZ‐fused gene library and (ii) high‐density membrane hybridization. Changes in transcriptome profile were examined in the presence and in the absence of Yap1p, as well as under normal and H2O2‐mediated stress conditions. Both approaches gave coherent results, leading to the identification of many genes that appear to be directly or indirectly regulated by Yap1p. Promoter sequence analysis of target genes revealed that this regulatory effect is not always dependent upon the presence of a Yap1p binding site. The results show that the regulatory role of Yap1p is not restricted to the activation of stress response but that this factor can act as a positive or a negative regulator, both under normal and oxidative stress conditions. Among the targets, a few genes participating in growth control cascades were detected. In particular, the RPI1 gene, a repressor of the ras‐cAMP pathway, was found to be downregulated by Yap1p during the early phase of growth, but upregulated in the stationary phase or after oxidative stress.
Functional polarization of the Escherichia coli chromosome terminus: the dif site acts in chromosome dimer resolution only when located between long stretches of opposite polarity
Authors: Pérals Koryn , Cornet François , Merlet Yann , Delon Isabelle , Louarn Jean‐Michel
Published: March 2003

In Escherichia coli, chromosome dimers are generated by recombination between circular sister chromosomes. Dimers are lethal unless resolved by a system that involves the XerC, XerD and FtsK proteins acting at a site (dif) in the terminus region. Resolution fails if dif is moved from its normal position. To analyse this positional requirement, dif was transplaced to a variety of positions, and deletions and inversions of portions of the dif region were constructed. Resolution occurs only when dif is located at the convergence of multiple, oppositely polarized DNA sequence elements, inferred to lie in the terminus region. These polar elements may position dif at the cell septum and be general features of chromosome organization with a role in nucleoid dynamics.
Transcription activation at the Escherichia coli melAB promoter: the role of MelR and the cyclic AMP receptor protein
Authors: Belyaeva Tamara A. , Wade Joseph T. , Webster Christine L. , Howard Victoria J. , Thomas Mark S. , Hyde Eva I. , Busby Stephen J. W.
Published: March 2003

MelR is a melibiose‐triggered transcription activator that belongs to the AraC family of transcription factors. Using purified Escherichia coli RNA polymerase and a cloned DNA fragment carrying the entire melibiose operon intergenic region, we have demonstrated in vitro open complex formation and activation of transcription initiation at the melAB promoter. This activation is dependent on MelR and melibiose. These studies also show that the cyclic AMP receptor protein (CRP) interacts with the melAB promoter and increases MelR‐dependent transcription activation. DNAase I footprinting has been exploited to investigate the location of MelR‐and CRP‐binding sites at the melAB promoter. We showed previously that MelR binds to two identical 18 bp target sequences centred at position −100.5 (Site 1) and position −62.5 (Site 2). In this work, we show that MelR additionally binds to two other related 18 bp sequences: Site 1′, centred at position −120.5, located immediately upstream of Site 1, and Site R, at position −238.5, which overlaps the transcription start site of the divergent melR promoter. MelR can bind to Site 1′, Site 1, Site 2 and Site R, in both the absence and the presence of melibiose. However, in the presence of melibiose, MelR also binds to a fifth site (Site 2′, centred at position −42.5) located immediately downstream of Site 2, and overlapping the −35 region of the melAB promoter. Additionally, although CRP is unable to bind to the melAB promoter in the absence of MelR, in the presence of MelR, it binds to a site located between MelR binding Site 1 and Site 2. Thus, tandem‐bound MelR recruits CRP to the MelR. We propose that expression from the melAB promoter has an absolute requirement for MelR binding to Site 2′. Optimal expression of the melAB promoter requires Sites 1′, Site 1, Site 2 and Site 2′; CRP acts as a ‘bridge’ between MelR bound at Sites 1′ and 1 and at Sites 2 and 2′, increasing expression from the melAB promoter. In support of this model, we show that improvement of the base sequence of Site 2′ removes the requirement for Site 1′ and Site 1, and short circuits the effects of CRP.
Oxygenated mycolic acids are necessary for virulence of Mycobacterium tuberculosis in mice
Authors: Dubnau Eugenie , Chan John , Raynaud Catherine , Mohan Vellore P. , Lanéelle Marie‐Antoinette , Yu Keming , Quémard Annaik , Smith Issar , Daffé Mamadou
Published: March 2003

Members of the Mycobacterium tuberculosis group synthesize a family of long‐chain fatty acids, mycolic acids, which are located in the cell envelope. These include the non‐oxygenated α‐mycolic acid and the oxygenated keto‐ and methoxymycolic acids. The function in bacterial virulence, if any, of these various types of mycolic acids is unknown. We have constructed a mutant strain of M. tuberculosis with an inactivated hma (cmaA, mma4) gene; this mutant strain no longer synthesizes oxygenated mycolic acids, has profound alterations in its envelope permeability and is attenuated in mice.
Genes required for cytochrome c synthesis inBacillus subtilis
Authors: Le Brun Nick E. , Bengtsson Jenny , Hederstedt Lars
Published: March 2003

Cytochromes of c‐type contain covalently bound haem and in bacteria are located on the periplasmic side of the cytoplasmic membrane. More than eight different gene products have been identified as being specifically required for the synthesis of cytochromes c in Gram‐negative bacteria. Corresponding genes are not found in the genome sequences of Gram‐positive bacteria. Using two random mutagenesis approaches, we have searched for cytochrome c biogenesis genes in the Gram‐positive bacterium Bacillus subtilis. Three genes, resB, resC and ccdA, were identified. CcdA has been found previously and is required for a late step in cytochrome c synthesis and also plays a role in spore synthesis. No function has previously been assigned for ResB and ResC but these predicted membrane proteins show sequence similarity to proteins required for cytochrome c synthesis in chloroplasts. Attempts to inactivate resB and resC in B. subtilis have indicated that these genes are essential for growth. We demonstrate that various nonsense mutations in resB or resC can block synthesis of cytochromes c with no effect on other types of cytochromes and little effect on sporulation and growth. The results strongly support the recent proposal that Gram‐positive bacteria, cyanobacteria, ε‐proteobacteria, and chloroplasts have a similar type of machinery for cytochrome c synthesis (System II), which is very different from those of most Gram‐negative bacteria (System I) and mitochondria (System III).
Identification of genes affecting selenite toxicity and resistance in Saccharomyces cerevisiae
Authors: Pinson Benoît , Sagot Isabelle , Daignan‐Fornier Bertrand
Published: March 2003

Recent studies associating dietary selenium with reduced cancer susceptibility have aroused interest in this substance. In the millimolar range, selenite is toxic and slightly mutagenic for yeast. We show that selenite‐treated yeast cells tend to arrest as large budded cells and that this arrest is abolished in a rad9 mutant that is significantly sensitive to selenite. Interestingly, a rev3 mutant affected in the error‐prone repair pathway is also sensitive to selenite, whereas mutations in the other DNA repair pathways do not strongly affect resistance to selenite. We propose that selenite treatment leads to DNA damage inducing the RAD9‐dependent cell cycle arrest. Selenite‐induced DNA damage could be converted to mutations by the Rev3p‐dependent lesion bypass system, thus allowing the cell cycle to progress. We have also investigated the selenite detoxification mechanisms and identified three genes involved in this process. In the present study, we show that lack of the cadmium glutathione‐conjugate vacuolar pump Ycf1p or overexpression of the sulphite resistance membrane protein Ssu1p enhance the capacity of yeast cells to resist selenite treatment. Finally, we show that overexpression of the glutathione reductase Glr1p increases resistance to selenite, suggesting that selenite toxicity in yeast is closely linked to its oxidative capacity.
Growth phase‐dependent expression and degradation of histones in the thermophilic archaeon Thermococcus zilligii
Authors: Dinger Marcel E. , Baillie Gregory J. , Musgrave David R.
Published: March 2003

HTz is a member of the archaeal histone family. The archaeal histones have primary sequences and structural similarity to the eukaryal histone fold domain, and are thought to resemble the archetypal ancestor of the eukaryal nucleosome core histones. The effects of growth phase on the total soluble proteins from Thermococcus zilligii, isolated after various stages of growth from mid‐logarithmic to late stationary phase, were examined by denaturing polyacrylamide gel electrophoresis. On entry into stationary phase, at least 11 proteins were detected that changed considerably in level. One of these proteins was identified by Western hybridization as HTz. The level of HTz decreased dramatically as cells entered stationary phase, and it could not be detected by late stationary phase. Unexpectedly, the Western hybridization detected a second protein, with an estimated molecular mass of approximately 14 kDa, which paralleled the decrease in level of HTz. Native purified HTz was shown to retain complete activity after prolonged incubation at the growth temperature of the organism, suggesting that the decrease in HTz was a specific cell‐regulated process. Analysis of native purified HTz by electrospray ionization mass spectrometry revealed the molecular masses of HTz1 and HTz2 to be 7204 ± 3 Da and 7016 ± 3 Da respectively. The only non‐covalent species that was detected corresponded to the molecular mass of an HTz1–HTz2 heterodimer. Northern analyses of T. zilligii total RNA with an htz1 gene probe indicated a rapid decrease in expression of htz1 with progression of the growth phase, and complete repression of htz1 transcript synthesis by late logarithmic phase. Three proteins that changed in level with growth phase were identified by N‐terminal sequence analysis. The first was homologous to a hypothetical protein conserved in all Archaea sequenced to date, the second to the Sac10b family of archaeal DNA‐binding proteins and the third to the C‐terminal region of the leucine‐responsive regulatory family of DNA‐binding proteins (LRPs).
Variable small protein (Vsp)‐dependent and Vsp‐independent pathways for glycosaminoglycan recognition by relapsing fever spirochaetes
Authors: Magoun Loranne , Zückert Wolfram R. , Robbins Douglas , Parveen Nikhat , Alugupalli Kishore R. , Schwan Tom G. , Barbour Alan G. , Leong John M.
Published: March 2003

Tick‐borne relapsing fever, caused by pathogenic Borrelia such as B. hermsii and B. turicatae, features recurrent episodes of bacteraemia, each of which is caused by a population of spirochaetes that expresses a different variable major protein. Relapsing fever is also associated with the infection of a variety of tissues, such as the central nervous system. In this study, we show that glycosaminoglycans (GAGs) mediate the attachment of relapsing fever spirochaetes to mammalian cells. B. hermsii strain DAH bound to immobilized heparin, and heparin and dermatan sulphate blocked bacterial binding to host cells. Bacterial binding was diminished by inhibition of host cell GAG synthesis or sulphation, or by the enzymatic removal of GAGs. GAGs mediated the attachment of relapsing fever spirochaetes to potentially relevant target cells, such as endothelial and glial cells. B. hermsii was able to attach to GAGs independently of variable major proteins, because strains expressing the variable major proteins Vsp33, Vlp7 or no variable major protein at all each recognized GAGs. Nevertheless, we found that a variable major protein of B. turicatae directly promoted GAG binding by this relapsing fever spirochaete. B. turicatae strain Oz1 serotype B, which expresses the variable major protein VspB, bound to GAGs more efficiently than did B. turicatae Oz1 serotype A, which expresses VspA. Recombinant VspB, but not VspA, bound to heparin and dermatan sulphate. Previous studies have shown that strain Oz1 serotype B grows to higher concentrations in the blood than does Oz1 serotype A. Thus, relapsing fever spirochaetes have the potential to express Vsp‐dependent and Vsp‐independent GAG‐binding activities and, for one pair of highly related B. turicatae strains, differences in GAG binding correlate with differences in tissue tropism.
Assembly of aryl‐capped siderophores by modular peptide synthetases and polyketide synthases
Authors: Quadri Luis E. N.
Published: March 2003

Bacterial siderophores assist pathogens in iron acquisition inside their hosts. They are often essential for achieving a successful infection, and their biosynthesis represents an attractive antibiotic target. Recently, several siderophore biosynthetic loci have been identified, and in vitro studies have advanced our knowledge of the biosynthesis of aryl‐capped peptide and peptide–polyketide siderophores from Mycobacterium spp., Pseudomonas spp., Yersinia spp. and other bacteria. These studies also provided insights into the assembly of related siderophores and many secondary metabolites of medical relevance. Assembly of aryl‐capped peptide and peptide–polyketide siderophores involves non‐ribosomal peptide synthetase, polyketide synthase and non‐ribosomal‐peptide polyketide hybrid subunits. Analysis of these subunits suggests that their domains and modules are functionally and structurally independent. It appears that nature has selected a set of functional domains and modules that can be rearranged in different order and combinations to biosynthesize different products. Although much remains to be learned about modular synthetases and synthases, it is already possible to conceive strategies to engineer these enzymes to generate novel products.
Rns, a virulence regulator within the AraC family, requires binding sites upstream and downstream of its own promoter to function as an activator
Authors: Munson George P. , Scott June R.
Published: March 2003

Strains of enterotoxigenic Escherichia coli that express CS1 and CS2 pili require the transcriptional activator Rns, a member of the AraC family, for the expression of the pilin genes. Rns is also an activator of its own expression. However, the arrangement of its binding sites near its own promoter is unusual for a prokaryotic activator. Most activators have at least one binding site 30–80 nucleotides upstream of the transcription start site, but Rns has a single upstream binding site centred at −227. Rns also has two binding sites downstream of the transcription start site centred at +43 and +82, a region generally thought to be reserved for repressors. In vitro, the binding of a MBP::Rns fusion protein to each of these sites facilitates the binding of RNA polymerase to the rns promoter and the formation of an open complex. In vivo, the upstream binding site and one downstream site are required for Rns‐dependent activation of its promoter despite the atypical location of these binding sites for an activator. This suggests that Rns may represent a new class of prokaryotic activators.
The regulatory locus cinRI in Rhizobium leguminosarum controls a network of quorum‐sensing loci
Authors: Lithgow James K. , Wilkinson Adam , Hardman Andrea , Rodelas Belen , Wisniewski‐Dyé Florence , Williams Paul , Downie J. Allan
Published: March 2003

N‐(3‐hydroxy‐7‐cis‐tetradecenoyl)‐l‐homoserine lactone (3OH,C14:1‐HSL) is a quorum‐sensing signalling molecule produced by Rhizobium leguminosarum. It is unusual in that it inhibits the growth of several strains of R. leguminosarum and was previously known as ‘small bacteriocin’. The cinRI locus responsible for the production of 3OH,C14:1‐HSL has been characterized; it is predicted to be on the chromosome, based on DNA hybridization. The cinR and cinI genes are in different transcriptional units, separated by a predicted transcription terminator. CinR regulates cinI expression to a very high level in a cell‐density dependent manner, and cinI expression is positively autoregulated by 3OH,C14:1‐HSL, the only identified N‐acyl homoserine lactone (AHL) produced by CinI. No other AHLs were identified that strongly induced cinI expression. Mutation of cinI or cinR abolishes the production of 3OH,C14:1‐HSL and also reduces the production of several other AHLs. This is thought to result from the expression of three other AHL production loci being affected by the absence of 3OH,C14:1‐HSL. AHLs produced by these other loci include N‐hexanoyl‐ and N‐octanoyl‐l‐homoserine lactones and, unexpectedly, N‐heptanoyl‐l‐homoserine lactone (C7‐HSL). The expression of the rhiI gene on the symbiotic plasmid is greatly reduced in a cinI mutant, and the major regulatory effect appears to be mediated at least in part as a result of an effect on expression of RhiR, the regulator of rhiI. Thus, cinR and cinI appear to be at the top of a regulatory cascade or network that influences several AHL‐regulated quorum‐sensing loci. The expression of cinI–lacZ fusions is significantly reduced (but not abolished) when the symbiosis plasmid pRL1JI is present, resulting in a reduction in the level of 3OH,C14:1‐HSL produced. Mutation of cinI had little effect on growth or nodulation. However, plasmid transfer was affected, and the results obtained indicate that 3OH,C14:1‐HSL produced by either the donor or the recipient in mating experiments can stimulate transfer of pRL1JI.
Trehalose is required for conformational repair of heat‐denatured proteins in the yeast endoplasmic reticulum but not for maintenance of membrane traffic functions after severe heat stress
Authors: Simola Mari , Hänninen Anna‐Liisa , Stranius Satu‐Maarit , Makarow Marja
Published: March 2003

Saccharomyces cerevisiae cells grown at physiological temperature 24°C require preconditioning at 37°C to acquire tolerance towards brief exposure to 48–50°C. During preconditioning, the cytosolic trehalose content increases remarkably and in the absence of trehalose synthesis yeast cannot acquire thermotolerance. It has been speculated that trehalose protects proteins and membranes under environmental stress conditions, but recently it was shown to assist the Hsp104 chaperone in refolding of heat‐damaged proteins in the yeast cytosol. We have demonstrated that heat‐denatured proteins residing in the endoplasmic reticulum (ER) also can be refolded once the cells are returned to physiological temperature. Unexpectedly, not only ER chaperones but also the cytosolic Hsp104 chaperone is required for conformational repair events in the ER lumen. Here we show that trehalose facilitates refolding of glycoproteins in the ER after severe heat stress. In the absence of Tps1p, a subunit of trehalose synthase, refolding of heat‐damaged glycoproteins to bioactive and secretion‐competent forms failed or was retarded. In contrast, membrane traffic operated many hours after severe heat stress even in the absence of the TPS1 gene, demonstrating that trehalose had no role in thermoprotection of membranes engaged in vesicular traffic. However, cytosolic proteins were aggregated and protein synthesis abolished, resulting finally in cell death.
Genes encoding synthetases of cyclic depsipeptides, anabaenopeptilides, in Anabaena strain 90
Authors: Rouhiainen Leo , Paulin Lars , Suomalainen Sini , Hyytiäinen Heidi , Buikema William , Haselkorn Robert , Sivonen Kaarina
Published: March 2003

Anabaena strain 90 produces three hepatotoxic heptapeptides (microcystins), two seven‐residue depsipeptides called anabaenopeptilide 90A and 90B, and three six‐residue peptides called anabaenopeptins. The anabaenopeptilides belong to a group of cyanobacterial depsipeptides that share the structure of a six‐amino‐acid ring with a side‐chain. Despite their similarity to known cyclic peptide toxins, no function has been assigned to the anabaenopeptilides. Degenerate oligonucleotide primers based on the conserved amino acid sequences of other peptide synthetases were used to amplify DNA from Anabaena 90, and the resulting polymerase chain reaction (PCR) products were used to identify a peptide synthetase gene cluster. Four genes encoding putative anabaenopeptilide synthetase domains were characterized. Three genes, apdA, apdB and apdD, contain two, four and one module, respectively, encoding a total of seven modules for activation and peptide bond formation of seven l‐amino acids. Modules five and six also carry methyltransferase‐like domains. Before the first module, there is a region similar in amino acid sequence to formyltransferases. A fourth gene (apdC), between modules six and seven, is similar in sequence to halogenase genes. Thus, the order of domains is co‐linear with the positions of amino acid residues in the finished peptide. A mutant of Anabaena 90 was made by inserting a chloramphenicol resistance gene into the apdA gene. DNA amplification by PCR confirmed the insertion. Mass spectrometry analysis showed that anabaenopeptilides are not made in the mutant strain, but other peptides, such as microcystins and anabaenopeptins, are still produced by the mutant.
Characterization of the 2‐ketogluconate utilization operon in Pseudomonas aeruginosa PAO1
Authors: Swanson Britta L. , Hager Paul , Phibbs Paul , Ochsner Urs , Vasil Michael L. , Hamood Abdul N.
Published: March 2003

The Pseudomonas aeruginosa protein PtxS negatively regulates its own synthesis by binding to the upstream region of its gene. We have recently identified a 14 bp palindromic sequence within the ptxS upstream region as the PtxS operator site (OP1). In this study, we searched the P. aeruginosa genomic sequence to determine whether this 14 bp sequence exists in other regions of the P. aeruginosa chromosome. Another PtxS operator site (OP2) was located 47 bp downstream of ptxS. DNA gel shift experiments confirmed that PtxS specifically binds to a 520 bp fragment that carries OP2. The DNA segment 3′ of OP2 contains four open reading frames (ORF1–ORF4), which code for 29, 32, 48 and 35 kDa proteins respectively. The molecular weight of the products of ORFs 2 and 3 were confirmed by T7 expression experiments. Computer analyses suggest that ORF2 encodes an ATP‐dependent kinase; ORF3, a transporter; and ORF4, a dehydrogenase. The predicted product of ORF1 showed no homology to previously identified proteins and contains all the conserved amino acids within the aldose 1‐epimerase protein motif. Examination of the ptxs–ORF1 intergenic region (using promoter fusion experiments) showed that no potential promoter exists. An isogenic mutant defective in ORF1 was constructed in the P. aeruginosa strain PAO1. In contrast to its parent strain, the mutant failed to grow on a minimal medium in which 2‐ketogluconate was the sole carbon source. Similarly, a previously constructed ptxS isogenic mutant of PAO1 did not grow in a minimal medium containing 2‐ketogluconate as the sole carbon source. Furthermore, a plasmid carrying a fragment that contains ptxS and ORFs 1–4 complemented the defect of the previously described P. aeruginosa 2‐ketogluconate‐negative mutant. In the presence of 10 mM 2‐ketogluconate, the in vitro binding of PtxS to a DNA fragment that carries either OP1 or OP2 was inhibited. These results suggest that: (i) ptxS together with the other four ORFs constitute the 2‐ketogluconate utilization operon (kgu) in P. aeruginosa. Therefore, ORFs 1–4 were designated kguE, kguK, kguT and kguD respectively. (ii) PtxS regulates the expression of the kgu operon by binding to two operators (OP1 and OP2) within the operon; and (iii) 2‐ketogluconate is the molecular inducer of the kgu operon or the molecular effector of PtxS.
Identification of transcription activators that regulate gonococcal adaptation from aerobic to anaerobic or oxygen‐limited growth
Authors: Lissenden Sarah , Mohan Sudesh , Overton Tim , Regan Tess , Crooke Helen , Cardinale Jean A. , Householder Tracey C. , Adams Phillip , O'conner C. David , Clark Virginia L. , Smith Harry , Cole Jeffrey A.
Published: March 2003

Analysis of the Neisseria gonorrhoeae DNA sequence database revealed the presence of two genes, one encoding a protein predicted to be 37.5% identical (50% similar) in amino acid sequence to the Escherichia coli FNR protein and the other encoding a protein 41% and 42% identical (54 and 51% sequence similarity) to the E. coli NarL and NarP proteins respectively. Both genes have been cloned into E. coli and insertionally inactivated in vitro. The mutated genes have been transformed into gonococci and recombined into the chromosome. The fnr mutation totally abolished and the narP mutation severely diminished the ability of gonococci to: (i) grow anaerobically; (ii) adapt to oxygen‐limited growth; (iii) initiate transcription from the aniA promoter (which directs the expression of a copper‐containing nitrite reductase, AniA, in response to the presence of nitrite); and (iv) reduce nitrite during growth in oxygen‐limited media. The product of nitrite reduction was identified to be nitrous oxide. Immediately upstream of the narL/narP gene is an open reading frame that, if translated, would encode a homologue of the E. coli nitrate‐ and nitrite‐sensing proteins NarX and NarQ. As transcription from the aniA promoter was not activated during oxygen‐limited growth in the presence of nitrate, the gonococcal two‐component regulatory system is designated NarQ–NarP rather than NarX–NarL. As far as we are aware, this is the first well‐documented example of a two‐component regulatory system working in partnership with a transcription activator in pathogenic neisseria. A 45 kDa c‐type cytochrome that was synthesized during oxygen‐limited, but not during oxygen sufficient, growth was identified as a homologue of cytochrome c peroxidases (CCP) of other bacteria. The gene for this cytochrome, designated ccp, was located, and its regulatory region was cloned into the promoter probe vector pLES94. Transcription from the ccp promoter was repressed during aerobic growth and induced during oxygen‐limited growth and was totally FNR dependent, suggesting that the gonococcal FNR protein is a transcription activator of at least two genes. However, unlike AniA, synthesis of the CCP homologue was insensitive to the presence of nitrite during oxygen‐limited growth.
Rifampicin‐resistant initiation of chromosome replication from oriC in ihf mutants
Authors: Von Freiesleben Ulrik , Rasmussen Knud V. , Atlung Tove , Hansen Flemming G.
Published: March 2003

IHF (integration host factor) mutants exhibit asynchronous initiation of chromosome replication from oriC as determined from flow cytometric analysis of cultures where RNA synthesis was inhibited with rifampicin. However, the run‐out kinetics of chromosome replication in ihf mutants shows that they continue to produce oriCs for some time in the absence of RNA synthesis resulting in a twofold increase in the oriC per mass ratio. An ihf dnaA double mutant did not exhibit this continued increase of the oriC per mass ratio. This indicates that ihf mutants can initiate replication from oriC in a rifampicin‐resistant initiation mode but requires fully functional DnaA protein. The origin per mass ratio, determined by a quantitative Southern blotting technique, showed that the ihf mutants had an origin per mass ratio that was 60% of the wild type although it had a normal DnaA protein concentration. This shows that the initiation mass was substantially higher in the ihf mutants. The oriC per terminus ratio, which was also determined by Southern blotting, was very low in the ihf mutant, although it grew with the same doubling times as the wild‐type strain. This indicates that cells lacking IHF replicate their chromosome(s) very fast.
AmtR, a global repressor in the nitrogen regulation system of Corynebacterium glutamicum
Authors: Jakoby Marc , Nolden Lars , Meier‐Wagner Jana , Krämer Reinhard , Burkovski Andreas
Published: March 2003

The uptake and assimilation of nitrogen sources is effectively regulated in bacteria. In the Gram‐negative enterobacterium Escherichia coli, the NtrB/C two‐component system is responsible for the activation of transcription of different enzymes and transporters, depending on the nitrogen status of the cell. In this study, we investigated regulation of ammonium uptake in Corynebacterium glutamicum, a Gram‐positive soil bacterium closely related to Mycobacterium tuberculosis. As shown by Northern blot hybridizations, regulation occurs on the level of transcription upon nitrogen starvation. In contrast to enterobacteria, a repressor protein is involved in regulation, as revealed by measurements of methylammonium uptake and β‐galactosidase activity in reporter strains. The repressor‐encoding gene, designated amtR, was isolated and sequenced. Deletion of amtR led to deregulation of transcription of amt coding for the C. glutamicum (methyl)ammonium uptake system. E. coli extracts from amtR‐expressing cells were applied in gel retardation experiments, and binding of AmtR to the amt upstream region was observed. By deletion analyses, a target motif for AmtR binding was identified, and binding of purified AmtR protein to this motif, ATCTATAGN1−4ATAG, was shown. Furthermore, the binding of AmtR to this sequence was proven in vivo using a yeast one‐hybrid system. Subsequent studies showed that AmtR not only regulates transcription of the amt gene but also of the amtB–glnK–glnD operon encoding an amt paralogue, the signal transduction protein PII and the uridylyltransferase/uridylyl‐removing enzyme, key components of the nitrogen regulatory cascade. In summary, regulation of ammonium uptake and assimilation in the high G+C content Gram‐positive bacterium C. glutamicum differs significantly from the mechanism found in the low G+C content Gram‐positive model organism Bacillus subtilis and from the paradigm of nitrogen control in the Gram‐negative enterobacteria.
Role of two arginine residues in domain II, loop 2 of Cry1Ab and Cry1Ac Bacillus thuringiensisδ‐endotoxin in toxicity and binding to Manduca sexta and Lymantria dispar aminopeptidase N
Authors: Lee Mi Kyong , Rajamohan Francis , Jenkins Jeremy L. , Curtiss April S. , Dean Donald H.
Published: March 2003

Two arginine residues (368–369) of Cry1Ab and Cry1Ac were mutated to alanine, glutamic acid and lysine by site‐directed mutagenesis. Insecticidal activities of the mutant toxins on Manduca sexta and Lymantria dispar larvae were examined. Cry1Ac mutant toxins (c)RR‐AA and (c)RR‐EE and Cry1Ab mutant toxins (b)RR‐AA and (b)RR‐EE showed great reductions in toxicity against both insects. In contrast, conservatively changed (c)RR‐KK and (b)RR‐KK mutants did not alter toxicity to either insect. Binding assays with brush border membrane vesicles (BBMVs) prepared from L. dispar midguts demonstrated that (c)RR‐AA, (c)RR‐EE, (b)RR‐AA and (b)RR‐EE bound with lower affinities compared with their respective wild‐type toxins. To M. sexta BBMVs, (c)RR‐AA and (c)RR‐EE showed great reductions in BBMV binding. However, (b)RR‐AA and (b)RR‐EE did not alter BBMV competition patterns, despite their reduced toxicity. Further binding assays were performed with aminopeptidase N (APN) purified from L. dispar and M. sexta BBMVs using surface plasmon resonance (BIAcore). Direct correlation between toxicity and APN binding was observed for the mutant toxins using this technique. The inconsistency between BBMV and APN binding data with Cry1Ab to M. sexta suggests the possibility of a different Cry1Ab toxin‐binding mechanism or the importance of another receptor in M. sexta.
The processing of a Benzo(a)pyrene adduct into a frameshift or a base substitution mutation requires a different set of genes in Escherichia coli
Authors: Lenne‐Samuel Nathalie , Janel‐Bintz Regine , Kolbanovskiy Aleksandr , Geacintov Nicholas E. , Fuchs Robert P. P.
Published: March 2003

Replication through a single DNA lesion may give rise to a panel of translesion synthesis (TLS) events, which comprise error‐free TLS, base substitutions and frameshift mutations. In order to determine the genetic control of the various TLS events induced by a single lesion, we have chosen the major N2‐dG adduct of (+)‐anti‐Benzo(a)pyrene diol epoxide [(+)‐anti‐BPDE] adduct located within a short run of guanines as a model lesion. Within this sequence context, in addition to the major event, i.e. error‐free TLS, the adduct also induces base substitutions (mostly G → T transversions) and −1 frameshift mutations. The pathway leading to G → T base substitution mutagenesis appears to be SOS independent, suggesting that TLS is most probably performed by the replicative Pol III holoenzyme itself. In contrast, both error‐free and frameshift TLS pathways are dependent upon SOS‐encoded functions that belong to the pool of inducible DNA polymerases specialized in TLS (translesional DNA polymerases), namely umuDC (Pol V) and dinB (Pol IV). It is likely that, given the diversity of conformations that can be adopted by lesion‐containing replication intermediates, cells use one or several translesional DNA polymerases to achieve TLS.
Expression of the virulence‐related Sca (Mn2+) permease in Streptococcus gordonii is regulated by a diphtheria toxin metallorepressor‐like protein ScaR
Authors: Jakubovics Nicholas S. , Smith Anthony W. , Jenkinson Howard F.
Published: March 2003

The acquisition of transition metal ions by pathogenic bacteria is crucial to their growth and survival within the human host, however, the mechanisms of metal ion homeostasis in streptococci are unknown. The scaCBA operon in the human oral bacterium Streptococcus gordonii encodes the components of an ABC‐type transporter for manganese (Mn2+). Production of substrate‐binding lipoprotein ScaA was increased approximately fivefold in cells cultured in low Mn2+ medium (< 0.1 µM Mn2+), but not in iron (Fe2+/Fe3+)‐limited medium, and was enhanced in the presence of human saliva or serum. mRNA analysis revealed that under low Mn2+ conditions, levels of scaCBA transcript (2.6 kb) were increased > 20‐fold. The Mn2+‐responsive transcriptional regulator of the sca operon was purified and characterized as a 215‐amino‐acid residue polypeptide, designated ScaR, with 26% identity to the Corynebacterium diphtheriae diphtheria toxin repressor (DtxR). Inactivation of scaR in S. gordonii DL1 (Challis) resulted in constitutive derepression of sca operon transcription. Expression of tpx, located immediately downstream of scaA and encoding a putative thiol peroxidase, was not subject to ScaR regulation. Purified ScaR protein bound to the scaC promoter region in vitro in the presence of Mn2+ (Kd∼ 80 nM) and, to a lesser extent, in the presence of Ni2+ or Zn2+. The metalloregulator protein binding region was localized by DNA protection analysis to a 46 bp sequence encompassing the −35 and −10 promoter signatures. This sequence was well conserved within the promoters of corresponding virulence‐related permease operons in other streptococci. The results identify a new Mn2+‐sensing regulator of Mn2+ transport in streptococci, important for Mn2+ homeostasis during infection of the human host.
Glucose‐induced cAMP signalling in yeast requires both a G‐protein coupled receptor system for extracellular glucose detection and a separable hexose kinase‐dependent sensing process
Authors: Rolland Filip , De Winde Johannes H. , Lemaire Katleen , Boles Eckhard , Thevelein Johan M. , Winderickx Joris
Published: March 2003

In Saccharomyces cerevisiae, glucose activation of cAMP synthesis requires both the presence of the G‐protein‐coupled receptor (GPCR) system, Gpr1‐Gpa2, and uptake and phosphorylation of the sugar. In a hxt‐null strain that lacks all physiologically important glucose carriers, glucose transport as well as glucose‐induced cAMP signalling can be restored by constitutive expression of the galactose permease. Hence, the glucose transporters do not seem to have a regulatory function but are only required for glucose uptake. We established a system in which the GPCR‐dependent glucose‐sensing process is separated from the glucose phosphorylation process. It is based on the specific transport and hydrolysis of maltose providing intracellular glucose in the absence of glucose transport. Preaddition of a low concentration (0.7 mM) of maltose to derepressed hxt‐null cells and subsequent addition of glucose restored the glucose‐induced cAMP signalling, although there was no glucose uptake. Addition of a low concentration of maltose itself does not increase the cAMP level but enhances Glu6P and apparently fulfils the intracellular glucose phosphorylation requirement for activation of the cAMP pathway by extracellular glucose. This system enabled us to analyse the affinity and specificity of the GPCR system for fermentable sugars. Gpr1 displayed a very low affinity for glucose (apparent Ka = 75 mM) and responded specifically to extracellular α and βd‐glucose and sucrose, but not to fructose, mannose or any glucose analogues tested. The presence of the constitutively active Gpa2val132 allele in a wild‐type strain bypassed the requirement for Gpr1 and increased the low cAMP signal induced by fructose and by low glucose up to the same intensity as the high glucose signal. Therefore, the low cAMP increases observed with fructose and low glucose in wild‐type cells result only from the low sensitivity of the Gpr1‐Gpa2 system and not from the intracellular sugar kinase‐dependent process. In conclusion, we have shown that the two essential requirements for glucose‐induced activation of cAMP synthesis can be fulfilled separately: an extracellular glucose detection process dependent on Gpr1 and an intracellular sugar‐sensing process requiring the hexose kinases.
The trans‐activation domain of the sporulation response regulator Spo0A revealed by X‐ray crystallography
Authors: Lewis Richard J. , Krzywda Szymon , Brannigan James A. , Turkenburg Johan P. , Muchová Katarína , Dodson Eleanor J. , Barák Imrich , Wilkinson Anthony J.
Published: March 2003

Sporulation in Bacillus involves the induction of scores of genes in a temporally and spatially co‐ordinated programme of cell development. Its initiation is under the control of an expanded two‐component signal transduction system termed a phosphorelay. The master control element in the decision to sporulate is the response regulator, Spo0A, which comprises a receiver or phosphoacceptor domain and an effector or transcription activation domain. The receiver domain of Spo0A shares sequence similarity with numerous response regulators, and its structure has been determined in phosphorylated and unphosphorylated forms. However, the effector domain (C‐Spo0A) has no detectable sequence similarity to any other protein, and this lack of structural information is an obstacle to understanding how DNA binding and transcription activation are controlled by phosphorylation in Spo0A. Here, we report the crystal structure of C‐Spo0A from Bacillus stearothermophilus revealing a single α‐helical domain comprising six α‐helices in an unprecedented fold. The structure contains a helix–turn–helix as part of a three α‐helical bundle reminiscent of the catabolite gene activator protein (CAP), suggesting a mechanism for DNA binding. The residues implicated in forming the σA‐activating region clearly cluster in a flexible segment of the polypeptide on the opposite side of the structure from that predicted to interact with DNA. The structural results are discussed in the context of the rich array of existing mutational data.