bluuebunny/crossref_metadata_2025 huggingface dataset:
Global analysis of transcription kinetics during competence development in Streptococcus pneumoniae using high density DNA arrays
Authors: Rimini Rebecca , Jansson Birger , Feger Georg , Roberts Tracy C. , De Francesco Massimo , Gozzi Alessandro , Faggioni Federico , Domenici Enrico , Wallace Donald M. , Frandsen Niels , Polissi Alessandra
Published: March 2003

The kinetics of global changes in transcription patterns during competence development in Streptococcus pneumoniae was analysed with high‐density arrays. Four thousand three hundred and one clones of a S. pneumoniae library, covering almost the entire genome, were amplified by PCR and gridded at high density onto nylon membranes. Competence was induced by the addition of CSP (competence stimulating peptide) to S. pneumoniae cultures grown to the early exponential phase. RNA was extracted from samples at 5 min intervals (for a period of 30 min) after the addition of CSP. Radiolabelled cDNA was generated from isolated total RNA by random priming and the probes were hybridized to identical high‐density arrays. Genes whose transcription was induced or repressed during competence were identified. Most of the genes previously known to be competence induced were detected together with several novel genes that all displayed the characteristic transient kinetics of competence‐induced genes. Among the newly identified genes many have suggested functions compatible with roles in genetic transformation. Some of them may represent new members of the early or late competence regulons showing competence specific consensus sequences in their promoter regions. Northern experiments and mutational analysis were used to confirm some of the results.
Functional analysis of promoters involved in quorum sensing‐based regulation of bacteriocin production in Lactobacillus
Authors: Risøen Per Arne , Brurberg May Bente , Eijsink Vincent G. H. , Nes Ingolf F.
Published: March 2003

Bacteriocin production in Lactobacillus sake LTH673 involves at least four operons: a regulatory operon (sppIPKR); two operons encoding bacteriocins and their immunity proteins (sppAiA and orfX); and an operon needed for secretion (sppTE). We show here that the response regulator encoded by sppR in L. sake LTH673, as well as the homologous response regulators encoded by plnC and plnD in Lactobacillus plantarum C11, bind to characteristic repeats found in the −80 to −40 regions of spp operons. The promoters controlling bacteriocin operons are strictly regulated, and their activity is increased more than 1000‐fold upon activation. Constitutive expression for the regulatory and transport operons is driven, at least in part, by promoters upstream of the −80 to −40 regions. Peak promoter activity of the regulatory and transporter operons precedes that of the two bacteriocin operons. The results reveal how promoters involved in quorum sensing‐based regulation of bacteriocin production in Lactobacillus differ in strength, leakiness and timing of their activity.
Escherichia coli cells defective for the recN gene display constitutive elevation of mutagenesis at 3,N4‐ethenocytosine via an SOS‐induced mechanism
Authors: Dunman Paul M. , Ren Li , Rahman M. Sayeedur , Palejwala Vaseem A. , Murphy Holly S. , Volkert Michael R. , Humayun M. Zafri
Published: March 2003

The Escherichia coli UVM (UV Modulation of mutagenesis) response is a DNA damage‐inducible mutagenic pathway detected as significantly increased mutagenesis at 3,N4‐ethenocytosine (εC) lesions borne on transfected single‐stranded M13 vector DNA. All major classes of DNA‐damaging agents can induce UVM, and the phenomenon is independent of previously characterized mutagenic responses in E. coli. To understand this phenomenon further, we set out to identify and characterize mutants in the UVM response. Screening a mutant bank of cells defective for 1‐methyl‐3‐nitro‐1‐nitrosoguanidine‐inducible genes revealed that defects in the recN gene cause a constitutive elevation of mutagenesis at εC residues. In contrast to normal cells that show ≈ 6% mutagenesis at εC lesions, but ≈ 60% upon UVM induction, recN‐defective strains display approximately 50% mutagenesis at εC lesion sites in untreated cells. However, the recN‐mediated mutagenesis response was found to require the recA gene and the umuDC genes, and could be suppressed in the presence of a plasmid harbouring the SOS transcriptional repressor LexA. These results imply that recN cells are constitutively active for SOS mutagenesis functions. The observation that εC mutagenesis is enhanced in recN cells confirms previous findings that mutagenesis at εC can also be independently elevated by the SOS pathway.
Differential functionalities of amphiphilic peptide segments of the cell‐septation penicillin‐binding protein 3 of Escherichia coli
Authors: Marrec‐Fairley Monique , Piette André , Gallet Xavier , Brasseur Robert , Hara Hiroshi , Fraipont Claudine , Ghuysen J. M. , Nguyen‐Distèche M.
Published: March 2003

The class B M1‐V577 penicillin‐binding protein (PBP) 3 of Escherichia coli consists of a M1–L39 membrane anchor (bearing a cytosolic tail) that is linked via a G40–S70 intervening peptide to an R71–I236 non‐catalytic module (containing the conserved motifs 1–3) itself linked via motif 4 to a D237–V577 catalytic module (containing the conserved motifs 5–7 of the penicilloyl serine transferases superfamily). It has been proposed that during cell septation the peptidoglycan crosslinking activity of the acyl transferase module of PBP3 is regulated by the associated M1–I236 polypeptide itself in interaction with other components of the divisome. The fold adopted by the R71–V577 polypeptide of PBP3 has been modelled by reference to the corresponding R76–S634 polypeptide of the class B Streptococcus pneumoniae PBP2x. Based on these data and the results of site‐directed mutagenesis of motifs 1–3 and of peptide segments of high amphiphilicity (identified from hydrophobic moment plots), the M1–I236 polypeptide of PBP3 appears to be precisely designed to work in the way proposed. The membrane anchor and the G40–S70 sequence (containing the G57–Q66 peptide segment) upstream from the non‐catalytic module have the information ensuring that PBP3 undergoes proper insertion within the divisome at the cell septation site. Motif 1 and the I74–L82 overlapping peptide segment, motif 2 and the H160–G172 overlapping peptide segment, and the G188–D197 motif 3 are located at or close to the intermodule junction. They contain the information ensuring that PBP3 folds correctly and the acyl transferase catalytic centre adopts the active configuration. The E206–V217 peptide segment is exposed at the surface of the non‐catalytic module. It has the information ensuring that PBP3 fulfils its cell septation activity within the fully complemented divisome.
Comparison of the sequences of the Aspergillus nidulans hxB and Drosophila melanogaster ma‐l genes with nifS from Azotobacter vinelandii suggests a mechanism for the insertion of the terminal sulphur atom in the molybdopterin cofactor
Authors: Amrani Laïla , Primus Jann , Glatigny Annie , Arcangeli Loretta , Scazzocchio Claudio , Finnerty Victoria
Published: March 2003

The molybdopterin cofactor (MoCF) is required for the activity of a variety of oxidoreductases. The xanthine oxidase class of molybdoenzymes requires the MoCF to have a terminal, cyanolysable sulphur ligand. In the sulphite oxidase/nitrate reductase class, an oxygen is present in the same position. Mutations in both the ma‐l gene of Drosophila melanogaster and the hxB gene of Aspergillus nidulans result in loss of activities of all molybdoenzymes that necessitate a cyanolysable sulphur in the active centre. The ma‐l and hxB genes encode highly similar proteins containing domains common to pyridoxal phosphate‐dependent cysteine transulphurases, including the cofactor binding site and a conserved cysteine, which is the putative sulphur donor. Key similarities were found with NifS, the enzyme involved in the generation of the iron–sulphur centres in nitrogenase. These similarities suggest an analogous mechanism for the generation of the terminal molybdenum‐bound sulphur ligand. We have identified putative homologues of these genes in a variety of organisms, including humans. The human homologue is located in chromosome 18.q12.
Characterization of the MFα pheromone of the human fungal pathogen Cryptococcus neoformans
Authors: Davidson Robert C. , Moore Tracey D. E. , Odom Audrey R. , Heitman Joseph
Published: March 2003

Cryptococcus neoformans is an important human pathogenic fungus with a defined sexual cycle and well‐developed molecular and genetic approaches. C. neoformans is predominantly haploid and has two mating types, MATa and MATα. Mating is known to be regulated by nutritional limitation and thought also to be regulated by pheromones. Previously, a portion of the MATα locus was cloned, and a presumptive pheromone gene, MFα1, was identified by its ability to induce conjugation tube‐like filaments when introduced by transformation into MATa cells. Here, the ability of the MFα1 gene to induce these morphological changes in MATa cells was used as a phenotypic assay to perform a structure–function analysis of the gene. We show that the MFα1 open reading frame is required for the morphological response of MATa cells. We also find that the cysteine residue of the C‐terminal CAAX motif is required for activity of the MFα1 pheromone. In addition, we use a reporter system to measure the expression levels of the MFα1 pheromone gene and find that two signals, nutrient starvation and the presence of factors secreted by mating partner cells, impinge on this promoter and regulate MFα1 expression. We identify a second pheromone gene, MFα2, and show phenotypically that this gene is also expressed. Finally, we have synthesized the MFα1 pheromone and show that only the predicted mature modified form of the α‐factor peptide triggers morphological responses in MATa cells.
Pse1/Kap121‐dependent nuclear localization of the major yeast multidrug resistance (MDR) transcription factor Pdr1
Authors: Delahodde Agnès , Pandjaitan Rudy , Corral‐Debrinski Marisol , Jacq Claude
Published: March 2003

Pdr1 and Pdr3 are two very similar transcription factors that mainly control membrane biogenesis by adjusting the production of different membrane proteins, such as different ABC or major facilitator superfamily (MFS) transporters. We observed that the pse1‐1 mutation in the importin/β‐karyopherin Pse1/Kap121 specifically induced the cytoplasmic localization of Pdr1, but not that of Pdr3. Interactions between Pse1 and Pdr1 could be observed in vivo, and a short peptide of 44 amino acids from Pdr1 was shown to contain the information necessary and sufficient for Pse1‐dependent nuclear import. This Pdr1‐NLS sequence, absent in Pdr3, although rich in serine and tyrosine, is different from the Pse1‐dependent nuclear localization signal (NLS) of Pho4. Furthermore, we showed that Pse1/Kap121 is likely to be the sole import receptor for the regulator Pdr1. Together, these new observations underscore the diversity of cellular processes that address to the nucleus two very similar transcription factors involved in the control of the same phenotype, thus securing their function in the cell.
Dermatan sulphate is released by proteinases of common pathogenic bacteria and inactivates antibacterial α‐defensin
Authors: Schmidtchen Artur , Frick Inga‐Maria , Björck Lars
Published: March 2003

Defensins represent an evolutionarily conserved group of small peptides with potent antibacterial activities. We report here that extracellular proteinases secreted by the human pathogens Pseudomonas aeruginosa, Enterococcus faecalis and Streptococcus pyogenes release dermatan sulphate by degrading dermatan sulphate‐containing proteoglycans, such as decorin. Dermatan sulphate was found to bind to neutrophil‐derived α‐defensin, and this binding completely neutralized its bactericidal activity. During infection, proteoglycan degradation and release of dermatan sulphate may therefore represent a previously unknown virulence mechanism, which could serve as a target for novel antibacterial strategies.
LcrV is a channel size‐determining component of the Yop effector translocon of Yersinia
Authors: Holmström Anna , Olsson Jan , Cherepanov Peter , Maier Elke , Nordfelth Roland , Pettersson Jonas , Benz Roland , Wolf‐Watz Hans , Forsberg Åke
Published: March 2003

Delivery of Yop effector proteins by pathogenic Yersinia across the eukaryotic cell membrane requires LcrV, YopB and YopD. These proteins were also required for channel formation in infected erythrocytes and, using different osmolytes, the contact‐dependent haemolysis assay was used to study channel size. Channels associated with LcrV were around 3 nm, whereas the homologous PcrV protein of Pseudomonas aeruginosa induced channels of around 2 nm in diameter. In lipid bilayer membranes, purified LcrV and PcrV induced a stepwise conductance increase of 3 nS and 1 nS, respectively, in 1 M KCl. The regions important for channel size were localized to amino acids 127–195 of LcrV and to amino acids 106–173 of PcrV. The size of the channel correlated with the ability to translocate Yop effectors into host cells. We suggest that LcrV is a size‐determining structural component of the Yop translocon.
Activation of heat shock transcription factor in yeast is not influenced by the levels of expression of heat shock proteins
Authors: Hjorth‐Sørensen Bjørn , Hoffmann Eva R. , Lissin Nikolai M. , Sewell Andrew K. , Jakobsen Bent K.
Published: March 2003

Heat shock transcription factor (HSF) transiently induces the expression of a universally conserved set of proteins, the heat shock proteins (Hsps), when cells are exposed to elevated temperatures as well as to a wide range of other environmental stresses. The tight control of heat shock gene expression has prompted a model, according to which HSF activity and ‘free’ heat shock protein levels are tied up in a regulatory loop. Other data have indicated that HSF senses stress directly. Here, we report that yeast cells in which the basal expression levels of Hsps have been significantly increased exhibit improved thermotolerance but display no detectable difference in the temperature required for transient activation of HSF. In a separate experiment, overexpression of SSA2, a member of the Hsp70 family and a prominent candidate for the feedback regulation of HSF, did not inhibit the heat shock response. Our findings challenge the dogma that relief of the suppression of HSF activity by Hsps can account for the acute heat shock response.
SoxR‐dependent response to oxidative stress and virulence of Erwinia chrysanthemi: the key role of SufC, an orphan ABC ATPase
Authors: Nachin Laurence , El Hassouni Mohammed , Loiseau Laurent , Expert Dominique , Barras Frédéric
Published: March 2003

Erwinia chrysanthemi causes soft‐rot disease in a great variety of plants. In addition to the depolymerizing activity of plant cell wall‐degrading enzymes, iron acquisition and resistance to oxidative stress contribute greatly to the virulence of this pathogen. Here, we studied the pin10 locus originally thought to encode new virulence factors. The sequence analysis revealed six open reading frames that were homologous to the Escherichia coli sufA, sufB, sufC, sufD, sufS and sufE genes. Sequence similarity searching predicted that (i) SufA, SufB, SufD, SufS and SufE proteins are involved in iron metabolism and possibly in Fe–S cluster assembly; and (ii) SufC is an ATPase of an ABC transporter. The reverse transcription–polymerase chain reaction procedure showed that the sufABCDSE genes constitute an operon. Expression of a sufB::uidA fusion was found to be induced in iron‐deficient growth conditions and to be repressed by the iron‐sensing Fur repressor. Each of the six suf genes was inactivated by the insertion of a cassette generating a non‐polar mutation. The intracellular iron level in the sufA, sufB, sufC, sufS and sufE mutants was higher than in the wild type, as assessed by increased sensitivity to the iron‐activated antibiotic streptonigrin. In addition, inactivation of sufC and sufD led to increased sensitivity to paraquat. Virulence tests showed that sufA and sufC mutants exhibited reduced ability to cause maceration of chicory leaves, whereas a functional sufC gene was necessary for the bacteria to cause systemic invasion of Saintpaulia ionantha. The E. coli sufC homologue was inactivated by reverse genetic. This mutation was found to modify the soxR‐dependent induction of soxS gene expression. We discuss the possibility that SufC is a versatile ATPase that can associate either with the other Suf proteins to form a Fe–S cluster‐assembling machinery or with membrane proteins encoded elsewhere in the chromosome to form an Fe–S ABC exporter. Overall, these results stress the importance of the connection between iron metabolism and oxidative stress during the early steps of infection by E. chrysanthemi.
Bacillus subtilis mutations that alter the pathway of phosphorylation of the anti‐anti‐σF factor SpoIIAA lead to a Spo− phenotype
Authors: Lee Chung‐Sheng , Clarkson Joanna , Shu Jwu‐Ching , Campbell Iain D. , Yudkin Michael D.
Published: March 2003

Sigma‐F, the first sporulation‐specific transcription factor of Bacillus subtilis, is regulated by an anti‐sigma factor SpoIIAB, which can also act as a protein kinase that phosphorylates the anti‐anti‐sigma factor SpoIIAA. The time course of phosphorylation reaction is biphasic, a fact that has been interpreted in terms of a mechanism for sequestering SpoIIAB away from σF and thus allowing activation of σF when needed. Site‐directed mutagenesis of SpoIIAA has allowed us to isolate two mutants that cannot activate σF and which are therefore Spo−. The two mutant SpoIIAA proteins, SpoIIAAL61A and SpoIIAAL90A, are phosphorylated with linear kinetics; in addition they are less able to form the stable non‐covalent complex that wild‐type SpoIIAA makes with SpoIIAB in the presence of ADP. The phosphorylated form of SpoIIAAL90A was hydrolysed by the specific phosphatase SpoIIE at the same rate as wild‐type SpoIIAA‐P, but the rate of hydrolysis of SpoIIAAL61A‐P was much slower. The secondary structure and the global fold of the mutant proteins were unchanged from the wild type. The results are interpreted in terms of a model for the wild type in which SpoIIAB, after phosphorylating SpoIIAA, is released in a form that is tightly bound to ADP and which then makes a ternary complex with an unreacted SpoIIAA. We propose that it is the inability to make this ternary complex that deprives the mutant cells of a means of keeping SpoIIAB from inhibiting σF.
Heterologous expression of archaeal selenoprotein genes directed by the SECIS element located in the 3′ non‐translated region
Authors: Rother M. , Resch A. , Gardner W. L. , Whitman W. B. , Böck A.
Published: March 2003

Previous in silico analysis of selenoprotein genes in Archaea revealed that the selenocysteine insertion (SECIS) motif necessary to recode UGA with selenocysteine was not adjacent to the UGA codon as is found in Bacteria. Rather, paralogous stem–loop structures are located in the 3′ untranslated region (3′ UTR), reminiscent of the situation in Eukarya. To assess the function of such putative SECIS elements, the Methanococcus jannaschii MJ0029 (fruA, which encodes the A subunit of the coenzyme F420‐reducing hydrogenase) mRNA was mapped in vivo and probed enzymatically in vitro. It was shown that the SECIS element is indeed transcribed as part of the respective mRNA and that its secondary structure corresponds to that predicted by RNA folding programs. Its ability to direct selenocysteine insertion in vivo was demonstrated by the heterologous expression of MJ0029 in Methanococcus maripaludis, resulting in the synthesis of an additional selenoprotein, as analysed by 75Se labelling. The selective advantage of moving the SECIS element in the untranslated region may confer the ability to insert more than one selenocysteine into a single polypeptide. Evidence for this assumption was provided by the finding that the M. maripaludis genome contains an open reading frame with two in frame TGA codons, followed by a stem–loop structure in the 3′ UTR of the mRNA that corresponds to the archaeal SECIS element.
The genetic organization of Desulfovibrio desulphuricans ATCC 27774 bacterioferritin and rubredoxin‐2 genes: involvement of rubredoxin in iron metabolism
Authors: Da Costa Patrícia N. , Romão Célia V. , LeGall Jean , Xavier António V. , Melo Eurico , Teixeira Miguel , Saraiva Lígia M.
Published: March 2003

The anaerobic bacterium Desulfovibrio desulphuricans ATCC 27774 contains a unique bacterioferritin, isolated with a stable di‐iron centre and having iron‐coproporphyrin III as its haem cofactor, as well as a type 2 rubredoxin with an unusual spacing of four amino acid residues between the first two binding cysteines. The genes encoding for these two proteins were cloned and sequenced. The deduced amino acid sequence of the bacterioferritin shows that it is among the most divergent members of this protein family. Most interestingly, the bacterioferritin and rubredoxin‐2 genes form a dicistronic operon, which reflects the direct interaction between the two proteins. Indeed, bacterioferritin and rubredoxin‐2 form a complex in vitro, as shown by the significant increase in the anisotropy and decay times of the fluorescence of rubredoxin‐2 tryptophan(s) when mixed with bacterioferritin. In addition, rubredoxin‐2 donates electrons to bacterioferritin. This is the first identification of an electron donor to a bacterioferritin and shows the involvement of rubredoxin‐2 in iron metabolism. Furthermore, analysis of the genomic data for anaerobes suggests that rubredoxins play a general role in iron metabolism and oxygen detoxification in these prokaryotes.
Role of activator site position and a distal UP‐element half‐site for sigma factor selectivity at a CRP/H‐NS‐activated σS‐dependent promoter in Escherichia coli
Authors: Germer Jens , Becker Gisela , Metzner Martin , Hengge‐Aronis Regine
Published: March 2003

Transcription initiation by the stress‐associated σS‐containing RNA polymerase holoenzyme (EσS) in Escherichia coli is often subject to complex regulation that involves multiple additional regulators and histone‐like proteins. csiD is a stationary phase‐inducible σS‐dependent gene in E. coli that requires activation by cAMP‐CRP (bound to a site centred at −68.5 nucleotides upstream of the transcriptional start site) and is positively modulated by the abundant nucleoid‐associated proteins H‐NS and Lrp. By shifting the CRP box to positions between −80.5 and −60.5, we could demonstrate that: (i) activation is equally helix phase dependent as at classic class I promoters; (ii) EσS prefers a CRP box location at −68.5/−70.5, whereas Eσ70 is nearly inactive with such an arrangement; and (iii) with the CRP site moved to −60.5, transcription can be initiated efficiently by both holoenzymes. The csiD promoter region also contains a distal UP‐element half‐site located downstream of the CRP box, as demonstrated by mutational studies, in which this element was either eliminated or completed to a full UP‐element. The UP‐element half‐site favours EσS‐mediated expression, whereas with the full UP‐element, nearly wild‐type levels of csiD transcription were observed in the absence of σS. Finally, we show that the two histone‐like proteins, H‐NS and Lrp, both act by influencing activation by cAMP‐CRP, but do so by different mechanisms. In particular, H‐NS directly or indirectly increases positional stringency for the CRP binding site. The implications of these findings with respect to sigma factor selectivity, activation mechanisms used by the two holoenzymes and the architecture of σS‐dependent promoters are discussed.
The CRP–cAMP complex and downregulation of the glnAp2 promoter provides a novel regulatory linkage between carbon metabolism and nitrogen assimilation in Escherichia coli
Authors: Tian Zhe‐Xian , Li Quan‐Sheng , Buck Martin , Kolb Annie , Wang Yi‐Ping
Published: March 2003

In Escherichia coli, glnA (encoding glutamine synthetase) is transcribed from two promoters (glnAp1 and glnAp2). The glnAp1 is a σ70‐dependent promoter that is activated by the cAMP receptor protein (CRP). Under nitrogen‐deficient growth conditions, glnAp1 is repressed by NtrC‐phosphate. The downstream glnAp2 promoter is σ54‐dependent and is activated by NtrC‐phosphate. Here, we show that glnAp2 expression is affected by different carbon sources and that the CRP–cAMP complex inhibits the glnAp2 promoter activity. Primer extension and KMnO4 footprinting analysis indicate that the inhibitory effect is at the transcriptional level in vivo. When glnAp2 is activated by NifA, a similar inhibitory effect by CRP–cAMP is observed. Site‐directed mutagenesis and deletion analysis indicate that the characterized and putative CRP‐binding sites located in the upstream region of the glnAp2 promoter are not essential for the inhibitory effect. CRP–cAMP inhibits σ54‐dependent glnAp2 strongly, by 21‐fold. By activating glnAp1 and downregulating glnAp2, the overall effect of CRP–cAMP on glnA expression is an approximately fourfold reduction, which correlates with the reduction of γ‐glutamyl transferase activities in the cells. We propose therefore that a physiological role of CRP–cAMP activation of glnAp1 is to partially compensate for CRP–cAMP downregulation of glnAp2, allowing a low but non‐negligible level of expression of the important genes transcribed from it. A novel regulatory linkage between carbon and nitrogen regulons is proposed.
A complex role for the γ‐butyrolactone SCB1 in regulating antibiotic production in Streptomyces coelicolor A3(2)
Authors: Takano Eriko , Chakraburtty Rekha , Nihira Takuya , Yamada Yashuhiro , Bibb Mervyn J.
Published: March 2003

Many streptomycetes produce extracellular γ‐butyrolactones. In several cases, these have been shown to act as signals for the onset of antibiotic production. Synthesis of these molecules appears to require a member of the AfsA family of proteins (AfsA is required for A‐factor synthesis of the γ‐butyrolactone A‐factor and consequently for streptomycin production in Streptomyces griseus). An afsA homologue, scbA, was identified in Streptomyces coelicolor A3(2) and was found to lie adjacent to a divergently transcribed gene, scbR, which encodes a γ‐butyrolactone binding protein. Gel retardation assays and DNase I footprinting studies revealed DNA binding sites for ScbR at − 4 to − 33 nt with respect to the scbA transcriptional start site, and at − 42 to − 68 nt with respect to the scbR transcriptional start site. Addition of the γ‐butyrolactone SCB1 of S. coelicolor resulted in loss of the DNA‐binding ability of ScbR. A scbA mutant produced no γ‐butyrolactones, yet overproduced two antibiotics, actinorhodin (Act) and undecylprodigiosin (Red), whereas a deletion mutant of scbR also failed to make γ‐butyrolactones and showed delayed Red production. These phenotypes differ markedly from those expected by analogy with the S. griseus A‐factor system. Furthermore, transcription of scbR increased, and that of scbA was abolished, in an scbR mutant, indicating that ScbR represses its own expression while activating that of scbA. In the scbA mutant, expression of both genes was greatly reduced. Addition of SCB1 to the scbA mutant induced transcription of scbR, but did not restore scbA expression, indicating that the deficiency in scbA transcription in the scbA mutant is not solely due to the inability to produce SCB1, and that ScbA is a positive autoregulator in addition to being required for γ‐butyrolactone production. Overall, these results indicate a complex mechanism for γ‐butyrolactone‐mediated regulation of antibiotic biosynthesis in S. coelicolor.
How do cyanobacteria sense and respond to light?
Authors: Mullineaux Conrad W.
Published: March 2003

Cyanobacteria exhibit numerous responses to changes in the intensity and spectral quality of light. What sensors do cyanobacteria use to detect light and what are the mechanisms of signal transduction? The publication in 1996 of the complete genome sequence of the cyanobacterium Synechocystis 6803 provided a tremendous stimulus for research in this field, and many light‐sensors and signal transducers have now been identified. However, our knowledge of cyanobacterial light‐signal transduction remains fragmentary. This review summarizes what we know about the ways in which cyanobacteria perceive light, some of the ways which they respond to light signals and some recent achievements in elucidating the signal transduction mechanisms. Some problems in characterizing cyanobacterial signal transduction pathways are outlined and alternative experimental strategies are discussed.
Structure of the Lrp‐regulated serA promoter of Escherichia coli K‐12
Authors: Yang Li , Lin Rong Tuan , Newman Elaine B.
Published: March 2003

Expression of the Escherichia coli serA gene is activated in vivo by the product of the lrp gene, leucine‐responsive regulatory protein (Lrp), an effect partially reversed by L‐leucine. We show here that serA is transcribed from two promoters, P1 45 bp upstream of the translation start site, and P2 92 bp further upstream. Lrp binds to a long AT‐rich sequence from –158 to –82 from the start of the coding region, i.e. upstream of P1 and overlapping P2. It activates transcription from P1 and represses expression from P2. A second regulator, cAMP/CRP, activates P2, an effect that is largely inhibited by Lrp, such that catabolite repressor protein (Crp) and Lrp are rival activators of serA transcription.
Proteolysis and its regulation at the surface of Streptococcus pyogenes
Authors: Rasmussen Magnus , Björck Lars
Published: March 2003

Pathogenic bacteria often produce proteinases that are believed to be involved in virulence. Moreover, several host defence systems depend on proteolysis, demonstrating that proteolysis and its regulation play an important role during bacterial infections. Here, we discuss how proteolytical events are regulated at the surface of Streptococcus
pyogenes during infection with this important human pathogen.◊Streptococcus
pyogenes produces proteinases, and host protein‐ases are produced and released
as a result of the infection. Streptococcus pyogenes also recruits host proteinase
inhibitors to its surface, suggesting that proteolysis is tightly regulated at the
bacterial surface. We propose that the initial phase of a S. pyogenes infection
is characterized by inhibition of proteolysis and complement activity at the bacterial
surface. This is achieved mainly through binding of host proteinase inhibitors and
complement regulatory proteins to bacterial surface proteins. In a later phase of
the infection, massive proteolytic activity will release bacterial surface proteins
and degrade human tissues, thus facilitating bacterial spread. These proteolytic
events are regulated both temporally and spatially, and should influence virulence
and the outcome of S. pyogenes infections.
Involvement of surface polysaccharides in the organic acid resistance of Shiga Toxin‐producing Escherichia coli O157:H7
Authors: Barua Soumitra , Yamashino Takafumi , Hasegawa Tadao , Yokoyama Keiko , Torii Keizo , Ohta Michio
Published: March 2003

In general, wild Escherichia coli strains can grow effectively under moderately acidic organic acid‐rich conditions. We found that the Shiga Toxin‐producing E. coli (STEC) O157:H7 NGY9 grows more quickly than a K‐12 strain in Luria–Bertani (LB)‐2‐morpholinoethanesulphonic acid (MES) broth supplemented with acetic acid (pH 5.4). Hypothesizing that the resistance of STEC O157:H7 to acetic acid is as a result of a mechanism(s) other than those known, we screened for STEC mutants sensitive to acetic acid. NGY9 was subjected to mini‐Tn5 mutagenesis and, from 50 000 colonies, five mutants that showed a clear acetic acid‐sensitive phenotype were isolated. The insertion of mini‐Tn5 in three mutants occurred at the fcl, wecA (rfe) and wecB (rffE) genes and caused loss of surface O‐polysaccharide, loss of both O‐polysaccharide and enterobacterial common antigen (ECA) and loss of ECA respectively. The other two mutants showed inactivation of the waaG (rfaG) gene but at different positions that caused a deep rough mutant with loss of the outer core oligosaccharide of lipopolysaccharide (LPS) as well as phenotypic loss of O‐polysaccharide and ECA. With the introduction of plasmids carrying the fcl, wecA, wecB and waaG genes, respectively, all mutants were complemented in their production of O‐polysaccharide and ECA, and normal growth was restored in organic acid‐rich culture conditions. We also found that the growth of Salmonella LPS mutants Ra, Rb1, Rc, Rd1, Rd2 and Re was suppressed in the presence of acetic acid compared with that of the parents. These results suggest that the full expression of LPS (including O‐polysaccharide) and ECA is indispensable to the resistance against acetic acid and other short chain fatty acids in STEC O157:H7 and Salmonella. To the best of our knowledge, this is a newly identified physiological role for O‐polysaccharide and ECA as well as an acid resistance mechanism.
Translational readthrough of the PDE2 stop codon modulates cAMP levels in Saccharomyces cerevisiae
Authors: Namy Olivier , Duchateau‐Nguyen Guillemette , Rousset Jean‐Pierre
Published: March 2003

The efficiency of translation termination in yeast can vary several 100‐fold, depending on the context around the stop codon. We performed a computer analysis designed to identify yeast open reading frames (ORFs) containing a readthrough motif surrounding the termination codon. Eight ORFs were found to display inefficient stop codon recognition, one of which, PDE2, encodes the high‐affinity cAMP phosphodiesterase. We demonstrate that Pde2p stability is very impaired by the readthrough‐dependent extension of the protein. A 20‐fold increase in readthrough of PDE2 was observed in a [PSI+] as compared with a [psi–] strain. Consistent with this observation, an important increase in cAMP concentration was observed in suppressor backgrounds. These results provide a molecular explanation for at least some of the secondary phenotypes associated with suppressor backgrounds.
Activation of Helicobacter pylori CagA by tyrosine phosphorylation is essential for dephosphorylation of host cell proteins in gastric epithelial cells
Authors: Püls Jürgen , Fischer Wolfgang , Haas Rainer
Published: March 2003

Helicobacter pylori type I strains harbour the cag pathogenicity
island (cag‐PAI), a 37 kb sequence, which encodes the components of a type
IV secretion system. CagA, the first identified effector protein of the cag‐PAI,
is translocated into eukaryotic cells and tyrosine phosphorylated (CagAP‐tyr)
by a host cell tyrosine kinase. Translocation of CagA induces the dephosphorylation
of a set of phosphorylated host cell proteins of unknown identity. CagA proteins
of independent H. pylori strains vary in sequence and thus in the number and
composition of putative tyrosine phosphorylation motifs (TPMs). The CagA protein
of H. pylori strain J99 (CagAJ99) does not carry any of three putative
tyrosine phosphorylation motifs (TPM‐A, TPM‐B or TPM‐C) predicted by the MOTIF
algorithm in CagA proteins. CagAJ99 is not tyrosine phosphorylated and
is inactive in the dephosphorylation of host cell proteins. By site‐specific mutagenesis,
we introduced a TPM‐C into CagAJ99 by replacing a single lysine with a
tyrosine. This slight modification resulted in tyrosine phosphorylation of CagAJ99
and host cell protein dephosphorylation. In contrast, the removal of the indigenous
TPM‐C from CagAP12 did not abolish its tyrosine phosphorylation, suggesting
that further phosphorylated sites are present in CagAP12. By generation
of hybrid CagA proteins, a phosphorylation of the most N‐terminal TPM‐A could be
excluded. Our data suggest that tyrosine phosphorylation at TPM‐C is sufficient,
but not exclusive, to activate translocated CagA. Activated CagAP‐tyr might either convert into a phosphatase itself or activate a cellular phosphatase to dephosphorylate cellular phosphoproteins and modulate cellular signalling cascades of the host.
Inactivation of the srtA gene in Listeria monocytogenes inhibits anchoring of surface proteins and affects virulence
Authors: Bierne Hélène , Mazmanian Sarkis K. , Trost Matthias , Pucciarelli M. Graciela , Liu Gwen , Dehoux Pierre ,   the European Listeria Genome Consortium, Jänsch Lothar , Portillo Francisco Garcia‐del , Schneewind Olaf , Cossart Pascale
Published: March 2003

During infection of their hosts, Gram‐positive bac‐teria express surface proteins that serve multiple biological functions. Surface proteins harbouring a C‐terminal sorting signal with an LPXTG motif are covalently linked to the cell wall peptidoglycan by a transamidase named sortase. Two genes encoding putative sortases, termed srtA
and srtB, were identified in the genome of the intracellular pathogenic bacterium
Listeria monocytogenes. Inactivation of srtA abolishes anchoring of
the invasion protein InlA to the bacterial surface. It also prevents the proper sorting
of several other peptidoglycan‐associated LPXTG proteins. Three were identified by
a mass spectrometry approach. The ΔsrtA mutant strain is defective
in entering epithelial cells, similar to a ΔinlA mutant. In contrast
to a ΔinlA mutant, the ΔsrtA mutant is impaired
for colonization of the liver and spleen after oral inoculation in mice. Thus, L.
monocytogenes srtA is required for the cell wall anchoring of InlA and, presumably, for the anchoring of other LPXTG‐containing proteins that are involved in listerial infections.
Rpa12p, a conserved RNA polymerase I subunit with two functional domains
Authors: Mullem Vincent Van , Landrieux Emilie , Vandenhaute Jean , Thuriaux Pierre
Published: March 2003

Rpa12p is a subunit of RNA polymerase I formed of two zinc‐binding domains. The N‐terminal zinc region (positions 1–60) is poorly conserved from yeast to man. The C‐terminal domain contains an invariant Q.RSADE..T.F motif shared with the TFIIS elongation factor of RNA polymerase II and its archaeal counterpart. Deletions removing the N‐terminal domain fail to grow at 34°C, are sensitive to
nucleotide‐depleting drugs and become lethal in rpa14‐Δ mutants
lacking the non‐essential RNA polymerase I subunit Rpa14p. They also strongly alter
the immunofluorescent properties of RNA polymerase I in the nucleolus. Finally, they
prevent the binding of Rpa12p to immunopurified polymerase I and impair a specific
two‐hybrid interaction with the second largest subunit. In all these respects, N‐terminal
deletions behave like full deletions. In contrast, C‐terminal deletions retaining
only the first N‐terminal 60 amino acids are indistinguishable from wild type. Thus,
the N‐terminal zinc domain of Rpa12p determines its anchoring to RNA polymerase I
and is the only critical part of that subunit in vivo.
Charting and unzipping the surface layer of Corynebacterium glutamicum with the atomic force microscope
Authors: Scheuring Simon , Stahlberg Henning , Chami Mohamed , Houssin Christine , Rigaud Jean‐Louis , Engel Andreas
Published: March 2003

Bacterial surface layers (S‐layers) are extracellular protein networks that act as molecular sieves and protect a large variety of archaea and bacteria from hostile environments. Atomic force microscopy (AFM) was used to asses the S‐layer of Coryne‐bacterium

glutamicum formed of PS2 proteins that assemble into hexameric complexes within

a hexagonal lattice. Native and trypsin‐treated S‐layers were studied. Using the

AFM stylus as a nanodissector, native arrays that adsorbed to mica as double layers

were separated. All surfaces of native and protease‐digested S‐layers were imaged

at better than 1 nm lateral resolution. Difference maps of the topographies of native

and proteolysed samples revealed the location of the cleaved C‐terminal fragment

and the sidedness of the S‐layer. Because the corrugation depths determined from

images of both sides span the total thickness of the S‐layer, a three‐dimensional

reconstruction of the S‐layer could be calculated. Lattice defects visualized at

1 nm resolution revealed the molecular boundaries of PS2 proteins. The combination

of AFM imaging and single molecule force spectroscopy allowed the mechanical properties

of the Corynebacterium glutamicum S‐layer to be examined. The results provide a basis for understanding the amazing stability of this protective bacterial surface coat.
The fate of the BlaI repressor during the induction of the Bacillus licheniformis BlaP β‐lactamase
Authors: Filée Patrice , Benlafya Kamal , Delmarcelle Michaël , Moutzourelis Georgios , Frère Jean‐Marie , Brans Alain , Joris Bernard
Published: March 2003

The induction of the Staphylococcus aureus BlaZ and Bacillus licheniformis
749/I BlaP β‐lactamases by β‐lactam antibiotics occurs according
to similar processes. In both bacteria, the products of the blaI and blaR1
genes share a high degree of sequence homology and act as repressors and penicillin‐sensory
transducers respectively. It has been shown in S. aureus that the BlaI repressor,
which controls the expression of BlaZ negatively, is degraded after the addition
of the inducer. In the present study, we followed the fate of BlaI during β‐lactamase
induction in B. licheniformis 749/I and in a recombinant Bacillus subtilis
168 strain harbouring the pDML995 plasmid, which carries the B. licheniformis
blaP, blaI and blaR1 genes. In contrast to the situation in B.
licheniformis 749/I, β‐lactamase induction in B. subtilis 168/pDML995
was not correlated with the proteolysis of BlaI. To exclude molecular variations
undetectable by SDS–PAGE, two‐dimensional gel electrophoresis was performed
with cellular extracts from uninduced or induced B. subtilis 168/pDML995 cells.
No variation in the BlaI isoelectric point was observed in induced cells, whereas
the DNA‐binding property was lost. Cross‐linking experiments with dithiobis(succimidylpropionate)
confirmed that, in uninduced recombinant B. subtilis cells, BlaI was present as a homodimer and that this situation was not altered in induced conditions. This latter result is incompatible with a mechanism of inactivation of BlaI by proteolysis and suggests that the inactivation of BlaI results from a non‐covalent modification by a co‐activator and that the subsequent proteolysis of BlaI might be a secondary phenomenon. In addition to the presence of this co‐activator, our results show that the presence of penicillin stress is also required for full induction of β‐lactamase biosynthesis.
Cytoplasmic dynein intermediate chain and heavy chain are dependent upon each other for microtubule end localization in Aspergillus nidulans
Authors: Zhang Jun , Han Gongshe , Xiang Xin
Published: March 2003

The multisubunit microtubule motor, cytoplasmic dynein, targets to various subcellular locations in eukaryotic cells for various functions. The cytoplasmic dynein heavy chain (HC) contains the microtubule binding and ATP binding sites for motor function, whereas the intermediate chain (IC) is implicated in the in vivo targeting of the HC. Concerning any targeting event, it is not known whether the IC has to form a complex with the HC for targeting or whether the IC can target to a site independently of the HC. In the filamentous fungus Aspergillus nidulans, the dynein HC is localized to the ends of microtubules near the hyphal tip. In this study, we demonstrate that our newly identified dynein IC in A. nidulans is also localized to microtubule ends and is required for HC’s localization to microtubule ends in living cells. With the combination of two reagents, an HC loss‐of function mutant and the green fluorescent protein (GFP)‐fused IC that retains its function, we show that the IC’s localization to microtubule ends also requires HC, suggesting that cytoplasmic dynein HC–IC complex formation is important for microtubule end targeting. In addition, we show that the HC localization is not apparently altered in the deletion mutant of NUDF, a LIS1‐like protein that interacts directly with the ATP‐binding domain of the HC. Our study suggests that, although HC–IC association is important for the targeting of dynein to microtubule ends, other essential components, such as NUDF, may interact with the targeted dynein complex to produce full motor activities in vivo.
A basic helix–loop–helix protein with similarity to the fungal morphological regulators, Phd1p, Efg1p and StuA, controls conidiation but not dimorphic growth in Penicillium marneffei
Authors: Borneman Anthony R. , Hynes Michael J. , Andrianopoulos Alex
Published: March 2003

Members of the APSES protein group are basic helix–loop–helix (bHLH) proteins that regulate processes such as mating, asexual sporulation and dimorphic growth in fungi. Penicillium marneffei is a human pathogen and is the only
member of its genus to display a dimorphic growth transition. At 25°C, P.
marneffei grows with a filamentous morphology and produces asexual spores from
multicellular con‐idiophores. At 37°C, the filamentous morphology is replaced
by yeast cells that reproduce by fission. We have cloned and characterized an APSES
protein‐encoding gene from P. marneffei that has a high degree of similarity
to Aspergillus nidulans stuA. Deletion of stuA in P. marneffei
showed that it is required for metula and phialide formation during conidiation but
is not required for dimorphic growth. This suggests that APSES proteins may control
processes that require budding (formation of the metulae and phialides, pseudohyphal
growth in Saccharomyces cerevisiae and dimorphic growth in Candida albicans)
but not those that require fission (dimorphic growth in P. marneffei). The
A. nidulansΔstuA mutant has defects in both conidiation
and mating. The P. marneffei stuA gene was capable of complementing the conidiation
defect but could only inefficiently complement the sexual defects of the A. nidulans
mutant. This suggests that the P. marneffei gene, which comes from an asexual
species, has diverged significantly from the A. nidulans gene with respect to sexual but not asexual development.
Nitrate and the GATA factor AreA are necessary for in vivo binding of NirA, the pathway‐specific transcriptional activator of Aspergillus nidulans
Authors: Narendja Frank , Goller Sabine P. , Wolschek Markus , Strauss Joseph
Published: March 2003

In Aspergillus nidulans, the genes coding for nitrate reductase (niaD) and nitrite reductase (niiA), are transcribed divergently from a common promoter region of 1200 basepairs. We have previously characterized the relevant cis‐acting elements for the two synergistically acting transcriptional activators NirA and AreA. We have further shown that AreA is constitutively bound to a central cluster of four GATA sites, and is involved in opening the chromatin structure over the promoter region thus making additional cis‐acting binding sites accessible. Here we show that the asymmetric mode of NirA–DNA interaction determined in vitro is also found in vivo. Binding of the NirA transactivator is not constitutive as in other binuclear C6‐Zn2+‐cluster proteins but depends on nitrate induction and, additionally, on the presence of a wild‐type areA allele. Dissecting the role of AreA further, we found that it is required for intracellular nitrate accumulation and therefore could indirectly exert its effect on NirA via inducer exclusion. We have tested this possibility in a strain accumulating nitrate in the absence of areA. We found that in such a strain the intracellular presence of inducer is not sufficient to promote either chromatin rearrangement or NirA binding, implying that both processes are directly dependent on AreA.