bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of a replicon of the moderately promiscuous plasmid, pGSH5000, with features of both the mini‐replicon of pCU1 and the ori‐2 of F
Authors: Da Silva‐Tatley Fernanda M. , Steyn Lafras M.
Published: October 2006

The dominant, polA1‐independent replicon of pGSH500, repβ (1.8 kb), consists of a cis‐acting oriV region of 245 bp; a repB gene that is essential for autonomous replication and 18, 30 to 36 bp iterons which constitute the inc/cop region. The molecular organization of repβ resembles that of mini‐pCU1 (IncN). Furthermore, there is a 58% identity between the Rep proteins of these replicons. RepB also shows a 31% identity with RepE of mini‐F. In addition, an 80% identity over 200 bp was identified between the cis‐acting βoriV region and the equivalent region of ori‐2 (mini‐F). Replicons with deletions of repB could be complemented by Rep (pCU1) and RepE (mini‐F) in trans, supporting the hypothesis that repβ is a natural hybrid between a pCU1‐like and F‐like replicon.
Stationary‐phase production of the antibiotic actinorhodin in Streptomyces coelicolor A3(2) is transcriptionally regulated
Authors: Gramajo Hugo C. , Takano Eriko , Bibb Mervyn J.
Published: October 2006

Production of actinorhodin, a polyketide antibiotic made by Streptomyces coelicolor A3(2), normally occurs only in stationary‐phase cultures. S1 nuclease protection experiments showed that transcription of actII‐ORF4, the activator gene required for expression of the biosynthetic structural genes, increased dramatically during the transition from exponential to stationary phase. The increase in actII‐ORF4 expression was followed by transcription of the biosynthetic structural genes actIII and actVI‐ORF1, and by the production of actinorhodin. The presence of actII‐ORF4 on a multicopy plasmid resulted in enhanced levels of actII‐oRF4 mRNA, and transcription of actIII and actinorhodin production during exponential growth, suggesting that actinorhodin synthesis in rapidly growing cultures is normally limited only by the availability of enough of the activator protein. bldA, which encodes a tRNALeuUUA that is required for the efficient translation of a single UUA codon in the actII‐ORF4 mRNA, was transcribed throughout growth. Moreover, translational fusions of the 5prime; end of actII‐ORF4 that included the UUA codon to the ermE reporter gene demonstrated the presence of functional bldA tRNA in young, exponentially growing cultures and no increase in the efficiency of translation of UUA codons, relative to UUG codons, was observed during growth. The normal growth‐phase‐dependent production of actinorhodin in the liquid culture conditions used in these experiments appears to be mediated at the transcriptional level through activation of the actII‐ORF4 promoter.
Specific binding sites in the alcR and alcA promoters of the ethanol regulon for the CREA repressor mediating carbon cataboiite repression in Aspergillus nidulans
Authors: Kulmburg P. , Mathieu M. , Dowzer C. , Kelly J. , Felenbok B.
Published: October 2006

The CREA repressor responsible for carbon catabolite repression in Aspergillus nidulans represses the transcription of the ethanol regulon. The N‐terminal part of the CREA protein encompassing the two zinc fingers (C2H2 class family) and an alanine‐rich region was expressed in Escherichia colias a fusion protein with giutathione‐S‐transferase. Our results show that CREA is a DNA‐binding protein able to bind to the promoters of both the specific trans‐acting gene, alcR, and of the structural gene, alcA, encoding the alcohol dehydrogenase I. DNase I protection foot‐printing experiments revealed several specific binding sites in the alcR and in the alcA promoters having the consensus sequence 5′‐G/CPyGGGG‐3′. The disruption of one of these CREA‐binding sites in the alcR promoter overlapping the induction target for the trans‐activator ALCR results in a partially derepressed alc phenotype and derepressed alcR transcription, showing that this binding site is functional in vivo. Our data suggest that CREA represses the ethanol regulon by a double lock mechanism repressing both the trans‐acting gene, alcR, and the structural gene, alc A.
NAD+ ‐dependent malic enzyme of Rhizobium meliloti is required for symbiotic nitrogen fixation
Authors: Driscoll Brian T. , Finan Turlough M.
Published: October 2006

DEAE‐cellulose chromatography of extracts of free‐living Rhizobium meliloti cells revealed separate NAD+‐dependent and NADP+‐dependent malic enzyme activities. The NAD+ malic enzyme exhibited more activity with NAD+ as cofactor, but also showed some activity with NADP+. The NADP+ malic enzyme only showed activity when NADP+ was supplied as cofactor. Three independent transposon‐induced mutants of R. meliloti which lacked NADP+ malic enzyme activity (dme) but retained NADP+ malic enzyme activity were isolated. In an otherwise wild‐type background, the dme mutations did not alter the carbon utilization phenotype; however, nodules induced by these mutants failed to fix N2. Structurally, these nodules appeared to develop like wild‐type nodules up to the stage where N2‐fixation would normally begin. These results support the proposal that NAD+ malic enzyme, together with pyruvate dehydrogenase, functions in the generation of acetyl‐CoA required for TCA cycle function in N2‐fixing bacteroids which metabolize C4‐dicarboxylic acids supplied by the plant.
Positive transcriptional control of mry regulates virulence in the group A streptococcus
Authors: Okada Nobuhiko , Geist Robert T. , Caparon Michael G.
Published: October 2006

Transcription of the antiphagocytic M protein in the group A streptococcus (Streptococcus pyogenes) is environmentally regulated in response to CO2 and requires Mry, a trans‐acting positive regulatory protein. We have examined the role of Mry in environmental regulation by analysing the factors that regulate expression of the gene that encodes Mry (mry). By employing a strategy that utilizes integrational plasmids, it was found that expression of mry requires the participation of DNA sequences that extend 473 base pairs upstream of the Mry coding region. Transcription of mry, as analysed in S1 nuclease protection assays, is initiated from two separate promoters located within this extended regulatory region. Construction and analysis of transcriptional fusions between the mry promoters and a promoterless chloramphenicol acetyltransferase gene demonstrated that mry is autoregulated and environmentally regulated in response to the level of CO2. These data suggest a model for the regulation of virulence in S. pyogenes where positive transcriptional control of mry in response to environmental stimuli regulates the expression of the M protein.
The Bacillus subtilis addAB genes are fully functional in Escherichia coli
Authors: Kooistra Jan , Haijema Bert Jan , Venema Gerard
Published: October 2006

An Escherichia coli recBCD deletion mutant was transformed with plasmids containing the Bacillus subtilis add genes. The transformants had relatively high ATP‐dependent exonuclease‐ and ATP‐dependent helicase activities, and their viability, the ability to repair u.v.‐damaged DNA and the recombination in conjugation were nearly completely restored. The B. subtilis Add enzyme did not show Chi‐activity in phage lambda recombination. The individual B. subtilis Add proteins were not able to form an enzymatically active complex with the E. coli RecB,C,D proteins, and they could not complement the recB,C,D deficiency.

Evidence is presented that only two subunits are involved in the B. subtilis ATP‐dependent exonuclease. This is in contrast to E. coli in which the RecBCD enzyme consists of three subunits.
Transcriptional organization of the F1845 fimbrial adhesin determinant of Escherichia coli
Authors: Bilge Sima S. , Apostol John M. , Fullner Karia Jean , Moseley Steve L.
Published: October 2006

The transcriptional organization of the gene cluster encoding the F1845 fimbrial adhesin of a diarrhoea‐associated Escherichia coli was investigated. Genes daaA to daaE were determined to constitute a single transcriptional unit under the control of the daaA promoter. The nucleotide sequence of daaA and that of an upstream open reading frame encoded on the opposite strand, designated daaF, were determined to share limited homology with the papB and papi genes of the P fimbrial adhesin, respectively. The 5′ termini of the daaF and daaABCDE transcripts were mapped by primer extension and nuclease protection analyses. The promoters for these transcripts were associated with potential regulatory sequences including two consensus leucine‐responsive regulatory protein (Lrp)‐binding sites which contained differentially methylated GATC sequences, a cAMP‐CRP‐binding site, and an integration host factor (IHF)‐binding site. Expression of the daa locus was determined to be dependent on Lrp, subject to catabolite repression, and dependent on IHF.
The Klebsiella pneumoniae nifJ promoter: analysis of promoter elements regulating activation by the Nif A promoter
Authors: Charlton Wayne , Cannon Wendy , Buck Martin
Published: October 2006

The nifJ and nifH promoters of Klebsiella pneumoniae are divergently transcribed σ;54‐dependent promoters that are positively activated by the NifA protein. NifA binds to upstream activator sequences (UASs), usually located 60‐200 bp upstream of the start of transcription. Bound NifA is presented to the RNA polymerase‐σ54 complex (Eσ54) via DNA loop formation, mediated by the binding of integration host factor protein (IHF) between Eσ54 and NifA. The nifJ promoter sequence contains three potential NifA binding sites (UAS1, 2 and 3) and two potential RNA polymerase‐σ54‐binding sites (downstream promoter elements, DPEs 1 and 2). DPE2 is located 420 bp into the coding region and DPE1 overlaps UAS1 by 5 bp.

Mutational and footprinting analyses have shown efficient activation of the nifJ promoter requires that NifA is bound at UAS 2 and 3. Transcription is initiated at DPE1. Only a weak interaction of NifA with the UAS overlapping DPE1 was detected. Footprints demonstrated that Eσ54 forms a closed complex at DPE1 but not DPE2 and that bound Eσ54 closely approaches the −15 region of DPE1. Stimulation of nifJ promoter activity by IHF was not as great as that observed for other nif promoters. In the absence of IHF nifH promoter sequences stimulated activation of the nifJ promoter. This appeared to require NifA bound at the nifH UAS. Thus, one additional role of IHF may be to partition NifA between the two promoters by constraining the topology of the DNA.
DNA sequencing and expression of the gene rnb encoding Escherichia coli ribonuclease II
Authors: Zilhão R. , Camelo L. , Arraiano C. M.
Published: October 2006

The Escherichia coli ribonuclease II (RNase II) is an exonuclease involved in mRNA degradation that hydrolyses single‐stranded polyribonucleotides processively in the 3′ to 5′ direction. Sequencing of a 2.2 kb Msel–Rsal fragment containing the rnb gene revealed an open reading frame of 1794 nucleotides that encodes a protein of 598 amino acid residues, whose calculated molecular mass is 67 583 Da. This value is in good agreement with that obtained by sodium dodecyl sulphate/ polyacrylamide gel electrophoresis of polypeptides synthesized by expression with the T7 RNA polymerase/promoter system. This system was also used to confirm the correct orientation of rnb. Translation initiation was confirmed by rnb–lacZ fusions. The mRNA start site was determined by S1 nuclease mapping. Two E. coli mutants harbouring different rnb alleles deficient in RNase II activity were complemented with the expressed fragment carrying the rnb gene.
Properties of FNR proteins substituted at each of the five cysteine residues
Authors: Green J. , Sharrocks A. D. , Green B. , Geisow M. , Guest J. R.
Published: October 2006

FNR is a transcriptional regulator controlling the expression of a number of Escherichia coli genes in response to anoxia. It is structurally‐related to CRP (the cyclic AMP receptor protein) except for the presence of a cysteine‐rich N‐terminal extension, which may form part of an iron‐binding, redox‐sensing domain in FNR. Site‐directed substitution has previously shown that four of the cysteine residues (C20, C23, C29 and C122) are essential for FNR function, whereas the fifth (C16) is not. The FNR protein exists in two forms separable by non‐reducing SOS‐PAGE, and in studies with altered FNR proteins containing single substitutions at each of the five cysteine residues it was concluded that the faster‐migrating form (FNR(27)), possesses an intramolecular disulphide bond linking C122 to one of the cysteines near the N‐terminus. FNR(27)) was more abundant in aerobic cells but the physiological significance of this was not established. Footprint studies indicated that FNR proteins lacking essential cysteine residues are impaired in their ability to protect FNR sites in the ndh promoter. The non‐essential cysteine residue (C16) was identified as the source of the most reactive sulphydryl group and all of the inactive proteins exhibited different sulphydryl reactivities. The iron content of the C122A‐substituted protein was much reduced but those of the other proteins were less affected. Electrospray mass spectrometry confirmed the accuracy of the gene‐derived amino acid composition of FNR with a mutant protein and it showed that a fraction of the wild‐type protein may carry a 78 Da substituent which could not be removed with dithio‐threitol or β‐mercaptoethanol.
Multisensory activation of the phosphorelay initiating sporulation in Bacillus subtilis: identification and sequence of the protein kinase of the alternate pathway
Authors: Trach Kathleen A. , Hoch James A.
Published: October 2006

The phosphorelay is the signal‐transduction system recognizing and integrating environmental signals to initiate sporulation. The major signal input to the phosphorelay is an ATP‐dependent kinase, KinA, responsible for phosphorylating the Spo0F protein. Mutants lacking KinA, however, still sporulate, suggesting that other kinases can fulfil its role. In order to identify these kinases, genes for kinases were isolated by hybridization using a degenerate oligonucleotide probe designed for common regions of this class of kinases. A gene for a second kinase, KinB, was isolated which gave a sporulation negative phenotype when inactivated in a kinA background. The kinB locus was sequenced and found to be a small operon consisting of the kinB gene and another gene, kapB, transcribed from a single σ;A‐dependent promoter. Inactivation of either kinB or kapB in a kinA strain led to severe sporulation deficiency. The kinB gene coded for a 47774 Mr protein with the carboxyl half of this protein highly homologous to the same domain of KinA. The amino‐terminal domain of KinB was hydro‐phobic with six recognizable membrane‐spanning regions. The kapB gene coded for a moderately charged, probably soluble, protein of 14666 Mr, with no homology to any known protein. Genetic evidence suggests that KapB is required either for the function of KinB or for its expression. Although double mutants kinA kinB cannot sporulate and assume a stage 0 phenotype, the SpoA∼P‐dependent regulation of the abrB gene is normal in these strains, suggesting that low levels of SpoA∼P accumulate even in the absence of both kinases. This accumulation is dependent on functional spoOF and spoOB genes and its source is unknown. The KinA and KinB pathways are the only pathways capable of producing sufficient SpoOA∼P to allow initiation and completion of sporulation under laboratory conditions.
In vivo processing of an intron‐containing archael tRNA
Authors: Nieuwlandt Daniel T. , Carr Mary Beth , Daniels Charles J.
Published: October 2006

In vitro studies on the processing of halobacterial tRNA introns have led to the proposal that archaeal and eukaryotic tRNA intron endonucleases have distinctly different requirements for the recognition of pre‐tRNAs. Using a Haloferax volcanii in vivo expression vector we have examined the in vivo processing of modified forms of the halobacterial intron‐containing tRNATrp gene. As observed in vitro, changes in the exon–intron boundary structure of this pre‐tRNA block processing. Intron sequences, other than those at the exon–intron boundaries, are not essential for processing in vivo. We also show that conversion of the tryptophan anticodon to an opal suppressor anticodon is tolerated when the exon‐intron boundary structure is maintained.
SycE, a chaperone‐like protein of Yersinia enterocolitica involved in the secretion of YopE
Authors: Wattiau Pierre , Cornells Guy R.
Published: October 2006

Pathogenic yersiniae secrete a set of 11 anti‐host proteins called Yops. The yop genes, scattered around the pYV plasmid, constitute a thermoinduced regulon controlled by the product of virF gene. The secretion of the Yops also requires the presence of the products of the other vir genes and operons, namely virA, virB and virC. The large virC operon and presumably some genes of the virA region encode a new secretion system. Mutations in any of these vir genes impair the production of all the Yops. In contrast, mutations in the yerA locus, located close to yopf, specifically abolish the expression of the cyto‐toxin YopE. We describe here the counterpart of yerA in Yersinia enterocolitica W22703. We demonstrate that the gene product of yerA regulates the production of YopE at a post‐transcriptional level. It specifically binds the YopE protein. We consider that it acts as a specific chaperone and we call it SycE (for specific YopE chaperone). We hypothesize that SycE is a link between translation and the specific Yop export machinery. It is the first representative of a new family of pYV‐encoded proteins.
Sucrose‐derived exopolysaccharides of Streptococcus mutans V403 contribute to infectivity in endocarditis
Authors: Munro Cindy L. , Macrina Francis L.
Published: October 2006

We used an isogenic mutant of Streptococcus mutans V403, which differs from the wild‐type V403 in genes involved in glucan and fructan production, to examine the importance of these exopolysaccharides as factors affecting infectivity in endocarditis. Rats inoculated with V403 developed endocarditis more frequently than animals inoculated with the mutant strain which produced neither glucan nor fructan (58% versus 12%, P<0.01). In phagocytosis assays, both strains were found to be associated with the human granulocytes but a greater number of live V403 than of mutant organisms could be recovered. Colony counts recovered from fibrin plates incubated with the mutant were lower than those incubated with V403. These experiments indicate that exopolysaccharides produced by Streptococcus mutans contribute to its infectivity in endocarditis.
Dual roles of plcA in Listeria monocytogenes pathogenesis
Authors: Camilli Andrew , Tilney Lewis G. , Portnoy Daniel A.
Published: October 2006

The plcA gene of Listeria monocytogenes encodes a secreted phosphatidylinositol‐speciftc phospholi‐pase C (PI‐PLC). Recent studies have established that transposon mutations within plcA result in avirulence for mice and pleiotropic effects when examined in tissue‐culture models of infection. Genetic analysis reveals that many of the effects of the transposon insertions are due to loss of readthrough transcription from plcA into the downstream gene prfA, which encodes an essential transcription factor of numerous L. monocytogenes virulence genes. Construction of an in‐frame deletion within plcA had no effect on expression of prfA thus allowing direct assignment of a role of the PI‐PLC in pathogenesis. PI‐PLC was shown to play a significant role in mediating escape of L. monocytogenes from phagosomes of primary murine macrophages. Interestingly, this defect manifested itself in vivo in the liver but not in the spleen of infected mice.
Bacterial ureases: structure, regulation of expression and role in pathogenesis
Authors: Collins Carleen M. , D'Orazio Sarah E. F.
Published: October 2006

The nickel metalloenzyme urease catalyses the hydrolysis of urea to ammonia and carbamate, and thus generates the preferred nitrogen source of many organisms. When produced by bacterial pathogens in either the urinary tract or the gastroduodenal region, urease acts as a virulence factor. At both sites of infection urease is known to enhance the survival of the infecting bacteria. Ammonia resulting from the action of urease is believed to increase the pH of the environment to one more favourable for growth, and to injure the surrounding epithelial cells. In addition, in the urinary tract urease activity can result in the formation of urinary calculi. Bacterial urease gene clusters contain from seven to nine genes depending upon the species. These genes encode the urease structural subunits and accessory polypeptides involved in the biosynthesis of the nickel metallocentre. So far, three distinct mechanisms of urease gene expression have been described for ureolytic bacteria. Some species constitutively produce urease; some species produce urease only if urea is present in the growth medium; and some species produce urease only during nitrogen‐limiting growth conditions. For either the urea‐inducible genes or the nitrogen‐regulated genes transcription appears to be positively regulated. In the nitrogen‐regulated systems, urease gene expression requires Nac (nitrogen assimilation control), a member of the LysR family of transcriptional activators. Urea dependent expression of urease requires UreR (urease regulator), a member of the AraC family of transcriptional activators. An evolutionary tree for urease genes of eight bacterial species is proposed.
Aeromonas spp. can secrete Escherichia coli alkaline phosphatase into the culture supernatant, and its release requires a functional general secretion pathway
Authors: Wong Kevin R. , Buckley J. Thomas
Published: October 2006

Aerolysin is a channel‐forming protein secreted by Aeromonas hydrophila. To determine if regions of aerolysin could direct the secretion of another protein, portions of aerA were fused to phoA, the Escherichia coli alkaline phosphatase gene and cloned into E. coli, Aeromonas salmonicida, and A. hydrophila. We were surprised to find that secretion of the enzyme by both Aeromonas spp. was independent of the aerolysin segments fused to it. The smallest fusion product contained only the signal sequence and two amino acids of aerolysin. The largest had more than 90% of the aerolysin molecule. The fusion proteins were found in the periplasms of E. coli and A. salmonicida grown in LB medium containing glucose, as well as in the shocked cells. Aerolysin itself was secreted by A. salmonicida under these conditions. In contrast, when A. salmonicida containing any of the fused genes was grown in LB medium without glucose, most of the alkaline phosphatase activity was extracellular, whereas β‐lactamase remained in its normal periplasmic location. Similar results were obtained with A. hydrophila. The change in location of the enzyme in A. salmonicida appeared to be related to the pH of the growth medium. A. salmonicida and A. hydrophila also secreted native E. coli alkaline phosphatase, but A. hydrophila strains with mutations in the general secretion pathway were unable to release the enzyme. We conclude that the Aeromonas secretion system can recognize the E. coli enzyme as an extracellular protein and direct it outside the cell.
Targeted interruption of the psaA and psaB genes encoding the reaction‐centre proteins of photosystem I in the filamentous cyanobacterium Anabaena variabilis ATCC 29413
Authors: Nyhus Karin J. , Thiel Teresa , Pakrasi Himadri B.
Published: October 2006

The two reaction‐centre proteins of the photosystem I (PSI) complex are encoded by two adjacent genes named psaA and psaB. We have performed targeted mutagenesis to insertionally inactivate each of these genes in the filamentous cyanobacterium Anabaena variabilis ATCC 29413. The resulting mutant strains, termed psaA:: NmR and psaB:: NmR, were blue because of a high ratio of phycobilin to chlorophyll and were unable to grow in light. These mutant cells also lacked chemically reducible P700 (the reaction‐centre chlorophylls of PSI) and as a consequence did not exhibit any PSI‐mediated photochemical activity. However, their photosystem II (PSII) complexes were fully active. The loss of the PsaA and PsaB proteins and their associated chlorophyll molecules resulted in a five‐ to sevenfold decrease in the chlorophyll/PSII ratio in the mutant cells relative to the wild‐type cells. Interestingly, the psaS:: NmR and not the psaA:: NmR mutant strain retained a small fluorescence peak (77K) at 721 nm originating from chlorophyll molecule(s) presumably bound to a small amount of the PsaA protein present in the psaB mutant. These results demonstrate that this organism is suitable for the manipulation of PSI reaction‐centre proteins.
Repeat sequences in the Bordetella pertussis adenylate cyclase toxin can be recognized as alternative carboxy‐proximal secretion signals by the Escherichia coliα‐haemolysin translocator
Authors: Šebo Peter , Ladant Daniel
Published: October 2006

The 1706‐residue adenylate cyclase toxin (CyaA) of Bordetella pertussis is an RTX protein with extensive carboxy‐proximai glycine and aspartate‐rich repeats. CyaA does not have a cleavable amino‐terminal signal peptide and can be secreted across both bacterial membranes of the Escherichia coli cell envelope by the α‐haemolysin (HlyA) translocator (HlyBD/TolC). We performed deletion mapping of secretion signals recognized in CyaA by this heterologous translocator. Truncated proteins with N–terminal and internal deletions were secreted at levels up to 10 times higher than intact CyaA and similar to HlyA. A secretion signal recognized by HlyBD/ToiC was found within the last 74 residues of CyaA. However, secretion of CyaA was reduced but not abolished upon deletion of the last 75 or 217 residues, indicating that at least two additional secretion signals recognized by HlyBD/TolC are within CyaA. One of them was localized to the repeat sequence between residues Asp‐1587 to lle‐1631. Interestingly, a conserved acidic' motif (Glu/Asp)‐(X)11‐Asp‐(X)3/5‐(Glu/Asp)‐(X)14‐Asp was found in the C‐terminal sequences of HlyA, CyaA and the two secreted CyaA derivatives. We speculate that the presence and spacing of acidic residues may be an important feature of secretion signals recognized by the haemolysin translocator.
Escherichia coli hha mutants, DNA supercoiling and expression of the haemolysin genes from the recombinant plasmid pANN202‐312
Authors: Carmona M. , Balsalobre C. , Muñoa F. , Mouriño M. , Jubete Y. , De la Cruz F. , Juárez A.
Published: October 2006

The hha gene of Escherichia coli was identified as modulating the expression of the haemolysin (hly) genes encoded by the recombinant plasmid pANN202‐312. hha mutants harbouring plasmid pANN202‐312 showed increased haemolysin production. The product of the hha gene, the Hha protein, shows strong homology to the YmoA protein of Yersinia enterocolitica, which plays a role in the thermoregulation of various V. enterocolitica virulence genes. We show in this study that the Hha protein modulates the expression of haemolysin at the transcriptional level in ceils harbouring plasmid pANN202‐312. In addition, hha mutants show alterations in the level of plasmid DNA supercolting. This suggests that the hha mutation increases haemolysin expression through changes in the DNA topology. This hypothesis is supported by our finding that gyr mutations, inhibitors of DNA gyrase such as novo‐biocin, or growth in conditions reported to reduce levels of negative supercoiling, such as low osmolarity medium, increase haemolysin production.
Interchange of functional domains switches enzyme specificity: construction of a chimeric pneumococcal‐clostridial cell wall lytic enzyme
Authors: Croux C. , Ronda C. , Lopez R. , Garcia J. L.
Published: October 2006

Bacterial autolysins are endogenous enzymes that specifically cleave covalent bonds in the cell wall. These enzymes show both substrate and bond specificities. The former is related to their interaction with the insoluble substrate whereas the latter determine their site of action. The bond specificity allows their classification as muramidases (lysozymes), glucosaminldases, amidases, and endopeptidases. To demonstrate that the autolysin (LYC muramidase) of Clostridium acetobutylicum ATCC824 presents a domainal organization, a chimeric gene (clc) containing the regions coding for the catalytic domain of the LYC muramidase and the choline‐binding domain of the pneumococcal phage CPL1 muramidase has been constructed by in vitro recombination of the corresponding gene fragments. This chimeric construction codes for a choline‐binding protein (CLC) that has been purified using affinity chromatography on DEAE‐cellulose. Several biochemical tests demonstrate that this rearrangement of domains has generated an enzyme with a choline‐dependent muramidase activity on pneumococcal cell walls. Since the parental LYC muramidase was cholineindependent and unable to degrade pneumococcal cell walls, the formation of this active chimeric enzyme by exchanging protein domains between two enzymes that specifically hydrolyse cell walls of bacteria belonging to different genera shows that a switch on substrate specificity has been achieved. The chimeric CLC muramidase behaved as an autolytic enzyme when it was adsorbed onto a live autolysin‐defective mutant of Streptococcus pneumoniae. The construction described here provides experimental support for the theory of modular evolution which assumes that novel proteins have evolved by the assembly of preexisting polypeptide units.
The role of the lipoprotein sorting signal (aspartate +2) in pullulanase secretion
Authors: Poquet Isabelle , Kornacker Michael G. , Pugsley Anthony P.
Published: October 2006

The analyses of hybrid proteins and of deletion and insertion mutations reveal that the only amino acid at the amino‐proximal end of the cell surface lipoprotein pullulanase that is specifically required for its extracellular secretion is an aspartate at position +2, immediately after the fatty acylated amino‐terminal cysteine. To see whether the requirement for this amino acid is related to its proposed role as a cyto‐plasmic membrane lipoprotein sorting signal, we used sucrose gradient floatation analysis to determine the subcellular location of pullulanase variants (with or without the aspartate residue) that accumulated in cells lacking the pullulanase‐specific secretion genes. A non‐secretable pullulanase variant with a serine at position +2 cofractionated mainly with the major peak of outer membrane porin. In contrast, most (55%) of a pullulanase variant with an aspartate at position +2 cofractionated with slightty lighter fractions that contained small proportions of both outer membrane porin and the cytoplasmic membrane marker NADH oxidase. Only 5% of this pullulanase variant cofractionated with the major NADH oxidase peak, while the rest (c. 40%) remained at the bottom of the gradient in fractions totally devoid of porin and NADH oxidase. When analysed by sedimentation through sucrose gradients, however, a large proportion of this variant was recovered from fractions near the top of the gradient that also contained the major NADH oxidase peak. When this peak fraction was applied to a floatation gradient, the pullulanase activity remained at the bottom while the NADH oxidase floated to the top. Thus, there is no evidence that lipoproteins that cofractionate with the cytoplasmic membrane under certain conditions are actually associated with the membrane. Instead, the results support our previous proposal that lipoproteins with an aspartate +2 residue are specifically enriched in a distinct domain of the cell envelope that contains material from both the cytoplasmic and the outer membranes. Possible explanations for the requirement for the aspartate residue in pullulanase secretion are discussed.
Construction and pathogenicity of Aspergillus fumigatus mutants that do not produce the ribotoxin restrictocin
Authors: Smith J. M. , Davies J. E. , Holden D. W.
Published: October 2006

Aspergillus fumigatus, the most common cause of invasive pulmonary aspergillosis (IPA), produces a potent cytotoxjn called restrictocin. To investigate the role of restrictocin in (PA, we have constructed fungal strains in which the res gene has been inactivated by gene disruption. These disruptants lack the specific extracellular ribonucleolytic activity associated with restrictocin, as measured by an in vitro rabbit reticulocyte lysate assay. Western blot analysis of one drsruptant, using an anti‐restrictocin monoclonal antibody, confirmed that the toxin is not produced. The growth characteristics of the disruptants could not be distinguished from those of their parental isolates on a variety of culture media. The pathogenicity of two disruptants was assessed in a murine model of IPA. There were no significant differences in mortality when these strains were compared with the parental isolates and an ectopic transformant. In addition, histological examination of infectediung tissue did not reveal any obvious differences in the number or size of fungal colonies or in the polymorphonuclearleucocyte response. Our results demonstrate that restrictocin is not an important virulence factor in this model of IPA.
Translational initiation at the coat‐protein gene of phage MS2: native upstream RNA relieves inhibition by local secondary structure
Authors: de Smit Maarten H. , van Duin Jan
Published: October 2006

Maximal translation of the coat‐protein gene from RNA bacteriophage MS2 requires a contiguous stretch of native MS2 RNA that extends hundreds of nucleotides upstream from the translational start site. Deletion of these upstream sequences from MS2 cDNA plasmids results in a 30‐fold reduction of translational efficiency. By site‐directed mutagenesis, we show that this low level of expression is caused by a hairpin structure centred around the initiation codon. When this hairpin is destabilized by the introduction of mismatches, expression from the truncated messenger increases 20‐fold to almost the level of the full‐length construct. Thus, the translational effect of hundreds of upstream nucleotides can be mimicked by a single substitution that destabilizes the structure. The same hairpin is also present in full‐length MS2 RNA, but there it does not Impair ribosome binding. Apparently, the upstream RNA somehow reduces the inhibitory effect of the structure on translational initiation. The upstream MS2 sequence does not stimulate translation when cloned in front of another gene, nor can unrelated RNA segments activate the coat‐protein gene. Several possible mechanisms for the activation are discussed and a function in gene regulation of the phage is suggested.
ColE1 multimer formation triggers inhibition of Escherichia coli cell division
Authors: Patient Michaela E. , Summers David K.
Published: October 2006

Multimer formation and consequent copy number depression are acknowledged causes of multicopy plasmid instability. Multimer resolution sites (among which ColE1 cer is best‐characterized) have been identified in a variety of plasmids. They participate in the conversion of multimers to monomers, maximizing the number of independently segregating molecules and minimizing the frequency of plasmid loss. We show that multimer resolution alone is insufficient to ensure stable maintenance of ColE1‐like plasmids in a recombination‐proficient host. The expression of Red, a transcript encoded within cer and expressed in multimer‐containing cells, is also required. The appearance of Red correlates with the inhibition of division of multimer‐containing cells, presumably allowing time for the conversion of multimers to monomers by site‐specific recombination.
Pore‐forming and haemolytic properties of the Gardnerella vaginalis cytoiysin
Authors: Cauci Sabina , Monte Rossella , Ropele Monica , Missero Caterina , Not Tarcisio , Quadrifoglio Franco , Menestrina Gianfranco
Published: October 2006

The pleomorphic bacterium Gardnerella vaginalis releases in the culture broth a haemolytic exotoxin (Gvh) which is probably a virulence determinant of this unique bacterium, implicated in gynaecological and urological disorders. This 59kDa cytolysin was purified to homogeneity in just one chromatographic step directly from the culture supernatant, a final specific activity up to 1.9 × 106 HU mg−1 being obtained. The toxin‐induced lesion on human erythrocytes results from the formation of a pore whose radius is approximately 2.4 nm. The damage is inhibited by osmotic protectants and shows a sigmoidal dose‐response profile suggesting an aggregation process of haemolysin molecules on the target membrane to create the functional lesion. The extent and the kinetics of haemolysis are strongly dependent on temperature and an activation energy of 64.0 kJ mol−1 has been derived. Lipid membranes can be very efficient inhibitors of Gvh‐haemolysis, being able to bind the toxin quite avidly. The inhibitory effect requires the presence of cholesterol and it is stronger when cholesterol is mixed with negatively charged phospholipids rather than with zwitterionic phospholipids, suggesting that a negative surface potential increases the affinity of the toxin for the lipid bilayer. The functional properties of Gvh have been compared with those of Clostridium perfringens theta‐toxin (PFO) and Escherichia coli haemolysin (HlyA), which are representative of widespread haemolysins produced by Gram‐positive and Gram‐negative bacteria, respectively. The toxin shares several features with the family of the so‐called ‘sulphydryl‐activated’ cytolysins produced by Gram‐positive bacteria, although Gvh does not truly belong to this family, being deactivated by β‐mercaptoethanol and being antigenically distinct from them. We report here for the first time the detection in the vaginal fluid of infected women of a specific IgA response against the toxin.
An unusual promoter controls cell‐cycle regulation and dependence on DNA replication of the Caulobacter fliLM early flagellar operon
Authors: Stephens Craig M. , Shapiro Lucille
Published: October 2006

Transcription of flagellar genes in Caulobacter crescentus is programmed to occur during the predivisional stage of the cell cycle. The mechanism of activation of Class II flagellar genes, the highest identified genes in the Caulobacter flagellar hierarchy, is unknown. As a step toward understanding this process, we have defined cis‐acting sequences necessary for expression of a Class II flagellar operon, fliLM. Deletion analysis indicated that a 55 bp DNA fragment was sufficient for normal, temporally regulated promoter activity. Transcription from this promoter‐containing fragment was severely reduced when chromosomal DNA replication was inhibited. Extensive mutational analysis of the promoter region from ‐42 to ‐5 identified functionally important nucleotides at ‐36 and ‐35, between ‐29 and ‐22 and at ‐12, which correlates well with sequences conserved between fliLM and the analogous regions of two other Class II flagellar operons. The promoter sequence does not resemble that recognized by any known bacterial sigma factor. Models for regulation of Caulobacter early flagellar promoters are discussed in which RNA polymerase containing a novel Sigma subunit interacts with an activation factor bound to the central region of the promoter.
Amino acid replacements in the Serratia marcescens haemolysin ShlA define sites involved in activation and secretion
Authors: Schönherr Roland , Tsolis Renée , Focareta Tony , Braun Volkmar
Published: October 2006

The haemolysin of Serratia marcescens (ShlA) is translocated through the cytoplasmic membrane by the signal peptide‐dependent export apparatus. Translocation across the outer membrane (secretion) is mediated by the ShIB protein. Only the secreted form of ShlA is haemolytic. ShIB also converts in vitro inactive ShlA (ShlA*), synthesized in the absence of ShIB, into the haemolytic form (a process termed activation). To define regions in ShlA involved in both processes, ShlA derivatives were isolated and tested for secretion and activation. Analysis of C‐terminally truncated proteins (ShlA) assigned the secretion signal to the amino‐terminal 238 residues of ShlA. Trypsin cleavage of a secreted ShlA' derivative yielded a 15kDa N‐terminal fragment, by which a haemolytically inactive ShlA* protein could be activated in vitro. It is suggested that the haemolysin activation site is located in this N‐terminal fragment. Replacement of asparagine‐69 and asparagine‐109 by isoleucine yielded inactive haemolysin derivatives. Both asparagine residues are part of two short sequence motifs, reading Ala‐Asn‐Pro‐Asn, which are critical to both activation and secretion. These point mutants as well as N‐terminal deletion derivatives which were not activated by ShIB were activated by adding a non‐haemolytic N‐terminal fragment synthesized in an ShIB+ strain (complementation). Apparently the activated N‐terminal fragment substituted for the missing activation of the ShlA derivatives and directed them into the erythrocyte membrane, where they formed pores. It is concluded that activation is only required for initiation of pore formation, and that in vivo activation and secretion are tightly coupled processes. Complementation may also indicate that haemolysin oligomers form the pores.
Regulation and sequence of the structural gene for cytochrome C552 from Escherichia coli: not a hexahaem but a 50kDa tetrahaem nitrite reductase
Authors: Darwin Andrew , Hussain Haitham , Griffiths Lesley , Grove Jane , Sambongi Yoshihiro , Busby Stephen , Cole Jeff
Published: October 2006

The structural gene, nrfA, for cytochrome C552, which is the terminal reductase of the formate‐dependent pathway for nitrite reduction to ammonia, has been located at co‐ordinate 4366 on the physical map of the Escherichia coli chromosome. The DNA sequence of nrfA encodes a tetrahaem c‐type cytochrome with a predicted Mr for the unprocessed product of 53788. Cleavage of the putative signal peptide at Ala‐26 would result in a mature, periplasmic cytochrome of Mr 50580 rather than a larger hexahaem cytochrome, as has been widely reported previously. A cytochrome of this size was detected by staining SDS‐polyacryla‐mide gels for covalently bound haem. This cytochrome was partially purified by anion exchange chromatography and confirmed to be cytochrome C552 by difference spectroscopy. Similar cytochromes were detected in five other E. coli strains including strain ST 249, which was used previously to purify and characterize the protein. A plasmid with an in‐phase deletion within nrfA directed the synthesis of a truncated haemoprotein of the predicted mass. In‐phase translational fusions to lacZ were used to locate the nrfA translation start, and the transcription start site was found by S1 mapping.

Expression from the FNR‐dependent nrfA promoter was almost totally repressed during aerobic growth, partially induced during anaerobic growth in the absence of nitrite or in the presence of nitrate, but fully induced only during anaerobic growth in the presence of nitrite. No nitrate repression was detected in a narL mutant, but nitrite induction was unaffected, indicating that the nitrite‐sensing mechanism is independent of the NarL protein. Expression from the nrfA promoter was subject to glucose repression but regulation was independent of the CRP‐cAMP complex.
Activation of potassium channels during metabolite detoxification in Escherichia coli
Authors: Ferguson Gail P. , Munro Andrew W. , Douglas Roseileen M. , McLaggan Debra , Booth Ian R.
Published: October 2006

In bacteria the detoxification of compounds as diverse as methylglyoxal and chlorodinitrobenzene proceeds through the formation of a glutathione adduct. In the Gram‐negative bacteria, e.g. Escherichia coli, such glutathione adducts activate one, or both, of a pair of potassium efflux systems KefB and KefC. These systems share many of the properties of cation‐translocating channels in eukaryotes. The activity of these systems has been found to be present in a range of Gram‐negative bacteria, but not in the glutathione‐deficient species of Gram‐positive organisms. The conservation of the activity of these systems in a diverse range of organisms suggested a physiological role for these systems. Here we demonstrate that in E. coli cells activation of the KefB efflux system is essential for the survival of exposure to methylglyoxal. Methylglyoxal can be added to the growth medium or its synthesis can be stimulated in the cytoplasm. Under both sets of conditions survival is aided by the activity of KefB. Inhibition of KefB activity by the addition of 10 mM potassium to the growth medium stimulates methylglyoxal‐induced cell death. This establishes an essential physiological function for the KefB system.
Trehalose metabolism in Escherichia coli: stress protection and stress regulation of gene expression
Authors: Strom A. R. , Kaasen I.
Published: October 2006

Endogenously synthesized trehalose is a stress protectant in Escherichia coli. Externally supplied trehalose does not serve as a stress protectant, but it can be utilized as the sole source of carbon and energy. Mutants defective in trehalose synthesis display an impaired osmotic tolerance in minimal growth media without glycine betaine, and an impaired stationary‐phaseinduced heat tolerance. Mechanisms for stress protection by trehalose are discussed. The genes for trehalose‐6‐phosphate synthase (otsA) and anabolic trehalose‐6‐phosphate phosphatase (otsB) constitute an operon. Their expression is induced both by osmotic stress and by growth into the stationary phase and depend on the sigma factor encoded by rpoS (katF). rpoS is amber‐mutated in E. coli K‐12 and its DNA sequence varies among K‐12 strains. For trehalose catabolism under osmotic stress E. coli depends on the osmoticcally inducible periplasmic trehalase (TreA). In the absence of osmotic stress, trehalose induces the formation of an enzyme IITre (TreB) of the group translocation system, a catabolic trehalose‐6‐phosphate phosphatase (TreE), and an amylotrehalase (TreC) which converts trehalose to free glucose and a glucose polymer.
Spatial and temporal controls of the Aspergillus brlA developmental regulatory gene
Authors: Aguirre Jesus
Published: October 2006

The Aspergillus nidulans brlA gene encodes a transcriptional regulator of central importance in controlling conidiophore development. I have determined the effects of mutations in other developmental regulatory genes on expression of a brlA‐lacZ fusion gene. Deletion of brlA reduced ß;‐galactosidase levels by half and led to delocalization of enzyme accumulation. The medA 26 and abaA2 developmental mutations led to overexpression of the fusion gene without altering spatial specificity. In contrast, the stuA1 mutation did not affect the timing or levels of brlA expression during induction, but Instead resulted in spatial derangement of expression. These results and the phenotypes of the mutants suggest a model in which subsets of morphogenetic loci are controlled by differing levels and combinations of regulatory gene products, which are themselves determined by interactions among the regulatory genes.