bluuebunny/crossref_metadata_2025 huggingface dataset:
The N-terminal segment of pulmonary surfactant lipopeptide SP-C has intrinsic propensity to interact with and perturb phospholipid bilayers
Authors: PLASENCIA Ines , RIVAS Luis , KEOUGH Kevin M. W. , MARSH Derek , PÉREZ-GIL Jesús
Published: January 2004

In the present study, 13-residue peptides with sequences corresponding to the native N-terminal segment of pulmonary SP-C (surfactant protein C) have been synthesized and their interaction with phospholipid bilayers characterized. The peptides are soluble in aqueous media but associate spontaneously with bilayers composed of either zwitterionic (phosphatidylcholine) or anionic (phosphatidylglycerol) phospholipids. The peptides show higher affinity for anionic than for zwitterionic membranes. Interaction of the peptides with both zwitterionic and anionic membranes promotes phospholipid vesicle aggregation, and leakage of the aqueous content of the vesicles. The lipid–peptide interaction includes a significant hydrophobic component for both zwitterionic and anionic membranes, although the interaction with phosphatidylglycerol bilayers is also electrostatic in nature. The effects of the SP-C N-terminal peptides on the membrane structure are mediated by significant perturbations of the packing order and mobility of phospholipid acyl chain segments deep in the bilayer, as detected by differential scanning calorimetry and spin-label ESR. These results suggest that the N-terminal region of SP-C, even in the absence of acylation, possesses an intrinsic propensity to interact with and perturb phospholipid bilayers, thereby potentially facilitating SP-C promoting bilayer-monolayer transitions at the alveolar spaces.
Cobatoxin 1 from Centruroides noxius scorpion venom: chemical synthesis, three-dimensional structure in solution, pharmacology and docking on K+ channels
Authors: JOUIROU Besma , MOSBAH Amor , VISAN Violeta , GRISSMER Stephan , M'BAREK Sarrah , FAJLOUN Ziad , VAN RIETSCHOTEN Jurphaas , DEVAUX Christiane , ROCHAT Hervé , LIPPENS Guy , AYEB Mohamed EL , DE WAARD Michel , MABROUK Kamel , SABATIER Jean-Marc
Published: January 2004

CoTX1 (cobatoxin 1) is a 32-residue toxin with three disulphide bridges that has been isolated from the venom of the Mexican scorpion Centruroides noxius Hoffmann. Here we report the chemical synthesis, disulphide bridge organization, 3-D (three-dimensional) solution structure determination, pharmacology on K+ channel subtypes (voltage-gated and Ca2+-activated) and docking-simulation experiments. An enzyme-based cleavage of the synthetic folded/oxidized CoTX1 indicated half-cystine pairs between Cys3-Cys22, Cys8-Cys27 and Cys12-Cys29. The 3-D structure of CoTX1 (solved by 1H-NMR) showed that it folds according to the common α/β scaffold of scorpion toxins. In vivo, CoTX1 was lethal after intracerebroventricular injection to mice (LD50 value of 0.5 µg/mouse). In vitro, CoTX1 tested on cells expressing various voltage-gated or Ca2+-activated (IKCa1) K+ channels showed potent inhibition of currents from rat Kv1.2 (Kd value of 27 nM). CoTX1 also weakly competed with 125I-labelled apamin for binding to SKCa channels (small-conductance Ca2+-activated K+ channels) on rat brain synaptosomes (IC50 value of 7.2 µM). The 3-D structure of CoTX1 was used in docking experiments which suggests a key role of Arg6 or Lys10, Arg14, Arg18, Lys21 (dyad), Ile23, Asn24, Lys28 and Tyr30 (dyad) residues of CoTX1 in its interaction with the rat Kv1.2 channel. In addition, a [Pro7,Gln9]-CoTX1 analogue (ACoTX1) was synthesized. The two residue replacements were selected aiming to restore the RPCQ motif in order to increase peptide affinity towards SKCa channels, and to alter the CoTX1 dipole moment such that it is expected to decrease peptide activity on Kv channels. Unexpectedly, ACoTX1 exhibited an activity similar to that of CoTX1 towards SKCa channels, while it was markedly more potent on IKCa1 and several voltage-gated K+ channels.
Redundancy in the function of mitochondrial phosphate transport in Saccharomyces cerevisiae and Arabidopsis thaliana
Authors: Hamel Patrice , Saint‐Georges Yann , De Pinto Brigida , Lachacinski Nicole , Altamura Nicola , Dujardin Geneviève
Published: January 2004

Most cellular ATP is produced within the mitochondria from ADP and Pi which are delivered across the inner‐membrane by specific nuclearly encoded polytopic carriers. In Saccharomyces cerevisiae, some of these carriers and in particular the ADP/ATP carrier, are represented by several related isoforms that are distinct in their pattern of expression. Until now, only one mitochondrial Pi carrier (mPic) form, encoded by the MIR1 gene in S. cerevisiae, has been described. Here we show that the gene product encoded by the YER053C ORF also participates in the delivery of phosphate to the mitochondria. We have called this gene PIC2 for Pi carrier isoform 2. Overexpression of PIC2 compensates for the mitochondrial defect of the double mutant Δmir1 Δpic2 and restores phosphate transport activity in mitochondria swelling experiments. The existence of two isoforms of mPic does not seem to be restricted to S. cerevisiae as two Arabidopsis thaliana cDNAs encoding two different mPic‐like proteins are also able to complement the double mutant Δmir1 Δpic2. Finally, we demonstrate that Pic2p is a mitochondrial protein and that its steady state level increases at high temperature. We propose that Pic2p is a minor form of mPic which plays a role under specific stress conditions.
Thermoregulation of the Escherichia coli O157:H7 pO157 ecf operon and lipid A myristoyl transferase activity involves intrinsically curved DNA
Authors: Yoon Jang W. , Minnich Scott A. , Ahn Jong S. , Park Yong H. , Paszczynski Andrzej , Hovde Carolyn J.
Published: January 2004

Escherichia coli O157:H7 survives in diverse environments from the ruminant gastrointestinal tract to cool nutrient‐dilute water. We hypothesized that the gene regulation required for this flexibility includes intrinsically curved DNA that responds to environmental changes. Three intrinsically curved DNAs were cloned from the E. coli O157:H7 virulence plasmid (pO157), sequenced and designated Bent 1 through Bent 3 (BNT1, BNT2 and BNT3). Compared to BNT1 and BNT3, BNT2 had characteristics typical of intrinsically curved DNA including electrophoretic gel retardation at 4°C, six partially phased adenine:thymine tracts and transcriptional activation. BNT2::lacZ operon fusions showed that BNT2 activated transcription at 24°C compared to 37°C and was partially repressed by a bacterial nucleoid‐associated protein H‐NS. BNT2 regulated the E. coli attaching and effacing gene‐positive conserved fragments 1–4 (ecf1–4) that are conserved in Shiga toxin‐producing E. coli associated with human disease. Experimental analyses showed that ecf1–4 formed an operon. ecf1, 2 and 3 encoded putative proteins associated with bacterial surface polysaccharide biosynthesis and invasion and ecf4 complemented a chromosomal deletion of lpxM encoding lipid A myristoyl transferase. Mass spectrometric analysis of lipid A from ecf and lpxM single and double mutants showed that myristoylation was altered at lower temperature.
Evidence that putrescine acts as an extracellular signal required for swarming in Proteus mirabilis
Authors: Sturgill Gwen , Rather Philip N.
Published: January 2004

In a search for Proteus mirabilis genes that were regulated by cell‐to‐cell signalling, a lacZ fusion (cmr437::mini‐Tn5lacZ) was identified that was repressed 10‐fold by a self‐produced extracellular signal from wild‐type cells. However, the cmr437::mini‐Tn5lacZ insertion itself led to a marked reduction in this extracellular repressing signal. The cmr437::mini‐Tn5lacZ insertion was mapped to a speA homologue in P. mirabilis. Sequence analysis indicated that a speB homologue was encoded downstream of speA. Products of the SpeA and SpeB enzymes (agmatine and putrescine) were tested for repression of cmr437::lacZ. Agmatine did not have repressing activity. However, putrescine was an effective repressing molecule at concentrations down to 30 µM. A second prominent phenotype of the cmr437 (speA)::mini‐Tn5lacZ insertion was a severe defect in swarming motility. This swarming defect was also observed in a strain containing a disruption of the downstream speB gene. Differentiation of the speB mutant to swarmer cells was delayed by two hours relative to wild‐type cells. Furthermore, the speB mutant was unable to migrate effectively across agar surfaces and formed very closely spaced swarming rings. Exogenous putrescine restored both the normal timing of swarmer cell differentiation and the ability to migrate to speB mutants.
Mutations of the CK2 phosphorylation site of Sic1 affect cell size and S‐Cdk kinase activity in Saccharomyces cerevisiae
Authors: Coccetti Paola , Rossi Riccardo L. , Sternieri Flora , Porro Danilo , Russo Gian Luigi , Di Fonzo Andrea , Magni Fulvio , Vanoni Marco , Alberghina Lilia
Published: January 2004

By sequence analysis we found an amino acid stretch centred on Serine201 matching a stringent CK2 consensus site within the C‐terminal, inhibitory domain of Sic1. Here we show by direct mass spectrometry analysis that Sic1, but not a mutant protein whose CK2 phospho‐acceptor site has been mutated to alanine, Sic1S201A, is actually phosphorylated in vitro by CK2 on Serine 201. Mutation of Serine 201 alters the coordination between growth and cell cycle progression. A significant increase of average protein content and of the average protein content at the onset of DNA synthesis is observed for exponentially growing cells harbouring the Sic1S201A protein. A strong reduction of the same parameters is observed in cells harbouring Sic1S201E. The deregulated coordination between cell size and cell cycle is also apparent at the level of S‐Cdk activity.
Kinetics of plasmid segregation in Escherichia coli
Authors: Gordon Scott , Rech Jerôme , Lane David , Wright Andrew
Published: January 2004

Low copy‐number bacterial replicons occupy specific locations in their host cells. Production of a GFP‐Lac repressor hybrid protein in cells carrying F or P1 plasmids tagged with a lac operator array reveals that in smaller (younger) cells these plasmids are seen mainly as a single fluorescent focus at mid‐cell, whereas larger cells tend to have two foci, one at each quarter‐cell position. Duplication of the central focus is presumed to represent active partition of plasmid copies. We report here our investigation by time‐lapse microscopy of the subsequent movement of these copies to the quarter positions. Following duplication of the central focus, the new foci migrated rapidly and directly to their quarter‐cell destinations, where they remained until the next cell cycle. The speed of movement was about five times faster than poleward migration of oriC and 50 times faster than cell elongation. Aberrant positioning of mini‐F lacking its sopC centromere demonstrated the requirement for the partition system in this localization process. From the measured number of F plasmid copies per cell it appears that each migrating focus contains two or more plasmid molecules. The molecular basis of this clustering, and evidence for phasing of the partition event in the cell cycle, are discussed.
From the inside out – processing of the Chlamydial autotransporter PmpD and its role in bacterial adhesion and activation of human host cells
Authors: Wehrl Wolfgang , Brinkmann Volker , Jungblut Peter R. , Meyer Thomas F. , Szczepek Agnes J.
Published: January 2004

Polymorphic membrane protein (Pmp)21 otherwise known as PmpD is the longest of 21 Pmps expressed by Chlamydophila pneumoniae. Recent bioinformatical analyses annotated PmpD as belonging to a family of exported Gram‐negative bacterial proteins designated autotransporters. This prediction, however, was never experimentally supported, nor was the function of PmpD known. Here, using 1D and 2D PAGE we demonstrate that PmpD is processed into two parts, N‐terminal (N‐pmpD), middle (M‐pmpD) and presumably third, C‐terminal part (C‐pmpD). Based on localization  of  the  external  part  on  the  outer  membrane as shown by immunofluorescence, immuno‐electron microscopy and immunoblotting combined with trypsinization, we demonstrate that N‐pmpD translocates to the surface of bacteria where it non‐covalently binds other components of the outer membrane. We propose that N‐pmpD functions as an adhesin, as antibodies raised against N‐pmpD blocked  chlamydial  infectivity  in  the  epithelial  cells. In addition, recombinant N‐pmpD activated human monocytes in vitro by upregulating their metabolic activity and by stimulating IL‐8 release in a dose‐dependent manner. These results demonstrate that N‐PmpD is an autotransporter component of chlamydial outer membrane, important for bacterial invasion and host inflammation.
A CDP‐choline pathway for phosphatidylcholine biosynthesis in Treponema denticola
Authors: Kent Claudia , Gee Patricia , Lee Si Young , Bian Xuelin , Fenno J. Christopher
Published: January 2004

The genomes of Treponema denticola and Treponema pallidum contain a gene, licCA, which is predicted to encode a fusion protein containing choline kinase and CTP:phosphocholine cytidylyltransferase activities. Because both organisms have been reported to contain phosphatidylcholine, this raises the possibility that they use a CDP‐choline pathway for the biosynthesis of phosphatidylcholine. This report shows that phosphatidylcholine is a major phospholipid in T. denticola, accounting for 35–40% of total phospholipid. This organism readily incorporated [14C]choline into phosphatidylcholine, indicating the presence of a choline‐dependent biosynthetic pathway. The licCA gene was cloned, and recombinant LicCA had choline kinase and CTP:phosphocholine cytidylyltransferase activity. The licCA gene was disrupted in T. denticola by erythromycin cassette mutagenesis, resulting in a viable mutant. This disruption completely blocked incorporation of either [14C]choline or 32Pi into phosphatidylcholine. The rate of production of another phospholipid in T. denticola, phosphatidylethanolamine, was elevated considerably in the licCA mutant, suggesting that the elevated level of this lipid compensated for the loss of phosphatidylcholine in the membranes. Thus it appears that T. denticola does contain a licCA‐dependent CDP‐choline pathway for phosphatidylcholine biosynthesis.
Salmonella type III secretion‐associated chaperones confer secretion‐pathway specificity
Authors: Lee Sang Ho , Galán Jorge E.
Published: January 2004

Type III protein secretion systems (TTSSs) are ancestrally related to the flagellar export system and are essential for the virulence of many bacteria pathogenic for humans, animals and plants. Most proteins destined to travel the TTSS pathway possess at least two domains that specifically target them to the secretion apparatus. One of the domains is located within the amino terminal first ∼20 amino acids and the second domain, located within the first ∼140 amino acids, serves as a binding site for specific chaperones. It has been previously proposed that these two secretion signals are capable of operating independently of one another to facilitate secretion into the extracellular environment. We have found that in the absence of their chaperone‐binding domains, the Salmonella typhimurium TTSS‐secreted proteins SptP and SopE are no longer targeted for secretion through their cognate TTSS and, instead, are secreted through the flagellar export pathway. These results indicate the existence of an ‘ancestral’ flagellar secretion signal within TTSS‐exported proteins that is revealed in the absence of the chaperone‐binding domain. Furthermore, we found that secretion into culture supernatants as well as translocation into host cells by the cognate TTSS require both, the amino terminal and chaperone‐binding domains. We conclude from these studies that a critical function for the TTSS‐associated chaperones is to confer secretion‐pathway specificity to their cognate secreted proteins.
Flavonoids induce temporal shifts in gene‐expression of nod‐box controlled loci in Rhizobium sp. NGR234
Authors: Kobayashi Hajime , Graven Yamama Naciri , Broughton William J. , Perret Xavier
Published: January 2004

Rhizobia, soil bacteria of the Rhizobiales, enter the roots of homologous legumes, where they induce the formation of nitrogen‐fixing nodules. Signals emanating from both symbiotic partners control nodule development. Efficient nodulation requires precise, temporal regulation of symbiotic genes. Roots continuously release flavonoids that interact with transcriptional activators of the LysR family. NodD proteins, which are members of this family, act both as sensors of the environment and modulate the expression of genes preceded by conserved promoter sequences called nod‐boxes. The symbiotic plasmid of the broad host‐range Rhizobium sp. NGR234 caries 19 nod‐boxes (NB1 to NB19), all of which were cloned upstream of a lacZ‐reporter gene. A flavonoid, daidzein was able to induce 18 of the 19 nod‐boxes in a NodD1‐dependent manner. Interestingly, induction of four nod‐boxes (NB6, NB15, NB16 and NB17) is highly dependent on NodD2 and was delayed  in  comparison  with  the  others.  In  turn, NodD2 is involved in the repression of the NB8 nodABCIJnolOnoeI operon. Activation of transcription of nodD2 is also dependent on flavonoids despite  the  absence  of  a  nod‐box  like  sequence  in the upstream promoter region. Mutational analysis showed that syrM 2 (another member of the LysR family), which is controlled by NB19, is also necessary for expression of nodD 2. Thus, NodD1, NodD2 and SyrM2 co‐modulate a flavonoid‐inducible regulatory cascade that coordinates the expression of symbiotic genes with nodule development.
Hyperinitiation of DNA replication in Escherichia coli leads to replication fork collapse and inviability
Authors: Simmons Lyle A. , Breier Adam M. , Cozzarelli Nicholas R. , Kaguni Jon M.
Published: January 2004

Elevated dnaA expression from a multicopy plasmid induces more frequent initiation from the Escherichia coli replication origin, oriC, but viability is maintained. In comparison, chromosomally encoded dnaAcos also stimulates initiation, but this is lethal. By quantitative methods, we show that the level of initiation induced by elevated dnaA expression leads to collapsed replication forks that are mostly within 10 map units of oriC. Because forks collapse randomly, nucleoprotein complexes at specific sites such as datA are not the cause. When replication restart is blocked by a mutation in recB or priA, the increased initiations via elevated dnaA expression causes inviability. The amount of collapsed forks is substantially higher under elevated expression of dnaAcos compared to that of dnaA. We propose that the lethal phenotype of chromosomally encoded dnaAcos is a result of hyperinitiation that overwhelms the repair capacity of the cell.
Staphylococcus aureus multiresistance plasmid pSK41: analysis of the replication region, initiator protein binding and antisense RNA regulation
Authors: Kwong Stephen M. , Skurray Ronald A. , Firth Neville
Published: January 2004

The vast majority of large staphylococcal plasmids characterized to date appear to possess an evolutionarily common replication system, which has clearly had a major impact on the evolution of antimicrobial resistant staphylococci worldwide. Related systems have also been found in plasmids from other Gram‐positive genera, including enterococci, streptococci and bacilli. The 46.4 kb plasmid pSK41 is the prototype of a family of conjugative staphylococcal multiresistance plasmids. The replication region of pSK41 encodes a protein product, Rep, which was shown to be essential for replication; mutations that truncated Rep could be complemented in trans. Rep was found to bind in vitro to four tandem repeat sequences located centrally within the rep coding region. An A + T‐rich inverted repeat sequence upstream of rep was required for efficient replication, whereas no sequences downstream of rep were necessary. An antisense countertranscript, RNAI, encoded upstream of rep was identified and transcriptional start points for both RNAI and the rep‐mRNA were defined.
Individual RD1‐region genes are required for export of ESAT‐6/CFP‐10 and for virulence of Mycobacterium tuberculosis
Authors: Guinn Kristi M. , Hickey Mark J. , Mathur Sanjeev K. , Zakel Kelly L. , Grotzke Jeff E. , Lewinsohn David M. , Smith Sherilyn , Sherman David R.
Published: January 2004

The RD1 genomic region is present in virulent strains of Mycobacterium tuberculosis (MTB), missing from the vaccine strain M. bovis BCG, and its importance to virulence has been established experimentally. Based on in silico analysis, it has been suggested that RD1 may encode a novel secretion system, but the mechanism by which this region affects virulence is unknown. Here we examined mutants disrupted in five individual RD1 genes. Both in vitro and in vivo, each mutant displayed an attenuated phenotype very similar to a mutant missing the entire RD1 region. Genetic complementation of individual genes restored virulence. Attenuated mutants could multiply within THP‐1 cells, but they were unable to spread to uninfected macrophages. We also examined export of two immunodominant RD1 proteins, CFP‐10 and ESAT‐6. Export of these proteins was greatly reduced or abolished in each attenuated mutant. Again, genetic complementation restored a wild‐type phenotype. Our results indicate that RD1 genes work together to form a single virulence determinant, and argue that RD1 encodes a novel specialized secretion system that is required for pathogenesis of MTB.
An ER packaging chaperone determines the amino acid uptake capacity and virulence of Candida albicans
Authors: Martínez Paula , Ljungdahl Per O.
Published: January 2004

The Candida albicans CSH3 gene encodes a functional and structural homologue of Shr3p, a yeast protein that is specifically required for proper uptake and sensing of extracellular amino acids in Saccharomyces cerevisiae. A Candida csh3Δ/csh3Δ null mutant has a reduced capacity to take up amino acids, and is unable to switch morphologies on solid and in liquid media in response to inducing amino acids. CSH3/csh3Δ heterozygous strains display normal amino acid induced morphological switching. However, although heterozygous cells apparently sense and properly react to amino acid induced signals they cannot take up amino acids at wild‐type rates. Strikingly, both CSH3/csh3Δ heterozygous and csh3Δ/csh3Δ homozygous strains are unable to efficiently mount virulent infections in a mouse model. The haploinsufficiency phenotypes indicate that both CSH3 alleles contribute to maintain high‐capacity amino acid uptake in wild‐type strains. These results strongly suggest that C. albicans cells use amino acids, presumably as nitrogen sources, during growth in mammalian hosts.
Chromosomal constraints in Gram‐positive bacteria revealed by artificial inversions
Authors: Campo N. , Dias M. J. , Daveran‐Mingot M. ‐L. , Ritzenthaler P. , Le Bourgeois P.
Published: January 2004

We used artificial chromosome inversions to investigate the chromosomal constraints that preserve genome organization in the Gram‐positive bacterium Lactococcus lactis. Large inversions, 80–1260 kb in length, disturbing the symmetry of the origin and terminus of the replication axis to various extents, were constructed using the site‐specific Cre‐loxP recombination system. These inversions were all mechanistically feasible and fell into various classes according to stability and effect on cell fitness. The L. lactis chromosome supports only to some extent unbalance in length of its replication arms. The location of detrimental inversions allowed identification of two constrained chromosomal regions: a large domain covering one fifth of the genome that encompasses the origin of replication (Ori domain), and a smaller domain located at the opposite of the chromosome (Ter domain).
ThevirFpromoter inShigella: more than just a curved DNA stretch
Authors: Prosseda Gianni , Falconi Maurizio , Giangrossi Mara , Gualerzi Claudio O. , Micheli Gioacchino , Colonna Bianca
Published: January 2004

In the human enteropathogenShigellatranscription ofvirF, the primary regulator of the invasion functions, is strictly temperature‐dependent and is antagonistically mediated by H‐NS and FIS, which bind to specific sites on thevirFpromoter. Here we report on the relevance of DNA geometry to the themoregulation ofvirFand demonstrate that thevirFpromoter hosts a major DNA bend halfway between two H‐NS sites. The bent region has been mutagenizedin vitroto mimic temperature‐induced changes of DNA curvature. Functional analysis of curvature mutants and of promoter constructs in which the two H‐NS sites are phased‐out by a half–helix turn reveals that modifying the spatial relationships between these sites severely affects the interaction of H‐NS with thevirFpromoter, as well as itsin vivoandin vitrotemperature‐dependent activity. The role of promoter curvature as thermosensor is also compatible with the present observation that, with increasing temperature, thevirFbending centre moves downstream at a rate having its maximum around the transition temperature, abruptly unmasking a binding site for the transcriptional activator FIS.
Host processing of branched DNA intermediates is involved in targeted transposition of IS911
Authors: Loot Celine , Turlan Catherine , Chandler Michael
Published: January 2004

A simplified system using bacterial insertion sequence IS911 has been developed to investigate targeted insertion next to DNA sequences resembling IS ends. We show here that these IR‐targeted events occur by an unusual mechanism. In the circular IS911 transposition intermediate the two IRs are abutted to form an IR/IR junction. IR‐targeted insertion involves transfer of a single end of the junction to the target IR to generate a branched DNA structure. The single‐end transfer (SET) intermediate, but not the final insertion product, can be detected in an in vitro reaction. SET intermediates must be processed by the bacterial host to obtain the final insertion products. Sequence analysis of these IR‐targeted insertion products and of those obtained in vivo revealed high levels of DNA sequence conversion in which mutations from one IR were transferred to another. These sequence changes cannot be explained by the classic transposition pathway. A model is presented in which the four‐way Holliday‐like junction created by SET is processed by host‐mediated branch migration, resolution, repair and replication. This pathway resembles those described for processing other branched DNA structures such as stalled replication forks.
Molybdate transport and its effect on nitrogen utilization in the cyanobacterium Anabaena variabilis ATCC 29413
Authors: Zahalak Marta , Pratte Brenda , Werth Kelly J. , Thiel Teresa
Published: January 2004

Molybdenum is an essential component of the cofactors of many metalloenzymes including nitrate reductase and Mo‐nitrogenase. The cyanobacterium Anabaena variabilis ATCC 29413 uses nitrate and atmospheric N2 as sources of nitrogen for growth. Two of the three nitrogenases in this strain are Mo‐dependent enzymes, as is nitrate reductase; thus, transport of molybdate is important for growth of this strain. High‐affinity transport of molybdate in A. variabilis was mediated by an ABC‐type transport system encoded by the products of modA and modBC. The modBC gene comprised a fused orf including components corresponding to modB and modC of Escherichia coli. The deduced ModC part of the fused gene lacked a recognizable molybdate‐binding domain. Expression of modA and modBC was induced by starvation for molybdate. Mutants in modA or modBC were unable to grow using nitrate or Mo‐nitrogenase. Growth using the alternative V‐nitrogenase was not impaired in the mutants. A high concentration of molybdate (10 µM) supported normal growth of the modBC mutant using the Nif1 Mo‐nitrogenase, indicating that there was a low‐affinity molybdate transport system in this strain. The modBC mutant did not detectably transport low concentrations of 99Mo (molybdate), but did transport high concentrations. However, such transport was observed only after cells were starved for sulphate, suggesting that an inducible sulphate transport system might also serve as a low‐affinity molybdate transport system in this strain.
Chromosome 1 trisomy compromises the virulence of Candida albicans
Authors: Chen Xi , Magee B. B. , Dawson Dean , Magee P. T. , Kumamoto Carol A.
Published: January 2004

Although increases in chromosome copy number typically have devastating developmental consequences in mammals, fungal cells such as Saccharomyces cerevisiae seem to tolerate trisomies without obvious impairment of growth. Here, we demonstrate that two commonly used laboratory strains of the yeast Candida albicans, CAI‐4 and SGY‐243, can carry three copies of chromosome 1. Although the trisomic strains grow well in the laboratory, Ura+ derivatives of CAI‐4, carrying three copies of chromosome 1, are avirulent in the intravenously inoculated mouse model, unlike closely related strains carrying two copies of chromosome 1. Furthermore, changes in chromosome copy number occur during growth in an animal host and during growth in the presence of growth‐inhibiting drugs. These results suggest that chromosome copy number variation provides a mechanism for genetic variation in this asexual organism.
RcaE is a complementary chromatic adaptation photoreceptor required for green and red light responsiveness
Authors: Terauchi Kazuki , Montgomery Beronda L. , Grossman Arthur R. , Lagarias J. Clark , Kehoe David M.
Published: January 2004

The recent discovery of large numbers of phytochrome photoreceptor genes in both photosynthetic and non‐photosynthetic prokaryotes has led to efforts to understand their physiological roles in environmental acclimation. One receptor in this class, RcaE, is involved in controlling complementary chromatic adaptation, a process that regulates the transcription of operons encoding light‐harvesting proteins in cyanobacteria. Although all previously identified phytochrome responses are maximally sensitive to red and far red light, complementary chromatic adaptation is unique in that it is responsive to green and red light. Here, we present biochemical and genetic evidence demonstrating that RcaE is a photoreceptor and that it requires the cysteine at position 198 to ligate an open chain tetrapyrrole covalently in a manner analogous to chromophore attachment in plant phytochromes. Furthermore, although the wild‐type rcaE gene can rescue red and green light photoresponses of an rcaE null mutant, a gene in which the codon for cysteine 198 is converted to an alanine codon rescues the red light but not the green light response. Thus, RcaE is a photoreceptor that is required for both green and red light responsiveness during complementary chromatic adaptation and is the first identified phytochrome class sensor that is involved in sensing and responding to green and red light rather than red and far red light.
Nucleoid restructuring in stationary‐state bacteria
Authors: Frenkiel‐Krispin Daphna , Ben‐Avraham Irit , Englander Joseph , Shimoni Eyal , Wolf Sharon G. , Minsky Abraham
Published: January 2004

The textbook view of the bacterial cytoplasm as an unstructured environment has been overturned recently by studies that highlighted the extent to which non‐random organization and coherent motion of intracellular components are central for bacterial life‐sustaining activities. Because such a dynamic order critically depends on continuous consumption of energy, it cannot be perpetuated in starved, and hence energy‐depleted, stationary‐state bacteria. Here, we show that, at the onset of the stationary state, bacterial chromatin undergoes a massive reorganization into ordered toroidal structures through a process that is dictated by the intrinsic properties of DNA and by the ubiquitous starvation‐induced DNA‐binding protein Dps. As starvation proceeds, the toroidal morphology acts as a structural template that promotes the formation of DNA–Dps crystalline assemblies through epitaxial growth. Within the resulting condensed assemblies, DNA is effectively protected by means of structural sequestration. We thus conclude that the transition from bacterial active growth to stationary phase entails a co‐ordinated process, in which the energy‐dependent dynamic order of the chromatin is sequentially substituted with an equilibrium crystalline order.
In vivo studies on putative Shine–Dalgarno sequences of the halophilic archaeon Halobacterium salinarum
Authors: Sartorius‐Neef Simone , Pfeifer Felicitas
Published: January 2004

The involvement of Shine–Dalgarno sequences in the translation of mRNA in halophilic archaea was investigated for two gvp genes involved in gas vesicle formation in Halobacterium salinarum PHH1. With the exception of gvpA and gvpO, all reading frames of the p‐gvpDEFGHIJKLM and p‐gvpACNO mRNAs contained upstream of the AUG start codon a putative Shine–Dalgarno (SD) sequence that is complementary to the 3′‐end of the small ribosomal subunit RNA. The importance of the SD sequences of gvpG and gvpH was investigated in Haloferax volcanii transformants, and an alteration of the SD sequence resulted in a reduction of the amount of the GvpG or GvpH protein. For a more quantitative analysis the region upstream of gvpH was fused to the bgaH reading frame encoding an enzyme with β‐galactosidase activity as reporter. Scanning mutagenesis within the mRNA leader demonstrated that mutations adjacent to the putative SD sequence GGAGGUCA did not influence the efficiency of translation, whereas constructs harbouring an altered SD sequence yielded only 5–50% of the β‐galactosidase activities obtained with the wild‐type SD element. A complete mutation of the SD sequence still yielded 20% of the wild‐type activity. Alterations in the spacing of the SD sequence and the translation initiation codon of gvpH indicated that a distance of 4 or 10 nucleotides yielded a similar β‐galactosidase activity as found with the 7 nt spacing of the SD element in wild type, whereas a distance of 1 nt resulted in the loss of translation. A complete deletion of the 5′‐UTR resulting in a leaderless mRNA yielded an enhanced β‐galactosidase activity in transformants implying that the initiation of translation involved a mechanism other than a specific mRNA–rRNA interaction.
Cellulolysis is severely affected in Clostridium cellulolyticum strain cipCMut1
Authors: Maamar Hédia , Valette Odile , Fierobe Henri‐Pierre , Bélaich Anne , Bélaich Jean‐Pierre , Tardif Chantal
Published: January 2004

Progress towards understanding the molecular basis of cellulolysis by Clostridium cellulolyticm was obtained through the study of the first cellulolysis defective mutant strain, namely cipCMut1. In this mutant, a 2 659 bp insertion element, disrupts the cipC gene at the sequence encoding the seventh cohesin of the scaffoldin CipC. cipC is the first gene in a large ‘cel’ gene cluster, encoding several enzymatic subunits of the cellulosomes, including the processive cellulase Cel48F, which is the major component. Physiological and biochemical studies showed that the mutant strain was affected in cellulosome synthesis and severely impaired in its ability to degrade crystalline cellulose. It produced small amounts of a truncated CipC protein (P120), which had functional cohesin domains and assembled complexes which did not contain any of the enzymes encoded by genes of the ‘cel’ cluster. The mutant cellulolytic system was mainly composed of three proteins designated P98, P105 and P125. Their N‐termini did not match any of the known cellulase sequences from C. cellulolyticum. A large amount of entire CipC produced in the cipCMut1 strain by trans‐complementation with plasmid pSOScipC did not restore the cellulolytic phenotype, in spite of the assembly of a larger amount of complexes. The complexes produced in the mutant and complemented strains contained at least 12 different dockerin‐containing proteins encoded by genes located outside of the ‘cel’ cluster. The disturbances observed in the mutant and trans‐complemented strains were the result of a strong polar effect resulting from the cipC gene disruption. In conclusion, this study provided genetic evidence that the cellulases encoded by the genes located in the ‘cel’ cluster are essential for the building of cellulosomes efficient in crystalline cellulose degradation.
Use of antisense RNA to modify the composition of cellulosomes produced by Clostridium cellulolyticum
Authors: Perret Stéphanie , Maamar Hédia , Bélaich Jean‐Pierre , Tardif Chantal
Published: January 2004

The enzymatic composition of the cellulosomes produced by Clostridium cellulolyticum was modified by inhibiting the synthesis of Cel48F that is the major cellulase of the cellulosomes. The strain ATCC 35319 (pSOSasrF) was developed to over‐produce a 469 nucleotide‐long antisense‐RNA (asRNA) directed against the ribosome‐binding site region and the beginning of the coding region of the cel48F mRNAs. The cellulolytic system secreted by the asRNA‐producing strain showed a markedly lower amount of Cel48F, compared to the control strain transformed with the empty plasmid (pSOSzero). This was correlated with a 30% decrease of the specific activity of the cellulolytic system on Avicel cellulose, indicating that Cel48F plays an important role in the recalcitrant cellulose degradation. However, only minor effects were observed on the growth parameters on cellulose. In both transformant strains, cellulosome production was found to be reduced and two unknown proteins (P105 and P98) appeared as major components of their cellulolytic systems. These proteins did not contain any dockerin domain and were shown to be not included into the cellulosomes; they are expected to participate to the non‐cellulosomal cellulolytic system of C. cellulolyticum.
Bacillus anthracis requires siderophore biosynthesis for growth in macrophages and mouse virulence
Authors: Cendrowski Stephen , MacArthur William , Hanna Philip
Published: January 2004

Systemic anthrax infections can be characterized as proceeding in stages, beginning with an early intracellular establishment stage within phagocytes that is followed by extracelluar stages involving massive bacteraemia, sepsis and death. Because most bacteria require iron, and the host limits iron availability through homeostatic mechanisms, we hypothesized that B. anthracis requires a high‐affinity mechanism of iron acquisition during its growth stages. Two putative types of siderophore synthesis operons, named Bacillus anthracis catechol, bac (anthrabactin), and anthrax siderophore biosynthesis, asb (anthrachelin), were identified. Directed gene deletions in both anthrabactin and anthrachelin pathways were generated in a B. anthracis (Sterne) 34F2 background resulting in mutations in asbA and bacCEBF. A decrease in siderophore production was observed during iron‐depleted growth in both the ΔasbA and ΔbacCEBF strains, but only the ΔasbA strain was attenuated for growth under these conditions. In addition, the ΔasbA strain was severely attenuated both for growth in macrophages (MΦ) and for virulence in mice. In contrast, the ΔbacCEBF strain did not differ phenotypically from the parental strain. These findings support a requirement for anthrachelin but not anthrabactin in iron assimilation during the intracellular stage of anthrax.
Evolution of fungal sex chromosomes
Authors: Fraser James A. , Heitman Joseph
Published: January 2004

Sexual reproduction enables organisms to shuffle two parental genomes to produce recombinant progeny, and to purge the genome of deleterious mutations. Sex is conserved in virtually all organisms, from bacteria and fungi to plants and animals, and yet the mechanisms by which sexual identity are established share both conserved general features and are remarkably diverse. In animals, sexual identity is established by dimorphic sex chromosomes, whereas in fungi a specialized region of the genome, known as the mating‐type locus, governs the establishment of cell type identity and differs in DNA sequence between cells of different mating‐types. Recent studies on the mating‐type loci of fungi and algae reveal features shared with the mammalian X and Y chromosomes, suggesting that these represent early steps in the evolution of sex chromosomes.
The lysozyme of the starfish Asterias rubens
Authors: Bachali Sana , Bailly Xavier , Jollès Jacqueline , Jollès Pierre , Deutsch Jean S.
Published: January 2004

On the basis of a partial N‐terminal sequence, Jollès and Jollès [Jollès, J., & Jollès, P. (1975) Eur. J. Biochem.54, 19–23] previously proposed that the lysozyme from the starfish Asterias rubens represents a new form of lysozyme, called type i (invertebrate) lysozyme. Indeed, it differed from both the types c (chicken) and g (goose) known in other animals, as well as from plant and phage lysozymes. Recently, several proteins belonging to the same family have been isolated from protostomes. Here we report the complete mature protein sequence and cDNA sequence of the lysozyme from Asterias. These sequences vindicate the previously proposed homology between the starfish, a deuterostome, and protostome lysozymes. In addition, we present a structural analysis that allows us to postulate upon the function of several conserved residues.
Mechanisms of internalization and recycling of the chemokine receptor, CCR5
Authors: Mueller Anja , Strange Philip G.
Published: January 2004

CCR5 is a G protein‐coupled receptor that binds several natural chemokines but it is also a coreceptor for the entry of M tropic strains of HIV‐1 into cells. Levels of CCR5 on the cell surface are important for the rate of HIV‐1 infection and are determined by a number of factors including the rates of CCR5 internalization and recycling. Here we investigated the involvement of the actin cytoskeleton in the control of ligand‐induced internalization and recycling of CCR5. Cytochalasin D, an actin depolymerizing agent, inhibited chemokine‐induced internalization of CCR5 and recycling of the receptor in stably transfected CHO cells and in the monocytic cell line, THP‐1. CCR5 internalization and recycling were inhibited by Toxin B and C3 exoenzyme treatment in CHO and THP‐1 cells, confirming activation of members of the RhoGTPase family by CCR5. The specific Rho kinase inhibitor Y27632, however, had no effect on CCR5 internalization or recycling. Ligand‐induced activation of CCR5 leads to Rho kinase‐dependent formation of focal adhesion complexes. These data indicate that CCR5 internalization and recycling are regulated by actin polymerization and activation of small G proteins in a Rho‐dependent manner.
Mutational and computational analysis of the role of conserved residues in the active site of a family 18 chitinase
Authors: Synstad Bjørnar , Gåseidnes Sigrid , van Aalten Daan M. F. , Vriend Gert , Nielsen Jens E , Eijsink Vincent G. H.
Published: January 2004

Glycoside hydrolysis by retaining family 18 chitinases involves a catalytic acid (Glu) which is part of a conserved DXDXE sequence motif that spans strand four of a (βα)8 barrel (TIM barrel) structure. These glycoside hydrolases are unusual in that the positive charge emerging on the anomeric carbon after departure of the leaving group is stabilized by the substrate itself (the N‐acetyl group of the distorted −1 sugar), rather than by a carboxylate group on the enzyme. We have studied seven conserved residues in the catalytic center of chitinase B from Serratia marcescens. Putative roles for these residues are proposed on the basis of the observed mutational effects, the pH‐dependency of these effects, pKa calculations and available structural information. The results indicate that the pKa of the catalytic acid (Glu144) is ‘cycled’ during catalysis as a consequence of substrate‐binding and release and, possibly, by a back and forth movement of Asp142 between Asp140 and Glu144. Rotation of Asp142 towards Glu144 also contributes to an essential distortion of the N‐acetyl group of the −1 sugar. Two other conserved residues (Tyr10 and Ser93) are important because they stabilize the charge on Asp140 while Asp142 points towards Glu144. Asp215, lying opposite Glu144 on the other side of the scissile glycosidic bond, contributes to catalysis by promoting distortion of the −1 sugar and by increasing the pKa of the catalytic acid. The hydroxyl group of Tyr214 makes a major contribution to the positioning of the N‐acetyl group of the −1 sugar. Taken together, the results show that catalysis in family 18 chitinases depends on a relatively large number of (partly mobile) residues that interact with each other and the substrate.
Structural properties of the protein SV‐IV
Authors: Caporale Carlo , Caruso Carla , Colonna Giovanni , Facchiano Angelo , Ferranti Pasquale , Mamone Gianfranco , Picariello Gianluca , Colonna Flavia , Metafora Salvatore , Stiuso Paola
Published: January 2004

We have investigated the molecular mechanisms that produce different structural and functional behavior in the monomeric and trimeric forms of seminal vesicle protein no. 4, a protein with immunomodulatory, anti‐inflammatory, and procoagulant activity secreted from the rat seminal vesicle epithelium. The monomeric and trimeric forms were characterized in solution by CD. Details of the self‐association process and structural changes that accompany aggregation were investigated by different experimental approaches: trypsin proteolysis, sequence analysis, chemical modification, and computer modeling. The self‐association process induces conformational change mainly in the 1–70 region, which appears to be without secondary structure in the monomer but contains α‐helix in the trimer. In vivo, proteolysis of seminal vesicle protein no. 4 generates active peptides and this is affected by the monomer/trimer state, which is regulated by the concentration of the protein. The information obtained shows how conformational changes between the monomeric and trimeric forms represent a crucial aspect of activity modulation.
Localization, purification and properties of a tetrathionate hydrolase from Acidithiobacillus caldus
Authors: Bugaytsova Zhanna , Lindström E. Börje
Published: January 2004

The moderately thermophilic bacterium Acidithiobacillus caldus is found in bacterial populations in many bioleaching operations throughout the world. This bacterium oxidizes elemental sulfur and other reduced inorganic sulfur compounds as the sole source of energy. The purpose of this study was to purify and characterize the tetrathionate hydrolase of A. caldus. The enzyme was purified 16.7‐fold by one step chromatography using a SP Sepharose column. The purified enzyme resolved into a single band in 10% polyacrylamide gel, both under denaturing and native conditions. Its homogeneity was confirmed by N‐terminal amino acid sequencing. Tetrathionate hydrolase was shown to be a homodimer with a molecular mass of 103 kDa (composed from two 52 kDa monomers). The purified enzyme had optimum activity at pH 3.0 and 40 °C and an isoelectric point of 9.8. The periplasmic localization of the enzyme was determined by differential fractionation of A. caldus cells. Detected products of the tetrathionate hydrolase reaction were thiosulfate and pentathionate as confirmed by RP‐HPLC analysis. The activity of the purified enzyme was drastically enhanced by divalent metal ions.