bluuebunny/crossref_metadata_2025 huggingface dataset:
HtrA is the unique surface housekeeping protease in Lactococcus lactis and is required for natural protein processing
Authors: Poquet Isabelle , Saint Véronique , Seznec Erwan , Simoes Nathalie , Bolotin Alexander , Gruss Alexandra
Published: March 2003

We identified an exported protease in Lactococcus lactis ssp. lactis strain IL1403 belonging to the HtrA/DegP family. Inactivation of the chromosomal gene (htrALl) encoding this protease (HtrALl) results in growth thermo‐sensitivity at very high temperatures (above 37°C for L. lactis). The role of htrALl in extracellular proteolysis under normal growth conditions was examined by testing the stability of different exported proteins (i.e. fusions, a heterologous pre‐pro‐protein or a native protein containing repeats), having different locations. In the wild‐type (wt) strain, degradation products, including the C‐terminal protein ends, were present in the medium, indicating that proteolysis occurs during or after export to the cell surface; in one case, degradation was nearly total. In contrast, proteolysis was totally abolished in the htrA strain for all five proteins tested, and the yield of full‐length products was significantly increased. These results suggest that HtrALl is the sole extracellular protease that degrades abnormal exported proteins. In addition, our results reveal that HtrALl is needed for the pro‐peptide processing of a natural pro‐protein and for maturation of a native protein. We propose that in lactococci, and possibly in other Gram‐positive organisms with small sized‐genomes, a single surface protease, HtrA, is totally responsible for the housekeeping of exported proteins.
The putative iron transport system SitABCD encoded on SPI1 is required for full virulence of Salmonella typhimurium
Authors: Janakiraman Anuradha , Slauch James M.
Published: March 2003

Salmonella typhimurium is an invasive pathogen that causes diseases ranging from mild gastroenteritis to enteric fever. During the infection process, S. typhimurium induces a number of virulence genes required to circumvent host defences and/or acquire nutrients in the host. We have used the in vivo expression technology (IVET) system to select for S. typhimurium genes that are induced after invasion of a murine cultured cell line. We have characterized a putative iron transporter in Salmonella pathogenicity island 1, termed sitABCD. The sitABCD operon is induced under iron‐deficient conditions in vitro and is repressed by Fur. This locus is induced in the animal specifically after invasion of the intestinal epithelium. We show that a sit null mutant is significantly attenuated in BALB/c mice, suggesting that SitABCD plays an important role in iron acquisition in the animal.
The ChiA (YheB) protein of Escherichia coli K‐12 is an endochitinase whose gene is negatively controlled by the nucleoid‐structuring protein H‐NS
Authors: Francetic˜ Olivera , Badaut Cyril , Rimsky Sylvie , Pugsley Anthony P.
Published: March 2003

The chromosome of Escherichia coli K‐12 contains a putative gene, yheB (chiA), at centisome 74.7, whose product shows sequence similarity with chitinases of bacterial and viral origin. We cloned the chiA (yheB) gene and demonstrated that it codes for a 94.5 kDa periplasmic protein with endochitinase/lysozyme activity. Under standard laboratory growth conditions, chiA expression is very low, as shown by the Lac− phenotype of a chiA transcriptional fusion to a promoterless lacZ reporter. To identify factors that control chitinase gene expression, we generated random Tn10 insertions in the chromosome of the fusion‐containing strain, selecting for a Lac+ phenotype. The majority of the mutations that caused a Lac+ phenotype mapped to the hns gene, encoding the nucleoid‐structuring protein H‐NS. Transcription of chiA in vivo is driven by a single σ70 promoter and is derepressed in an hns mutant. Using a competitive gel retardation assay, we demonstrated that H‐NS binds directly and with high affinity to the chiA promoter region. In addition to hns, other E. coli mutations causing defects in global regulatory proteins, such as fis, crp or stpA in combination with hns, increased chiA expression to different extents, as did decreasing the growth temperature from 37°C to 30°C. A possible physiological function of ChiA (YheB) endochitinase in E. coli K‐12 is discussed.
Localization of the histidine kinase PilS to the poles of Pseudomonas aeruginosa and identification of a localization domain
Authors: Boyd Jessica M.
Published: March 2003

Transcription of the type IV pilus subunit gene of Pseudomonas aeruginosa is controlled by a two‐component signal transduction system. PilS, the histidine kinase, is membrane bound and PilR, its cognate response regulator, is cytoplasmic. The signal that activates PilS is unknown. PilS has three domains: (i) The N‐terminus, predicted to form six transmembrane (TM) helices; (ii) a central linker domain; and (iii) the C‐terminal transmitter domain containing all the conserved residues of sensor kinases. A translational fusion of the gfp gene (green fluorescent protein) to the 3′ end of pilS was used to determine the position of PilS in the bacterial cell. Epifluorescence microscopy revealed that PilS is retained to the poles of P. aeruginosa but is distributed evenly about the membrane of Escherichia coli. Deletions of the PilS–GFP fusion revealed that the TM domain was sufficient and necessary to bring GFP to the membrane of P. aeruginosa and E. coli but was not sufficient to confine GFP to the poles. Retention to the poles of P. aeruginosa required both the TM and linker domains. Replacement of the PilS TM domain with an E. coli membrane protein, MalG, still allowed polar localization. Therefore, the PilS TM domain positions the linker domain close to the membrane allowing it to interact with the putative polar anchor which is specific to P. aeruginosa.
Dual regulation of mucoidy in Pseudomonas aeruginosa and sigma factor antagonism
Authors: Boucher J. C. , Schurr M. J. , Deretic V.
Published: March 2003

The conversion to mucoid, exopolysaccharide alginate‐overproducing phenotype in Pseudomonas aeruginosa during chronic respiratory infections in cystic fibrosis patients occurs via mutations that activate the alternative sigma factor AlgU (σE). In this study, we demonstrate that conversion to mucoidy can be caused via a second, algU‐independent pathway, in which alginate production and transcription of the critical algD promoter depend on another alternative σ factor, RpoN (σ54). The algD promoters dependent on σ54 and σE showed a complete overlap resulting in identical mRNA 5′ ends. The two pathways were not independent, as σ54 also repressed σE‐dependent transcription of algD both in vitro and in vivo. The negative regulatory effect of σ54 on σE‐dependent algD expression was based on σ54 binding to the algD promoter and its interference with σE‐dependent transcription. This phenomenon, referred to here as σ factor antagonism, reflects the unique properties of σ54, which lacks an intrinsic ability to form open transcription initiation complexes. We propose that this peculiar feature of σ54 has evolved in part to allow its recruitment as a repressor of certain promoter subsets. The repression of algD by σ54 also depends on environmental conditions, supporting the notion that σ factor antagonism plays a physiological role in controlling alginate production in P. aeruginosa during adaptation to different ecological sites (e.g. biofilm development, stress and other growth conditions) and unique environments in the chronically infected host.
SC3 and SC4 hydrophobins have distinct roles in formation of aerial structures in dikaryons of Schizophyllum commune
Authors: Van Wetter Marie‐Anne , Wösten Han A. B. , Wessels Joseph G. H.
Published: March 2003

Two monokaryons of Schizophyllum commune can form a fertile dikaryon when the mating‐type genes differ. Monokaryons form sterile aerial hyphae, while dikaryons also form fruiting bodies that function in sexual reproduction. The SC3 hydrophobin gene is expressed both in monokaryons and in dikaryons. The SC4 hydrophobin is dikaryon specific. In the monokaryon, SC3 lowers the water surface tension, coats aerial hyphae with a hydrophobic layer and mediates attachment of hyphae to hydrophobic surfaces. The SC4 protein lines gas channels within fruiting bodies with a hydrophobic membrane. Using gene disruptions, in this study, we show that in dikaryons SC3 fulfils the same roles as in monokaryons. SC4, on the other hand, has a role within fruiting bodies. In contrast to gas channels in fruiting bodies of the wild type, those of a ΔSC4 strain easily filled with water. Thus, SC4 prevents gas channels filling with water under wet conditions, probably serving uninterrupted gas exchange. Other dikaryon‐specific hydrophobin genes, SC1 and SC6, apparently do not substitute for the SC4 gene. In addition, by expressing the SC4 gene behind the SC3 promoter in a ΔSC3 monokaryon, it was shown that SC4 cannot fully substitute for SC3, indicating that both hydrophobins evolved to fulfil specific functions.
A novel autoregulation mechanism of fnrN expression in Rhizobium leguminosarum bv viciae
Authors: Colombo María Victoria , Gutiérrez Delia , Palacios José Manuel , Imperial Juan , Ruiz‐Argüeso Tomás
Published: March 2003

The fnrN gene from Rhizobium leguminosarum UPM791 controls microaerobic expression of both nitrogen fixation and hydrogenase activities in symbiotic cells. Two copies of fnrN are present in this strain, one chromosomal (fnrN1) and the other located in the symbiotic plasmid (fnrN2). Their expression was studied by cloning the regulatory regions in lacZ promoter‐probe vectors. The fnrN genes were found to be autoregulated: they are expressed only at basal levels under aerobic conditions; they are highly expressed under microaerobic conditions; and they are expressed at basal levels in the double mutant DG2 (fnrN1 fnrN2) under any condition. The promoters of both genes contain two FnrN‐binding sequences (anaeroboxes), centred at positions −12.5 (proximal anaerobox) and −44.5 (distal anaerobox). Expression analysis and gel retardation experiments with fnrN1‐derivative promoter mutants altered in key bases of the anaerobox sequences demonstrated that binding of FnrN1 to the distal anaerobox is necessary for microaerobic activation of transcription, and that binding of FnrN1 to the proximal anaerobox results in transcriptional repression. The apparent affinity of FnrN1 for the proximal anaerobox was fivefold lower than for the distal anaerobox, resulting in repression of transcription of fnrN1 only at high‐FnrN1 concentrations. This positive and negative autoregulation mechanism ensures an equilibrated expression of fnrN in response to microaerobic conditions.
Increased sensitivity to the antimalarials mefloquine and artemisinin is conferred by mutations in the pfmdr1 gene of Plasmodium falciparum
Authors: Duraisingh Manoj T. , Roper Cally , Walliker David , Warhurst David C.
Published: March 2003

The declining efficacy of chloroquine and pyrimethamine/sulphadoxine in the treatment of human malaria has led to the use of newer antimalarials such as mefloquine and artemisinin. Sequence polymorphisms in the pfmdr1 gene, the gene encoding the plasmodial homologue of mammalian multidrug resistance transporters, have previously been linked to resistance to chloroquine in some, but not all, studies. In this study, we have used a genetic cross between the strains HB3 and 3D7 to study inheritance of sensitivity to the structurally unrelated drugs mefloquine and artemisinin, and to several other antimalarials. We find a complete allelic association between the HB3‐like pfmdr1 allele and increased sensitivity to these drugs in the progeny. Different pfmdr1 sequence polymorphisms in other unrelated lines were also associated with increased sensitivity to these drugs. Our results indicate that the pfmdr1 gene is an important determinant of susceptibility to antimalarials, which has major implications for the future development of resistance.
Oligomerization of the chromatin‐structuring protein H‐NS
Authors: Smyth Clare P. , Lundbäck Thomas , Renzoni Debora , Siligardi Giuliano , Beavil Rebecca , Layton Meredith , Sidebotham Julie M. , Hinton Jay C. D. , Driscoll Paul C. , Higgins Christopher F. , Ladbury John E.
Published: March 2003

H‐NS is a major component of the bacterial nucleoid, involved in condensing and packaging DNA and modulating gene expression. The mechanism by which this is achieved remains unclear. Genetic data show that the biological properties of H‐NS are influenced by its oligomerization properties. We have applied a variety of biophysical techniques to study the structural basis of oligomerization of the H‐NS protein from Salmonella typhimurium. The N‐terminal 89 amino acids are responsible for oligomerization. The first 64 residues form a trimer dominated by an α‐helix, likely to be in coiled–coil conformation. Extending this polypeptide to 89 amino acids generated higher order, heterodisperse oligomers. Similarly, in the full‐length protein no single, defined oligomeric state is adopted. The C‐terminal 48 residues do not participate in oligomerization and form a monomeric, DNA‐binding domain. These N‐ and C‐terminal domains are joined via a flexible linker which enables them to function independently within the context of the full‐length protein. This novel mode of oligomerization may account for the unusual binding properties of H‐NS.
Sialic acid metabolism's dual function in Haemophilus influenzae
Authors: Vimr Eric , Lichtensteiger Carol , Steenbergen Susan
Published: March 2003

Many bacterial commensals and pathogens use the sialic acids as carbon and nitrogen sources. In Escherichia coli, the breakdown of these sugars is catalysed by gene products of the nan (Nacylneuraminate) operon; other microorganisms may use a similar catabolic strategy. Despite the known ligand and antirecognition functions of the sialic acids, the contribution of their catabolism to infection or host colonization has never been directly investigated. We addressed these questions with Haemophilus influenzae type b, which metabolizes relatively few carbohydrates, using the infant‐rat infection model. The predicted H. influenzae homologue (HI0142) of the E. coli sialic acid aldolase structural gene, nanA, was subcloned and mutagenized by insertion of a kanamycin resistance cassette. Phenotypic investigation of the resulting H. influenzae aldolase mutants showed that: (i) HI0142 is essential for sialic acid degradation; (ii) the products of the open reading frames (ORFs) flanking HI0142 (HI0140, 41, 44 and 45) are likely to have the same functions as those of their counterparts in E. coli; (iii) sialylation of the lipooligosaccharide (LOS) epitope recognized by monoclonal antibody 3F11 is dependent on an environmental source of sialic acid; (iv) a nanA mutant hypersialylates its LOS sialyl acceptor, corresponding to an apparent increased fitness of the mutant in the infant‐rat model; and (v) expression of the LOS sialyl acceptor is altered in cells grown without exogenous sialic acid, indicating the direct or indirect effect of sialic acid metabolism on LOS antigenicity. Taken together the data show the dual role of sialic acid catabolism in nutrition and cell surface modulation.
Morphogenesis in Aspergillus nidulans requires Dopey (DopA), a member of a novel family of leucine zipper‐like proteins conserved from yeast to humans
Authors: Pascon Renata Castiglioni , Miller Bruce L.
Published: March 2003

DopA is the founding member of a novel protein family required for correct cell morphology and spatiotemporal organization of multicellular structures in the filamentous fungus Aspergillus nidulans. DopA homologues from Saccharomyces cerevisiae (Dop1), Candida albicans, Caenorhabditis elegans, Rattus norvegicus and Homo sapiens have been identified from genome sequencing projects. S. cerevisiae DOP1 is essential for viability and, like DopA, affects cellular morphogenesis. dopA encodes a large protein (207 kDa) containing several putative domains, including three leucine zipper‐like domains. Strains with either the temperature‐sensitive dopA1ts allele, which alters one of the leucine zippers, or the null ΔdopA allele, had abnormal morphology of the vegetative hyphae, delayed and asynchronous initiation of asexual development, aberrant morphogenesis of the conidiophore and an early block in the sexual cycle. The expression patterns of key transcriptional regulators of the asexual and sexual cycle (brlA, abaA and steA) are altered in a ΔdopA background, suggesting that DopA functions upstream in the developmental pathway. Double mutant analysis showed that dopA interacts genetically with constitutively active and inactive forms of A. nidulans Aras to modulate hyphal morphogenesis and asexual development.
Fusidic acid‐resistant EF‐G perturbs the accumulation of ppGpp
Authors: Macvanin Mirjana , Johanson Urban , Ehrenberg Måns , Hughes Diarmaid
Published: March 2003

Reductions in growth rate caused by fusidic acid‐resistant EF‐G mutants in Salmonella typhimurium correlate strongly with increased mean cell size. This is unusual because growth rate and cell size normally correlate positively. The global transcription regulator molecule ppGpp has a role in co‐ordinating growth rate and division, and its basal level normally correlates inversely with cell size at division. We show that fusidic acid‐resistant EF‐G mutants have perturbed ppGpp basal levels during steady‐state growth and perturbed induced levels during starvation. One mutation, fusA1, associated with the slowest growth rate and largest cell size, causes a reduction in the basal level of ppGpp to one‐third of that found in the wild‐type strain. Other fusA mutants with intermediate or wild‐type growth rates and cell sizes have either normal or increased basal levels of ppGpp. There is an inverse relationship between the basal level of ppGpp in vivo and the degree to which translation dependent on mutant EF‐G is inhibited by ppGpp in vitro. This enhanced interaction between mutant EF‐G and ppGpp correlates with an increased KM for GTP. Our results suggest that mutant EF‐G modulates the production of ppGpp by the RelA (PSI) pathway. In conclusion, fusidic acid‐resistant EF‐G mutations alter the level of ppGpp and break the normal relationship between growth rate and cell size at division. It would not be surprising if other phenotypes associated with these mutants, such as loss of virulence, were also related to perturbations in ppGpp levels effected through altered transcription patterns.
Pathways for the utilization of N‐acetyl‐galactosamine and galactosamine in Escherichia coli
Authors: Brinkkötter Andreas , Klöß Holger , Alpert C.‐A. , Lengeler Joseph W.
Published: March 2003

Among enteric bacteria, the ability to grow on N‐acetyl‐galactosamine (GalNAc or Aga) and on d‐galactosamine (GalN or Gam) differs. Thus, strains B, C and EC3132 of Escherichia coli are Aga+ Gam+ whereas E. coli K‐12 is Aga− Gam−, similarly to Klebsiella pneumoniae KAY2026, Klebsiella oxytoca M5a1 and Salmonella typhimurium LT2. The former strains carry a complete aga/kba gene cluster at 70.5 min of their gene map. These genes encode an Aga‐specific phosphotransferase system (PTS)or IIAga (agaVWE) and a GalN‐specific PTS or IIGam (agaBCD). Both PTSs belong to the mannose–sorbose family, i.e. the IIB, IIC and IID domains are encoded by different genes, and they share a IIA domain (agaF). Furthermore, the genes encode an Aga6P‐deacetylase (agaA), a GalN6P deaminase (agaI), a tagatose‐bisphosphate aldolase comprising two different peptides (kbaYZ) and a putative isomerase (agaS), i.e. complete pathways for the transport and degradation of both amino sugars. The genes are organized in two adjacent operons (kbaZagaVWEFA and agaS kbaYagaBCDI) and controlled by a repressor AgaR. Its gene agaR is located upstream of kbaZ, and AgaR responds to GalNAc and GalN in the medium. All Aga− Gam− strains, however, carry a deletion covering genes agaW′EF′A; consequently they lack active IIAga and IIGam PTSs, thus explaining their inability to grow on the two amino sugars. Remnants of a putative recombination site flank the deleted DNA in the various Aga− Gam− enteric bacteria. Derivatives with an Aga+ Gam− phenotype can be isolated from E. coli K‐12. These retain the ΔagaW′EF′A deletion and carry suppressor mutations in the gat and nag genes for galactitol and N‐acetyl‐glucosamine metabolism, respectively, that allow growth on Aga but not on GalN.
LcrQ/YscM1, regulators of the Yersinia yop virulon, are injected into host cells by a chaperone‐dependent mechanism
Authors: Cambronne Eric D. , Cheng Luisa W. , Schneewind Olaf
Published: March 2003

Pathogenic Yersinia species employ type III machines to secrete YopBDR into the extracellular milieu. After attaching to host cells, yersiniae transform the type III machinery into an injection device and target YopEHMNOPT into eukaryotic cells. Yersinia pseudotuberculosis LcrQ is a transcriptional regulator that prevents the expression of yop genes. We report that LcrQ is injected into eukaryotic cells. YscM1, the transciptional regulator of Yersinia enterocolitica, is also injected into eukaryotic cells, whereas the related YscM2 protein remains associated with bacterial cells. Type III targeting of YscM1 requires binding to the SycH chaperone. Chaperone binding as well as depletion of YscM1 and YscM2 from the cytoplasm of Y. enterocolitica causes an increase in yop expression, whereas a block in regulator export reduces expression. We propose a model whereby the chaperone‐mediated injection of LcrQ/YscM1 functions as a regulatory switch for bacteria that are attached to host cells, triggering the expression of Yops that travel the type III targeting pathway.
Control of plasmid DNA replication by iterons: no longer paradoxical
Authors: Chattoraj Dhruba K.
Published: March 2003

Replication origins of a family of bacterial plasmids have multiple sites, called iterons, for binding a plasmid‐specific replication initiator protein. The iteron–initiator interactions are essential for plasmid replication as well as for inhibition of plasmid over‐replication. The inhibition increases with plasmid copy number and eventually shuts plasmid replication off completely. The mechanism of inhibition appears to be handcuffing, the coupling of origins via iteron‐bound initiators that block origin function. The probability of a trans‐reaction such as handcuffing is expected to increase with plasmid copy number and diminish with increases in cell volume, explaining how the copy number can be maintained in a growing cell. Control is also exerted at the level of initiator synthesis and activation by chaperones. We propose that increases in active initiators promote initiation by overcoming handcuffing, but handcuffing dominates when the copy number reaches a threshold. Handcuffing should be ultrasensitive to copy number, as the negative control by iterons can be stringent (switch‐like).
Bacteriophage PRD1 DNA entry uses a viral membrane‐associated transglycosylase activity
Authors: Rydman Pia S. , Bamford Dennis H.
Published: March 2003

Amino acid sequence analyses have indicated that the amino‐terminal part of bacteriophage PRD1 structural protein P7 carries a conserved transglycosylase domain. We analysed wild‐type PRD1 and different mutant particles in zymograms and found a glycolytic activity that was associated with protein P7. This is the first time a putative bacteriophage or plasmid lytic transglycosylase has been shown to have an enzymatic activity. In the absence of protein P7, the phage DNA replication and host cell lysis were delayed. Gene VII of PRD1 is known to encode proteins P7 and P14. In this investigation, the open reading frame coding for P14 was mapped to the 3′ end of gene VII. Proteins P7 and P14 probably form a heteromultimeric complex, which is located at the particle vertices and is involved in the early steps of the PRD1 life cycle
Genetic analysis of the strong gyrase site (SGS) of bacteriophage Mu: localization of determinants required for promoting Mu replication
Authors: Pato M. L. , Banerjee M.
Published: March 2003

The Mu strong gyrase site (SGS), located in the centre of the Mu genome, is required for efficient Mu replication, as it promotes synapsis of the prophage termini. Other gyrase sites tested, even very strong ones, were unable to substitute for the SGS in Mu replication. To determine the features required for its unique properties, a deletion analysis was performed on the SGS. For this analysis, we defined the 20 bp centred on the midpoint of the 4 bp staggered cleavage made by gyrase to be the ‘core’ and the flanking sequences to be the ‘arms’. The deletion analysis showed that (i) ≈ 40 bp of the right arm is required, in addition to core sequences, for both efficient Mu replication and gyrase cleavage; and (ii) the left arm was not required for efficient Mu replication, although it was required for efficient gyrase cleavage. These observations implicated the right arm as the unique feature of the SGS. The second observation showed that strong gyrase cleavage and Mu replication could be dissociated and suggested that even weak gyrase sites, if supplied with the right arm of the SGS, could promote Mu replication. Hybrid sites were constructed with gyrase sites that could not support efficient Mu replication. The SGS right arm was used to replace one arm of the strong pSC101 gyrase site or the weaker pBR322 site. The pSC101 hybrid site allowed efficient Mu replication, whereas the pBR322 hybrid site allowed substantial, but reduced, replication. Hence, it appears that optimal Mu replication requires a central strong gyrase site with the properties imparted by the right arm sequences. Possible roles for the SGS right arm in Mu replication are addressed.
Social motility in Myxococcus xanthus requires FrzS, a protein with an extensive coiled‐coil domain
Authors: Ward Mandy J. , Lew Helen , Zusman David R.
Published: March 2003

Gliding motility in the developmental bacterium Myxococcus xanthus involves two genetically distinct motility systems, designated adventurous (A) and social (S). Directed motility responses, which facilitate both vegetative swarming and developmental aggregation, additionally require the ‘frizzy’ (Frz) signal transduction pathway. In this study, we have analysed a new gene (frzS), which is positioned upstream of the frzA–F operon. Insertion mutations in frzS caused both vegetative spreading and developmental defects, including ‘frizzy’ aggregates in the FB strain background. The ‘frizzy’ phenotype was previously considered to result only from defective directed motility responses. However, deletion of the frzS gene in an A−S+ motility background demonstrated that FrzS is a new component of the S‐motility system, as the A−frzS double mutant was non‐spreading (A−S−). Compared with known S‐motility mutants, the frzS mutants appear similar to pilT mutants, in that both produce type IV pili, extracellular fibrils and lipopolysaccharide (LPS) O‐antigen, and both agglutinate rapidly in a cohesion assay. The FrzS protein has an unusual domain composition for a bacterial protein. The N‐terminal domain shows similarity to the receiver domains of the two‐component response regulator proteins. The C‐terminal domain is composed of up to 38 heptad repeats (a b c d e f g)38, in which residues at positions a and d are predominantly hydrophobic, whereas residues at positions e and g are predominantly charged. This periodic disposition of specific residues suggests that the domain forms a long coiled‐coil structure, similar to those found in the α‐fibrous proteins, such as myosin. Overexpression of this domain in Escherichia coli resulted in the formation of an unusual striated protein lattice that filled the cells. We speculate on the role that this novel protein could play in gliding motility.
Aromatic components of two ferric enterobactin binding sites in Escherichia coli FepA
Authors: Cao Zhenghua , Qi Zengbiao , Sprencel Cathy , Newton Salete M. C. , Klebba Phillip E.
Published: March 2003

Ferric enterobactin is a catecholate siderophore that binds with high affinity (Kd ≈ 10−10 M) to the Escherichia coli outer membrane protein FepA. We studied the involvement of aromatic amino acids in its uptake by determining the binding affinities, kinetics and transport properties of site‐directed mutants. We replaced seven aromatic residues (Y260, Y272, Y285, Y289, W297, Y309 and F329) in the central part of FepA primary structure with alanine, individually and in double combinations, and determined the ability of the mutant proteins to interact with ferric enterobactin and the protein toxins colicins B and D. All the constructs showed normal expression and localization. Among single mutants, Y260A and F329A were most detrimental, reducing the affinity between FepA and ferric enterobactin 100‐ and 10‐fold respectively. Double substitutions involving Y260, Y272 and F329 impaired (100‐ to 2500‐fold) adsorption of the iron chelate more strongly. For Y260A and Y272A, the drop in adsorption affinity caused commensurate decreases in transport efficiency, suggesting that the target residues primarily act in ligand binding. F329A, like R316A, showed greater impairment of transport than binding, intimating mechanistic involvement during ligand internalization. Furthermore, immunochemical studies localized F329 in the FepA ligand binding site. The mutagenesis results suggested the existence of dual ligand binding sites in the FepA vestibule, and measurements of the rate of ferric enterobactin adsorption to fluoresceinated FepA mutant proteins confirmed this conclusion. The initial, outermost site contains aromatic residues and probably functions through hydrophobic interactions, whereas the secondary site exists deeper in the vestibule, contains both charged and aromatic residues and probably acts through hydrophobic and electrostatic bonds.
Disruption of the F plasmid partition complex in vivo by partition protein SopA
Authors: Lemonnier M. , Bouet J.‐Y. , Libante V. , Lane D.
Published: March 2003

The SopA protein plays an essential, though so far undefined, role in partition of the mini‐F plasmid but, when overproduced, it causes loss of mini‐F from growing cells. Our investigation of this phenomenon has revealed that excess SopA protein reduces the linking number of mini‐F. It appears to do so by disturbing the partition complex, in which SopB normally introduces local positive supercoiling upon binding to the sopC centromere, as it occurs only in plasmids carrying sopC and in the presence of SopB protein. SopA‐induced reduction in linking number is not associated with altered sop promoter activity or levels of SopB protein and occurs in the absence of changes in overall supercoil density. SopA protein mutated in the ATPase nucleotide‐binding site (K120Q) or lacking the presumed SopB interaction domain does not induce the reduction in linking number, suggesting that excess SopA disrupts the partition complex by interacting with SopB to remove positive supercoils in an ATP‐dependent manner. Destabilization of mini‐F also depends on sopC and SopB, but the K120Q mutant retains some capacity for destabilizing mini‐F. SopA‐induced destabilization thus appears to be complex and may involve more than one SopA activity. The results are interpreted in terms of a regulatory role for SopA in the oligomerization of SopB dimers bound to the centromere.
RuvABC‐dependent double‐strand breaks in dnaBts mutants require RecA
Authors: Seigneur Marie , Ehrlich S. Dusko , Michel Bénédicte
Published: March 2003

Replication fork arrest can cause DNA double‐strand breaks (DSBs). These DSBs are caused by the action of the Holliday junction resolvase RuvABC, indicating that they are made by resolution of Holliday junctions formed at blocked forks. In this work, we study the homologous recombination functions required for RuvABC‐mediated breakage in cells deficient for the accessory replicative helicase Rep or deficient for the main Escherichia coli replicative helicase DnaB. We show that, in the rep mutant, RuvABC‐mediated breakage occurs in the absence of the homologous recombination protein RecA. In contrast, in dnaBts mutants, most of the RuvABC‐mediated breakage depends on the presence of RecA, which suggests that RecA participates in the formation of Holliday junctions at forks blocked by the inactivation of DnaB. This action of RecA does not involve the induction of the SOS response and does not require any of the recombination proteins essential for the presynaptic step of homologous recombination, RecBCD, RecF or RecO. Consequently, our observations suggest a new function for RecA at blocked replication forks, and we propose that RecA acts by promoting homologous recombination without the assistance of known presynaptic proteins.
A novel NO‐responding regulator controls the reduction of nitric oxide in Ralstonia eutropha
Authors: Pohlmann Anne , Cramm Rainer , Schmelz Karin , Friedrich Bärbel
Published: March 2003

Ralstonia eutropha H16 mediates the reduction of nitric oxide (NO) to nitrous oxide (N2O) with two isofunctional single component membrane‐bound NO reductases (NorB1 and NorB2). This reaction is integrated into the denitrification pathway that involves the successive reduction of nitrate to dinitrogen. The norB1 gene is co‐transcribed with norA1 from a σ54 (RpoN)‐dependent promoter, located upstream of norA1. With the aid of norA1′–lacZ transcriptional fusions and the generation of regulatory mutants, it was shown that norB1 gene transcription requires a functional rpoN gene and the regulator NorR, a novel member of the NtrC family of response regulators. The regulator gene maps adjacent to norAB, is divergently transcribed and present in two copies on the megaplasmid pHG1 (norR1) and the chromosome (norR2). Transcription activation by NorR responds to the availability of NO. A nitrite reductase‐deficient mutant that is incapable of producing NO endogenously, showed a 70% decrease of norA1 expression. Addition of the NO‐donating agent sodium nitroprusside caused induction of norA1′–lacZ transcription. Truncation of the N‐terminal receiver domain of NorR1 interrupted the NO signal transduction and led to a constitutive expression of norA1′–lacZ. The results indicate that NorR controls the reductive conversion of NO in R. eutropha. This reaction is not strictly co‐ordinated on the regulatory level with the other nitrogen oxide‐reducing steps of the denitrification chain that are independent of NorR.
Novel fungal transcriptional activators, Cmr1p of Colletotrichum lagenarium and Pig1p of Magnaporthe grisea, contain Cys2His2 zinc finger and Zn(II)2Cys6 binuclear cluster DNA‐binding motifs and regulate transcription of melanin biosynthesis genes in a developmentally specific manner
Authors: Tsuji Gento , Kenmochi Youki , Takano Yoshitaka , Sweigard James , Farrall Leonard , Furusawa Iwao , Horino Osamu , Kubo Yasuyuki
Published: March 2003

Colletotrichum lagenarium and Magnaporthe grisea are plant pathogenic fungi that produce melanin during the appressorial differentiation stage of conidial germination and during the late stationary phase of mycelial growth. Here, we report the identification of genes for two unique transcription factors, CMR1 (Colletotrichummelanin regulation) and PIG1 (pigment of Magnaporthe), that are involved in melanin biosynthesis. Both Cmr1p and Pig1p contain two distinct DNA‐binding motifs, a Cys2His2 zinc finger motif and a Zn(II)2Cys6 binuclear cluster motif. The presence of both these motifs in a single transcriptional regulatory protein is unique among known eukaryotic transcription factors. Deletion of CMR1 in C. lagenarium caused a defect in mycelial melanization, but not in appressorial melanization. Also, cmr1Δ mutants do not express the melanin biosynthetic structural genes SCD1 and THR1 during mycelial melanization, although the expression of these two genes was not affected during appressorial melanization.
The psp locus of Yersinia enterocolitica is required for virulence and for growth in vitro when the Ysc type III secretion system is produced
Authors: Darwin Andrew J. , Miller Virginia L.
Published: March 2003

The phage shock protein locus (pspFpspABCDE) of Escherichia coli has proved to be something of an enigma since its discovery. The physiological functions of the psp locus, including those of the predicted effector protein PspA, are unknown. In a previous genetic screen, we determined that a Yersinia enterocolitica pspC mutant was severely attenuated for virulence. In this study, the psp locus of Y. enterocolitica was characterized further. The pspC gene of Y. enterocolitica was found to be important for normal growth when the Ysc type III secretion system was expressed in the laboratory. This growth defect was specifically caused by production of the secretin protein, YscC. Expression of the psp genes was induced when the type III secretion system was functional or when only the yscC gene was expressed. This induction of psp gene expression required a functional pspC gene. Most significantly, evidence suggests that the expression of at least one gene that is not part of the psp locus is regulated by Psp proteins. This unidentified gene (or genes) may also be important for growth when the type III secretion system is expressed. These conclusions are supported by the effects of various psp mutations on virulence. This is the first indication that Psp proteins might be involved in the regulation of genes besides the psp locus itself.
Requirement of nickel metabolism proteins HypA and HypB for full activity of both hydrogenase and urease in Helicobacter pylori
Authors: Olson Jonathan W. , Mehta Nalini S. , Maier Robert J.
Published: March 2003

The nickel‐containing enzymes hydrogenase and urease require accessory proteins in order to incorporate properly the nickel atom(s) into the active sites. The Helicobacter pylori genome contains the full complement of both urease and hydrogenase accessory proteins. Two of these, the hydrogenase accessory proteins HypA (encoded by hypA) and HypB (encoded by hypB), are required for the full activity of both the hydrogenase and the urease enzymes in H. pylori. Under normal growth conditions, hydrogenase activity is abolished in strains in which either hypA (HypA:kan) or hypB (HypB:kan) have been interrupted by a kanamycin resistance cassette. Urease activity in these strains is 40 (HypA:kan)‐ and 200 (HypB:kan)‐fold lower than for the wild‐type (wt) strain 43504. Nickel supplementation in the growth media restored urease activity to almost wt levels. Hydrogenase activity was restored to a lesser extent, as has been observed for hyp mutants in other (H2‐oxidizing) bacteria. Expression levels of UreB (the urease large subunit) were not affected by inactivation of either hypA or hypB, as determined by immunoblotting. Urease activity was not affected by lesions in the genes for either the hydrogenase accessory proteins HypD or HypF or the hydrogenase large subunit structural gene, indicating that the urease deficiency was not caused by lack of hydrogenase activity. When crude extracts of wt, HypA:kan and HypB:kan were separated by anion exchange chromatography, the urease‐containing fractions of the mutant strains contained about four (HypA:kan)‐ and five (HypB:kan)‐fold less nickel than did the urease from wt, indicating that the lack of urease activity in these strains results from a nickel deficiency in the urease enzyme.
Role of the glutathione/glutaredoxin and thioredoxin systems in yeast growth and response to stress conditions
Authors: Grant Chris M.
Published: March 2003

Sulphydryl groups (‐SH) play a remarkably broad range of roles in the cell, and the redox status of cysteine residues can affect both the structure and the function of numerous enzymes, receptors and transcription factors. The intracellular milieu is usually a reducing environment as a result of high concentrations of the low‐molecular‐weight thiol glutathione (GSH). However, reactive oxygen species (ROS), which are the products of normal aerobic metabolism, as well as naturally occurring free radical‐generating compounds, can alter this redox balance. A number of cellular factors have been implicated in the regulation of redox homeostasis, including the glutathione/glutaredoxin and thioredoxin systems. Glutaredoxins and thioredoxins are ubiquitous small heat‐stable oxidoreductases that have proposed functions in many cellular processes, including deoxyribonucleotide synthesis, repair of oxidatively damaged proteins, protein folding and sulphur metabolism. This review describes recent findings in the lower eukaryote Saccharomyces cerevisiae that are leading to a better understanding of their role in redox homeostasis in eukaryotic cell metabolism.
Sugar transport in Sulfolobus solfataricus is mediated by two families of binding protein‐dependent ABC transporters
Authors: Elferink Marieke G. L. , Albers Sonja‐V. , Konings Wil N. , Driessen Arnold J. M.
Published: March 2003

The extreme thermoacidophilic archaeon Sulfolobus solfataricus grows optimally at 80°C and pH 3 and uses a variety of sugars as sole carbon and energy source. Glucose transport in this organism is mediated by a high‐affinity binding protein‐dependent ATP‐binding cassette (ABC) transporter. Sugar‐binding studies revealed the presence of four additional membrane‐bound binding proteins for arabinose, cellobiose, maltose and trehalose. These glycosylated binding proteins are subunits of ABC transporters that fall into two distinct groups: (i) monosaccharide transporters that are homologous to the sugar transport family containing a single ATPase and a periplasmic‐binding protein that is processed at an unusual site at its amino‐terminus; (ii) di‐ and oligosaccharide transporters, which are homologous to the family of oligo/dipeptide transporters that contain two different ATPases, and a binding protein that is synthesized with a typical bacterial signal sequence. The latter family has not been implicated in sugar transport before. These data indicate that binding protein‐dependent transport is the predominant mechanism of transport for sugars in S. solfataricus.
Chs1 of Candida albicans is an essential chitin synthase required for synthesis of the septum and for cell integrity
Authors: Munro Carol A. , Winter Ken , Buchan Arlene , Henry Keith , Becker Jeffrey M. , Brown Alistair J. P. , Bulawa Christine E. , Gow Neil A. R.
Published: March 2003

CaCHS1 of the fungal pathogen Candida albicans encodes an essential chitin synthase that is required for septum formation, viability, cell shape and integrity. The CaCHS1 gene was inactivated by first disrupting one allele using the ura‐blaster protocol, then placing the remaining allele under the control of the maltose‐inducible, glucose‐repressible MRP1 promoter. Under repressing conditions, yeast cell growth continued temporarily, but daughter buds failed to detach from parents, resulting in septumless chains of cells with constrictions defining contiguous compartments. After several generations, a proportion of the distal compartments lysed. The conditional Δchs1 mutant also failed to form primary septa in hyphae; after several generations, growth stopped, and hyphae developed swollen balloon‐like features or lysed at one of a number of sites including the hyphal apex and other locations that would not normally be associated with septum formation. CHS1 therefore synthesizes the septum of both yeast and hyphae and also maintains the integrity of the lateral cell wall. The conditional mutant was avirulent under repressing conditions in an experimental model of systemic infection. Because this gene is essential in vitro and in vivo and is not present in humans, it represents an attractive target for the development of antifungal compounds.
Cell cycle regulation in the hyperthermophilic crenarchaeon Sulfolobus acidocaldarius
Authors: Hjort Karin , Bernander Rolf
Published: March 2003

The regulation and co‐ordination of the cell cycle of the hyperthermophilic crenarchaeon Sulfolobus acidocaldarius was investigated with antibiotics. We provide evidence for a core regulation involving alternating rounds of chromosome replication and genome segregation. In contrast, multiple rounds of replication of the chromosome could occur in the absence of an intervening cell division event. Inhibition of the elongation stage of chromosome replication resulted in cell division arrest, indicating that pathways similar to checkpoint mechanisms in eukaryotes, and the SOS system of bacteria, also exist in archaea. Several antibiotics induced cell cycle arrest in the G2 stage. Analysis of the run‐out kinetics of chromosome replication during the treatments allowed estimation of the minimal rate of replication fork movement in vivo to 250 bp s−1. An efficient method for the production of synchronized Sulfolobus populations by transient daunomycin treatment is presented, providing opportunities for studies of cell cycle‐specific events. Possible targets for the antibiotics are discussed, including topoisomerases and protein glycosylation.
Biogenesis of inner membrane proteins in Escherichia coli
Authors: De Gier Jan‐Willem , Luirink Joen
Published: March 2003

For a long time, it was generally assumed that the biogenesis of inner membrane proteins in Escherichia coli occurs spontaneously, and that only the translocation of large periplasmic domains requires the aid of a protein machinery, the Sec translocon. However, evidence obtained in recent years indicates that most, if not all, inner membrane proteins require the assistance of protein factors to reach their native conformation in the membrane. Here, we review and discuss recent advances in our understanding of the biogenesis of inner membrane proteins in E. coli.
Molecular analysis of the pRA2 partitioning region: ParB autoregulates parAB transcription and forms a nucleoprotein complex with the plasmid partition site, parS
Authors: Kwong Stephen M. , Yeo Chew Chieng , Poh Chit Laa
Published: March 2003

The partitioning locus (par) of plasmid pRA2 belongs to a recently discovered subgroup of plasmid partitioning systems that are evolutionarily distinct from the P1, F and R1/NR1 prototypes. The pRA2 par region was effective in stabilizing both pRA2 and F mini‐replicons. Analysis of the nucleotide sequence revealed three potential coding regions that were designated parA, parB and parC. Through mutagenesis, parA and parB were found to be essential for partitioning function, whereas parC did not appear to be required. Using transcriptional reporter systems, it was demonstrated in vivo that ParB repressed par promoter activity by 60‐fold and that ParA had little effect on transcriptional activity. Primer extension analysis revealed that the par transcriptional start point was located 47 nucleotides upstream of the parA translational start codon. Based on this information, putative −10 and −35 transcriptional signals were identified, and their subsequent deletion resulted in a dramatic reduction in promoter activity. The par promoter region was also demonstrated to exert incompatibility towards a plasmid with an active pRA2 par system. Nested deletions in this region allowed the incompatibility determinant, designated parS, to be localized. Recombinant ParA and ParB proteins were overexpressed and purified by affinity chromatography. Through in vitro binding experiments, purified ParB was shown to interact specifically with the par promoter region. DNase I footprinting revealed that ParB not only binds to the conserved sequence 5′‐TCA AA(T/C) (G/C)CT CAA (A/T)A, which is present in three copies in the par promoter region, but also binds to the pRA2 partitioning site, parS. It appears that ParB has a dual role in pRA2 partitioning, being responsible for both the regulation of par transcription and the formation of a partition nucleoprotein complex at parS.