bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization and modelling of VanT: a novel, membrane‐bound, serine racemase from vancomycin‐resistant Enterococcus gallinarum BM4174
Authors: Arias Cesar A. , Martín‐Martinez Mercedes , Blundell Tom L. , Arthur Michel , Courvalin Patrice , Reynolds Peter E.
Published: March 2003

Sequence determination of a region downstream from the vanXYC gene in Enterococcus gallinarum BM4174 revealed an open reading frame, designated vanT, that encodes a 698‐amino‐acid polypeptide with an amino‐terminal domain containing 10 predicted transmembrane segments. The protein contained a highly conserved pyridoxal phosphate attachment site in the C‐terminal domain, typical of alanine racemases. The protein was overexpressed in Escherichia coli, and serine racemase activity was detected in the membrane but not in the cytoplasmic fraction after centrifugation of sonicated cells, whereas alanine racemase activity was located almost exclusively in the cytoplasm. When the protein was overexpressed as a polypeptide lacking the predicted transmembrane domain, serine racemase activity was detected in the cytoplasm. The serine racemase activity was partially (64%) inhibited by d‐cycloserine, whereas host alanine racemase activity was almost totally inhibited (97%). Serine racemase activity was also detected in membrane preparations of constitutively vancomycin‐resistant E. gallinarum BM4174 but not in BM4175, in which insertional inactivation of the vanC‐1d‐Ala:d‐Ser ligase gene probably had a polar effect on expression of the vanXYC and vanT genes. Comparative modelling of the deduced C‐terminal domain was based on the alignment of VanT with the Alr alanine racemase from Bacillus stearothermophilus. The model revealed that almost all critical amino acids in the active site of Alr were conserved in VanT, indicating that the C‐terminal domain of VanT is likely to adopt a three‐dimensional structure similar to that of Alr and that the protein could exist as a dimer. These results indicate that the source of d‐serine for peptidoglycan synthesis in vancomycin‐resistant enterococci expressing the VanC phenotype involves racemization of l‐ to d‐serine by a membrane‐bound serine racemase.
Overexpression of yeast karyopherin Pse1p/Kap121p stimulates the mitochondrial import of hydrophobic proteins in vivo
Authors: Corral‐Debrinski M. , Belgareh N. , Blugeon C. , Claros M. G. , Doye V. , Jacq C.
Published: March 2003

During evolution, cellular processes leading to the transfer of genetic information failed to send all the mitochondrial genes into the nuclear genome. Two mitochondrial genes are still exclusively located in the mitochondrial genome of all living organisms. They code for two highly hydrophobic proteins: the apocytochrome b and the subunit I of cytochrome oxidase. Assuming that the translocation machinery could not efficiently transport long hydrophobic fragments, we searched for multicopy suppressors of this physical blockage. We demonstrated that overexpression of Pse1p/Kap121p or Kap123p, which belong to the superfamily of karyopherin β proteins, facilitates the translocation of chimeric proteins containing several stretches of apocytochrome b fused to a reporter mitochondrial gene. The effect of PSE1/KAP121 overexpression (in which PSE1 is protein secretion enhancer 1) on mitochondrial import of the chimera is correlated with an enrichment of the corresponding transcript in cytoplasmic ribosomes associated with mitochondria. PSE1/KAP121 overexpression also improves the import of the hydrophobic protein Atm1p, an ABC transporter of the mitochondrial inner membrane. These results suggest that in vivo PSE1/KAP121 overexpression facilitates, either directly or indirectly, the co‐translational import of hydrophobic proteins into mitochondria.
Protonmotive force, ExbB and ligand‐bound FepA drive conformational changes in TonB
Authors: Larsen Ray A. , Thomas Michael G. , Postle Kathleen
Published: March 2003

TonB couples the cytoplasmic membrane protonmotive force (pmf) to active transport across the outer membrane, potentially through a series of conformational changes. Previous studies of a TonB transmembrane domain mutant (TonB‐ΔV17) and its phenotypical suppressor (ExbB‐A39E) suggested that TonB is conformationally sensitive. Here, two new mutations of the conserved TonB transmembrane domain SHLS motif were isolated, TonB‐S16L and ‐H20Y, as were two new suppressors, ExbB‐V35E and ‐V36D. Each suppressor ExbB restored at least partial function to the TonB mutants, although TonB‐ΔV17, for which both the conserved motif and the register of the predicted transmembrane domain α‐helix are affected, was the most refractory. As demonstrated previously, TonB can undergo at least one conformational change, provided both ExbB and a functional TonB transmembrane domain are present. Here, we show that this conformational change reflects the ability of TonB to respond to the cytoplasmic membrane proton gradient, and occurs in proportion to the level of TonB activity attained by mutant–suppressor pairs. The phenotype of TonB‐ΔV17 was more complex than the ‐S16L and ‐H20Y mutations, in that, beyond the inability to be energized efficiently, it was also conditionally unstable. This second defect was evident only after suppression by the ExbB mutants, which allow transmembrane domain mutants to be energized, and presented as the rapid turnover of TonB‐ΔV17. Importantly, this degradation was dependent upon the presence of a TonB‐dependent ligand, suggesting that TonB conformation also changes following the energy transduction event. Together, these observations support a dynamic model of energy transduction in which TonB cycles through a set of conformations that differ in potential energy, with a transition to a higher energy state driven by pmf and a transition to a lower energy state accompanying release of stored potential energy to an outer membrane receptor.
The regulator gene phoB mediates phosphate stress‐controlled synthesis of the membrane lipid diacylglyceryl‐N,N,N‐trimethylhomoserine in Rhizobium (Sinorhizobium) meliloti
Authors: Geiger Otto , Röhrs Viola , Weissenmayer Barbara , Finan Turlough M. , Thomas‐Oates Jane E.
Published: March 2003

Bacteria react to phosphate starvation by activating genes involved in the transport and assimilation of phosphate as well as other phosphorous compounds. Some soil bacteria have evolved an additional mechanism for saving phosphorus. Under phosphate‐limiting conditions, they replace their membrane phospholipids by lipids not containing phosphorus. Here, we show that the membrane lipid pattern of the free‐living microsymbiotic bacterium Rhizobium (Sinorhizobium) meliloti is altered at low phosphate concentrations. When phosphate is growth limiting, an increase in sulpholipids, ornithine lipids and the de novo synthesis of diacylglyceryl trimethylhomoserine (DGTS) lipids is observed. Rhizobium meliloti phoCDET mutants, deficient in phosphate uptake, synthesize DGTS constitutively at low or high medium phosphate concentrations, suggesting that reduced transport of phosphorus sources to the cytoplasm causes induction of DGTS biosynthesis. Rhizobium meliloti phoU or phoB mutants are unable to form DGTS at low or high phosphate concentrations. However, the functional complementation of phoU or phoB mutants with the phoB gene demonstrates that, of the two genes, only intact phoB is required for the biosynthesis of the membrane lipid DGTS.
Starvation‐induced Mucts62‐mediated coding sequence fusion: a role for ClpXP, Lon, RpoS and Crp
Authors: Lamrani Sabah , Ranquet Caroline , Gama Marie‐José , Nakai Hiroshi , Shapiro James A. , Toussaint Ariane , Maenhaut‐Michel Geneviève
Published: March 2003

The formation of araB–lacZ coding sequence fusions in Escherichia coli is a particular type of chromosomal rearrangement induced by Mucts62, a thermoinducible mutant of mutator phage Mu. Fusion formation is controlled by the host physiology. It only occurs after aerobic carbon starvation and requires the phage‐encoded transposase pA, suggesting that these growth conditions trigger induction of the Mucts62 prophage. Here, we show that thermal induction of the prophage accelerated araB–lacZ fusion formation, confirming that derepression is a rate‐limiting step in the fusion process. Nonetheless, starvation conditions remained essential to complete fusions, suggesting additional levels of physiological regulation. Using a transcriptional fusion indicator system in which the Mu early lytic promoter is fused to the reporter E. coli lacZ gene, we confirmed that the Mucts62 prophage was derepressed in stationary phase (S derepression) at low temperature. S derepression did not apply to prophages that expressed the Mu wild‐type repressor. It depended upon the host ClpXP and Lon ATP‐dependent proteases and the RpoS stationary phase‐specific σ factor, but not upon Crp. None of these four functions was required for thermal induction. Crp was required for fusion formation, but only when the Mucts62 prophage encoded the transposition/replication activating protein pB. Finally, we found that thermally induced cultures did not return to the repressed state when shifted back to low temperature and, hence, remained activated for accelerated fusion formation upon starvation. The maintenance of the derepressed state required the ClpXP and Lon host proteases and the prophage Ner‐regulatory protein. These observations illustrate how the cts62 mutation in Mu repressor provides the prophage with a new way to respond to growth phase‐specific regulatory signals and endows the host cell with a new potential for adaptation through the controlled use of the phage transposition machinery.
The protein kinase C‐mediated MAP kinase pathway involved in the maintenance of cellular integrity in Saccharomyces cerevisiae
Authors: Heinisch Jürgen J. , Lorberg Anja , Schmitz Hans‐Peter , Jacoby Jörg J.
Published: March 2003

Signal transduction mediated by the single yeast isozyme of protein kinase C (Pkc1p) is essential for the maintenance of cellular integrity in this model eukaryote. The past few years have seen a dramatic increase in our knowledge of the upstream regulatory factors that modulate Pkc1p activity (e.g. Tor2p, Rom1p, Rom2p, Rho1p, Slg1p, Mid2p) and of the downstream targets of the MAP kinase cascade triggered by it (e.g. Rlm1p, SBF complex). The picture that has emerged connects this pathway to a variety of other cellular processes, such as cell cycle progression (Cdc28p, Swi4p), mating (Ste20p), nutrient sensing (Ira1p), calcium homeostasis (calcineurin, Mid2p, Fks2p) and the structural dynamics of the cytoskeleton (Spa1p, Bni1p).
Localization of the Yersinia PTPase to focal complexes is an important virulence mechanism
Authors: Persson Cathrine , Nordfelth Roland , Andersson Kerstin , Forsberg Åke , Wolf‐Watz Hans , Fällman Maria
Published: March 2003

The protein tyrosine phosphatase YopH, produced by the pathogen Yersinia pseudotuberculosis, is an essential virulence determinant involved in antiphagocytosis. Upon infection, YopH is translocated into the target cell, where it recognizes focal complexes. Genetic analysis revealed that YopH harbours a region that is responsible for specific localization of this PTPase to focal complexes in HeLa cells and professional phagocytes. This region is a prerequisite for blocking an immediate–early Yersinia‐induced signal within target cells. The region is also essential for antiphagocytosis and virulence, illustrating the biological significance of localization of YopH to focal complexes during Yersinia infection. These results also indicate that focal complexes play a role in the general phagocytic process.
Structure of the Mesorhizobium huakuii and Rhizobium galegae Nod factors: a cluster of phylogenetically related legumes are nodulated by rhizobia producing Nod factors with α,β‐unsaturated N‐acyl substitutions
Authors: Yang Guo‐Ping , Debellé Frédéric , Savagnac Arlette , Ferro Myriam , Schiltz Odile , Maillet Fabienne , Promé Danielle , Treilhou Michel , Vialas Corinne , Lindstrom Kristina , Dénarié Jean , Promé Jean‐Claude
Published: March 2003

Rhizobia are symbiotic bacteria that synthesize lipochitooligosaccharide Nod factors (NFs), which act as signal molecules in the nodulation of specific legume hosts. Based on the structure of their N‐acyl chain, NFs can be classified into two categories: (i) those that are acylated with fatty acids from the general lipid metabolism; and (ii) those (= αU‐NFs) that are acylated by specific α,β‐unsaturated fatty acids (containing carbonyl‐conjugated unsaturation(s)). Previous work has described how rhizobia that nodulate legumes of the Trifolieae and Vicieae tribes produce αU‐NFs. Here, we have studied the structure of NFs from two rhizobial species that nodulate important genera of the Galegeae tribe, related to Trifolieae and Vicieae. Three strains of Mesorhizobium huakuii, symbionts of Astragalus sinicus, produced as major NFs, pentameric lipochitooligosaccharides O‐sulphated and partially N‐glycolylated at the reducing end and N‐acylated, at the non‐reducing end, by a C18:4 fatty acid. Two strains of Rhizobium galegae, symbionts of Galega sp., produced as major NFs, tetrameric O‐carbamoylated NFs that could be O‐acetylated on the glucosamine residue next to the non‐reducing terminal glucosamine and were N‐acylated by C18 and C20 α,β‐unsaturated fatty acids. These results suggest that legumes nodulated by rhizobia synthesizing αU‐NFs constitute a phylogenetic cluster in the Galegoid phylum.
Modulating quorum sensing by antiactivation: TraM interacts with TraR to inhibit activation of Ti plasmid conjugal transfer genes
Authors: Hwang Ingyu , Smyth Audra J. , Luo Zhao‐Qing , Farrand Stephen K.
Published: March 2003

Conjugal transfer of the Ti plasmid pTiC58 is regulated by a quorum‐sensing system involving the transcriptional activator TraR and the acyl homoserine lactone autoinducer N‐(3‐oxo‐octanoyl)‐l‐homoserine lactone (AAI). Activation of tra gene expression by TraR and AAI is inhibited by TraM, an 11 kDa protein also coded for by the Ti plasmid. Previous studies suggested that TraM interferes with TraR activity by directly interacting with the activator protein. Using the yeast two‐hybrid system, constructs of Saccharomyces cerevisiae containing a fusion of traR to the B42 domain of the prey plasmid pJG4.5 and a fusion of traM to the lexA gene of the bait plasmid pEG202 produced β‐galactosidase and grew on medium lacking leucine, both phenotypes indicative of an interaction between the two proteins. Early termination mutants and substitution mutants mapping to the C‐terminus of TraM were isolated by screening for alleles unable to interfere with TraR activity in Agrobacterium tumefaciens. These mutants all failed to interact with the TraR fusion in the two‐hybrid system. An N‐terminal deletion mutant of TraM lacking the first 27 residues weakly interacted with TraR in the two‐hybrid system whereas deletions of 48 amino acids or more abolished the interaction. As assessed by Western blot analysis, the mutant fusion proteins were produced at levels indistinguishable from that of the wild‐type TraM in the yeast tester strain. Mutants of TraR that were not inhibited by TraM in A. tumefaciens were isolated and fell into two classes. In the first, the mutation resulted in increased expression of wild‐type TraR. In the second, a proline residue at position 176 was changed to serine (P176 → S) or to leucine (P176 → L). The P176 → S mutant interacted with wild‐type TraM, but at a detectably lower level, in the two‐hybrid assay. Mutants of TraR with N‐terminal deletions as large as 105 amino acids interfered with the ability of TraM to inhibit wild‐type TraR in A. tumefaciens. Two‐hybrid assays indicated that these mutants, as well as a C‐terminal 49 residue fragment of TraR, can interact with TraM. We conclude that TraM and TraR interact in vivo and that this interaction is responsible for inhibition of TraR‐mediated activation. We also conclude that the two proteins interact with each other through domains located at their respective C‐termini.
Ralstonia solanacearum produces Hrp‐dependent pili that are required for PopA secretion but not for attachment of bacteria to plant cells
Authors: Van Gijsegem Frédérique , Vasse Jacques , Camus Jean‐Christophe , Marenda Marc , Boucher Christian
Published: March 2003

As in many other Gram‐negative plant pathogenic bacteria, the Ralstonia solanacearum hrp genes are involved in the production of a type III secretion apparatus that allows the translocation of PopA protein to the external medium. Here, we show that hrp genes are also involved in the biogenesis of pili that are mainly composed of the HrpY protein. These pili are produced at one pole of the bacterium and are also released into the external medium where they can form very long straight bundles. An hrpY mutant is defective in pilus production, impaired in interactions with plants and in secretion of the PopA protein but not in attachment to plant cells.
Salmonella Pathogenicity Island 2
Authors: Hensel Michael
Published: March 2003

Systemic infections by Salmonella enterica, such as typhoid fever, are a significant threat to human health. Recent studies indicate that the function of a type III secretion system encoded by Salmonella Pathogenicity Island 2 (SPI2) is central for the ability of S. enterica to cause systemic infections and for intracellular pathogenesis. This review summarizes approaches leading to the identification of SPI2, the molecular genetics and evolution of SPI2, and the current understanding of the regulation of gene expression. Recent studies have indicated that SPI2 is used by intracellular Salmonella to actively modify functions of the host cells. The role of SPI2 during pathogenesis of salmonellosis and current models regarding function will be discussed.
Gonococcal lipooligosaccharide is a ligand for the asialoglycoprotein receptor on human sperm
Authors: Harvey H. A. , Porat N. , Campbell C. A. , Jennings M. , Gibson B. W. , Phillips N. J. , Apicella M. A. , Blake M. S.
Published: March 2003

In the present study, we show that Neisseria gonorrhoeae lipooligosaccharide (LOS) can bind to the asialoglycoprotein receptor (ASGP‐R) on human sperm. This work demonstrates the presence of ASGP‐R on human sperm. Binding of purified ASGP‐R ligand decreased in the presence of gonococci. Binding of purified iodinated gonococcal LOS identified a protein of molecular weight corresponding to that of human ASGP‐R. The presence of excess unlabelled LOS blocked binding of iodinated gonococcal LOS. Binding of wild‐type gonococcal LOS to sperm was higher than that of mutant LOS lacking the galactose ligand for ASGP‐R. These data suggest that the ASGP‐R on human sperm cells recognizes and binds wild‐type gonococcal LOS. This interaction may contribute to the transmission of gonorrhea from infected males to their sexual partners.
Interdependence of environmental factors influencing reciprocal patterns of gene expression in virulent Borrelia burgdorferi
Authors: Yang Xiaofeng , Goldberg Martin S. , Popova Taissia G. , Schoeler George B. , Wikel Stephen K. , Hagman Kayla E. , Norgard Michael V.
Published: March 2003

The paradigm for differential antigen expression in Borrelia burgdorferi, the agent of Lyme disease, is the reciprocal expression of its outer surface (lipo)proteins (Osp) A and C; as B. burgdorferi transitions from its arthropod vector into mammalian tissue, ospC is upregulated, and ospA is downregulated. In the current study, using B. burgdorferi cultivated under varying conditions in BSK‐H medium, we found that a decrease in pH, in conjunction with increases in temperature (e.g. 34°C or 37°C) and cell density, acted interdependently for the reciprocal expression of ospC and ospA. The lower pH (6.8), which induced the reciprocal expression of ospC and ospA in BSK‐H medium, correlated with a drop in pH from 7.4 to 6.8 of tick midgut contents during tick feeding. In addition to ospC and ospA, other genes were found to be regulated in reciprocal fashion. Such genes were either ospC‐like (e.g. ospF, mlp‐8 and rpoS) (group I) or ospA‐like (lp6.6 and p22) (group II); changes in expression occurred at the mRNA level. That the expression of rpoS, encoding a putative stress‐related alternative sigma factor (σs), was ospC‐like suggested that the expression of some of the group I genes may be controlled through σs. The combined results prompt a model that allows for predicting the regulation of other B. burgdorferi genes that may be involved in spirochaete transmission, virulence or mammalian host immune responses.
The role of the interdomain B linker in the activation of the XylR protein of Pseudomonas putida
Authors: Garmendia Junkal , De Lorenzo Víctor
Published: March 2003

In the presence of toluene and other structural analogues, the enhancer binding protein XylR activates the σ54 promoter Pu of the TOL (toluene degradation) plasmid pWW0 of Pseudomonas putida. Introduction of amino acid changes Val‐219Asp and Ala‐220Pro, which enter a proline kink at the interdomain region (B linker) between the A (signal reception) module and the central portion of XylR, originated a protein with unforeseen properties. These included a minor ability to activate Pu in the absence of aromatic effectors, a much higher responsiveness to m‐xylene and a significant response to a large collection of aromatic inducers. Such changes could not be attributed to variations in XylR expression levels or to the fortuitous creation of a novel promoter, but to a genuine change in the properties of the activator. Structural predictions suggested that the mutation entirely disrupted an otherwise probable coiled‐coil structure. A second directed mutant within the same region consisting of a major replacement of amino acids A220–N221 by the peptide HHHR produced an even more exacerbated phenotype. These data support a model in which the linker B region influences the effector profile by modifying at a distance the operative shape of the effector pocket and fixing the protein in an intermediate step of the activation process.
Genetic and transcriptional analysis of absA, an antibiotic gene cluster‐linked two‐component system that regulates multiple antibiotics in Streptomyces coelicolor
Authors: Anderson Todd B. , Brian Paul , Champness Wendy C.
Published: March 2003

In Streptomyces coelicolor, the AbsA1–AbsA2 two‐component system regulates the expression of multiple antibiotic gene clusters. Here, we show that the response regulator encoded by the absA2 gene is a negative regulator of these antibiotic gene clusters. A genetic analysis shows that the phosphorylated form of the AbsA2 response regulator (phospho‐AbsA2), generated by the cognate AbsA1 sensor histidine kinase, is required for normal growth phase regulation of antibiotic synthesis. In the absence of phospho‐AbsA2, antibiotics are produced earlier and more abundantly. Overexpression of AbsA1 also deregulates antibiotic synthesis, apparently shifting the AbsA1 protein from a kinase‐active to a phospho‐AbsA2 phosphatase‐active form. The absA1 and absA2 genes, which are adjacent, are located in one of the antibiotic gene clusters that they regulate, the cluster for the calcium‐dependent antibiotic (CDA). The absA genes themselves are growth phase regulated, with phospho‐AbsA2 responsible for growth phase‐related positive autoregulation. We discuss the possible role and mechanism of AbsA‐mediated regulation of antibiotic synthesis in the S. coelicolor life cycle.
NucA is required for DNA cleavage during transformation of Bacillus subtilis
Authors: Provvedi Roberta , Chen Inês , Dubnau David
Published: March 2003

We have re‐examined the roles of nucA and nin, in the transformation of Bacillus subtilis as conflicting accounts have been presented concerning the importance of these genes for transformation. The present report demonstrates that nucA deficiency lowers the rate of DNA transport and that NucA is needed for the double‐strand cleavage of transforming DNA, probably acting directly as an endonuclease. A relative paucity of DNA termini, resulting from the absence of this endonuclease activity, most probably accounts for the decreased transport rate. NucA is a bitopic integral membrane protein, with its C‐terminus external to the membrane where it is appropriately located to effect the cleavage of bound transforming DNA. We have also investigated the roles of the known competence genes in the DNA processing that accompanies transformation in B. subtilis. The genes that are required for DNA transport (comEA, comEC and comFA) are also required for the degradation of the non‐transforming strand that accompanies internalization, but comEC and comFA are not needed for the double‐strand cleavage that occurs external to the cell membrane.
Overlapping sense and antisense transcription units in Trypanosoma brucei
Authors: Liniger Matthias , Bodenmüller Kurt , Pays Etienne , Gallati Sabina , Roditi Isabel
Published: March 2003

Procyclins are the major surface glycoproteins of insect‐form Trypanosoma brucei. The procyclin expression sites are polycistronic and are transcribed by an α‐amanitin‐resistant polymerase, probably RNA polymerase I (Pol I). The expression sites are flanked by transcription units that are sensitive to α‐amanitin, which is a hallmark of Pol II‐driven transcription. We have analysed a region of 9.5 kb connecting the EP/PAG2 expression site with the downstream transcription unit. The procyclin expression site is longer than was previously realized and contains an additional gene, procyclin‐associated gene 4 (PAG4), and a region of unknown function, the T region, that gives rise to trans‐spliced, polyadenylated RNAs containing small open reading frames (ORFs). Two new genes, GU1 and GU2, were identified in the downstream transcription unit on the opposite strand. Unexpectedly, the 3′ untranslated region of GU2 and the complementary T transcripts overlap by several hundred base pairs. Replacement of GU2 by a unique tag confirmed that sense and antisense transcription occurred from a single chromosomal locus. Overlapping transcription is stage specific and may extend ≥ 10 kb in insect‐form trypanosomes. The nucleotide composition of the T. brucei genome is such that antisense ORFs occur frequently. If stable mRNAs can be derived from both strands, the coding potential of the genome may be substantially larger than has previously been suspected.
Poly(A) polymerase activity and RNA polyadenylation in Streptomyces coelicolor A3(2)
Authors: Bralley Patricia , Jones George H.
Published: March 2003

The Streptomyces coelicolor genome sequence was searched for open reading frames (ORFs) similar to Escherichia coli poly(A) polymerase I, revealing an ORF with 36% amino acid sequence identity to that protein. Mycelial extracts prepared from S. coelicolor cultures incorporated radioactive ATP into an acid‐insoluble form, and some of the products of this incorporation had the properties expected of poly(A). [3H]‐uridine and [3H]‐adenosine were used to label the RNA in S. coelicolor cultures of different ages, and total RNA was fractionated by oligo dT cellulose chromatography. Approximately 3% of the total uridine‐labelled RNA and 11% of the adenosine‐labelled RNA were retained by the oligo dT cellulose columns. Enzymatic digestion of the retained RNA supported the conclusion that a significant fraction of the adenosine label was present in 3′‐poly(A) chains. Measurement of poly(A) tail lengths by end labelling of total RNA and RNase digestion revealed a maximum length of ≈ 18 residues. Radioactive cDNA prepared from the RNA fraction retained by oligo dT cellulose hybridized to the 16S and 23S genes from a streptomycete ribosomal RNA operon but not to the 5S gene. Reverse transcription–polymerase chain reaction (RT–PCR) revealed the presence of mRNAs in the RNA fraction retained by oligo dT cellulose.
Unique regulation of SclB – a novel collagen‐like surface protein of Streptococcus pyogenes
Authors: Rasmussen Magnus , Björck Lars
Published: March 2003

Slipped‐strand mispairing at sites containing so‐called coding repeats (CRs) can lead to phase variation of surface proteins in Gram‐negative bacteria. This mechanism, believed to contribute to virulence, has so far not been identified in a Gram‐positive bacterium. In the genome of the Gram‐positive human pathogen Streptococcus pyogenes, we identified pentanucleotide CRs within a putative signal sequence of an open reading frame (ORF) encoding a novel collagen‐like surface protein, denoted SclB. In 12 S. pyogenes strains, the number of CRs in the sclB gene varied from three to 19, rendering the start codon in frame with the downstream ORF in four strains and out of frame in eight strains. A protein reacting with anti‐SclB antibodies could only be solubilized from three strains, all containing an intact sclB gene. Variations in the number of CRs were observed within strains of the same M serotype and occurred during growth of S. pyogenes in fresh human blood, but not in medium. The SclB protein has a hypervariable N‐terminal part, a collagen‐like central part and a typical cell wall sorting sequence containing the LPXTGX motif. SclB is related to the collagen‐like SclA and is, like SclA, involved in the adhesion of S. pyogenes bacteria to human cells. However, the Mga protein, known to upregulate sclA and several additional genes encoding virulence factors of S. pyogenes, downregulates sclB transcription. This observation and the potential of SclB to phase vary by slipped‐strand mispairing emphasize the unique regulation of this novel S. pyogenes surface protein.
Identification of Salmonella typhi genes expressed within macrophages by selective capture of transcribed sequences (SCOTS)
Authors: Daigle France , Graham James E. , Curtiss Roy
Published: March 2003

Salmonella enterica serovar Typhi (S. typhi) is a human‐restricted pathogen which causes typhoid fever. Relatively little is known about S. typhi host interaction as animal models of this disease are severely limited by the lack of virulence of S. typhi in other hosts. The virulence of other Salmonella serovars in animal models is dependent on the abilities of these bacteria to survive within host macrophages. We have used selective capture of transcribed sequences (SCOTS) to identify S. typhi genes expressed during growth in human macrophages. This positive cDNA selection technique identified 28 distinct clones representing previously identified as well as novel, uncharacterized and hypothetical gene sequences that are expressed intracellularly. Transcripts for the Vi capsular antigen and genes whose products are involved in stress responses and nutrient acquisition were obtained from intracellular bacteria using SCOTS. Most of these clones are present in the S. typhimurium genome and are also expressed in murine macrophages. Nineteen of these gene sequences were disrupted insertionally in S. typhi, and most of the resulting mutants exhibited a lower level of survival within macrophages compared with the wild‐type parent strain. Mutant strains, transformed with a copy of a wild‐type gene, exhibited a macrophage survival level similar to that of the parental strain.
Bacteriophage T7 protein kinase phosphorylates RNase E and stabilizes mRNAs synthesized by T7 RNA polymerase
Authors: Marchand Isabelle , Nicholson Allen W. , Dreyfus Marc
Published: March 2003

The T7 protein encoded by the early gene 0.7 exhibits bifunctional activity. Whereas its C‐terminal one‐third participates in host transcription shut‐off, the N‐terminal two‐thirds bears a protein kinase (‘PK’) activity that can phosphorylate a number of host proteins in addition to itself. Here, we show that, when PK is expressed in uninfected Escherichia coli cells, the C‐terminal half of RNase E and the associated RNA helicase RhlB are heavily phosphorylated. Meanwhile, a subset of RNase E substrates, including the lac and cat mRNAs synthesized by bacteriophage T7 RNA polymerase (RNAP), are stabilized. These mRNAs are genuinely less stable than their counterparts synthesized by E. coli RNAP, because T7 RNAP outpaces translating ribosomes, creating naked, RNase E‐sensitive mRNA stretches behind itself. Thus, PK alleviates this effect of desynchronizing transcription and translation. The relationship between the modification of RNase E and RhlB and these mRNA stabilization effects, which may be relevant to the stability of late T7 mRNAs during infection, is discussed.
Overexpression of the alternative oxidase restores senescence and fertility in a long‐lived respiration‐deficient mutant of Podospora anserina
Authors: Lorin Séverine , Dufour Eric , Boulay Jocelyne , Begel Odile , Marsy Sophie , Sainsard‐Chanet Annie
Published: March 2003

Several lines of evidence have implicated reactive oxygen species (ROS) in the pathogenesis of various degenerative diseases and in organismal ageing. Furthermore, it has been shown recently that the alternative pathway respiration present in plants lowers ROS mitochondrial production. An alternative oxidase (AOXp) also occurs in the filamentous fungus Podospora anserina. We show here that overexpression of this oxidase does not decrease ROS production and has no effect on longevity, mitochondrial stability or ageing in this fungus. In the same way, inactivation of the gene has no effect on these parameters. In contrast, overexpression of the alternative oxidase in the long‐lived cox5::BLE mutant, deficient in cytochrome c oxidase, considerably increases ROS production of the mutant. It rescues slow growth rate and female sterility, indicating an improved energy level. This overexpression also restores senescence and mitochondrial DNA instability, demonstrating that these parameters are controlled by the energy level and not by the expression level of the alternative oxidase. We also suggest that expression of this oxidase in organisms naturally devoid of it could rescue respiratory defects resulting from cytochrome pathway dysfunctions.
Consequences of RNase E scarcity in Escherichia coli
Authors: Jain Chaitanya , Deana Atilio , Belasco Joel G.
Published: March 2003

The endoribonuclease RNase E plays an important role in RNA processing and degradation in Escherichia coli. The construction of an E. coli strain in which the cellular concentration of RNase E can be precisely controlled has made it possible to examine and quantify the effect of RNase E scarcity on RNA decay, gene regulation and cell growth. These studies show that RNase E participates in a step in the degradation of its RNA substrates that is partially or fully rate‐determining. Our data also indicate that E. coli growth requires a cellular RNase E concentration at least 10–20% of normal and that the feedback mecha‐nism that limits overproduction of RNase E is also able to increase its synthesis when its concentration drops below normal. The magnitude of the in‐crease in RNA longevity under conditions of RNase E scarcity may be limited by an alternative pathway for RNA degradation. Additional experiments show that RNase E is a stable protein in E. coli. No other E. coli gene product, when either mutated or cloned on a multicopy plasmid, seems to be capable of compensating for an inadequate supply of this essential protein.
Functional assembly of the foreign carotenoid lycopene into the photosynthetic apparatus of Rhodobacter sphaeroides, achieved by replacement of the native 3‐step phytoene desaturase with its 4‐step counterpart from Erwinia herbicola
Authors: Garcia‐Asua Guillermo , Cogdell Richard J. , Hunter C. Neil
Published: March 2003

Photosynthetic organisms synthesize a diverse range of carotenoids. These pigments are important for the assembly, function and stability of photosynthetic pigment–protein complexes, and they are used to quench harmful radicals. The photosynthetic bacterium Rhodobacter sphaeroides was used as a model system to explore the origin of
carotenoid diversity. Replacing the native 3‐step phytoene desaturase (CrtI) with
the 4‐step enzyme from Erwinia herbicola results in significant flux down
the spirilloxanthin pathway for the first time in Rb. sphaeroides. In Rb.
sphaeroides, the completion of four desaturations to lycopene by the Erwinia
CrtI appears to require the absence of CrtC and, in a crtC background, even
the native 3‐step enzyme can synthesize a significant amount (13%) of lycopene,
in addition to the expected neurosporene. We suggest that the CrtC hydroxylase can
intervene in the sequence of reactions catalyzed by phytoene desaturase. We investigated
the properties of the lycopene‐synthesizing strain of Rb. sphaeroides. In
the LH2 light‐harvesting complex, lycopene transfers absorbed light energy to the
bacteriochlorophylls with an efficiency of 54%, which compares favourably
with other LH2 complexes that contain carotenoids with 11 conjugated double bonds.
Thus, lycopene can join the assembly pathway for photosynthetic complexes in Rb.
sphaeroides, and can perform its role as an energy donor to bacteriochlorophylls.
Uptake and intracellular transportation of a bacterial surface protein in lymphoid cells
Authors: Frick Inga‐Maria , Axcrona Karol , Härdig Ylva , Tapper Hans , Gustafsson Lotta , Kellner Roland , Leanderson Tomas , Björck Lars
Published: March 2003

Some strains of the human pathogen Streptococcus pyogenes express a surface protein called protein H, which is released from the streptococcal surface by a cysteine proteinase produced by the bacteria. Here, we find that soluble protein H binds to the surface of lymphocytes and granulocytes, and that the molecule is taken up by lymphocytes and transported to the perinuclear region. The translocation over the cell membrane is rapid, and the uptake and intracellular transportation is not dependent on actin polymerization. Protein H could be immunoprecipitated from cell extracts and nuclear preparations of lymphocytes, and analysis of molecular interactions between pro‐tein H and proteins of different cellular compart‐ments demonstrated a binding to nucleophosmin/ B23, a protein known to shuttle between the cytoplasm and the nucleus, and to the nuclear proteins SET and hnRNP A2/B1. Nucleophosmin/B23 was co‐immunoprecipitated with protein H from cell and nuclear extracts, and binding experiments, including kinetic analyses, suggest that protein H dissociating from nucleophosmin/B23 complexes in the perinuclear region or in the nucleus binds to proteins SET and hnRNP A2/B1. Finally, the uptake and intracellular transportation of protein H was found to result in a cytostatic effect on B and T lymphocytes.
Protein engineering modulates the transport properties and ion selectivity of the pores formed by staphylococcal γ‐haemolysins in lipid membranes
Authors: Comai Massimiliano , Serra Mauro Dalla , Coraiola Manuela , Werner Sandra , Colin Didier A. , Monteil Henri , Prévost Gilles , Menestrina Gianfranco
Published: March 2003

Staphylococcal γ‐haemolysins are bicomponent toxins in a family including other leucocidins and α‐toxin. Two active toxins are formed combining HlgA or HlgC with HlgB. Both open pores in lipid membranes with conductance, current voltage characteristics and stability similar to α‐toxin, but different selectivity (cation instead of anion). Structural analogies between γ‐haemolysins and α‐toxin indicate the presence, at the pore entry, of a conserved region containing four positive charges in α‐toxin, but either positive or negative in γ‐haemolysins. Four mutants were produced (HlgA D44K, HlgB D47K, HlgB D49K and HlgB D47K/D49K) converting those negative charges to positive in HlgA and HlgB. When all charges were positive, the pores had the same selectivity and conductance as α‐toxin, suggesting that the cluster may form an entrance electrostatic filter. As mutated HlgC‐HlgB pores were less affected, additional charges in the lumen of the pore were changed (HlgB E107Q, HlgB D121N, HlgB T136D and HlgA K108T). Removing a negative charge from the lumen made the selectivity of both HlgA‐HlgB D121N and HlgC‐HlgB D121N more anionic. Residue D121 of HlgB is compensated by a positive residue (HlgA K108) in the HlgA‐HlgB pore, but isolated in the more cation‐selective HlgC‐HlgB pore. Interestingly, the pore formed by HlgA K108T‐HlgB, in which the positive charge of HlgA was removed, was as cation selective as HlgC‐HlgB. Meanwhile, the pore formed by HlgA K108T‐HlgB D121N, in which the two charge changes compensated, retrieved the properties of wild‐type HlgA‐HlgB. We conclude that the conductance and selectivity of the γ‐haemolysin pores depend substantially on the presence and location of charged residues in the channel.
Both metal binding sites in the homodimer are required for metalloregulation by the CadC repressor
Authors: Sun Yan , Wong Marco D. , Rosen Barry P.
Published: March 2003

The cadCA operon of plasmid pI258, which confers resistance to the soft
metals Cd(II), Pb(II) and Zn(II), is regulated by CadC, a metal‐responsive transcriptional
repressor. CadC is a 27.6 kDa homodimer composed of two 122‐residue monomers. Three
cysteine residues, Cys‐7, Cys‐58 and Cys‐60, have been shown to be required for sensing
soft metals. Thus, the repressor has two potential inducer binding sites, one on
each monomer. However, it is not known whether both binding sites are required for
derepression or whether binding of metal to a single site would result in transcript.
In this study, heterodimers were purified in which one binding site was wild type
and the other had substitutions of the cysteine residues. The wild type–mutant
heterodimers retained the ability to bind to cad operator/promoter DNA but did not dissociate from the DNA upon addition of soft metal ions. The results indicate that both subunits in the dimer must have functional metal binding sites for metal sensing to lead to derepression.
CpeR is an activator required for expression of the phycoerythrin operon (cpeBA) in the cyanobacterium Fremyella diplosiphon and is encoded in the phycoerythrin linker‐polypeptide operon (cpeCDESTR)
Authors: Cobley John G. , Clark A. Clay , Weerasurya Siromi , Queseda F. Anne , Xiao Jimmy Y. , Bandrapali Nirmala , D’Silva Ian , Thounaojam Maya , Oda Jeffrey F. , Sumiyoshi Teiko , Chu Man‐Ho
Published: March 2003

In the cyanobacteria, phycobilisomes are assembled from (αβ)6
hexamers of the coloured phycobili‐ proteins, allophycocyanin, phycocyanin and phy‐coerythrin
(PE). The precise architecture of the phycobilisome is determined by the various
colourless linker proteins that bind to the biliprotein hexamers. Genes for β
and α subunits of PE make up one operon (cpeBA), whereas genes
for PE‐associated linker polypeptides are in a second operon. In the chromatically
adapting cyanobacterium Fremyella diplosiphon green light is required for
the transcription of both cpeBA and the operon encoding the PE‐associated
linkers (cpeCDE). From the genome of F. diplosiphon we have
identified an open reading frame, cpeR, which, when expressed from a shuttle
plasmid, is capable of suppressing various mutations that cause a decrease in PE
synthesis. The introduction of a shuttle plasmid bearing cpeR+
into wild‐ type F. diplosiphon caused PE expression in red light. Fremyella
diplosiphon cpeR−, created by in vitro mutagenesis and
in vivo homologous recombination, is fully PE and, in this strain, cpeCDE
is transcribed normally whereas the transcript from cpeBA is undetectable.
Polymerase chain reaction (PCR) amplification of cDNA showed that cpeR is
transcribed as part of the cpeCDE operon on an extended transcript. As CpeR
is an activator required for expression of the cpeBA operon, we propose that
at the onset of green light the operons cpeCDESTR and cpeBA are expressed in series as a genetic cascade.
Discovery of two novel families of proteins that are proposed to interact with prokaryotic SMC proteins, and characterization of the Bacillus subtilis family members ScpA and ScpB
Authors: Soppa Jörg , Kobayashi Kazuo , Noirot‐Gros Marie‐Françoise , Oesterhelt Dieter , Ehrlich S. Dusko , Dervyn Etienne , Ogasawara Naotake , Moriya Shigeki
Published: March 2003

Structural maintenance of chromosomes (SMC) proteins are present in all eukaryotes and in many prokaryotes. Eukaryotic SMC proteins form complexes with various non‐SMC subunits, which affect their function, whereas the prokaryotic homologues had no known non‐SMC partners and were thought to act as simple homodimers. Here we describe two novel families of proteins, widespread in archaea and (Gram‐positive) bacteria, which we denote ‘segregation and condensation proteins’ (Scps). ScpA genes are localized next to smc genes in nearly all SMC‐ containing archaea, suggesting that they belong to the same operon and are thus involved in a common process in the cell. The function of ScpA was studied in Bacillus subtilis, which also harbours a well characterized smc gene. Here we show that scpA mutants display characteristic phenotypes nearly identical to those of smc mutants, including temperature‐ sensitive growth, production of anucleate cells, formation of aberrant nucleoids, and chromosome splitting by the so‐called guillotine effect. Thus, both SMC and ScpA are required for chromosome segregation and condensation. Interestingly, mutants of another B. subtilis gene, scpB, which is localized downstream from scpA, display the same phenotypes, which indicate that ScpB is also involved in these functions. ScpB is generally present in species that also encode ScpA. The physical interaction of ScpA and SMC was proven (i) by the use of the yeast two‐hybrid system and (ii) by the isolation of a complex containing both proteins from cell extracts of B. subtilis. By extension, we speculate that interaction of orthologues of the two proteins is important for chromosome segregation in many archaea and bacteria, and propose that SMC proteins generally have non‐SMC protein partners that affect their function not only in eukaryotes but also in prokaryotes.
Molecular dissection of Salmonella FliH, a regulator of the ATPase FliI and the type III flagellar protein export pathway
Authors: González‐Pedrajo Bertha , Fraser Gillian M. , Minamino Tohru , Macnab Robert M.
Published: March 2003

FliH is a soluble component of the flagellar export apparatus that binds to the ATPase FliI, and negatively regulates its activity. The 235‐amino‐acid FliH dimerizes and interacts with FliI to form a hetero‐trimeric (FliH)2FliI complex. In the present work, the importance of different regions of FliH was examined. A set of 24 scanning deletions of 10 amino acids was constructed over the entire FliH sequence, along with several combined deletions of 40 amino acids and truncations of both N‐ and C‐termini. The mutant proteins were examined with respect to (i) complementation; (ii) dominance and multicopy effects; (iii) interaction with wild‐type FliH; (iv) interaction with FliI; (v) inhibition of the ATPase activity of FliI; and (vi) interaction with the putative general chaperone FliJ. Analysis of the deletion mutants revealed a clear functional demarcation between the FliH N‐ and C‐terminal regions. The 10‐amino‐acid deletions throughout most of the N‐terminal half of the sequence complemented and were not dominant, whereas those throughout most of the C‐terminal half did not complement and were dominant. FliI binding was disrupted by C‐terminal deletions from residue 101 onwards, indicating that the C‐terminal domain of FliH is essential for interaction with FliI. FliH dimerization was abolished by deletion of residues 101–140 in the centre of the sequence, as were complementation, dominance and interaction with FliI and FliJ. The importance of this region was confirmed by the fact that fragment FliHC2 (residues 99–235) interacted with FliH and FliI, whereas fragment FliHC1 (residues 119–235) did not. FliHC2 formed a relatively unstable complex with FliI and showed biphasic regulation of ATPase activity, suggesting that the FliH N‐terminus stabilizes the (FliH)2FliI complex. Several of the N‐terminal deletions tested permitted close to normal ATPase activity of FliI. Deletion of the last five residues of FliH caused a fivefold activation of ATPase activity, suggesting that this region of FliH governs a switch between repression and activation of FliI. Deletion of the first 10 residues of FliH abolished complementation, severely reduced its interaction with FliJ and uncoupled its role as a FliI repressor from its other export functions. Based on these data, a model is presented for the domain construction and function of FliH in complex with FliI and FliJ.