bluuebunny/crossref_metadata_2025 huggingface dataset:
ClpE, a novel member of the HSP100 family, is involved in cell division and virulence of Listeria monocytogenes
Authors: Nair Shamila , Frehel Claude , Nguyen Laurence , Escuyer Vincent , Berche Patrick
Published: March 2003

We identified, in the facultative intracellular pathogen Listeria monocytogenes, a previously unknown Clp ATPase, unique among the HSP100 proteins because of the presence of a short N‐terminal region with a potential zinc finger motif. This protein of 726 amino acids is highly homologous to ClpE of Bacillus subtilis, and is a member of a new subfamily of HSP100/Clp ATPases. The clpE gene is transcribed as a monocistronic mRNA from a typical consensus σA promoter. clpE is not stimulated by various stresses, but is upregulated in a clpC mutant. This is the first example of cross‐regulation between Clp ATPases. By constructing a clpE mutant of L. monocytogenes, we found that ClpE is required for prolonged survival at 42°C and is involved in the virulence of this pathogen. A double mutant deficient in both ClpE and ClpC was avirulent in a mouse model and completely eliminated in the liver. Electron microscopy studies did not show any morphological alterations in clpE or clpC mutants. In the clpE–clpC double mutant, however, cell division was affected, indicating that ClpE acts synergistically with ClpC in cell septation. These results show that the Clp chaperones play a crucial role in both cell division and virulence of L. monocytogenes.
Negative regulation of bacterial heat shock genes
Authors: Narberhaus Franz
Published: March 2003

The expression of eubacterial heat shock genes is efficiently controlled at the transcriptional level by both positive and negative mechanisms. Positive control operates by the use of alternative sigma factors that target RNA polymerase to heat shock gene promoters. Alternatively, bacteria apply repressor‐dependent mechanisms, in which transcription of heat shock genes is initiated from a classical housekeeping promoter and cis‐acting DNA elements are used in concert with a cognate repressor protein to limit transcription under physiological conditions. Eight examples of negative regulation will be presented, among them the widespread CIRCE/HrcA system and the control by HspR in Streptomyces. Both mechanisms are designed to permit simple feedback control at the level of gene expression. Many bacteria have established sophisticated regulatory networks, often combining positive and negative mechanisms, in order to allow fine‐tuned heat shock gene expression in an environmentally responsive way.
The gene bglH present in the bgl operon of Escherichia coli, responsible for uptake and fermentation of β‐glucosides encodes for a carbohydrate‐specific outer membrane porin
Authors: Andersen Christian , Rak Bodo , Benz Roland
Published: March 2003

The cryptic gene bglH from the Escherichia coli chromosome was cloned into a tacOP‐driven expression vector. The resulting plasmid was transferred into the porin‐deficient E. coli strain KS26 and the protein was expressed by addition of IPTG. The BglH protein was localized in the outer membrane. It was purified to homogeneity using standard methods. Reconstitution experiments with lipid bilayer membranes defined BglH as a channel‐forming component, i.e. it is an outer membrane porin. The single‐channel conductance of BglH (560 pS in 1 M KCl) was only one‐third of that of the general diffusion porins of E. coli outer membrane. The presence of carbohydrates in the aqueous phase led to a dose‐dependent block of ion transport through the channel, similar to that found for LamB (maltoporin) of E. coli and Salmonella typhimurium, which means that BglH is a porin specific for the uptake of carbohydrates. The binding constants of a variety of different carbohydrates were calculated from titration experiments of the BglH‐induced membrane conductance. The tightest binding was observed with the aromatic β‐D‐glucosides arbutin and salicin, and with gentibiose and cellobiose. Binding of maltooligosaccharides to BglH was in contrast to their binding to LamB in that it was much weaker, indicating that the binding site of BglH for carbohydrates is different from that of LamB (maltoporin). The kinetics of cellopentaose binding to BglH was investigated using the carbohydrate‐induced current noise and was compared with that of cellopentaose binding to LamB (maltoporin) and ScrY (sucroseporin).
A ketolide resistance mutation in domain II of 23S rRNA reveals the proximity of hairpin 35 to the peptidyl transferase centre
Authors: Xiong Liqun , Shah Sunita , Mauvais Pascale , Mankin Alexander S.
Published: March 2003

Ketolides represent a new generation of macrolide antibiotics. In order to identify the ketolide‐binding site on the ribosome, a library of Escherichia coli clones, transformed with a plasmid carrying randomly mutagenized rRNA operon, was screened for mutants exhibiting resistance to the ketolide HMR3647. Sequencing of the plasmid isolated from one of the resistant clones and fragment exchange demonstrated that a single U754A mutation in hairpin 35 of domain II of the E. coli 23S rRNA was sufficient to confer resistance to low concentrations of the ketolide. The same mutation also conferred erythromycin resistance. Both the ketolide and erythromycin protected A2058 and A2059 in domain V of 23S rRNA from modification with dimethyl sulphate, whereas, in domain II, the ketolide protected, while erythromycin enhanced, modification of A752 in the loop of the hairpin 35. Thus, mutational and footprinting results strongly suggest that the hairpin 35 constitutes part of the macrolide binding site on the ribosome. Strong interaction of ketolides with the hairpin 35 in 23S rRNA may account for the high activity of ketolides against erythromycin‐resistant strains containing rRNA methylated at A2058. The existence of macrolide resistance mutations in the central loop of domain V and in hairpin 35 in domain II together with antibiotic footprinting data suggest that these rRNA segments may be in close proximity in the ribosome and that hairpin 35 may be a constituent part of the ribosomal peptidyl transferase centre.
Probing conserved surfaces on PapD
Authors: Hung Danielle L. , Knight Stefan D. , Hultgren Scott J.
Published: March 2003

PapD is the periplasmic chaperone required for the assembly of P pili in pyelonephritic strains of Escherichia coli. It consists of two immunoglobulin‐like domains bisected by a subunit binding cleft. PapD is the prototype member of a superfamily of immunoglobulin‐like chaperones that work in concert with their respective ushers to assemble a plethora of adhesive organelles including pilus‐ and non‐pilus‐associated adhesins. Three highly conserved residue clusters have been shown to play critical roles in the structure and function of PapD, as determined by site‐directed mutagenesis. The in vivo stability of the chaperone depended on the formation of a buried salt bridge within the cleft. Residues along the G1 beta strand were required for efficient binding of subunits consistent with the crystal structure of PapD–peptide complexes. Finally, Thr‐53, a residue that is part of a conserved band of residues located on the amino‐terminal domain surface opposite the subunit binding cleft, was also found to be critical for pilus assembly, but mutations at Thr‐53 did not interfere with chaperone–subunit complex formation.
Site‐specific recombination at dif by Haemophilus influenzae XerC
Authors: Neilson Liam , Blakely Garry , Sherratt David J.
Published: March 2003

Xer site‐specific recombination at the Escherichia coli chromosomal site dif converts chromosomal dimers to monomers, thereby allowing chromosome segregation during cell division. dif is located in the replication terminus region and binds the E. coli site‐specific recombinases EcoXerC and EcoXerD. The Haemophilus influenzae Xer homologues, HinXerC and HinXerD, bind E. coli dif and exchange strands of dif Holliday junctions in vitro. Supercoiled dif sites are not recombined by EcoXerC and EcoXerD in vitro, possibly as a consequence of a regulatory process, which ensures that in vivo recombination at dif is confined to cells that can initiate cell division and contain dimeric chromosomes. In contrast, the combined action of HinXerC and EcoXerD supports in vitro recombination between supercoiled dif sites, thereby overcoming the barrier to dif recombination exhibited by EcoXerC and EcoXerD. The recombination products are catenated and knotted molecules, consistent with recombination occurring within synaptic complexes that have entrapped variable numbers of negative supercoils. Use of catalytically inactive recombinases provides support for a recombination pathway in which HinXerC‐mediated strand exchange between directly repeated duplex dif sites generates a Holliday junction intermediate that is resolved by EcoXerD to catenated products. These can undergo a second recombination reaction to generate odd‐noded knots.
Termination of DNA replication of bacterial and plasmid chromosomes
Authors: Bussiere Dirksen E. , Bastia Deepak
Published: March 2003

Sequence‐specific replication termini occur in many bacterial and plasmid chromosomes and consist of two components: a cis‐acting ter site and a trans‐acting replication terminator protein. The interaction of a terminator protein with the ter site creates a protein–DNA complex that arrests replication forks in a polar fashion by antagonizing the action of the replicative helicase (thereby exhibiting a contrahelicase activity). Terminator proteins also arrest RNA polymerases in a polar fashion. Passage of an RNA transcript through a terminus from the non‐blocking direction abrogates replication termination function, a mechanism that is likely to be used in conditional termini or replication check points.
The contribution of the O‐glycosylated protein Pir2p/Hsp150 to the construction of the yeast cell wall in wild‐type cells and β1,6‐glucan‐deficient mutants
Authors: Kapteyn J. C. , Van Egmond P. , Sievi E. , Van Den Ende H. , Makarow M. , Klis F. M.
Published: March 2003

The cell wall of yeast contains a major structural unit, consisting of a cell wall protein (CWP) attached via a glycosylphosphatidylinositol (GPI)‐derived structure to β1,6‐glucan, which is linked in turn to β1,3‐glucan. When isolated cell walls were digested with β1,6‐glucanase, 16% of all CWPs remained insoluble, suggesting an alternative linkage between CWPs and structural cell wall components that does not involve β1,6‐glucan. The β1,6‐glucanase‐resistant protein fraction contained the recently identified GPI‐lacking, O‐glycosylated Pir‐CWPs, including Pir2p/Hsp150. Evidence is presented that Pir2p/Hsp150 is attached to β1,3‐glucan through an alkali‐sensitive linkage, without β1,6‐glucan as an interconnecting moiety. In β1,6‐glucan‐deficient mutants, the β1,6‐glucanase‐resistant protein fraction increased from 16% to over 80%. This was accompanied by increased incorporation of Pir2p/Hsp150. It is argued that this is part of a more general compensatory mechanism in response to cell wall weakening caused by low levels of β1,6‐glucan.
Identification of sequences in human transferrin that bind to the bacterial receptor protein, transferrin‐binding protein B
Authors: Retzer Mark D. , Yu Rong‐hua , Schryvers Anthony B.
Published: March 2003

Alignment of amino‐acid sequences from the N‐terminal and C‐terminal halves of transferrin‐binding protein B revealed an underlying bilobed nature with several regions of identity. Based on this analysis, purified recombinant fusion proteins of maltose‐binding protein (Mbp) with intact TbpB, its N‐terminal half or C‐terminal half from the human pathogens Neisseria meningitidis and Moraxella catarrhalis were produced. Solid‐phase binding assays and affinity isolation assays demonstrated that the N‐terminal and C‐terminal halves of TbpB could bind independently to human transferrin (hTf). A solid‐phase overlapping synthetic peptide library representing the amino‐acid sequence of hTf was probed with soluble, labelled Mbp–TbpB fusions to localize TbpB‐binding regions on hTf. An essentially identical series of peptides from domains within both lobes of hTf was recognized by intact TbpB from both organisms, demonstrating a conserved TbpB–hTf interaction. Both halves of TbpB from N. meningitidis bound the same series of peptides, which included peptides from equivalent regions on the two hTf lobes, indicating that TbpB interacts with each lobe of hTf in a similar manner. Mapping of the peptide‐binding regions on a molecular model of hTf revealed a series of nearly adjacent surface regions that nearly encircled each lobe. Binding studies with chimeric hTf/bTf transferrins demonstrated that regions in the C‐lobe of hTf were preferentially recognized by the N‐terminal half of TbpB. Collectively, these results provide evidence that TbpB consists of two lobes, each with distinct yet homologous Tf‐binding regions.
Characterization of the Pseudomonas syringae pv. tomato AvrRpt2 protein: demonstration of secretion and processing during bacterial pathogenesis
Authors: Mudgett Mary Beth , Staskawicz Brian J.
Published: March 2003

Pseudomonas syringae pv. tomato strain DC3000 (Pst DC3000) expressing avrRpt2 is specifically recognized by plant cells expressing RPS2 activity, resulting in localized cell death and plant resistance. Furthermore, transient expression of this bacterial avrRpt2 gene in plant cells results in RPS2‐dependent cell death. This indicates that the AvrRpt2 protein is recognized inside RPS2 plant cells and is sufficient for the activation of disease resistance‐mediated cell death in planta. We explored the possibility that Pst DC3000 delivers AvrRpt2 protein to plant cells via the hrp (type III) secretion pathway. We now provide direct evidence that mature AvrRpt2 protein is secreted from Pst DC3000 and that secretion is hrp dependent. We also show that AvrRpt2 is N‐terminally processed when Arabidopsis thaliana plants are infected with Pst DC3000 expressing avrRpt2. Similar N‐terminal processing of AvrRpt2 occurred when avrRpt2 was stably expressed in A. thaliana. No cleavage of AvrRpt2 was detected in bacteria expressing avrRpt2 in culture or in the plant extracellular fluids. The N‐terminus of AvrRpt2 was not required for RPS2 recognition in planta. However, this region of AvrRpt2 was essential for Pst DC3000‐mediated elicitation of RPS2‐dependent cell death in A. thaliana leaves.
C‐terminal interactions between the XerC and XerD site‐specific recombinases
Authors: Spiers Andrew J. , Sherratt David J.
Published: March 2003

Studies of the site‐specific recombinase Cre suggest a key role for interactions between the C‐terminus of the protein and a region located about 30 residues from the C‐terminus in linking in a cyclical manner the four recombinase monomers present in a recombination complex, and in controlling the catalytic activity of each monomer. By extrapolating the Cre DNA recombinase structure to the related site‐specific recombinases XerC and XerD, it is predicted that the extreme C‐termini of XerC and XerD interact with α‐helix M in XerD and the equivalent region of XerC respectively. Consequently, XerC and XerD recombinases deleted for C‐terminal residues, and mutated XerD proteins containing single amino acid substitutions in αM or in the C‐terminal residues were analysed. Deletion of C‐terminal residues of XerD has no measurable effect on co‐operative interactions with XerC in DNA‐binding assays to the recombination site dif, whereas deletion of 5 or 10 residues of XerC reduces co‐operativity with XerD some 20‐fold. Co‐operative interactions between pairs of truncated proteins during dif DNA binding are reduced 20‐ to 30‐fold. All of the XerD mutants, except one, were catalytically proficient in vitro; nevertheless, many failed to mediate a recombination reaction on supercoiled plasmid in vivo or in vitro, implying that the ability to form a productive recombination complex and/or mediate a controlled recombination reaction is impaired.
Bacterial transcription factors involved in global regulation
Authors: Vicente Miguel , Chater Keith F. , De Lorenzo Víctor
Published: March 2003

The presence of intricate global cell regulation mechanisms may be one reason for the exceptional environmental and evolutionary success of microbes. Promoters, the cis‐acting signals, are responsive to several stimuli related to growth, stress and substrate specificity. Their response is mediated by a wide variety of trans‐acting regulators that sense the environment and the physiological state of the cell and adjust the transcription of specific genes. One of the main transcriptional regulation webs operates in the transition from affluent to barren conditions, with σS being the chief actor in a company of players that stage a competition for the sparsely available RNA polymerase molecules. In this role, σS may be assisted by several factors, including nucleoid‐related proteins and metabolites. In addition, the levels of σS itself are regulated by mechanisms that include inactivation and degradation. Several transcription factors, belonging to different regulatory pathways, may operate in the same promoter. In such a case, the final transcriptional output depends both on the interplay of effectors and on the properties of the recruitment of the effector–RNA polymerase complex to the promoter. RNA polymerase itself is also capable of establishing selective interactions with activators and specific promoter regions through the carboxy‐terminal domain of its alpha subunit (αCTD). Transcriptional regulation controls pervade such crucial events in the life of bacterial cells as Escherichia coli cell division, Bacillus subtilis sporulation and Caulobacter crescentus differentiation. These examples suggest that bacteria have been particularly inventive in adapting gene expression regulation to survive under a diversity of environments and have done so by exploiting the malleable molecular mechanisms involved in transcription, developing complexities that may match those found in eukaryotic cells.
The prepropeptide of vacuolar aminopeptidase I is necessary and sufficient to target the fluorescent reporter protein GFP to the vacuole of yeast by the Cvt pathway
Authors: Martinez Eduardo , Seguí‐Real Bartolomé , Silles Eduardo , Mazón Maria J. , Sandoval Ignacio V.
Published: March 2003

We have studied the capacity of the prepro amino extension of vacuolar protease leucine aminopeptidase I (API) to target the fluorescent reporter protein GFP to the vacuole of yeast. The preproGFP chimera constructed by extending the amino end of GFP with the prepro‐part of API is rapidly degraded in both wild‐type WCG cells and WCG 11/21a cells deficient in the proteasome. In contrast, the chimera expressed in WCG‐PP cells deficient in both proteasome activity and vacuolar proteinase A accumulates in the vacuole, where it remains stable. Replacement of Gly by Ile‐7, a substitution that prevents folding of the pre‐part into an amphipathic helix and inhibits the targeting of the API precursor to the vacuole, inhibits the targeting of preproGFP to the vacuole. The separated pre‐ and pro‐parts of the API precursor do not target GFP to the vacuole. Targeting of preproGFP to the vacuole is independent of its levels of expression, as the fluorescent protein localizes to the vacuole in cells expressing the protein under the control of both the GAL 1/10 or the API promoter. The preproGFP expressed under both promoters is recovered as monomers from cytosolic cell extracts. PreproGFP expressed under the API promoter is packed into cytoplasmic bodies that penetrate into the vacuolar lumen to release the protein. Altogether our results show that the prepro‐part of the API precursor is necessary and sufficient to target the green fluorescent reporter protein to the vacuole.
Expression of the phosphotransferase system both mediates and is mediated by Mlc regulation in Escherichia coli
Authors: Plumbridge Jacqueline
Published: March 2003

The ptsHIcrr operon encodes the cytoplasmic components of the phosphotransferase system (PTS). It is expressed from two major promoters, of which the upstream promoter has previously been shown to be induced by glucose and to be dependent upon cAMP/CAP. This promoter is now shown to be repressed by Mlc. Mlc is a transcriptional regulator controlling, among others, the gene ptsG, encoding EIICBGlc, the glucose‐specific transporter of the PTS. Transcription of ptsH p0 and ptsG are subject to the same regulatory pattern. In addition to induction by glucose and repression by Mlc, mutations in ptsHIcrr, which interrupt the PEP‐dependent phosphate transfer through the soluble components of the PTS, lead to high expression of both ptsH and ptsG, while mutations inactivating EIIBCGlc are non‐inducible. Mutations in mlc lead to high constitutive expression and are dominant, implying that Mlc is the ultimate regulator of ptsHI and ptsG expression. Growth on other PTS sugars, besides glucose, also induces ptsH and ptsG expression, suggesting that the target of Mlc regulation is the PTS. However, induction by these other sugars is only observed in the presence of ptsG+, thus confirming the importance of glucose and EIICBGlc in the regulation of the PTS. The ptsG22 mutation, although negative for glucose transport, shows a weak positive regulatory phenotype. The mutation has been sequenced and its effect on regulation investigated.
ZntR is an autoregulatory protein and negatively regulates the chromosomal zinc resistance operon znt of Staphylococcus aureus
Authors: Singh Vineet K. , Xiong Anming , Usgaard Thomas R. , Chakrabarti Swarup , Deora Rajendar , Misra Tapan K. , Jayaswal Radheshyam K.
Published: March 2003

A chromosomally encoded znt operon of Staphylococcus aureus consists of two consecutive putative genes designated zntR and zntA. The zntA gene encodes a transmembrane protein that facilitates extrusion of Zn2+ and Co2+, whereas the zntR gene encodes a putative regulatory protein that controls the expression of the znt operon. The zntR gene was amplified using the polymerase chain reaction, cloned into Escherichia coli for overexpression as His‐tagged ZntR and purified by Ni2+‐affinity column. His‐tag‐free ZntR was purified to near homogeneity after digestion with enterokinase. Electrophoretic mobility shift assays (EMSAs) indicated that the ZntR bound to a fragment of DNA corresponding to the chromosomal znt promoter region with an affinity of about 8.0 × 10−12 M. The addition of 25 μM Zn2+ or Co2+ in the binding reaction completely or significantly inhibited association of ZntR with the znt promoter. DNase I footprinting assays identified a ZntR binding site encompassing 49 nucleotides in the znt promoter region that contained repeated TGAA sequences. These sequences have been proposed to be the binding sites for SmtB, a metallorepressor protein from the cyanobacterium Synechococcus, to its corresponding operator/promoter. In vitro transcription assays, using S. aureus RNA polymerase, revealed that ZntR represses transcription from the znt promoter in a concentration‐dependent fashion. The EMSAs, DNase I footprinting and in vitro transcription assays indicate that ZntR is a trans‐acting repressor protein that binds to the znt promoter region and regulates its own transcription together with that of zntA.
The type IV bundle‐forming pilus of enteropathogenic Escherichia coli undergoes dramatic alterations in structure associated with bacterial adherence, aggregation and dispersal
Authors: Knutton Stuart , Shaw Robert K. , Anantha Ravi P. , Donnenberg Michael S. , Zorgani Aziz A.
Published: March 2003

BFP, a plasmid‐encoded type IV bundle‐forming pilus produced by enteropathogenic Escherichia coli (EPEC), has recently been shown to be associated with the aggregation of bacteria and dispersal of bacteria from bacterial microcolonies. In standard 3 h HEp‐2 cell assays, EPEC adhere in localized microcolonies; after 6 h, bacterial microcolonies are no longer present, indicating that bacterial aggregation and dispersal occurs in vitro during EPEC adhesion to cultured epithelial cells. To examine the role of BFP in EPEC aggregation and dispersal, we examined HEp‐2 cell adhesion of strain E2348/69 and defined E2348/69 mutants by immunofluorescence and immunoelectron microscopy. BFP was expressed initially as ≈ 40 nm diameter pilus bundles that promoted bacteria–bacteria interaction and microcolony formation. BFP subsequently underwent a striking alteration in structural organization with the formation of much longer and thicker (≈ 100 nm diameter) pilus bundles, which frequently aggregated laterally to form even thicker bundles often arranged in a loose three‐dimensional network; EPEC dispersal from bacterial microcolonies was associated with this transformation of BFP from thin to thick bundles. Bacterial dispersal and transformation of BFP from thin to thick bundles did not occur with a bfpF mutant of strain E2348/69. It is concluded that BFP promotes both the formation and the dispersal of EPEC microcolonies, that the dispersal phase requires BfpF and that dispersal is associated with dramatic alterations in the structure of BFP bundles.
Identification of the pH‐inducible, proton‐translocating F1F0‐ATPase (atpBEFHAGDC) operon of Lactobacillus acidophilus by differential display: gene structure, cloning and characterization
Authors: Kullen Martin J. , Klaenhammer Todd R.
Published: March 2003

The influence of low pH on inducible gene expression in Lactobacillus acidophilus was investigated by the use of differential display. Logarithmic phase cultures were exposed to pH 3.5 for various intervals, and RNA was isolated and reverse transcribed. The resultant cDNAs were subjected to PCR and the products were resolved by electrophoresis. Several cDNA products were induced after exposure to pH 3.5. One of these products, a 0.7 kb fragment, showed sequence similarity to bacterial atpBEF genes of the atp operon, whose genes encode the various subunits of the F1F0‐ATPase. With the 0.7 kb differential display product as a probe, hybridizations with total RNA from untreated and acid‐treated L. acidophilus verified the acid inducibility of this operon. The increase in atp mRNA induced by low pH was accompanied by an increase in the activity of the enzyme in membrane extracts. The full‐length atp operon was sequenced, and its genes were in the order of atpBEFHAGDC, coding for the a, c, b, δ, α, γ, β and ε subunits respectively. The operon contained no i gene, but was preceded by a 122 bp intergenic space, which contained putative extended −10 and −35 promoter regions. Primer extension analysis of RNA from cultures that were shifted from pH 5.6 to pH 3.5, and held for 0, 30 or 45 min, revealed that the transcriptional start site did not change position as a function of culture pH or time after exposure to pH 3.5. The primary structure and genetic organization indicated that the H+‐ATPase of L. acidophilus is a typical F1F0‐type ATPase. The similarity to streptococcal ATPases and the acid inducibility of this operon suggest that it may function in the ATP‐dependent extrusion of protons and maintenance of cytoplasmic pH. Finally, the use of differential display RT‐PCR was an effective approach to identify genes in L. acidophilus induced by an environmental stimulus.
A homologue of the Agrobacterium tumefaciens VirB and Bordetella pertussis Ptl type IV secretion systems is essential for intracellular survival of Brucella suis
Authors: O'Callaghan David , Cazevieille Chantal , Allardet‐Servent Annick , Boschiroli Maria Laura , Bourg Gisèle , Foulongne Vincent , Frutos Patrice , Kulakov Youri , Ramuz Michel
Published: March 2003

Analysis of a TnblaM mutant of Brucella suis 1330, identified as being unable to multiply in Hela cells, allowed us to identify a 11 860 bp region of the B. suis genome encoding a type IV secretion system, homologous to the VirB system of Agrobacterium tumefaciens and the Ptl system of Bordetella pertussis. DNA sequence revealed 12 open reading frames (ORFs) encoding homologues of the 11 VirB proteins present in the pTi plasmid of Agrobacterium with a similar genetic organization, and a twelfth ORF encoding a putative lipoprotein, homologous to a protein involved in mating pair formation during bacterial conjugation and to adhesins used by Pseudomonas species to bind to plant roots. Phylogenetic trees based on the sequences of VirB4 and VirB9 protein homologues suggest that evolution of the systems from DNA transfer towards protein secretion did not stem from a single event but that the protein secretion systems have evolved independently. Four independent mutants in virB5, virB9 or virB10 were highly attenuated in an in vitro infection model with human macrophages. The virulence was restored by complementation with a plasmid containing the full virB region. The virB region appears to be essential for the intracellular survival and multiplication of B. suis.
Distinct roles for Rev1p and Rev7p during translesion synthesis in Saccharomyces cerevisiae
Authors: Baynton K. , Bresson‐Roy A. , Fuchs Robert P. P.
Published: March 2003

Translesion synthesis (TLS) in Saccharomyces cerevisiae requires at least Rev1p and polymerase ζ (Pol ζ), a complex of the Rev3 polymerase and its accessory factor Rev7p. Although their precise role(s) are poorly characterized, in vitro studies suggest that each protein contributes to TLS in a manner dependent on the particular lesion and surrounding DNA sequence. In the present study, strand segregation analysis is used to attempt to identify the role(s) of the Rev1 and Rev7 proteins during TLS. This assay uses double‐stranded plasmids containing a genetic marker opposite to a replication blocking lesion (N‐2‐acetylaminofluorene; AAF) to measure TLS quantitatively and qualitatively in vivo. The AAF adduct is localized within a repetitive sequence in a manner that allows the formation of misaligned primer–template replication intermediates. Elongation from a misaligned intermediate fixes a frameshift mutation (slipped TLS), while extension of the correctly aligned lesion terminus yields error‐free (non‐slipped) TLS. The results indicate that there is a strong requirement for Rev7p during Pol ζ‐mediated TLS measured in vivo. Furthermore, Rev1p is needed only for non‐slipped TLS; slipped TLS remains efficient in its absence, revealing a previously uncharacterized Rev1p activity similar to Escherichia coli UmuDC function. Specifically, this activity is required for elongation from a correctly aligned lesion terminus.
Mutational analysis of the Pseudomonas syringae pv. tomato hrpA gene encoding Hrp pilus subunit
Authors: Taira Suvi , Tuimala Jarno , Roine Elina , Nurmiaho‐Lassila Eeva‐Liisa , Savilahti Harri , Romantschuk Martin
Published: March 2003

Plant pathogenic Pseudomonas syringae strains harbour a type III secretion pathway suggested to be involved in the delivery of effector proteins from the bacteria into plant cells. During plant interaction, the bacteria apparently produce surface appendages, termed Hrp pili, that are indispensable for the secretion process. We have created an insertion mutation library, as well as deletion mutations to hrpA, the structural gene encoding Hrp pilin. Analysis of the mutants revealed gene regions important for hrpA expression, pilus assembly and pilus‐dependent autoagglutination of the bacteria. The majority of insertions in the amino‐terminal half of the pilin were tolerated without bacterial interaction with plants being affected, while the carboxy‐terminus appeared to be needed for pilus assembly. Insertions in the 5′ non‐translated region and the first codons within the open reading frame affected mRNA production or stability and abolished protein production.
Recruitment of the yeast MADS‐box proteins, ArgRI and Mcm1 by the pleiotropic factor ArgRIII is required for their stability
Authors: El Bakkoury Mohamed , Dubois Evelyne , Messenguy Francine
Published: March 2003

Regulation of arginine metabolism requires the integrity of four regulatory proteins, ArgRI, ArgRII, ArgRIII and Mcm1. To characterize further the interactions between the different proteins, we used the two‐hybrid system, which showed that ArgRI and Mcm1 interact together, and with ArgRII and ArgRIII, without an arginine requirement. To define the interacting domains of ArgRI and Mcm1 with ArgRIII, we fused portions of ArgRI and Mcm1 to the DNA‐binding domain of Gal4 (GBD) and created mutations in GBD‐ArgRI and GBD‐Mcm1. The putative α helix present in the MADS‐box domain of ArgRI and Mcm1 is their major region of interaction with ArgRIII. Interactions between the two MADS‐box proteins and ArgRIII were confirmed using affinity chromatography. The requirement for ArgRIII in the control of arginine metabolism can be bypassed in vitro as well as in vivo by overproducing ArgRI or Mcm1, which indicates that ArgRIII is not present in the protein complex formed with the ‘arginine boxes’. We show that the impairment of arginine regulation in an argRIII deletant strain is a result of a lack of stability of ArgRI and Mcm1. A mutation in ArgRI, impairing its interaction with ArgRIII, leads to an unstable ArgRI protein in a wild‐type strain. ArgRIII integrity is crucial for Mcm1 function, as shown by the marked decreased expression of five genes controlled by Mcm1. However, ArgRIII is likely to recruit other proteins in the yeast cell, as overexpression of Mcm1 does not compensate some physiological defects observed in an argRIII deletant strain.
Multiple independent horizontal transfers of informational genes from bacteria to plasmids and phages: implications for the origin of bacterial replication machinery
Authors: Moreira David
Published: March 2003

In contrast to the universality of other central genetic mechanisms, the replication machinery of Bacteria is clearly different from those of Archaea and Eukaryotes. A large number of bacterial genes involved in DNA replication can also be found in plasmids and phages. Based on this, it has been recently proposed that the ancestral bacterial genes were displaced by non‐orthologous replication genes from plasmids and phages, which would explain the profound difference between Bacteria and the other domains of life. The alternative hypothesis is that these DNA replication genes have been frequently transferred from bacterial hosts to the genomes of their plasmids and phages. The phylogenetic analysis of the bacterial DNA replication proteins most abundant in databases (replicative helicase DnaB, single‐strand binding protein Ssb and topoisomerase TopB) presented here supports the latter hypothesis. Each protein tree shows that sequences from plasmids and phages branch close to their bacterial‐specific hosts, suggesting multiple independent horizontal transfers. Therefore, there is no evidence so far for non‐orthologous gene displacement of these genes.
A bacterial genome in flux: the twelve linear and nine circular extrachromosomal DNAs in an infectious isolate of the Lyme disease spirochete Borrelia burgdorferi
Authors: Casjens Sherwood , Palmer Nanette , Van Vugt René , Mun Huang Wai , Stevenson Brian , Rosa Patricia , Lathigra Raju , Sutton Granger , Peterson Jeremy , Dodson Robert J. , Haft Daniel , Hickey Erin , Gwinn Michelle , White Owen , M. Fraser Claire
Published: March 2003

We have determined that Borrelia burgdorferi strain B31 MI carries 21 extrachromosomal DNA elements, the largest number known for any bacterium. Among these are 12 linear and nine circular plasmids, whose sequences total 610 694 bp. We report here the nucleotide sequence of three linear and seven circular plasmids (comprising 290 546 bp) in this infectious isolate. This completes the genome sequencing project for this organism; its genome size is 1 521 419 bp (plus about 2000 bp of undetermined telomeric sequences). Analysis of the sequence implies that there has been extensive and sometimes rather recent DNA rearrangement among a number of the linear plasmids. Many of these events appear to have been mediated by recombinational processes that formed duplications. These many regions of similarity are reflected in the fact that most plasmid genes are members of one of the genome's 161 paralogous gene families; 107 of these gene families, which vary in size from two to 41 members, contain at least one plasmid gene. These rearrangements appear to have contributed to a surprisingly large number of apparently non‐functional pseudogenes, a very unusual feature for a prokaryotic genome. The presence of these damaged genes suggests that some of the plasmids may be in a period of rapid evolution. The sequence predicts 535 plasmid genes ≥300 bp in length that may be intact and 167 apparently mutationally damaged and/or unexpressed genes (pseudogenes). The large majority, over 90%, of genes on these plasmids have no convincing similarity to genes outside Borrelia, suggesting that they perform specialized functions.
The respiratory gene OXA1 has two fission yeast orthologues which together encode a function essential for cellular viability
Authors: Bonnefoy Nathalie , Kermorgant Michèle , Groudinsky Olga , Dujardin Geneviève
Published: March 2003

The Saccharomyces cerevisiae nuclear gene OXA1, which is conserved from prokaryotes to human, was shown to be essential for cytochrome c oxidase and F1F0–ATP synthase biogenesis. We have searched for an orthologue of OXA1 in Schizosaccharomyces pombe, another yeast that is highly diverged from S. cerevisiae and which could more closely model higher eukaryotes. In particular, S. pombe exhibits a limited growth under anaerobic conditions and is petite negative, that is it does not tolerate large deletions of its mitochondrial DNA. Surprisingly, two S. pombe cDNAs able to complement an S. cerevisiae oxa1 mutation were isolated. The corresponding genes have different chromosomal locations and intron contents. They encode distinct proteins, both sharing a weak sequence identity one with the other and with Oxa1p. A phenotypic analysis of both single inactivations demonstrates that only one gene is essential for respiration in S. pombe. However, the double inactivation is lethal. This work gives new insight into the dependence of S. pombe viability upon oxa1 function, providing evidence of a connection between petite negativity, a functional respiratory chain and F1F0–ATP synthase complex in S. pombe.
Functional genomics of Helicobacter pylori: identification of a β‐1,4 galactosyltransferase and generation of mutants with altered lipopolysaccharide
Authors: Logan S. M. , Conlan J. W. , Monteiro M. A. , Wakarchuk W. W. , Altman E.
Published: March 2003

A previously annotated open reading frame (ORF) (HP0826) from Helicobacter pylori was cloned and expressed in Escherichia coli cells and determined to be a β‐1,4‐galactosyltransferase that used GlcNAc as an acceptor. Mutational analysis in H. pylori strains demonstrated that this enzyme plays a key role in the biosynthesis of the type 2 N‐acetyl‐lactosamine (LacNAc) polysaccharide O‐chain backbone, by catalysing the addition of Gal to GlcNAc. To examine the potential role of this O‐chain structure in bacterial colonization of the host stomach, the mutation was introduced into H. pylori strain SS1 which is known to be capable of colonizing the gastric mucosa of mice. Compared with the parental strain, mutated SS1 was less efficient at colonizing the murine stomach.
The archaeal halophilic virus‐encoded Dam‐like methyltransferase M.φCh1‐I methylates adenine residues and complements dam mutants in the low salt environment of Escherichia coli
Authors: Baranyi U. , Klein R. , Lubitz W. , Krüger D. H. , Witte A.
Published: March 2003

The genome of the archaeal virus φCh1, infecting Natrialba magadii (formerly Natronobacterium magadii), is composed of 58.5 kbp linear ds DNA. Virus particles contain several RNA species in sizes of 100–800 nucleotides. A fraction of φCh1 genomes is modified within 5′‐GATC‐3′ and related sequences, as determined by various restriction enzyme digestion analyses. High performance liquid chromatography revealed a fifth base, in addition to the four nucleosides, which was identified as N6‐methyladenosine. Genetic analyses and subsequent sequencing led to the identification of a DNA (N6‐adenine) methyltransferase (mtase) gene. The protein product was designated M.φCh1‐I. By the localization of the most conserved motifs (a DPPY motif occurring before FxGxG), the enzyme was placed within the β‐subgroup of the (N6‐adenine) methyltransferase class. The mtase gene of φCh1 was classified as a ‘late’ gene, as determined by measuring the kinetics of mRNA and protein expression in N. magadii during the lytic cycle of φCh1. After infection of cells, M.φCh1‐I mRNA and protein could be detected in lower amounts than in the situation of virus induction from lysogenic cells. Consequently, only about 5% of the φCh1 progeny genomes after infection of N. magadii carry the M.φCh1‐I methylation in contrast to 50% of virus genomes generated by induction of φCh1‐lysogenic N. magadii cells. Heterologous expression of the mtase from a halophile with 3 M cytoplasmic salt concentration showed an unexpected feature: the protein was active in the low environment of Escherichia coli and was able to methylate DNA in vivo. Interestingly, it seemed to exhibit a higher sequence specificity in E. coli that resulted in adenine methylation exclusively in the sequence 5′‐GATC‐3′. Additionally, expression of M.φCh1‐I in dam–E. coli cells led to a complete substitution of the function of M.Dam in DNA mismatch repair.
Sxa2 is a serine carboxypeptidase that degrades extracellular P‐factor in the fission yeast Schizosaccharomyces pombe
Authors: Ladds Graham , Davey John
Published: March 2003

Stimulating the fission yeast Schizosaccharomyces pombe with mating pheromones brings about responses that lead to cell conjugation. Persistent stimulation does not, however, induce a continuous response as the cells become desensitized to the presence of the pheromone. One mechanism that contributes to desensitization in M‐cells is the release of a carboxypeptidase that inactivates the extracellular P‐factor pheromone. Production of the carboxypeptidase requires a functional sxa2 gene. In this study, we report the first molecular characterization of the Sxa2 protein and provide direct evidence that it is the carboxypeptidase that degrades P‐factor. Sxa2 is synthesized as a precursor that undergoes an internal cleavage event catalysed by a protease with specificity for basic residues. This generates a series of catalytically active N‐terminal fragments and an inactive C‐terminal fragment. Cleavage is essential for activation of the carboxypeptidase and, although the C‐terminal fragment is inactive, it is required for the N‐terminal fragment to attain activity.
PrfA mediates specific binding of RNA polymerase of Listeria monocytogenes to PrfA‐dependent virulence gene promoters resulting in a transcriptionally active complex
Authors: Böckmann Regine , Dickneite Carmen , Goebel Werner , Bohne Jutta
Published: March 2003

There is accumulating evidence that the coordinate transcription of the virulence genes in Listeria monocytogenes constitutes a very complex regulation mechanism which might require other factors in addition to PrfA. We previously described an unknown proteinaceous component from crude bacterial cell extracts, which, together with PrfA, formed a specific complex (CI) in electrophoretic mobility shift assays (EMSA) with an hly promoter probe. Here we identify the RNA polymerase (RNAP) of L. monocytogenes as an essential component of the CI complex. Addition of purified RNAP plus PrfA to the hly promoter probe allowed reconstitution of a complex migrating at the same height as CI. By using EMSA and DNaseI footprint experiments it could be shown that PrfA leads to an enhanced and specific binding of RNAP. Transcriptional activity of RNAP in vitro, using the actA promoter, was strictly dependent on PrfA.
Species‐specific binding of human secretory component to SpsA protein of Streptococcus pneumoniae via a hexapeptide motif
Authors: Hammerschmidt Sven , Tillig Melanie P. , Wolff Sonja , Vaerman Jean‐Pierre , Chhatwal Gursharan S.
Published: March 2003

SpsA, a pneumococcal surface protein belonging to the family of choline‐binding proteins, interacts specifically with secretory immunglobulin A (SIgA) via the secretory component (SC). SIgA and free SC from mouse, rat, rabbit and guinea‐pig failed to interact with SpsA indicating species‐specific binding to human SIgA and SC. SpsA is the only pneumococcal receptor molecule for SIgA and SC as confirmed by complete loss of SIgA and SC binding to a spsA mutant. Analysis of recombinant SpsA fusion proteins showed that the binding domain is located in the N‐terminal region of SpsA. By the use of different truncated N‐terminal SpsA fusion proteins, the minimum binding domain was shown to be composed of 112 amino acids (residues 172–283). The sequence of this 112‐amino‐acids domain was used to spot synthesize 34 overlapping peptides, consisting of 15 amino acids each, with an offset of three amino acids on a cellulose membrane. One of the peptides reacted specifically with both SIgA and SC. By using a second membrane with immobilized synthetic peptides of decreasing length containing parts of the identified 15‐amino‐acid motif a hexapeptide, YRNYPT was identified as the binding motif for SC and SIgA. SpsA proteins with a size smaller than the assay‐positive domain of 112 amino acids were able to inhibit the interaction of SIgA and pneumococci provided they contained the binding motif. The results indicated that the hexapeptide YRNYPT located in SpsA of pneumococcal strain type 1 (ATCC 33400) between amino acids 198 and 203 is involved in SIgA and SC binding. Because synthetic peptides containing only parts of the hexapeptide also assayed positive, these results further suggest that at least the amino acids YPT of the identified hexapeptide are critical for binding to SC and SIgA. Amino acid substitutions in the identified putative binding motif abolished SC‐/SIgA‐binding activity of the mutated SpsA protein, confirming the functional activity of this hexapeptide and the critical role of the amino acids YPT in SC and SIgA binding. Identification of this motif, which is highly conserved in SpsA protein among different serotypes, might contribute towards a new peptide based vaccine strategy.
Novel type I restriction specificities through domain shuffling of HsdS subunits in Lactococcus lactis
Authors: O'sullivan David , Twomey Denis P. , Coffey Aidan , Hill Colin , Fitzgerald Gerald F. , Ross R. Paul
Published: March 2003

This study identifies a natural system in Lactococcus lactis, in which a restriction modification specificity subunit resident on a 6159 bp plasmid (pAH33) alters the specificity of a functional R/M mechanism encoded by a 20.3 kb plasmid, pAH82. The new specificity was identified after phenotypic and molecular analysis of a 26.5 kb co‐integrate plasmid (pAH90), which was detected after bacteriophage challenge of the parent strain. Analysis of the regions involved in the co‐integration revealed that two novel hybrid hsdS genes had been formed during the co‐integration event. The HsdS chimeras had interchanged the C‐ and N‐terminal variable domains of the parent subunits, generating two new restriction specificities. Comparison of the parent hsdS genes with other type I specificity determinants revealed that the region of the hsdS genes responsible for the co‐integration event is highly conserved among lactococcal type I hsdS determinants. Thus, as hsdS determinants are widespread in the genus Lactococcus, new restriction specificities may evolve rapidly after homologous recombination between these genes. This study demonstrates that, similar to previous observations in Gram‐negative bacteria, a Gram‐positive bacterium can acquire novel restriction specificities naturally through domain shuffling of resident HsdS subunits.