bluuebunny/crossref_metadata_2025 huggingface dataset:
A global repressor (Mlc) is involved in glucose induction of the ptsG gene encoding major glucose transporter in Escherichia coli
Authors: Kimata Keiko , Inada Toshifumi , Tagami Hideaki , Aiba Hiroji
Published: March 2003

Glucose stimulates the expression of ptsG encoding the major glucose transporter in Escherichia coli. We isolated Tn10 insertion mutations that confer constitutive expression of ptsG. The mutated gene was identified as mlc, encoding a protein that is known to be a repressor for transcription of several genes involved in carbohydrate utilization. Expression of ptsG was eliminated in a mlc crp double‐negative mutant. The Mlc protein was overproduced and purified. In vitro transcription studies demonstrated that transcription of ptsG is stimulated by CRP–cAMP and repressed by Mlc. The action of Mlc is dominant over that of CRP–cAMP. DNase I footprinting experiments revealed that CRP–cAMP binds at two sites centred at −40.5 and −95.5 and that Mlc binds at two regions centred around −8 and −175. The binding of CRP–cAMP stimulated the binding of RNA polymerase to the promoter while Mlc inhibited the binding of RNA polymerase but not the binding of CRP–cAMP. Gel‐mobility shift assay indicated that glucose does not affect the Mlc binding to the ptsG promoter. Our results suggest that Mlc is responsible for the repression of ptsG transcription and that glucose modulates the Mlc activity by unknown mechanism.
The relationship between hetero‐oligomer formation and function of the topological specificity domain of the Escherichia coli MinE protein
Authors: Zhang Yan , Rowland Susan , King Glenn , Braswell Emory , Rothfield Lawrence
Published: March 2003

MinE is an oligomeric protein that, in conjunction with other Min proteins, is required for the proper placement of the cell division site of Escherichia coli. We have examined the self‐association properties of MinE by analytical ultracentrifugation and by studies of hetero‐oligomer formation in non‐denaturing polyacrylamide gels. The self‐association properties of purified MinE predict that cytoplasmic MinE is likely to exist as a mixture of monomers and dimers. Consistent with this prediction, the C‐terminal MinE22–88 fragment forms hetero‐oligomers with MinE+ when the proteins are co‐expressed. In contrast, the MinE36–88 fragment does not form MinE+/MinE36–88 hetero‐oligomers, although MinE36–88 affects the topological specificity of septum placement as shown by its ability to induce minicell formation when co‐expressed with MinE+ in wild‐type cells. Therefore, hetero‐oligomer formation is not necessary for the induction of minicelling by expression of MinE36–88 in wild‐type cells. The interference with normal septal placement is ascribed to competition between MinE36–88 and the corresponding domain in the complete MinE protein for a component required for the topological specificity of septal placement.
Manifestation of the Kanagawa phenomenon, the virulence‐associated phenotype, of Vibrio parahaemolyticus depends on a particular single base change in the promoter of the thermostable direct haemolysin gene
Authors: Okuda Jun , Nishibuchi Mitsuaki
Published: March 2003

Thermostable direct haemolysin of Vibrio parahaemolyticus has been shown to be a major virulence factor. The Kanagawa phenomenon (KP), haemolysis induced by this haemolysin on a special blood agar medium, is strongly associated with clinical strains. We have been studying the expressions of various tdh genes encoding this haemolysin to elucidate the significance of the tdh genes possessed by KP‐negative strains isolated from patients. We examined the importance of the promoter sequence variation for expression level of the tdh gene in this study. Only the tdh2 gene, one of the two tdh genes (tdh1 and tdh2) present in a KP‐positive strain, was previously shown to be responsible for the haemolytic activity of the KP‐positive strain. The tdh1– and tdh2–lacZ fusions were used to determine and analyse the promoter sequence by primer extension and site‐directed mutagenesis methods. Two bases (positions −24 and −34) within the determined tdh2 promoter sequence were shown to be mostly responsible for the difference in the promoter strength between the tdh2 and tdh1 genes both in Escherichia coli and in V. parahaemolyticus backgrounds. Representative tdh promoters of KP‐negative strains are close to the tdh2 promoter; they differ at position −34 but have the same base at position −24 as the tdh2 promoter. We demonstrated that base substitution of the tdh promoters of KP‐negative strains only at position −34 is sufficient to increase the expression of these genes to the KP‐positive level. Therefore, the tdh genes of KP‐negative strains are considered to be potentially important because they can generate a KP‐positive subclone by a point mutation in their promoters.
Decorin‐binding adhesins from Borrelia burgdorferi
Authors: Guo Betty P. , Brown Eric L. , Dorward David W. , Rosenberg Lawrence C. , Höök Magnus
Published: March 2003

Lyme disease is a tick‐transmitted infection caused by the spirochete Borrelia burgdorferi. Ticks deposit B. burgdorferi into the dermis of the host, where they eventually become associated with collagen fibres. We demonstrated previously that B. burgdorferi is unable to bind collagen, but can bind the collagen‐associated proteoglycan decorin and expresses decorin‐binding proteins (Dbps). We have now cloned and sequenced two genes encoding the proteins, DbpA and DbpB, which have a similar structure, as revealed by circular dichroism (CD) spectroscopy of recombinant proteins. Competition experiments revealed a difference in binding specificity between DbpA and DbpB. Western blot analysis of proteinase K‐treated intact B. burgdorferi and transmission electron microscopy studies using antibodies raised against recombinant Dbps demonstrated that these proteins are surface exposed. DbpA effectively inhibits the attachment of B. burgdorferi to a decorin substrate, whereas DbpB had a marginal effect, suggesting a difference in substrate specificity between the two Dbps. Polystyrene beads coated with DbpA adhered to a decorin‐containing extracellular matrix produced by cultured skin fibroblasts, whereas beads coated with OspC did not. Taken together, these data suggest that Dbps are adhesins of the MSCRAMM (microbial surface component‐recognizing adhesive matrix molecule) family, which mediate B. burgdorferi attachment to the extracellular matrix of the host.
The FruA signal transduction protein provides a checkpoint for the temporal co‐ordination of inter‐ cellular signals in Myxococcus xanthus development
Authors: Ellehauge Eva , Nørregaard‐Madsen Mads , Søgaard‐Andersen Lotte
Published: March 2003

During fruiting body morphogenesis in Myxococcus xanthus, the intercellular C‐signal induces aggregation, sporulation and developmental gene expression. To understand how a single signal system may induce temporally separated processes, we have focused on the class II gene, which codes for an essential component in the C‐signal transduction pathway. We report that class II is identical to fruA and codes for a DNA binding response regulator. Transcription of fruA is developmentally regulated and depends on the early acting intercellular A‐ and E‐signals. However, fruA transcription is independent of C‐signal. Rather, genetic evidence suggests that C‐signal controls FruA activity post‐translationally. Genetic evidence strongly indicates that FruA is activated by phosphorylation. We propose that C‐signalling results in the phosphorylation of FruA, thus activating FruA to interact with downstream targets. In the motility branch of the C‐signalling pathway, FruA interacts with the Frz motility system; in the sporulation branch, we show that FruA is required for transcription of the sporulation locus devRS. On the basis of the two levels of control of FruA activity, we propose that FruA serves as a control point for the temporal co‐ordination of intercellular signals during M. xanthus development.
The E protein of satellite phage P4 acts as an anti‐repressor by binding to the C protein of helper phage P2
Authors: Liu Tao , Renberg Sara K. , Haggård‐Ljungquist Elisabeth
Published: March 2003

Temperate phage P2 has the capacity to function as a helper for the defective, unrelated, satellite phage P4. In the absence of a helper, P4 can either lysogenize its host or establish itself as a plasmid. For lytic growth, P4 requires the structural genes, packaging and lysis functions of the helper. P4 can get access to the late genes of prophage P2 by derepression, which is mediated by the P4 E protein. E has been hypothesized to function as an anti‐repressor. To locate possible epitopes interacting with E, an epitope display library was screened against E, and the most frequent sequence found had some identities to a region within P2 C. Using the yeast two‐hybrid system, a clear activation of a reporter gene was found, strongly supporting an interaction between E and C. The P2 C repressor is believed to act as a dimer, which is confirmed in this work using in vivo dimerization studies. The E protein was also found to form dimers in vivo. The E protein only affects dimerization of C marginally, but the presence of E enhances multimeric forms of C. Furthermore, binding of the C protein to its operator is inhibited by E in vitro, indicating that the anti‐repressor function of E is mediated by the formation of multimeric complexes of E and C that interfere with the binding of C to its operator.
Characterization of the C‐terminal domain essential for toxic activity of adenylate cyclase toxin
Authors: Bejerano Michal , Nisan Israel , Ludwig Albrecht , Goebel Werner , Hanski Emanuel
Published: March 2003

Adenylate cyclase toxin (CyaA) of Bordetella pertussis belongs to the RTX family of toxins. These toxins are characterized by a series of glycine‐ and aspartate‐rich nonapeptide repeats located at the C‐terminal half of the toxin molecules. For activity, RTX toxins require Ca2+, which is bound through the repeat region. Here, we identified a stretch of 15 amino acids (block A) that is located C‐terminally to the repeat region and is essential for the toxic activity of CyaA. Block A is required for the insertion of CyaA into the plasma membranes of host cells. Mixing of a short polypeptide composed of block A and eight Ca2+ binding repeats with a mutant of CyaA lacking block A restores toxic activity fully. This in vitro interpolypeptide complementation is achieved only when block A is present together with the Ca2+ binding repeats on the same polypeptide. Neither a short polypeptide composed of block A only nor a polypeptide consisting of eight Ca2+ binding repeats, or a mixture of these two polypeptides, complement toxic activity. It is suggested that functional complementation occurs because of binding between the Ca2+ binding repeats of the short C‐terminal polypeptide and the Ca2+ binding repeats of the CyaA mutant lacking block A.
Localization of FtsL to the Escherichia coli septal ring
Authors: Ghigo Jean‐Marc , Weiss David S. , Chen Joseph C. , Yarrow Justin C. , Beckwith Jon
Published: March 2003

In Escherichia coli, nine gene products are known to be essential for assembly of the division septum. One of these, FtsL, is a bitopic membrane protein whose precise function is not understood. Here we use fluorescence microscopy to study the subcellular localization of FtsL, both in a wild‐type strain and in a merodiploid strain that expresses a GFP–FtsL fusion protein. We show that FtsL localizes to the cell septum where it forms a ring analogous to the cytoplasmic FtsZ ring. FtsL localization is dependent upon the function of FtsZ, FtsA and FtsQ, but not FtsI. In a reverse approach, we use fusions of green fluorescent protein (GFP) to FtsZ, FtsA and ZipA to show that these proteins localize to the division site in an FtsL‐independent fashion. We propose that FtsL is a relatively late recruit to the ring structure that mediates septation.
Teichuronic acid operon of Bacillus subtilis 168
Authors: Soldo Blazenka , Lazarevic Vladimir , Pagni Marco , Karamata Dimitri
Published: March 2003

Sequence analysis reveals that the Bacillus subtilis 168 tuaABCDEFGH operon encodes enzymes required for the polymerization of teichuronic acid as well as for the synthesis of one of its precursors, the UDP‐glucuronate. Mutants deficient in any of the tua genes, grown in batch cultures under conditions of phosphate limitation, were characterized by reduced amounts of uronate in their cell walls. The teichuronic acid operon belongs to the Pho regulon, as phosphate limitation induces its transcription. Placing the tuaABCDEFGH operon under the control of the inducible Pspac promoter allowed its constitutive expression independently of the phosphate concentration in the medium; the level of uronic acid in cell walls was dependent on the concentration of the inducer. Apparently, owing to an interdependence between teichoic and teichuronic acid incorporation into the cell wall, in examined growth conditions, the balance between the two polymers is maintained in order to insure a constant level of the wall negative charge.
High‐frequency intracellular invasion of epithelial cells by serotype M1 group A streptococci: M1 protein‐mediated invasion and cytoskeletal rearrangements
Authors: Dombek Priscilla E. , Cue David , Sedgewick Jerry , Lam Hong , Ruschkowski Sharon , Finlay B. Brett , Cleary P. Patrick
Published: March 2003

A clonal variant of serotype M1 group A streptococcus (designated M1inv+) has been linked to severe and invasive infections, including sepsis, necrotizing fasciitis and toxic shock. High frequency internalization of cultured epithelial cells by the M1inv+ strain 90‐226 is dependent upon the M1 protein. Invasion of HeLa cells was blocked by an anti‐M1 antibody, invasion by an M1− strain (90‐226 emm1::km) was greatly reduced, and latex beads bound to M1 protein were readily internalized by HeLa cells. Beads coated with a truncated M1 protein were internalized far less frequently. Scanning electron microscopy indicated that streptococci invade by a zipper‐like mechanism, that may be mediated by interactions with host cell microvilli. Initially, internalized streptococci and streptococci undergoing endocytosis are associated with polymerized actin. Later in the internalization process, streptococcal‐containing vacuoles are associated with the lysosomal membrane glycoprotein, LAMP‐1.
Differential expression of an hsp70 gene during transition from the mycelial to the infective yeast form of the human pathogenic fungus Paracoccidioides brasiliensis
Authors: Da Silva Silvana Petrofeza , Borges‐Walmsley Maria Ines , Pereira Ildinete Silva , Soares Célia Maria de Almeida , Walmsley Adrian Robert , Felipe Maria Sueli Soares
Published: March 2003

We have isolated and characterized cDNA and genomic clones that encode a 70 kDa heat shock protein (Hsp70) from the dimorphic human pathogenic fungus Paracoccidioides brasiliensis. The gene encodes a 649‐amino‐acid protein showing high identity with other members of the hsp70 gene family. The hsp70 gene is induced during both heat shock of yeast cells at 42°C and the mycelial to yeast transition. A differential expression of this gene can be observed between mycelial and yeast forms, with a much higher level of expression in the yeast. We found two introns of 178 and 72 nucleotides in the P. brasiliensis hsp70 gene. Splicing of these introns is regulated during the heat shock process and possibly during infection. In order to analyse the differential accumulation of unspliced mRNA following cellular differentiation and/or heat shock, reverse transcriptase–polymerase chain reaction (RT–PCR) experiments were carried out. The temperature‐induced mycelial to yeast transition results in the transient accumulation of unspliced hsp70 mRNA transcripts. Yeast cells, after adaptation at 36°C, seem to be more proficient at splicing, at least with respect to hsp70 mRNA because, during a severe heat shock (42°C), the unspliced form of this mRNA does not accumulate. The mycelial to yeast differentiation will have the adaptational effect of increasing the resistance of the organism to environmental stress, which may be necessary for parasite survival in the mammalian host.
The prepilin peptidase is required for protein secretion by and the virulence of the intracellular pathogen Legionella pneumophila
Authors: Liles Mark R. , Edelstein Paul H. , Cianciotto Nicholas P.
Published: March 2003

Prepilin peptidases cleave, among other substrates, the leader sequences from prepilin‐like proteins that are required for type II protein secretion in Gram‐negative bacteria. To begin to assess the importance of type II secretion for the virulence of an intracellular pathogen, we examined the effect of inactivating the prepilin peptidase (pilD ) gene of Legionella pneumophila. Although the pilD mutant and its parent grew similarly in bacteriological media, they did differ in colony attributes and recoverability from late stationary phase. Moreover, at least three proteins were absent from the mutant's supernatant, indicating that PilD is necessary for the secretion of Legionella proteins. The absence of both the major secreted protein and a haemolytic activity from the mutant signalled that the L. pneumophila zinc metalloprotease is excreted via type II secretion. Most interestingly, the pilD mutant was greatly impaired in its ability to grow within Hartmannella vermiformis amoebae and the human macrophage‐like U937 cells. As reintroduction of pilD into the mutant restored infectivity and as a mutant lacking type IV pilin replicated like wild type, these data suggested that the intracellular growth of L. pneumophila is promoted by proteins secreted via a type II pathway. Intratracheal inoculation of guinea pigs revealed that the LD50 for the pilD mutant is at least 100‐fold greater than that for its parent, and the culturing of bacteria from infected animals showed a rapid clearance of the mutant from the lungs. This is the first study to indicate a role for PilD and type II secretion in intracellular parasitism.
Molecular characterization of Escherichia coli FtsE and FtsX
Authors: De Leeuw Erik , Graham Becky , Phillips Gregory J. , Ten Hagen‐Jongman Corinne M. , Oudega Bauke , Luirink Joen
Published: March 2003

The genes ftsE and ftsX are organized in one operon together with ftsY. FtsY codes for the receptor of the signal recognition particle (SRP) that functions in targeting a subset of inner membrane proteins. We have found no indications for a structural relationship between FtsE/X and FtsY. Evidence is presented that FtsE and FtsX form a complex in the inner membrane that bears the characteristics of an ATP‐binding cassette (ABC)‐type transporter. FtsE is a hydrophilic nucleotide‐binding protein that has a tendency to dimerize and associates with the inner membrane through an interaction with the integral membrane protein FtsX. An FtsE null mutant showed filamentous growth and appeared viable on high salt medium only, indicating a role for FtsE in cell division and/or salt transport.
Fps1p controls the accumulation and release of the compatible solute glycerol in yeast osmoregulation
Authors: Tamás Markus J. , Luyten Kattie , Sutherland F. Chris W. , Hernandez Agustin , Albertyn Jacobus , Valadi Hadi , Li Hong , Prior Bernard A. , Kilian Stephanus G. , Ramos José , Gustafsson Lena , Thevelein Johan M. , Hohmann Stefan
Published: March 2003

The accumulation of compatible solutes, such as glycerol, in the yeast Saccharomyces cerevisiae, is a ubiquitous mechanism in cellular osmoregulation. Here, we demonstrate that yeast cells control glycerol accumulation in part via a regulated, Fps1p‐mediated export of glycerol. Fps1p is a member of the MIP family of channel proteins most closely related to the bacterial glycerol facilitators. The protein is localized in the plasma membrane. The physiological role of Fps1p appears to be glycerol export rather than uptake. Fps1Δ mutants are sensitive to hypo‐osmotic shock, demonstrating that osmolyte export is required for recovery from a sudden drop in external osmolarity. In wild‐type cells, the glycerol transport rate is decreased by hyperosmotic shock and increased by hypo‐osmotic shock on a subminute time scale. This regulation seems to be independent of the known yeast osmosensing HOG and PKC signalling pathways. Mutants lacking the unique hydrophilic N‐terminal domain of Fps1p, or certain parts thereof, fail to reduce the glycerol transport rate after a hyperosmotic shock. Yeast cells carrying these constructs constitutively release glycerol and show a dominant hyperosmosensitivity, but compensate for glycerol loss after prolonged incubation by glycerol overproduction. Fps1p may be an example of a more widespread class of regulators of osmoadaptation, which control the cellular content and release of compatible solutes.
The XylS‐dependent Pm promoter is transcribed in vivo by RNA polymerase with σ32 or σ38 depending on the growth phase
Authors: Marqués Silvia , Manzanera Maximino , González‐Pérez María‐Mar , Gallegos María‐Trinidad , Ramos Juan L.
Published: March 2003

The Pm promoter of the TOL plasmid of Pseudomonas putida is expressed at high level along the growth curve. This transcription is dependent on the positive regulator XylS activated by 3‐methylbenzoate. The sigma factor σ38 is required for expression in early stationary phase and thereafter. To test whether σ70 was involved in Pm transcription in exponential phase, we have followed mRNA synthesis in a rpoD thermosensitive strain. No difference in Pm transcription was found between the wild type and the thermosensitive strain at the restrictive temperature of 42°C, indicating that transcription was independent of the sigma factor σ70. However, basal levels of mRNA expression from Pm in this strain in exponential phase were more than twofold higher at 42°C, suggesting involvement of σ32 in Pm transcription. In a rpoH background, no expression of Pm took place in the exponential phase, whereas it increased during stationary phase, and in a rpoH rpoS double mutant no activity from the Pm promoter was detected along the growth curve. We have shown that the increase in the amount of σ32 factor necessary for transcription in exponential phase is provided through induction of the heat shock response by the presence of the effector 3‐methylbenzoate, which is also required for activation of the positive regulator XylS. We conclude that activation of Pm transcription is achieved through a switch between two stress‐responsive factors, σ32 in exponential phase and σ38 in stationary phase. In both cases, transcription is dependent on the activator XylS and presents the same transcription start point.
Induction of an AP endonuclease activity in Streptococcus mutans during growth at low pH
Authors: Hahn K. , Faustoferri R. C. , Quivey R. G.
Published: March 2003

The oral microbe Streptococcus mutans uses adaptive mechanisms to withstand the fluctuating pH levels in its natural environment. The regulation of protein synthesis is part of the mechanism of acid adaptation and tolerance in S. mutans. Here, we demonstrate that the organism's acid‐inducible protein repertoire includes an AP endonuclease activity. This abasic site‐specific endonuclease activity is present at greater levels in cells grown at low pH than in cells grown at pH 7, and is apparently independent of the RecA protein. Experiments using tetrahydrofuran or α‐deoxyadenosine‐containing substrates indicate that the activity induced at low pH may be similar to the activity of exonuclease III from E. coli. Acid‐adapted S. mutans also shows an increased survival rate after exposure to near‐UV radiation in both the wild type and a recA strain. Far‐UV radiation resistance is observed in the wild type only. The endonuclease activity was purified ≈500‐fold from an S. mutans recA mutant strain grown at pH 5. Initial characterization revealed a 3′ to 5′ exonuclease activity, and showed additional functional similarities to DNA repair enzymes from other organisms.
ZapA, the IgA‐degrading metalloprotease of Proteus mirabilis, is a virulence factor expressed specifically in swarmer cells
Authors: Walker Kimberly E. , Moghaddame‐Jafari Sasan , Lockatell C. Virginia , Johnson David , Belas Robert
Published: March 2003

The IgA‐degrading metalloprotease, ZapA, of the urinary tract pathogen Proteus mirabilis is co‐ordinately expressed along with other proteins and virulence factors during swarmer cell differentiation. In this communication, we have used zapA to monitor IgA protease expression during the differentiation of vegetative swimmer cells to fully differentiated swarmer cells. Northern blot analysis of wild‐type cells and β‐galactosidase measurements using a zapA::lacZ fusion strain indicate that zapA is fully expressed only in differentiated swarmer cells. Moreover, the expression of zapA on nutrient agar medium is co‐ordinately regulated in concert with the cycles of cellular differentiation, swarm migration and consolidation that produce the bull's‐eye colonies typically associated with P. mirabilis. ZapA activity is not required for swarmer cell differentiation or swarming behaviour, as ZapA− strains produce wild‐type colony patterns. ZapA− strains fail to degrade IgA and show decreased survival compared with the wild‐type cells during infection in a mouse model of ascending urinary tract infection (UTI). These data underscore the importance of the P. mirabilis IgA‐degrading metalloprotease in UTI. Analysis of the nucleotide sequences adjacent to zapA reveals four additional genes, zapE, zapB, zapC and zapD, which appear to possess functions required for ZapA activity and IgA proteolysis. Based on homology to other known proteins, these genes encode a second metalloprotease, ZapE, as well as a ZapA‐specific ABC transporter system (ZapB, ZapC and ZapD). A model describing the function and interaction of each of these five proteins in the degradation of host IgA during UTI is presented.
The haemolysin‐secreting ShlB protein of the outer membrane of Serratia marcescens : determination of surface‐exposed residues and formation of ion‐permeable pores by ShlB mutants in artificial lipid bilayer membranes
Authors: Könninger Ulrich W. , Hobbie Silke , Benz Roland , Braun Volkmar
Published: March 2003

The ShlB protein in the outer membrane of Serratia marcescens is the only protein known to be involved in secretion of the ShlA protein across the outer membrane. At the same time, ShlB converts ShlA into a haemolytic and a cytolytic toxin. Surface‐exposed residues of ShlB were determined by reaction of an M2 monoclonal antibody with the M2 epitope DYKDDDDK inserted at 25 sites along the entire ShlB polypeptide. The antibody bound to the M2 epitope at 17 sites in intact cells, which indicated surface exposure of the epitope, and to 23 sites in isolated outer membranes. Two insertion mutants contained no ShlB(M2) protein in the outer membrane. The ShlB derivatives activated and/or secreted ShlA. To gain insights into the secretion mechanism, we studied whether highly purified ShlB and ShlB deletion derivatives formed pores in artificial lipid bilayer membranes. Wild‐type ShlB formed channels with very low single channel conductance that rarely assumed an open channel configuration. In contrast, open channels with a considerably higher single channel conductance were observed with the deletion mutants ShlB(Δ65–186), ShlB(Δ87–153), and ShlB(Δ126–200). ShlB(Δ126–200) frequently formed permanently open channels, whereas the conductance caused by ShlB(Δ65–186) and ShlB(Δ87–153) did not assume a stationary value, but fluctuated rapidly between open and closed configurations. The results demonstrate the orientation of large portions of ShlB in the outer membrane and suggest that ShlB may function as a specialized pore through which ShlA is secreted.
A putative two‐component signal transduction system regulates σE, a sigma factor required for normal cell wall integrity in Streptomyces coelicolor A3(2)
Authors: Paget Mark S. B. , Leibovitz Emmanuelle , Buttner Mark J.
Published: March 2003

The extracytoplasmic function (ECF) sigma factor, σE, is required for normal cell wall integrity in Streptomyces coelicolor. We have investigated the regulation of σE through a transcriptional and mutational analysis of sigE and the surrounding genes. Nucleotide sequencing identified three genes located downstream of sigE ; orf202, cseB and cseC (cse, control of sigE ). cseB and cseC encode a putative response regulator and a putative transmembrane sensor histidine protein kinase respectively. Although most sigE transcription appeared to be monocistronic, sigE was also transcribed as part of a larger operon, including at least orf202. sigE null mutants are sensitive to cell wall lytic enzymes, have an altered peptidoglycan muropeptide profile, and on medium deficient in Mg2+ they overproduce actinorhodin, sporulate poorly and form crenellated colonies. A constructed cseB null mutant appeared to have the same phenotype as a sigE null mutant, which was accounted for by the observed absolute dependence of the sigE promoter on cseB. It is likely that the major role of cseB is to regulate sigE transcription because expression of sigE alone from a heterologous promoter suppressed the cseB mutation. Mg2+ suppresses the CseB/SigE phenotype, probably by stabilizing the cell envelope, and sigE transcript levels were consistently higher in Mg2+‐deficient cultures than in high Mg2+‐grown cultures. We propose a model in which the CseB/CseC two‐component system modulates activity of the sigE promoter in response to signals from the cell envelope.
Heterotrimerization of PII‐like signalling proteins: implications for PII‐mediated signal transduction systems
Authors: Forchhammer Karl , Hedler Andrea , Strobel Heike , Weiss Verena
Published: March 2003

PII‐like signalling molecules are trimeric proteins composed of 12–13 kDa polypeptides encoded by the glnB gene family. Heterologous expression of a cyanobacterial glnB gene in Escherichia coli leads to an inactivation of E. coli's own PII signalling system. In the present work, we show that this effect is caused by the formation of functionally inactive heterotrimers between the cyanobacterial glnB gene product and the E. coli PII paralogues GlnB and GlnK. This led to the discovery that GlnK and GlnB of E. coli also form heterotrimers with each other. The influence of the oligomerization partner on the function of the single subunit was studied using heterotrimerization with the Synechococcus PII protein. Uridylylation of GlnB and GlnK was less efficient but still possible within these heterotrimers. In contrast, the ability of GlnB‐UMP to stimulate the adenylyl‐removing activity of GlnE (glutamine synthetase adenylyltransferase/removase) was almost completely abolished, confirming that rapid deadenylylation of glutamine synthetase upon nitrogen stepdown requires functional homotrimeric GlnB protein. Remarkably, however, rapid adenylylation of glutamine synthetase upon exposing nitrogen‐starved cells to ammonium was shown to occur in the absence of a functional GlnB/GlnK signalling system as efficiently as in its presence.
Helicobacter pylori cadA encodes an essential Cd(II)–Zn(II)–Co(II) resistance factor influencing urease activity
Authors: Herrmann Lutz , Schwan Dorothee , Garner Rachel , Mobley Harry L.T. , Haas Rainer , Schäfer Klaus P. , Melchers Klaus
Published: March 2003

Inactivation of Helicobacter pylori cadA, encoding a putative transition metal ATPase, was only possible in one of four natural competent H. pylori strains, designated 69A. All tested cadA mutants showed increased growth sensitivity to Cd(II) and Zn(II). In addition, some of them showed both reduced 63Ni accumulation during growth and no or impaired urease activity, which was not due to lack of urease enzyme subunits. Gene complementation experiments with plasmid (pY178)‐derived H. pylori cadA failed to correct the deficiencies, whereas resistance to Cd(II) and Zn(II) was restored. Moreover, pY178 conferred increased Co(II) resistance to both the cadA mutants and the wild‐type strain 69A. Heterologous expression of H. pylori cadA in an Escherichia coli zntA mutant resulted in an elevated resistance to Cd(II) and Zn(II). Expression of cadA in E. coli SE5000 harbouring H. pylori nixA, which encodes a divalent cation importer along with the H. pylori urease gene cluster, led to about a threefold increase in urease activity compared with E. coli control cells lacking the H. pylori cadA gene. These results suggest that H. pylori CadA is an essential resistance pump with ion specificity towards Cd(II), Zn(II) and Co(II). They also point to a possible role of H. pylori CadA in high‐level activity of H. pylori urease, an enzyme sensitive to a variety of metal ions.
The active site of the rolling circle replication protein Rep75 is involved in site‐specific nuclease, ligase and nucleotidyl transferase activities
Authors: Marsin Stéphanie , Forterre Patrick
Published: March 2003

The plasmid pGT5 from the hyperthermophilic archaeon Pyrococcus abyssi replicates via a rolling circle mechanism. The protein Rep75, encoded by this plasmid, exhibits a nicking–closing (NC) activity in vitro on single‐stranded oligonucleotides containing the pGT5 double‐stranded origin sequence. In addition, Rep75 catalyses a site‐specific nucleotidyl terminal transferase (NTT) activity, e.g. it can transfer one AMP or dAMP (from ATP or dATP) to the 3′‐OH of an oligonucleotide corresponding to the left part of the nicking site. The Rep75 sequence contains a motif similar to the active‐site motifs of Rep proteins from the ΦX174/pC194 superfamily. We show here that the tyrosine present in this motif is indeed essential for DNA cleavage by Rep75, but is dispensable for its NTT activity. However, a nearby arginine, which is not required for DNA cleavage, is involved in both NTT and closing, indicating that the same active site is involved in the NC and NTT activities of Rep75. For both NTT and NC, the G residue in 3′ of the nicking site is essential, whereas the A residue in 5′ is dispensable for NC, despite its conservation in RC plasmids of the ΦX174/pC194 superfamily. The NTT and closing activities have an optimal temperature lower than the nicking activity. These data indicate that the three reactions catalysed by Rep75 can be uncoupled, although they share part of their mechanisms. Finally, we show that NC is inhibited by ATP or dATP at concentrations that promote NTT. We propose a model in which the NTT activity of Rep75 plays a role in the regulation of pGT5 replication in vivo.
Molecular analysis of the role of streptococcal pyrogenic exotoxin A (SPEA) in invasive soft‐tissue infection resulting from Streptococcus pyogenes
Authors: Sriskandan Shiranee , Unnikrishnan Meera , Krausz Thomas , Cohen Jonathan
Published: March 2003

Epidemiological studies strongly implicate the bacterial superantigen, streptococcal pyrogenic exotoxin A (SPEA), in the pathogenesis of necrotizing soft‐tissue infection and toxic shock syndrome resulting from Streptococcus pyogenes. SPEA can act as a superantigen and cellular toxin ex vivo, but its role during invasive streptococcal infection is unclear. We have disrupted the wild‐type spea gene in an M1 streptococcal isolate. Supernatants from toxin‐negative mutant bacteria demonstrated a 50% reduction in pro‐mitogenic activity in HLA DQ‐positive murine splenocyte culture, and up to 20% reduction in activity in human PBMC culture. Mutant and wild‐type bacteria were then compared in mouse models of bacteraemia and streptococcal muscle infection. Disruption of spea was not associated with attenuation of virulence in either model. Indeed, a paradoxical increase in mutant strain‐induced mortality was seen after intravenous infection. Intramuscular infection with the SPEA‐negative mutant led to increased bacteraemia at 24 h and a reduction in neutrophils at the site of primary muscle infection. Purified SPEA led to a dose‐dependent increase in peritoneal neutrophils 6 h after administration. SPEA is not a critical virulence factor in invasive soft‐tissue infection or bacteraemia caused by S. pyogenes, and it could have a protective role in murine immunity to pyogenic infection. The role of this toxin may be different in hosts with augmented superantigen responsiveness.
Identification of DNA binding sites for ComE, a key regulator of natural competence in Streptococcus pneumoniae
Authors: Ween Ola , Gaustad Peter , Håvarstein Leiv Sigve
Published: March 2003

Natural competence in Streptococcus pneumoniae is regulated by a quorum‐sensing mechanism consisting of a competence‐stimulating peptide (CSP), its dedicated secretion apparatus (ComAB), its histidine kinase receptor (ComD) and a response regulator (ComE). In this report, we show that ComE is a DNA‐binding protein that acts autocatalytically by binding to a region in its own promoter. Two additional ComE binding sites were identified in the pneumococcal genome, one in the promoter region of comAB and the other upstream of an ABC transporter of unknown function. A comparison of the ComE‐binding sequences with the sequence motif previously found to be involved in the co‐ordinated expression of the late genes revealed that they are unrelated. These findings indicate that ComE activates transcription of the late genes indirectly, i.e. via one or more intermediate factors.
The farAB‐encoded efflux pump mediates resistance of gonococci to long‐chained antibacterial fatty acids
Authors: Lee Eun Hee , Shafer William M.
Published: March 2003

Gonococci often infect mucosal surfaces bathed in antibacterial fatty acids (FAs). Resistance of gonococci to FAs and other antibacterial hydrophobic agents has been attributed to the mtrCDE‐encoded efflux pump system and a heretofore undefined mechanism. This alternative resistance mechanism has been suggested to mediate gonococcal resistance to long‐chained FAs independently of the mtr efflux pump. We have now identified this alternative FA resistance system in gonococci and report that it bears significant similarity to the emrAB‐encoded efflux pump possessed by Escherichia coli and the vceAB‐encoded pump of Vibrio cholerae. We termed the gonococcal version of this efflux pump farAB (fatty acid resistance) to signify its involvement in FA resistance expressed by gonococci and to distinguish it from the emrAB‐ or vceAB‐encoded pumps that modulate bacterial susceptibility to uncoupling agents and certain antibiotics. Although the farAB system in gonococci was found to provide resistance to FAs independently of the mtrCDE‐encoded efflux pump, its function was dependent on the MtrE outer membrane protein. Moreover, expression of the tandemly linked farA and farB genes was positively associated with the presence of the MtrR transcriptional regulatory protein that normally downregulates the expression of mtrCDE. Thus, the data presented herein suggest that, while the mtrCDE‐ and farAB‐encoded systems act independently to mediate resistance of gonococci to host‐derived, hydrophobic antimicrobial agents, their capacity to export these agents is dependent on the same outer membrane protein (MtrE), and their expression may be differentially controlled by the same transcriptional regulatory protein (MtrR).
sbcB sbcC null mutations allow RecF‐mediated repair of arrested replication forks in rep recBC mutants
Authors: Bidnenko Vladimir , Seigneur Marie , Penel‐Colin Marion , Bouton Marie‐Florence , Dusko Ehrlich S. , Michel Bénédicte
Published: March 2003

We have proposed previously that, in Escherichia coli, blockage of replication forks can lead to the reversal of the fork. Annealing of the newly synthesized strands creates a double‐stranded end adjacent to a Holliday junction. The junction is migrated away from the DNA end by RuvAB and can be cleaved by RuvC, while RecBCD is required for the repair of the double‐stranded tail. Consequently, the rep mutant, in which replication arrests are frequent and fork reversal occurs, requires RecBCD for growth. We show here that the combination of sbcB sbcCD null mutations restores the viability to rep recBC mutants by activation of the RecF pathway of recombination. This shows that the proteins belonging to the RecF pathway are able to process the DNA ends made by the replication fork reversal into a structure that allows recombination‐dependent replication restart. However, we confirm that, unlike sbcB null mutations, sbcB15, which suppresses all other recBC mutant defects, does not restore the viability of rep recBC sbcCD strains. We also show that ruvAB inactivation suppresses the lethality and the formation of double‐stranded breaks (DSBs) in a rep recBC recF strain, totally deficient for homologous recombination, as well as in rep recBC mutants. This confirms that RuvAB processing of arrested replication forks is independent of the presence of recombination intermediates.
Norfloxacin‐induced DNA cleavage occurs at the dif resolvase locus in Escherichia coli and is the result of interaction with topoisomerase IV
Authors: Hojgaard Andrias , Szerlong Heather , Tabor Camille , Kuempel Peter
Published: March 2003

The dif locus is a site‐specific recombination site located within the terminus region of the chromosome of Escherichia coli. Recombination at dif resolves circular dimer chromosomes to monomers, and this recombination requires the XerC, XerD and FtsK proteins, as well as cell division. In order to characterize other enzymes that interact at dif, we tested whether quinolone‐induced cleavage occurs at this site. Quinolone drugs, such as norfloxacin, inhibit the type 2 topoisomerases, DNA gyrase and topoisomerase IV, and can cleave DNA at sites where these enzymes interact with the chromosome. Using strains in which either DNA gyrase or topoisomerase IV, or both, were resistant to norfloxacin, we determined that specific interactions between dif and topoisomerase IV caused cleavage at that site. This interaction required XerC and XerD, but did not require the C‐terminal region of FtsK or cell division.
Adaptation of Comamonas testosteroni TA441 to utilization of phenol by spontaneous mutation of the gene for a trans‐acting factor
Authors: Arai Hiroyuki , Akahira Saiko , Ohishi Tohru , Kudo Toshiaki
Published: March 2003

Comamonas testosteroni TA441 adapts to utilization of phenol upon incubation with phenol as the major carbon source. Strain TA441 has a cluster of genes (aphKLMNOPQB) encoding the catabolic enzymes phenol hydroxylase and catechol 2,3‐dioxygenase, and a divergently transcribed regulatory gene (aphR), but these genes are silent until adaptation occurs. We found another regulatory gene (aphS) downstream of aphR. AphS belongs to the GntR family of transcriptional regulators. All adapted strains were found to have mutations in the aphS gene or in the aphR–aphS intervening region. The adapted strains expressed phenol hydroxylase and catechol 2,3‐dioxygenase activity in the presence of phenol. The transcriptional activity of both the aphK and the aphR promoters was elevated in the adapted strains. A strain whose aphS gene was artificially disrupted was found to be able to grow using phenol, and the cells showed high levels of the above‐mentioned transcriptional and enzymatic activities, indicating that adaptation was caused only by the mutation in the aphS gene. Gel retardation analysis revealed that AphS bound to two specific sites in the promoter region between aphK and aphR. These results indicate that the active aphS gene product acts as a trans‐acting factor and represses transcription of the aph genes in strain TA441.
Effects of DksA and ClpP protease on sigma S production and virulence in Salmonella typhimurium
Authors: Webb Cathy , Moreno Matthew , Wilmes‐Riesenberg Mary , Curtiss Iii Roy , Foster John W.
Published: March 2003

Salmonella typhimurium responds to a variety of environmental stresses by accumulating the alternative sigma factor σS. The repertoire of σS ‐dependent genes that are subsequently expressed confers tolerance to a variety of potentially lethal conditions including low pH and stationary phase. The mechanism(s) responsible for triggering σS accumulation are of considerable interest, because they help to ensure survival of the organism during encounters with suboptimal environments. Two genes associated with regulating σS levels in S. typhimurium have been identified. The first is clpP, encoding the protease known to be responsible for degrading σS in Escherichia coli. The second is dksA, encoding a protein of unknown function not previously associated with regulating σS levels. As predicted, clpP mutants accumulated large amounts of σS even in log phase. However, dksA mutants failed to accumulate σS in stationary phase and exhibited lower accumulation during acid shock in log phase. DksA appears to be required for the optimal translation of rpoS based upon dksA mutant effects on rpoS transcriptional and translational lacZ fusions. The region of rpoS mRNA between codons 8 and 73 is required to see the effects of dksA mutations. This distinguishes the role of DksA from that of HF‐I (hfq ) in rpoS translation, as the HF‐I target area occurs well upstream of the rpoS start codon. DksA appears to be involved in the expression of several genes in addition to rpoS based on two‐dimensional SDS–PAGE analysis of whole‐cell proteins. As a result of their effects on gene expression, mutations in clpP and dksA decreased the virulence of S. typhimurium in mice, consistent with a role for σS in pathogenesis.
A MAP kinase encoded by theubc3gene ofUstilago maydisis required for filamentous growth and full virulence
Authors: Mayorga María E. , Gold Scott E.
Published: March 2003

Ustilago maydis, the causal agent of corn smut disease, displays dimorphic growth in which it alternates between a budding haploid saprophyte and a filamentous dikaryotic pathogen. We are interested in identifying the genetic determinants of filamentous growth and pathogenicity inU. maydis.To do this, we have taken a forward genetic approach. Previously, we showed that haploid adenylate cyclase (uac1) mutants display a constitutively filamentous phenotype. Mutagenesis of auac1disruption strain allowed the isolation of a large number of budding suppressor mutants. These mutants are namedubc, forUstilagobypass ofcyclase, as they no longer require the production of cAMP to grow in the budding morphology. Complementation of one of these suppressor mutants led to the identification ofubc3,which is required for filamentous growth and encodes a MAP kinase most similar to those of the yeast pheromone response pathway. In addition to filamentous growth, theubc3gene is required for pheromone response and for full virulence. Mutations in the earlier identifiedfuz7MAP kinase kinase also suppress the filamentous phenotype of theuac1disruption mutant, adding evidence that bothubc3andfuz7are members of this same MAP kinase cascade. These results support an important interplay of the cAMP and MAP kinase signal transduction pathways in the control of morphogenesis and pathogenicity inU. maydis.