bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of the propeptide in folding and secretion of elastase of Pseudomonas aeruginosa
Authors: Braun Peter , Tommassen Jan , Filloux Alain
Published: November 2003

Elastase of Pseudomonas aeruginosa is synthesized as a pre‐proprotein. The propeptide has been shown to inhibit the enzymatic activity of elastase. In this study, we investigated a possible additional role of the propeptide in the folding and secretion of the enzyme. When elastase was expressed in Escherichia coli without its propeptide, no active elastase was produced. The enzyme was poorly released from the cytoplasmic membrane and, depending on the expression level, it was either degraded or it accumulated in an inactive form in the cell envelopes, probably as aggregates. Since proper folding is required for the release of translocated proteins from the cytoplasmic membrane and for the acquirement of a stable and active conformation, these results suggest that the propeptide is involved in the proper folding of the elastase and that it functions as an intramolecular chaperone. When mature elastase was expressed without its propeptide in P. aeruginosa, the enzyme was not secreted, and it was degraded. Therefore, proper folding of mature elastase appears to be required for secretion of the enzyme. Expression of the propeptide, as a separate polypeptide, in trans with mature elastase resulted in the formation of active elastase. This active enzyme was secreted in P. aeruginosa. Apparently, the propeptide can also function as an intermolecular chaperone.
Signal sensing by σ54‐dependent regulators: derepression as a control mechanism
Authors: Shingler Victoria
Published: November 2003

Transcription by RNA polymerase utilizing the alternative sigma factor σ54 is regulated by a distinct class of positive activators designated the σ54‐dependent family. The activities of these regulators are themselves modulated in response to a wide variety of environmental signals. Factors that modulate the expression or the activity of the regulatory protein in response to chemical and metabolic changes are ultimately responsible for determining the level of expression of σ54‐dependent genes and hence the diverse bacterial functions that they encode. Many members of the σ54‐dependent family are part of two‐component sensor‐response systems. This MicroReview emphasizes recent data concerning the activities of a distinct subgroup of the σ54‐dependent regulators that directly sense and respond with transcriptional activation to the presence of small effector molecules in their environment. The functional consequences of effector activation in terms of regulation of the enzymatic (ATPase) activity of these transcriptional activators and interdomain interactions are discussed.
The two‐step lysis system of pneumococcal bacteriophage EJ‐1 is functional in Gram‐negative bacteria: triggering of the major pneumococcal autolysin in Escherichia coli
Authors: Díaz Eduardo , Munthali Mathildah , Lünsdorf Heinrich , Höltje Joachim‐Volker , Timmis Kenneth N.
Published: November 2003

The holin function Ejh of the pneumococcal bacteriophage EJ‐1 has been characterized. It shows structural features similar to, and functionally complemented, the prototype member of the holin family. In Escherichia coli and Pseudomonas putida the Ejh product caused cellular death, and changes in cell morphology could be accounted for by lesions in the cytoplasmic membrane. Expression of ejh resulted in the inhibition of growth in a variety of phylogenetically distant bacterial genera, suggesting a broad spectrum of action. Concomitant expression of the ejh and ejl (encodes a lysin) genes led to lysis of E. coli and P. putida cells. Remarkably, the Ejl lysin was able to attack murein from bacteria lacking choline in their sacculi, which suggests that pneumococcal lysins have a broader substrate specificity than previously assumed. Furthermore, the Ejh holin was able to trigger activity of the major pneumococcal autolysin cloned and expressed in E. coli, and this raised new questions about the regulation of this model autolysin. A new function for holins in systems where the phage lysin is supposed to be associated with the membrane is proposed.
Identification and characterization of ispA, a Shigella flexneri chromosomal gene essential for normal in vivo cell division and intercellular spreading
Authors: Mac Síomóin Ruairí A. , Nakata Noboru , Murai Tatuo , Yoshikawa Masanosuke , Tsuji Hiroyuki , Sasakawa Chihiro
Published: November 2003

The virulent phenotype of Shigella requires loci on the chromosome as well as on the large virulence plasmid, and is regulated via a complex web of interactions amongst various chromosomal and large plasmid genes. To further investigate the role of chromosomal loci in virulence, we performed random Tn10 mutagenesis in Shigella flexneri YSH6000T, and isolated an avirulent mutant (V3404) incapable of spreading throughout an epithelial cell monolayer. Although V3404 initially spread intercellularly at the same rate as the wild‐type, it gradually slowed down and ceased spreading as a result of increasing defects in cell division, leading to the formation of long filamentous bacteria lacking septa, trapped within cells. In addition, the mutation affected the ability of V3404 to polymerize actin, a prerequisite for intra‐ and inter‐cellular spreading ability. Sequencing of Tn10‐flanking DNA revealed that the mutated gene, designated ispA (intracellular septation), was equivalent to a previously sequenced but uncharacterised gene of Escherichia coli located between trp and tonB. Using E. coli sequence data, we cloned the ispA gene from the YSH6000T chromosome and found that it complemented the V3404 mutation. Nucleotide sequencing and in vitro expression experiments revealed that ispA coded for a small (21 kDa), very hydrophobic protein. These results thus show that ispA is an essential virulence gene affecting several functions of the virulence process.
Chemotactic motility is required for invasion of the host by the fish pathogen Vibrio anguillarum
Authors: O'Toole Ronan , Milton Debra L. , Wolf‐Watz Hans
Published: November 2003

The role of the flagellum and motility in the virulence of the marine fish pathogen Vibrio anguillarum was examined. Non‐motile mutants were generated by transposon mutagenesis. Infectivity studies revealed that disruption of the flagellum and subsequent loss of motility correlated with an approximate 500‐fold decrease in virulence when fish were inoculated by immersion in bacteria‐containing water. However, the flagellar filament and motility were not required for pathogenicity following intraperitoneal injection of fish. The transposon‐insertion site for six mutants was determined by cloning and sequencing of the Vibrio DNA flanking the transposon. V. anguillarum genes whose products showed strong homology to proteins with an established role in flagellum biosynthesis were identified. One of the aflagellate mutants had a transposon insertion in the rpoN gene of V. anguillarum. This rpoN mutant failed to grow at low concentrations of available iron and was avirulent by both the immersion and intraperitoneal modes of inoculation. A chemotaxis gene, cheR, was located upstream of one transposon insertion and an in‐frame deletion was constructed in the coding region of this gene. The resulting non‐chemotactic mutant exhibited wild‐type pathogenicity when injected intraperitoneally into fish but showed a decrease in virulence similar to that seen for the non‐motile aflagellate mutants following immersion infection. Hence, chemotactic motility is a required function of the flagellum for the virulence of V. anguillarum
Growth‐phase‐dependent expression of the osmotically inducible gene osmC of Escherichia coli K‐12
Authors: Gordia Sylvie , Gutierrez Claude
Published: November 2003

The transcription of the osmotically inducible gene osmC of Escherichia coli is initiated by two overlapping promoters, osmCp1 and osmCp2. The existence of these two promoters was confirmed by site‐directed mutagenesis. osmC transcription is regulated by the growth phase. In a medium of low osmotic pressure, expression of osmC is induced at the onset of decelerating phase and continues during the beginning of stationary phase. At elevated osmotic pressure, the induction occurs somewhat earlier during growth. Both promoters are repressed during early exponential phase. osmCp2 is induced during entry into stationary phase. Transcription from osmCp1, which is approximately 10‐fold lower than that of osmCp2 in rich medium, starts during the mid‐log phase and stops in early stationary phase. In the absence of σS, the stationary‐phase sigma factor encoded by rpoSosmCp2 expression is much reduced while expression of osmCp1 is unaffected. As a consequence, the regulation of osmC as a function of growth is at least partially independent of σS
The signal‐transduction network for Pho regulation in Bacillus subtilis
Authors: Hulett F. Marion
Published: November 2003

Depletion of nutrients, including phosphate, is a stress often encountered by a bacterial cell, and results in slowed growth, marking the cessation of exponential growth. Genes that are transcriptionally activated during phosphate starvation have been used to examine the signal‐transduction mechanisms governing the Pho regulon in Bacillus subtilis. Alkaline phosphatase, the traditional reporter protein for Pho regulation in prokaryotes, is encoded by a multigene family in B. subtilis. Characterization of the alkaline phosphatase family was a breakthrough in the study of regulation of the Pho regulon, especially the discovery of promoter elements exclusively responsive to phosphate‐starvation regulation. Current data suggest that at least three two‐component signal‐transduction systems interact, forming a regulatory network that controls the phosphate‐deficiency response in B. subtilis. The interconnected pathways involve the PhoP–PhoR system, whose primary role is to mediate the phosphate‐deficiency response; the Spo0 phosphorelay required for the initiation of sporulation; and a newly discovered signal‐transduction system, ResD–ResE, which also has a role in respiratory regulation during late growth. Parallel pathways positively regulate the Pho response via PhoP–PhoR. One pathway includes the ResD–ResE system, while the other involves a transition‐state regulator, AbrB. The Spo0 system represses the Pho response by negatively regulating both pathways. This review will discuss how the characterization of the APase multigene family made possible studies which show that the Pho regulon in B. subtilis is regulated by the integrated action of the Res, Pho and Spo signal‐transduction systems.
Three two‐component signal‐transduction systems interact for Pho regulation in Bacillus subtilis
Authors: Sun Guofu , Birkey Stephanie M. , Hulett F. Marion
Published: November 2003

The Pho regulon of Bacillus subtilis is controlled by three two‐component signal‐transduction systems: PhoP/PhoR, ResD/ResE, and the phosphorelay leading to the phosphorylation of Spo0A. Two of these systems act as positive regulators, while the third is involved in negative regulation of the Pho regulon. Under phosphate‐starvation‐induction conditions, the response regulator (RR) PhoP, and the histidine protein kinase (HK) PhoR, are involved in the induction of Pho‐regulon genes including the phoPR operon and genes encoding the major vegetative alkaline phosphatases, phoA and phoB. ResD (the RR) and ResE (the HK) are positive regulators of both aerobic and anaerobic respiration in B. subtilis. Current data suggest that they are also positive regulators of the Pho regulon, as is the transition‐state regulatory protein AbrB. Data presented reveal that ResDE and AbrB are involved in activation of the Pho regulon through separate regulatory pathways. Spo0A∼P (RR) exerts a negative effect on the Pho regulon through its repression of AbrB, and possibly through repression of ResDE. Both pathways converge to regulate transcription of the phoPR operon.
Promoter identification and expression analysis of Salmonella typhimurium and Escherichia coli nrdEF operons encoding one of two class I ribonucleotide reductases present in both bacteria
Authors: Jordan Albert , Aragall Eugeni , Gibert Isidre , Barbé Jordi
Published: November 2003

Salmonella typhimurium and Escherichia coli cells have two different class I ribonucleotide reductases encoded by the nrdEF and nrdAB operons. Despite the presence of one additional ribonucleotide reductase, the nrdAB‐encoded enzyme is essential to the aerobic growth of the cell because nrdAB‐defective mutants of both species are not viable in the presence of oxygen. Several factors controlling nrdAB gene transcription have been analysed intensively. Nothing is known about the expression of the nrdEF genes. To study this subject, and after cloning of E. coli nrdEF genes and sequencing of their 5′ ends, the promoter of this operon has been identified by primer extension in both bacterial species. The + 1 position was 691 bp and 692 bp upstream of the translational start points of the nrdE genes of S. typhimurium and E. coli, respectively. Downstream of the + 1 position, and before the nrdE gene, two open reading frames (ORFs) of 81 and 136 amino acid residues are present in both bacteria. The synthesis of a polypeptide with a molecular mass of 9 kDa, corresponding to the first of these two ORFs, was observed by using the T7 RNA polymerase expression system. Comparison of the amino acid predicted sequence of this ORF reveals a significant similarity with glutaredoxin proteins. Competitive, reverse‐transcription polymerase chain reaction experiments indicate that transcription from the nrdEF promoter normally takes place in wild‐type cells. nrdEF transcription is increased by hydroxyurea, which inhibits class I ribonucleotide reductase activity, in both RecA+ and RecA− cells. nrdAts mutants show a higher level of nrdEF transcription than wild‐type cells at either the permissive or the restrictive temperature. nrdEF expression was unaffected by changes in DNA supercoiling whether caused by the introduction of either topA ::Tn10 and hns ::Tn10 mutations or by the inhibition of DNA gyrase with the antibiotic novobiocin. In contrast to the nrdAB genes, the nrdEF operon is not essential to the cells because nrdEF‐defective mutants are viable under both aerobic and anaerobic conditions.
Phenylalanine 30 plays an important role in receptor binding of verotoxin‐1
Authors: Clark Clifford , Bast Darrin , Sharp Allan M. , St. Hilaire Phaedria M. , Agha Rummana , Stein Penelope E. , Toone Eric J. , Read Randy J. , Brunton James L.
Published: November 2003

The homopentameric B subunit of verotoxin 1 (VT1) binds to the glycosphingolipid receptor globotriaosylceramide (Gb3). We produced mutants with alanine substitutions for residues found near the cleft between adjacent subunits. Substitution of alanine for phenylalanine 30 (Phe‐30) resulted in a fourfold reduction in B subunit binding affinity for Gb3 and a 10‐fold reduction in receptor density in a solid‐phase binding assay. The interaction of wild‐type and mutant B subunits with Pk trisaccharide in solution was examined by titration microcalorimetry. The carbohydrate binding of the mutant was markedly impaired compared with that of the wild type and was too weak to allow calculation of a binding constant. These results demonstrate that the mutation significantly impaired the carbohydrate‐binding function of the B subunit. To ensure that the mutation had not caused a significant change in structure, the mutant B subunit was crystallized and its structure was determined by X‐ray diffraction. Difference Fourier analysis showed that its structure was identical to that of the wild type, except for the substitution of alanine for Phe‐30. The mutation was also produced in the VT1 operon, and mutant holotoxin was purified to homogeneity. The cytotoxicity of the mutant holotoxin was reduced by a factor of 105 compared to that of the wild type in the Vero cell cytotoxicity assay. The results suggest that the aromatic ring of Phe‐30 plays a major role in binding of the B subunit to the Galα1‐4Galβ1‐4Glc trisaccharide portion of Gb3. Examination of the VT1 B crystal structure suggests two potential carbohydrate‐binding sites which lie on either side of Phe‐30.
The initiation mess?
Authors: Herrick John , Kohiyama Masamichi , Atlung Tove , Hansen Flemming G.
Published: November 2003

This review concerns the mechanisms which control initiation of chromosome replication in enterobacteria with respect to cell growth. Initiation control is commonly separated into positive and negative regulatory mechanisms. Four main points are advanced concerning these different aspects of initiation control. (i) The average concentration of the initiator protein DnaA is proportional to the origin concentration, i.e. the origin per cell mass ratio and, thus, inversely proportional to the very often used term of the ‘initiation mass’. (ii) The time of initiation of chromosome replication in the cell cycle is set by DnaA protein accumulating to a threshold level, which in concert with a number of other factors allows for a co‐operative formation of the initiation complex. (iii) The time of initiation is not determined by the interaction with these other factors or by the transient interaction between newly replicated origins (oriC ) and the cell surface. (iv) The aberrant initiation phenotype observed in various mutants, including dnaA (ts) mutants, might be due to a defective pre‐initiation DnaA–oriC interaction or it might be due to a defect in the protection of newly initiated origins from reinitiation. Many of these points are discussed and evaluated in view of recent developments concerning the regulation of chromosome replication in Escherichia coli
Structure and function of the Bacillus SpoIIE protein and its localization to sites of sporulation septum assembly
Authors: Barák Imrich , Behari Jaideep , Olmedo Gabriela , Guzmán Plinio , Brown David P. , Castro Elda , Walker DeEtte , Westpheling Janet , Youngman Philip
Published: November 2003

Functioning of the spoIIE locus of Bacillus subtilis is required for formation of a normal polar septum during sporulation and for activation of the transcription factor σF, which directs early forespore‐specific gene expression. We have determined the DNA sequence of the wild type and several mutant alleles of the spoIIE gene of B. subtilis and sequenced a substantial portion of its presumptive homologue in Bacillus megaterium. We show that the spoIIE locus encodes a single large protein with a predicted molecular mass of 92 kDa. Each of five point‐mutation alleles, which have traditionally defined the locus, and two transposon‐generated mutations were shown to fall within the coding sequence for the 92 kDa gene product or within sequences expected to be required for its expression. The amino‐terminal portion of the predicted SpoIIE gene product, comprising approximately 40% of the protein, is extremely hydrophobic and is expected to contain up to 12 membrane‐spanning segments. The remainder of the protein contains no hydrophobic segments long enough to span a lipid bilayer and is therefore presumed to comprise one or more globular, aqueous‐phase exposed domains. An in‐frame fusion joining the 3′ end of the B. megaterium spoIIE coding sequence to the 5′ end of gfp, a gene encoding the green fluorescent protein (GFP) of Aquorea victoria, resulted in a strong, sporulation‐specific fluorescent signal localized to the sites of sporulation septum assembly. We speculate that SpoIIE plays a role in assembling the sporulation septum, perhaps determining the special properties of the structure that permit intercompartment signalling during development.
Two different mechanisms are involved in the heat‐shock regulation of chaperonin gene expression in Bradyrhizobium japonicum
Authors: Babst Markus , Hennecke Hauke , Fischer Hans‐Martin
Published: November 2003

Heat‐shock regulation was detected for three out of the five members of the groESL multigene family in Bradyrhizobium japonicum. The results uncovered the simultaneous presence of two distinct heat‐shock control systems which so far have not been reported to co‐exist in a single prokaryotic organism. The first system concerns groESL1 whose transcription is controlled in a σ32‐dependent manner similar to that known from work done with Escherichia coli. Heat‐shock control of groESL4 is mediated by the second system, which is characterized by an inverted‐repeat DNA structure originally described as a heat‐shock regulatory element (CIRCE) in Bacillus subtilis. This element represses expression of groESL4 under non‐stress conditions, as inferred from the increased expression of a groESL4′–′lacZ fusion suffering a 4 bp deletion within the CIRCE element. The two control systems clearly differ with respect to the temperature dependence and the kinetics of the heat‐shock response, and they also respond differently to the stress signal elicited by incorporation of the amino acid analogue p‐F‐phenylalanine into cellular protein. Knock‐out mutations in groEL4 resulted in an increased expression of groESL4, suggesting that repression via CIRCE depends, itself, upon the cellular level of GroEL4 protein.
The pilA regulatory gene modulates the pilus‐mediated adhesion of Neisseria meningitidis by controlling the transcription of pilC1
Authors: Taha Muhamed‐Kheir , Giorgini Dario , Nassif Xavier
Published: November 2003

Adherence to eukaryotic cells is essential in the pathogenesis of Neisseria meningitidis. Pilus‐mediated adhesion has been shown to play an essential role in this process. Pilin, the pilus major subunit, and two pilus associated proteins, PilC1 and PilC2, are key components in meningococcal adhesiveness. Phase and/or antigenic variation of these molecules are the only identified means by which N. meningitidis modulates pilus‐mediated adhesion. PilA/PilB is a pleiotropic regulatory system first characterized in Neisseria gonorrhoeae where it controls pilin gene transcription. Similar alleles are found in N. meningitidis. To address the role of this regulatory pathway in N. meningitidis, we engineered a meningococcal pilA mutant strain and analysed the consequences of this mutation on pilus‐mediated adhesion using epithelial Hec‐1‐B cells. This mutation resulted in a threefold reduction in adhesiveness. As no change in the amount of pilin nor in pilin gene mRNA was detected, we compared the expression of the pilC genes in both pilA and parental strains. Two transcriptional fusions pilC1–lacZ and pilC2–lacZ were constructed. A threefold reduction in β‐galactosidase activity was observed in the pilA mutant strain harbouring the pilC1–lacZ fusion. No effect of the pilA mutation on β‐galactosidase activity was observed in the strain carrying the pilC2–lacZ fusion. Gel retardation experiments confirmed that the PilA protein binds to the promoter region of pilC1 but not of pilC2. Taken together, these data demonstrate that PilA modulates meningococcal adhesiveness via the transcription of pilC1. Thus, in addition to phase variation, a more co‐ordinate and responsive system may allow a fine adaptation of adhesiveness of meningococci to various environmental signals.
Temperature‐dependent regulation of Yersinia enterocolitica Class III flagellar genes
Authors: Kapatral Vinayak , Olson John W. , Pepe Jeffrey C. , Miller Virginia L. , Minnich Scott A.
Published: November 2003

Temperature is a key environmental cue for Yersinia enterocolitica as well as for the two other closely related pathogens, Yersinia pestis and Yersinia pseudotuberculosis. Between the range of 30°C and 37°C, Y. enterocolitica phase‐varies between motility and plasmid‐encoded virulence gene expression. To determine how temperature regulates Y. enterocolitica motility, we have been dissecting the flagellar regulatory hierarchy to determine at which level motility is blocked by elevated temperature (37°C). Here we report the cloning, DNA sequences, and regulation of the two main regulators of Class III flagellar genes, fliA (σF) and flgM (anti‐σF), and a third gene, flgN, which we show is required for filament assembly. Identification of the Y. enterocolitica fliA and flgM genes was accomplished by functional complementation of both S. typhimurium and Y. enterocolitica mutations and by DNA sequence analysis. The Y. enterocolitica fliA gene, encoding the flagellar‐specific σ‐factor, σF, maps immediately downstream of the three flagellin structural genes. The flgM and flgN genes, encoding anti‐σF and a gene product required for filament assembly, respectively, map downstream of the invasin (inv) gene but are transcribed in the opposite (convergent) direction. By using Northern blot analyses we show that transcription of both fliA and flgM is immediately arrested when cells are exposed to 37°C, coincident with the timing of virulence gene induction. Unlike S. typhimurium flgM− mutants, Y. enterocolitica flgM− mutants are fully virulent.
Use of synthetic peptides to confirm that the Pseudomonas aeruginosa PAK pilus adhesin and the Candida albicans fimbrial adhesin possess a homologous receptor‐binding domain
Authors: Yu Lei , Lee Kok K. , Paranchych William , Hodges Robert S. , Irvin Randall T.
Published: November 2003

Pseudomonas aeruginosa PAK pili and Candida albicans fimbriae are adhesins present on the microbial cell surfaces which mediate binding to epithelial cell‐surface receptors. The receptor‐binding domain (adhesintope) of the PAK pilus adhesin has been shown previously to reside in the carboxy‐terminal disulphide‐bonded region of P. aeruginosa pilin (PAK128‐144). The delineation of the C. albicans fimbrial adhesintope was investigated in these studies using synthetic peptides which correspond to the whole (PAK128‐144) or part of (PAK134‐140) adhesintope of the PAK pilus and their respective anti‐peptide antisera and biotinylated PAK pili (Bt‐PAK pili), fimbriae (Bt‐fimbriae), P. aeruginosa whole cells (Bt‐P. aeruginosa) and C. albicans whole cells (Bt‐C. albicans). The results from these studies confirmed that a structurally conserved motif akin to the PAK(128‐144) peptide sequence is present in C. albicans fimbrial adhesin and that the seven‐amino‐acid residue PAK(134‐140) sequence plays an important role in forming the adhesintope for both P. aeruginosa PAK pilus and C. albicans fimbrial adhesins.
Two‐stage regulation of an anti‐sigma factor determines developmental fate during bacterial endospore formation
Authors: Kellner Ellen M. , Decatur Amy , Moran, Jr Charles P.
Published: November 2003

During endospore formation in Bacillus subtilis an asymmetric division produces two cells, forespore and mother cell, which follow different developmental paths. Commitment to the forespore‐specific developmental path is controlled in part by the activation of the forespore‐specific RNA polymerase sigma factor, σF. Activity of σF is inhibited in the mother cell by the anti‐sigma factor SpoIIAB. In the forespore, σF directs transcription of the structural gene for σG. However, σG does not become active until after engulfment of the forespore is complete. This σG activity is dependent upon the products of the spoIIIA operon. We showed that σG is present but mostly inactive in a spoIIIA mutant. We also demonstrated that the anti‐sigma factor SpoIIAB can bind to σGin vitro. Moreover, a mutant form of σG that binds SpoIIAB inefficiently in vitro was shown to function independently of SpoIIIA during sporulation. These and previously reported results support a model in which SpoIIAB functions as an inhibitor of σG activity during sporulation. Therefore, we propose that the anti‐sigma factor SpoIIAB antagonizes both σF and σG activities, and that this antagonism is relieved in the forespore in two stages. In the first stage, which follows septation, a SpoIIAA‐dependent mechanism partially relieves SpoIIAB inhibition of σF activity in the forespore. In the second stage, which follows forespore engulfment, a SpoIIIA‐dependent process inactivates SpoIIAB in the forespore, resulting in the activation of σG.
Back to log phase: σS as a global regulator in the osmotic control of gene expression in Escherichia coli
Authors: Hengge‐Aronis Regine
Published: November 2003

It is now well established that the σS subunit of RNA polymerase is a master regulator in a complex regulatory network that governs the expression of many stationary‐phase‐inducible genes in Escherichiacoli. In this review, more recent findings will be summarized that demonstrate that σS also acts as a global regulator for the osmotic control of gene expression, and actually does so in exponentially growing cells. Thus, many σS‐dependent genes are induced during entry into stationary phase as well as in response to osmotic upshift. K+ glutamate, which accumulates in hyperosmotically stressed cells, seems to specifically stimulate the activity of σS‐containing RNA polymerase at σS‐dependent promoters. Moreover, osmotic upshift results in an elevated cellular σS level similar to that observed in stationary‐phase cells. This increase is the result of a stimulation of rpoS translation as well as an inhibition of the turnover of σS, which in exponentially growing non‐stressed cells is a highly unstable protein. Whereas the RNA‐binding protein HF‐I, previously known as a host factor for the replication of phage Qβ RNA, is essential for rpoS translation, the recently discovered response regulator RssB, and ClpXP protease, have been shown to be required for σS degradation. The finding that the histone‐like protein H‐NS is also involved in the control of rpoS translation and σS turnover, sheds new light on the function of this protein in osmoregulation. Finally, preliminary evidence suggests that additional stresses, such as heat shock and acid shock, also result in increased cellular σS levels in exponentially growing cells. Taken together, σS function is clearly not confined to stationary phase. Rather, σS may be regarded as a sigma factor associated with general stress conditions.
Limited variation and maintenance of tight genetic linkage characterize heteroallelic pilE recombination following DNA transformation of Neisseria gonorrhoeae
Authors: Hill Stuart A.
Published: November 2003

Genetic recombination impacts on neisserial biology in two ways: (i) specific loci undergo rearrangement at high frequency leading to the formation of many different alleles; and (ii) Neisseria, being naturally competent for DNA transformation, provide a means to disseminate the novel alleles throughout a population. In this study pilE was used as a model system to examine heteroallelic recombination following DNA transformation. When gonococci were transformed with chromosomal donor DNA containing different pilE alleles, the majority of pilE recombinants arose through allelic replacement. Co‐conversion analysis across pilE showed that in ∼ 85–90% of recombination events encompassing pilE and an adjacent opa locus, linkage was maintained (i.e. ∼ 10–15% of recombination events terminated within the ∼ 1000 base pair pilE/opaE interval). In addition to those recombinants that arose through allelic replacement, a large pilus‐minus subpopulation was also observed (∼ 10% of all recombinants), indicating that many recombination events did not yield recombinant pilEs that could be assembled into functional pili. PilE mosaics increased following transformation with plasmid donor DNAs carrying pilE with limited flanking‐sequence homology, suggesting a potential role for flanking‐sequence homologies in mosaic formation. Overall, the data support the view that horizontal transmission of chromosomal DNA between gonococci will favour the spread of intact alleles, as opposed to expanding the allelic repertoire through the formation of gene mosaics.
Regulation of the expression of the traM gene of the F sex factor of Escherichia coli
Authors: Penfold Sonya S. , Simon John , Frost Laura S.
Published: November 2003

Conjugative F‐plasmid transfer is mediated by the transfer (tra ) region which encodes nearly 40 genes, 25 of which are essential for this process in Escherichia coli. TraM is required for conjugation and is encoded on a separate operon between the origin of transfer and the traJ gene. The traJ gene product is the positive regulator of transcription of the 30 kb tra operon, the first gene of which is traY. Using primer‐extension assays and immunoblots on the F plasmid itself and its derivatives, we demonstrate that F TraM regulates its own expression from two promoters and that it requires TraY as well as expression of the tra operon for maximal traM transcription. traY is the first gene in the tra operon under the control of the TraJ regulator, which is in turn negatively regulated by the antisense RNA, FinP, and the FinO protein. Thus, a control circuit has been established whereby traM is negatively regulated by the FinOP fertility inhibition system through its repression of TraJ expression, which adversely affects transcription of the traY gene.
Bacterial growth inhibition by overproduction of protein
Authors: Kurland C. G. , Dong Henjiang
Published: November 2003

Multicopy plasmids that have been engineered to produce large quantitites of a single gratuitous (non‐functional, non‐toxic) protein are often problematic. When fully induced, these engineered constructions produce very sick bacteria. The reasons for this may be found in the physiology of wild‐type laboratory strains that have been selected to grow at maximum rates with optimal quantities of their proteins. Such bacteria apparently experience the accumulation of gratuitous proteins as an internal shift down and they respond to this with a starvation response. Unlike the shift down associated with a change of growth media, the production of large quantities of gratuitous protein is not associated with a new pre‐programmed steady‐state of balanced growth. Consequently, the starvation response continues until the bacteria commit suicide by, among other things, destroying their ribosomes.
Replacement of the lysine residue in the consensus ATP‐binding sequence of the AddA subunit of AddAB drastically affects chromosomal recombination in transformation and transduction of Bacillus subtilis
Authors: Haijema Bert Jan , Noback Michiel , Hesseling Anne , Kooistra Jan , Venema Gerard , Meima Rob
Published: November 2003

The ATP‐dependent deoxyribonuclease enzyme complex (AddAB) of Bacillus subtilis possesses two consensus ATP‐binding sequences, located in the N‐terminal region of both subunits. The highly conserved lysine residues in both consensus ATP‐binding sequences were replaced by glycine, resulting in the mutant enzyme complexes AddAB‐A‐K36G (AddA*B) and AddAB‐B‐K14G (AddAB*). The mutation in subunit AddA reduced DNA repair and chromosomal transformation, and abolished bacteriophage PBS1‐mediated transduction. This mutation also resulted in a complete loss of the ATP‐dependent exonuclease and helicase activity. In contrast, the mutation in subunit AddB had only marginal effects. The recF and addAB genes are not required for transformation with plasmid DNA, but have overlapping activities in transformation with chromosomal DNA. By contrast to RecF, the AddAB enzyme is essential for PBS1‐mediated transduction. However, recF has a more important function with respect to DNA repair than addAB.
Yeast glutathione reductase is required for protection against oxidative stress and is a target gene for yAP‐1 transcriptional regulation
Authors: Grant Chris M. , Collinson Lindsay P. , Roe Jung‐Hye , Dawes Ian W.
Published: November 2003

Glutathione (GSH) is an abundant cellular thiol which has been implicated in many cellular processes including protection against xenobiotics, carcinogens and free radicals. Utilization of GSH in both enzymic and non‐enzymic defence mechanisms results in its conversion to the oxidized form (GSSG), and it must be recycled to GSH to maintain the high intracellular ratio of GSH to GSSG. Glutathione reductase (GLR) is a flavoenzyme, which catalyses reduction of GSSG to GSH using the reducing power of NADPH. We show that yeast mutants deleted for GLR1, encoding glutathione reductase, lack GLR activity and accumulate increased levels of GSSG. In addition, the glr1 mutant strain was unaffected in the inducible adaptive response to hydrogen peroxide, but showed increased sensitivity to oxidants including both peroxides and superoxide, indicating a requirement for GLR in protection against oxidative stress. Furthermore, GLR1 expression was elevated two to threefold in the presence of oxidants, and regulation was dependent upon the yAP‐1 transcriptional activator protein. Thus, GLR1 is one of a growing number of genes involved in the protection of yeast cells against oxidative stress and regulated by yAP‐1.
The role of the AUU initiation codon in the negative feedback regulation of the gene for translation initiation factor IF3 in Escherichia coli
Authors: Sacerdot C. , Chiaruttini C. , Engst K. , Graffe M. , Milet M. , Mathy N. , Dondon J. , Springer M.
Published: November 2003

The expression of the infC gene encoding translation initiation factor IF3 is negatively autoregulated at the level of translation, i.e. the expression of the gene is derepressed in a mutant infC background where the IF3 activity is lower than that of the wild type. The special initiation codon of infC, AUU, has previously been shown to be essential for derepression in vivo. In the present work, we provide evidence that the AUU initiation codon causes derepression by itself, because if the initiation codon of the thrS gene, encoding threonyl‐tRNA synthetase, is changed from AUG to AUU, its expression is also derepressed in an infC mutant background. The same result was obtained with the rpsO gene encoding ribosomal protein S15. We also show that derepression of infCthrS, and rpsO is obtained with other ‘abnormal’ initiation codons such as AUA, AUC, and CUG which initiate with the same low efficiency as AUU, and also with ACG which initiates with an even lower efficiency. Under conditions of IF3 excess, the expression of infC is repressed in the presence of the AUU or other ‘abnormal’ initiation codons. Under the same conditions and with the same set of ‘abnormal’ initiation codons, the repression of thrS and rpsO expression is weaker. This result suggests that the infC message has specific features that render its expression particularly sensitive to excess of IF3. We also studied another peculiarity of the infC message, namely the role of a GC‐rich sequence located immediately downstream of the initiation codon and conserved through evolution. This sequence was proposed to interact with a conserved region in 16S RNA and enhance translation initiation. Unexpectedly, mutating this GC‐rich sequence increases infC expression, indicating that this sequence has no enhancing role. Chemical and enzymatic probing of infC RNA synthesized in vitro indicates that this GC‐rich sequence might pair with another region of the mRNA. On the basis of our in vivo results we propose, as suspected from earlier in vitro results, that IF3 regulates the expression of its own gene by using its ability to differentiate between ‘normal’ and ‘abnormal’ initiation codons.
Characterization of the fibronectin‐attachment protein of Mycobacterium avium reveals a fibronectin‐binding motif conserved among mycobacteria
Authors: Schorey Jeffrey S. , Holsti Matthew A. , Ratliff Timothy L. , Allen Paul M. , Brown Eric J.
Published: November 2003

Mycobacterium avium is an intracellular pathogen and a major opportunistic infectious agent observed in patients with acquired immune deficiency syndrome (AIDS). Evidence suggests that the initial portal of infection by M. avium is often the gastrointestinal tract. However, the mechanism by which the M. avium crosses the epithelial barrier is unclear. A possible mechanism is suggested by the ability of M. avium to bind fibronectin, an extracellular matrix protein that is a virulence factor for several extracellular pathogenic bacteria which bind to mucosal surfaces. To further characterize fibronectin binding by M. avium, we have cloned the M. avium fibronectin‐attachment protein (FAP). The M. avium FAP (FAP‐A) has an unusually large number of Pro and Ala residues (40% overall) and is 50% identical to FAP of both Myco‐bacterium leprae and Mycobacterium tuberculosis. Using recombinant FAP‐A and FAP‐A peptides, we show that two non‐continuous regions in FAP‐A bind fibronectin. Peptides from these regions and homologous sequences from M. leprae FAP inhibit fibronectin binding by both M. avium and Mycobacterium bovis Bacillus Calmette–Guerin (BCG). These regions have no homology to eukaryotic fibronectin‐binding proteins and are only distantly related to fibronectin‐binding peptides of Gram‐positive bacteria. Nevertheless, these fibronectin‐binding regions are highly conserved among the mycobacterial FAPs, suggesting an essential function for this interaction in mycobacteria infection of their metazoan hosts.
FtsZ ring: the eubacterial division apparatus conserved in archaebacteria
Authors: Wang Xunde , Lutkenhaus Joe
Published: November 2003

FtsZ is a tubulin‐like protein that is essential for cell division in eubacteria. It functions by forming a ring at the division site that directs septation. The archaebacteria constitute a kingdom of life separate from eubacteria and eukaryotes. Like eubacteria, archaebacteria are prokaryotes, although they are phylogenetically closer to eukaryotes. Here it is shown that archaebacteria also possess FtsZ and that it is biochemically similar to eubacterial FtsZs. Significantly, FtsZ from the archaebacterium Haloferax volcanii is a GTPase that is localized to a ring that coincides with the division constriction. These results indicate that the FtsZ ring was part of the division apparatus of a common prokaryotic ancestor that was retained by both eubacteria and archaebacteria.
A comprehensive set of DnaA‐box mutations in the replication origin, oriC, of Escherichia coli
Authors: Langer Uwe , Richter Stefan , Roth Angelika , Weigel Christoph , Messer Walter
Published: November 2003

We probed the complex between the replication origin, oriC, and the initiator protein DnaA using different types of mutations in the five binding sites for DnaA, DnaA boxes R1–R4 and M: (i) point mutations in individual DnaA boxes and combinations of them; (ii) replacement of the DnaA boxes by a scrambled 9 bp non‐box motif; (iii) positional exchange; and (iv) inversion of the DnaA boxes. For each of the five DnaA boxes we found at least one type of mutation that resulted in a phenotype. This demonstrates that all DnaA boxes in oriC have a function in the initiation process. Most mutants with point mutations retained some origin activity, and the in vitro DnaA‐binding capacity of these origins correlated well with their replication proficiency. Inversion or scrambling of DnaA boxes R1 or M inactivated oriC‐dependent replication of joint replicons or minichromosomes under all conditions, demonstrating the importance of these sites. In contrast, mutants with inverted or scrambled DnaA boxes R2 or R4 could not replicate in wild‐type hosts but gave transformants in host strains with deleted or compromised chromosomal oriC at elevated DnaA concentrations. We conclude that these origins require more DnaA per origin for initiation than does wild‐type oriC. Mutants in DnaA box R3 behaved essentially like wild‐type oriC, except for those in which the low‐affinity box R3 was replaced by the high‐affinity box R1. Apparently, initiation is possible without DnaA binding to box R3, but high‐affinity DnaA binding to DnaA box R3 upsets the regulation. Taken together, these results demonstrate that there are finely tuned DnaA binding requirements for each of the individual DnaA boxes for optimal build‐up of the initiation complex and replication initiation in vivo
afsR is a pleiotropic but conditionally required regulatory gene for antibiotic production in Streptomyces coelicolor A3(2)
Authors: Floriano Belén , Bibb Mervyn
Published: November 2003

The N‐terminal region of AfsR, a putative pleiotropic regulatory protein for antibiotic production in Strepto‐myces coelicolor A3(2), is homologous to RedD and ActII‐ORF4, pathway‐specific regulatory proteins required for the production of the antibiotics undecylprodigiosin (Red) and actinorhodin (Act), respectively. The recent identification of afsS, which lies immediately 3′ of afsR and which stimulates antibiotic production when cloned at high copy number, questioned whether afsR was a pleiotropic regulatory gene. In this study we demonstrate that multiple copies of afsR can stimulate both Act and Red production and that, despite its homology, it cannot substitute for the pathway‐specific regulatory genes. Moreover, an in‐frame deletion that removed most of the afsR coding sequence resulted in loss of Act and Red production, and a marked reduction in the synthesis of the calcium‐dependent antibiotic (CDA), but only under some (non‐permissive) nutritional conditions. Although additional copies of afsR resulted in elevated levels of the actII‐ORF4 and redD transcripts, transcription of the pathway‐specific regulatory genes under non‐permissive conditions was unaffected by deletion of afsR. While afsR may operate independently of the pathway‐specific regulatory proteins to influence antibiotic production, the activity of ActII‐ORF4 and of RedD under non‐permissive conditions could depend on interaction with, or modification by, AfsR.
Structural determinants within the subunit protein of Ty1 virus‐like particles
Authors: Martin‐Rendon Enca , Marfany Gemma , Wilson Stuart , Ferguson David J. P. , Kingsman Susan M. , Kingsman Alan J.
Published: November 2003

The Ty virus‐like particles (VLPs) are functionally analogous to retroviral particles. They package the enzymes and the RNA necessary for retrotransposition, and mediate the integration of the reverse‐transcription product into the genome of the host cell. Here we map three structural determinants of particle assembly in the subunit protein. We have also identified key residues in these regions that seem to be involved in subunit interaction and particle morphology. In particular, two point mutations in putative amphipathic helices have remarkable effects on VLP morphology, increasing the diameter as much as eightfold.
Specific binding of the Listeria monocytogenes transcriptional regulator PrfA to target sequences requires additional factor(s) and is influenced by iron
Authors: Böckmann Regine , Dickneite Carmen , Middendorf Barbara , Goebel Werner , Sokolovic Zeljka
Published: November 2003

The PrfA protein, which is a member of the Crp/Fnr family of prokaryotic transcription activators, regulates the virulence genes of Listeria monocytogenes. In this work, specific binding of PrfA to its target DNA was determined by electrophoretic mobility‐shift assays (EMSAs) using cell‐free extracts from the two L. monocytogenes strains EGD and NCTC 7973. PrfA‐specific binding differs between the two strains, even when the concentration of PrfA was adjusted to similar levels. Both strains exhibited increased PrfA‐specific binding after a shift into minimal essential medium (MEM) without showing a significant change in the amount of PrfA protein, relative to extracts from bacteria grown in brain–heart infusion (BHI). The purified PrfA protein from strain EGD produced in Escherichia coli did not exhibit specific binding to the target DNA but did so upon addition of PrfA‐free extracts from various Listeria species and Bacillus subtilis. The observed activation of PrfA seems to be caused by a PrfA‐activating factor (Paf), which is probably a protein since elevated temperature, but not RNase treatment, destroyed the activation potential of such PrfA‐free extracts. Moreover, fractionation of these extracts by sucrose gradient centrifugation yielded the Paf activity in a fraction sedimenting at 3.2 S. Specific binding of PrfA‐containing extracts from strain EGD to the hly and actA promoter sequences was strongly inhibited by iron, whereas that of extracts from strain NCTC 7973 was only slightly reduced. The iron effect seems to be mediated by Paf rather than by PrfA itself.