bluuebunny/crossref_metadata_2025 huggingface dataset:
Involvement of the amino‐terminal phosphorylation module of UhpA in activation of uhpT transcription in Escherichia coli
Authors: Webber Carol A. , Kadner Robert J.
Published: November 2003

The UhpA protein is required for expression of the sugar phosphate transporter UhpT in Escherichia coli and is regulated by phosphate transfer from the transmembrane UhpBC sensor kinase complex. UhpA action requires the sensor kinase complex and the site of phosphorylation, Asp‐54, under normal conditions, but not when UhpA is overexpressed. Directed mutagenesis of the uhpA gene allowed examination of the role of several residues of UhpA in response to phosphorylation and in transcription activation. Residues Asp‐9, Asp‐54, and Lys‐101 are highly conserved and required for function in other response regulators. Changes at any of these residues in UhpA resulted in complete loss of phosphorylation‐dependent activity, but did not affect the high‐level, constitutive, UhpBC‐independent expression when the UhpA variants were overexpressed. Thus, these residues are important for the response to the phosphorylation pathway but not for transcription activation. Eight independent uhpA mutants selected for activity in the absence of UhpBC function carried the F17→V or H170→Y substitutions. Other substitutions for Phe‐17 conferred various phenotypes, ranging from inducible to high‐level constitutive behaviour. Residues in helix‐1 flanking Phe‐17 were converted to Ala or other residues. Alanine substitutions at Val‐13, Arg‐14, and Leu‐20 resulted in complete loss of phosphorylation‐dependent activation. Change of Gly‐16 to Ala had no effect, but changes to other residues resulted in loss of function. Alanine substitutions at Phe‐17 and at Gln‐19 resulted in high‐level constitutive expression, and changes at Ala‐18 and Leu‐21 had only modest effects. Most interesting was the L20→A substitution, which conferred low uhpT expression when overexpressed and interfered with action of the wild‐type chromosomal allele. The combination of the L20→A change with changes at Phe‐17, Asp‐54 and His‐170 indicated that the trans‐dominant action of L20→A occurred at several steps. The observations that UhpA can activate uhpT transcription in its unphosphorylated state are consistent with its occupancy of low‐affinity binding sites necessary for promoter function. We propose that the effect of phosphorylation of UhpA is to enhance its oligomerization on the DNA surface to extend to the low‐affinity sites, and that helix‐1 participates in the process of oligomer formation.
Regulation of the xcp secretion pathway by multiple quorum‐sensing modulons in Pseudomonas aeruginosa
Authors: Chapon‐Hervé Virginie , Akrim Mohammed , Latifi Amel , Williams Paul , Lazdunski Andrée , Bally Marc
Published: November 2003

The virulence of the opportunistic pathogen Pseudomonas aeruginosa is largely dependent upon the extracellular production of a number of secreted proteins with toxic or degradative activities. The synthesis of several exoenzymes is controlled in a cell‐density‐dependent manner by two interlinked quorum‐sensing systems. Their secretion across the outer membrane occurs through the Xcp translocation machinery. The xcp locus located at 40 min on the chromosome consists of two divergently transcribed operons, namely xcpPQ and xcpR to xcpZ. In this study, transcriptional fusions were constructed between the xcpP and xcpR genes and the lacZ reporter. Transcriptional activation of the xcpP and xcpR genes in P. aeruginosa is growth‐phase dependent and the lasR–lasI autoinduction system is required for this control. In the heterologous host Escherichia coli, the lasR gene product, together with its cognate autoinducer N‐(3‐oxododecanoyl)‐l‐homoserine lactone (OdDHL), activates both the xcpP–lacZ and the xcpR–lacZ gene fusion. The second P. aeruginosa quorum‐sensing modulon rhlR–rhlI (vsmR–vsmI ) is also involved in the control of the xcp genes. Expression of the lacZ fusions is strongly reduced in PANO67, a pleiotropic mutant defective in the production of N‐acyl‐homoserine lactones responsible for the activation of RhlR. Furthermore, introduction of the lasR mutation in PANO67 results in additional diminution of xcpR transcription, indicating that the two systems can regulate their target genes independently. These data demonstrate that expression of the xcp secretion system depends on a complex regulatory network involving cell–cell signalling which controls production and secretion of virulence‐associated factors.
Molecular organization of the genes required for the synthesis of type 1 capsular polysaccharide of Streptococcus pneumoniae: formation of binary encapsulated pneumococci and identification of cryptic dTDP‐rhamnose biosynthesis genes
Authors: Muñoz Rosario , Mollerach Marta , López Rubens , García Ernesto
Published: November 2003

We report here the molecular organization of the capsular locus (cap1 ) of the type 1 pneumococcus. This locus is located between dexB and aliA and flanked by IS1167 insertion elements. Sequence analysis showed that the cluster contains 11 genes (cap1A to cap1K ), which are apparently arranged as a single transcriptional unit. The presence of a functional promoter (cap1p ) located upstream of cap1A has been demonstrated and the transcription start point was mapped by primer‐extension analysis. A 14.3 kb fragment containing the genes cap1ABCDEFGHIJK and including cap1p was sufficient to allow the synthesis of a type 1 capsule in Streptococcus pneumoniae. An internal deletion of cap1E leads to an unencapsulated phenotype demonstrating that this gene is essential for capsular production. The cap1K gene has been expressed in Escherichia coli resulting in UDP‐glucose dehydrogenase (UDP‐GlcDH) activity. Moreover, this gene was able to restore the synthesis of type 3 capsule when cloned into a plasmid and introduced by transformation into S. pneumoniae cap3A mutants deficient in UDP‐GlcDH. In marked contrast with what was previously thought, recombination between cap1K and cap3A does occur. We provide data on the molecular mechanism that leads to the formation of binary encapsulated pneumococcal cells, i.e. strains that simultaneously produce type 1 and type 3 capsules. Downstream of cap1K, one truncated and three complete open reading frames homologous to those involved in the biosynthesis of dTDP‐rhamnose, a monosaccharide that does not participate in the formation of type 1 polysaccharide, have been identified in all the clinical strains of type 1 pneumococcus tested. Our results provide new insights into the generation of capsule diversity in pneumococci.
Genetic analysis of pertussis toxin promoter activation in Bordetella pertussis
Authors: Marques Ryan R. , Carbonetti Nicholas H.
Published: November 2003

Bordetella pertussis regulates expression of its virulence factors such as pertussis toxin (Ptx) via the bvg locus, which encodes a two‐component system composed of a sensor protein, BvgS, and a transcription activator, BvgA. We used a ptx–lac fusion on the B. pertussis chromosome to analyse promoter activation by alteration of specific sequences upstream of and within the promoter. Our data demonstrate that a pair of heptanucleotide inverted repeats separated by a turn of the DNA helix within the upstream repeat region (centred around nucleotide −136.5) are crucial cis‐activating elements, and probably represent the initial BvgA‐binding site. In addition, we demonstrate that the sequence between these repeats and the promoter plays a role in activation. Our data are most consistent with a model of co‐operative binding of BvgA dimers to this intervening region and interaction with RNA polymerase at the promoter to activate ptx transcription. In the core promoter region both the non‐consensus 21 bp spacing and the specific sequence between the −35 and −10 elements are crucial for promoter activity.
The Escherichia coli K‐12 sheA gene encodes a 34‐kDa secreted haemolysin
Authors: Del Castillo Francisco J. , Leal Sandra C. , Moreno Felipe , Castillo Ignacio del
Published: November 2003

Haemolytic toxins belong to one of several classes of virulence factors that contribute to bacterial pathogenicity. The non‐pathogenic Escherichia coli K‐12 laboratory strain was considered for years to be non‐haemolytic. However, overproduction of several transcriptional regulators induced the appearance of a haemolytic activity that is absent under usual laboratory growth conditions. In this work, we have cloned and characterized an E. coli K‐12 gene, sheA, whose overexpression results in a haemolytic phenotype. It maps to min 27 on the genetic map, and codes for a 34‐kDa polypeptide with at least one putative transmembrane segment. This polypeptide, which has neither signal peptide nor other known secretory motifs, is secreted to the medium during the exponential growth phase. In vitro coupled transcription/translation assays, using a plasmid carrying only the sheA gene as template, resulted in the production of a polypeptide with haemolytic activity per se. Our results demonstrate that the sheA gene actually encodes the E. coli K‐12 chromosomal haemolysin. The SheA haemolysin does not resemble other known cytolytic toxins, and it may represent the prototype of a novel family, as suggested by the presence of homologues in several E. coli pathogenic strains and in Shigella flexneri.
Nascent membrane and presecretory proteins synthesized in Escherichia coli associate with signal recognition particle and trigger factor
Authors: Valent Quido A. , De Gier Jan‐Willem L. , Heijne Gunnar von , Kendall Debra A. , Ten Hagen‐Jongman Corinne M. , Oudega Bauke , Luirink Joen
Published: November 2003

The Escherichia coli signal recognition particle (SRP) and trigger factor are cytoplasmic factors that interact with short nascent polypeptides of presecretory and membrane proteins produced in a heterologous in vitro translation system. In this study, we use an E. coli in vitro translation system in combination with bifunctional cross‐linking reagents to investigate these interactions in more detail in a homologous environment. Using this approach, the direct interaction of SRP with nascent polypeptides that expose particularly hydrophobic targeting signals is demonstrated, suggesting that inner membrane proteins are the primary physiological substrate of the E. coli SRP. Evidence is presented that the overproduction of proteins that expose hydrophobic polypeptide stretches, titrates SRP. In addition, trigger factor is efficiently cross‐linked to nascent polypeptides of different length and nature, some as short as 57 amino acid residues, indicating that it is positioned near the nascent chain exit site on the E. coli ribosome.
Alterations in the flow of one‐carbon units affect KinB‐dependent sporulation in Bacillus subtilis
Authors: Dartois Véronique , Liu Jian , Hoch James A.
Published: November 2003

Endospore formation in Bacillus subtilis is primarily dependent on the phosphorylation of the key transcription factor Spo0A by two major kinases, KinA and KinB, thought to be activated by distinct signals. Using a strategy designed to detect mutations that specifically affect the signalling pathway to KinB, we have isolated a Tn10 insertion mutant in one of two adjacent lrp‐like genes coding for homologues of the Escherichia coli leucine‐responsive regulatory protein (Lrp) and another mutant in the glyA gene encoding the serine hydroxymethyl transferase (SHMT). SHMT catalyses interconversion of serine and glycine while transferring the resulting one‐carbon unit into the C1 pool through methylene tetrahydrofolate. Sporulation experiments performed in a series of supplemented media indicated that the role of SHMT in the KinB pathway is to feed the pool of C1 units recruited for the biosynthesis of key metabolites, which include the methyl donor S‐adenosyl‐methionine (SAM). The results of experiments using l‐ethionine suggest that SAM is involved in post‐synthetic methylation reactions or biosynthesis of metabolites that serve to activate KinB. Truncated LrpA and LrpB peptides that have retained the DNA‐binding domain but have lost the C‐terminal half of the protein appear to act as repressors of glyA transcription and KinB‐dependent sporulation. However, deletions of lrpA, lrpB or lrpAB have little effect on glyA transcription or sporulation through KinB, suggesting that other effectors, such as additional Lrp homologues, may act in conjunction with LrpA and LrpB. Our results indicate that lrpA–lrpB together with the biosynthetic glyA gene lie on a common signalling pathway meant to activate the KinB sensor kinase.
Enzymic and genetic basis for bacterial growth on malonate
Authors: Dimroth Peter , Hilbi Hubert
Published: November 2003

Various bacteria are able to grow aerobically or anaerobically on malonate as sole source of carbon and energy. Independent of the mechanism for energy conservation, the decarboxylation of malonate is the key reaction in the decomposition of this compound. To achieve malonate decarboxylation under physiological conditions, the substrate must be converted into an activated (thioester) derivative. We report here on the malonate decarboxylases of Malonomonas rubra and Klebsiella pneumoniae. These enzymes perform an interesting substrate activation mechanism by generating a malonyl thioester with the enzyme. Formation of the malonyl‐S‐enzyme involves an ‘activation module’ that comprises the acetylation of a specific thiol group of an acyl carrier protein (ACP) and the transfer of the ACP moiety to malonate, yielding malonyl‐S‐ACP and acetate. The malonyl‐S‐ACP is subsequently decarboxylated with regeneration of the acetyl‐ACP. The malonate activation mechanism is related to the activation of citrate by citrate lyase. The relationship extends to the identical 2′‐(5′′‐phosphoribosyl)‐3′‐dephospho‐CoA thiol cofactor that is bound covalently to the corresponding ACP subunit. In Klebsiella pneumoniae, malonate is decarboxylated by a water‐soluble enzyme complex. In the anaerobic bacterium Malonomonas rubra, malonate decarboxylation is catalysed by a set of water‐soluble as well as membrane‐bound enzymes that function together in converting the free energy of the decarboxylation reaction into ΔμNa+. Therefore, this malonate decarboxylase includes a biotin carrier protein that accepts the CO2 moiety from malonyl‐S‐ACP and delivers it to a membrane‐bound decarboxylase acting as a Na+ pump. Genes encoding the individual protein components that perform the decarboxylation of malonate in K. pneumoniae or M. rubra have been identified within the mdc and mad gene clusters respectively. The function of most of the derived proteins could be envisaged from sequence similarities with proteins of known functions. The genetic evidence firmly supports the idea that malonate decarboxylation is carried out by the two different decarboxylases, as deduced from the biochemical studies of the enzymes.
The oxygen‐responsive transcriptional regulator FNR of Escherichia coli : the search for signals and reactions
Authors: Unden Gottfried , Schirawski Jan
Published: November 2003

The FNR (fumarate and nitrate reductase regulation) protein of Escherichia coli is an oxygen‐responsive transcriptional regulator required for the switch from aerobic to anaerobic metabolism. In the absence of oxygen, FNR changes from the inactive to the active state. The sensory and the regulatory functions reside in separate domains of FNR. The sensory domain contains a Fe–S cluster, which is of the [4Fe–4S]2+ type under anaerobic conditions. It is suggested that oxygen is supplied to the cytoplasmic FNR by diffusion and inactivates FNR by direct interaction. Reactivation under anoxic conditions requires cellular reductants. In vitro, the Fe–S cluster is converted to a [3Fe–4S]+ or a [2Fe–2S]2+ cluster by oxygen, resulting in FNR inactivation. After prolonged incubation with oxygen, the Fe–S cluster is destroyed. Reassembly of the [4Fe–4S]2+ cluster might require cellular proteins, such as the NifS‐like protein of E. coli. In this review, the rationale for regulation of alternative metabolic pathways by FNR and other oxygen‐dependent regulators is discussed. Only the terminal reductases of respiration, and not the dehydrogenases, are regulated in such a way as to achieve maximal H+/e− ratios and ATP yields.
The relaxosome protein MobC promotes conjugal plasmid mobilization by extending DNA strand separation to the nick site at the origin of transfer
Authors: Zhang Shuyu , Meyer Richard
Published: November 2003

The frequency of conjugal mobilization of plasmid R1162 is decreased approximately 50‐fold if donor cells lack MobC, one of the plasmid‐encoded proteins making up the relaxosome at the origin of transfer (oriT ). The absence of MobC has several different effects on oriT DNA. Site‐ and strand‐specific nicking by MobA protein is severely reduced, accounting for the lower frequency of mobilization. The localized DNA strand separation required for this nicking is less affected, but becomes more sensitive to the level of active DNA gyrase in the cell. In addition, strand separation is not efficiently extended through the region containing the nick site. These effects suggest a model in which MobC acts as a molecular wedge for the relaxosome‐induced melting of oriT DNA. The effect of MobC on strand separation may be partially complemented by the helical distortion induced by supercoiling. However, MobC extends the melted region through the nick site, thus providing the single‐stranded substrate required for cleavage by MobA.
Two abundant intramolecular transposition products, resulting from reactions initiated at a single end, suggest that IS2 transposes by an unconventional pathway
Authors: Lewis Leslie A. , Grindley Nigel D. F.
Published: November 2003

The Escherichia coli insertion sequence, IS2, is a member of the IS3 family of bacterial transposable elements. Its transposase is a fusion protein, OrfAB, made by a programmed −1 translational frameshift near to the end of orfA and just after the start of orfB. We have characterized two major products of IS2 intramolecular transposition, which accumulate in cells that express the IS2 OrfAB fusion protein at elevated levels. The more abundant product is a minicircle composed of the complete IS2 with just a single basepair (occasionally 2 bp) separating the two IS ends. In all cases, this basepair is derived from the vector sequence immediately adjacent to the left IS2 end (IRL). The second product is a figure‐eight molecule that contains all the IS2 and vector sequences present in the parental plasmid. One DNA strand contains the parental sequences unrearranged. The other contains a single‐stranded version of the minicircle junction — the precise 3′ end of IRR has been cleaved and joined to a target just outside the 5′ end of IRL; the remaining vector sequences have a free 5′ end, derived from cleavage at the 3′ end of IRR, and a free 3′ end, released upon cleavage of the target site adjacent to IRL. We propose that figure‐eight molecules are the precursor to IS2 minicircles and that the formation of these two products is the initial step in IS2 intermolecular transposition. This proposed transposition pathway provides a means for a transposase that can cleave only one strand at each IS end to produce simple insertions and avoid forming co‐integrates.
The LysR‐like regulator LeuO in Escherichia coli is involved in the translational regulation of rpoS by affecting the expression of the small regulatory DsrA‐RNA
Authors: Klauck Eberhard , Böhringer Jürgen , Hengge‐Aronis Regine
Published: November 2003

The translation of rpoS, which encodes the general stress sigma factor, σS, in Escherichia coli, is stimulated by various stress conditions. Regulatory factors involved in this control are the RNA‐binding Hfq (HF‐I) protein, the histone‐like protein H‐NS and the small regulatory DsrA‐RNA (with the last being specifically required for increased rpoS translation at low temperature). Here, we report the characterization of a transposon insertion mutant (Tn10‐8) with reduced σS levels that led to the identification of an additional factor involved in the regulation of rpoS translation, the LysR‐like regulator LeuO. Tn10‐8 decreases rpoS translation predominantly at low growth temperature. The mutation results in similarly strongly reduced DsrA‐RNA expression and does not affect rpoS expression in a dsrA null mutant background, indicating that it affects rpoS translation via DsrA‐RNA. Tn10‐8 is inserted 26 bp upstream of the leuO open reading frame, which encodes a putative LysR‐like regulator of unknown function. Instead of being a leuO null mutation, Tn10‐8 activates leuO expression as a result of the pout promoter on IS10L reading into leuO, indicating that LeuO represses dsrA and thereby reduces rpoS translation at low temperature. LeuO does not contribute to temperature regulation of dsrA since its own expression is rather low and not temperature dependent. In a mutant deficient for H‐NS, however, leuO is strongly derepressed. We conclude that rpoS translation is controlled by a regulatory network that includes Hfq, H‐NS, LeuO and DsrA‐RNA. In this network, H‐NS plays a dual role by interfering with rpoS translation in general and, via LeuO, influencing the synthesis of its own low‐temperature antagonist, DsrA‐RNA.
Trehalose synthesis is important for the acquisition of thermotolerance in Schizosaccharomyces pombe
Authors: Ribeiro Maria J. S. , Reinders Anke , Boller Thomas , Wiemken Andres , De Virgilio Claudio
Published: November 2003

Yeast cells show an adaptive response to a mild heat shock, resulting in thermotolerance acquisition. This is accompanied by induction of heat‐shock protein (hsp) synthesis and rapid accumulation of trehalose. Genetic approaches to determine the specific role of trehalose in heat‐induced thermotolerance in Saccharomyces cerevisiae have been hampered by the finding that deletion of TPS1, the gene encoding trehalose‐6‐phosphate synthase, causes a variety of pleiotropic effects, including inability to grow on glucose‐containing media. Here, we have studied a tps1 mutant of the yeast Schizosaccharomyces pombe that reportedly has no such growth defects. We show that tps1 mutants have a serious defect in heat shock‐induced acquisition of thermotolerance if conditioned at highly elevated temperatures (40–42.5°C), which, in wild‐type cells, prevent hsp but not trehalose synthesis. In contrast, hsp synthesis appears to become particularly important under conditions in which trehalose synthesis is either absent (in tps1 mutant strains) or not fully induced (conditioning at moderately elevated temperatures, i.e. 35°C). In addition, pka1 mutants deficient in cAMP‐dependent protein kinase were examined. Unconditioned pka1 cells had low levels of trehalose but a high basal level of thermotolerance. It was found that pka1 mutant cells, contrary to wild‐type cells, accumulated large amounts of trehalose, even during a 50°C treatment. pka1 tps1 double mutants lacked this ability and showed reduced intrinsic thermotolerance, indicating a particularly important role for trehalose synthesis, which takes place during the challenging heat shock.
Replacement of isoleucine‐47 by threonine in the HPr protein of Streptococcus salivarius abrogates the preferential metabolism of glucose and fructose over lactose and melibiose but does not prevent the phosphorylation of HPr on serine‐46
Authors: Gauthier Martine , Brochu Denis , Eltis Lindsay D. , Thomas Suzanne , Vadeboncoeur Christian
Published: November 2003

Phosphorylation of HPr on a serine residue at position 46 (Ser‐46) by an ATP‐dependent protein kinase has been reported in several Gram‐positive bacteria, and the resulting intermediate, HPr(Ser‐P), has been shown to mediate inducer exclusion in lactococci and lactobacilli and catabolite repression in Bacillus subtilis and Bacillus megaterium. We report here the phenotypic properties of an isogenic spontaneous mutant (G22.4) of Streptococcus salivarius ATCC 25975, in which a missense mutation results in the replacement of isoleucine at position 47 (Ile‐47) by threonine (Thr) in HPr. This substitution did not prevent the phosphorylation of HPr on Ser‐46, nor did it impede the phosphorylation of HPr on His‐15 by EI or the transfer of the phosphoryl group from HPr(His∼P) to other PTS proteins. However, the I47T substitution did perturb, in glucose‐grown but not in galactose‐grown cells, the cellular equilibrium between the various forms of HPr, resulting in an increase in the amount of free HPr at the expense of HPr(His∼P)(Ser‐P); the levels of HPr(His∼P) and HPr(Ser‐P) were not affected. Growth on melibiose was virtually identical for the wild‐type and mutant strains, whereas the generation time of the mutant on the other sugars tested (glucose, fructose, mannose, lactose and galactose) increased 1.2‐ to 1.5‐fold. The preferential metabolism of PTS sugars (glucose and fructose) over non‐PTS sugars (lactose and melibiose) that is observed in wild‐type cells was abolished in cells of mutant G22.4. Moreover, α‐ and β‐galactosidases were derepressed in glucose‐ and fructose‐grown cells of the mutant. The data suggest that HPr regulates the preferential metabolism of PTS sugars over the non‐PTS sugars, lactose and melibiose, through the repression of the pertinent catabolic genes. This HPr‐dependent repression, however, seems to occur solely when cells are growing on a PTS sugar.
Localization of the Escherichia coli cell division protein FtsI (PBP3) to the division site and cell pole
Authors: Weiss David S. , Pogliano Kit , Carson Michael , Guzman Luz‐Maria , Fraipont Claudine , Nguyen‐Distèche Martine , Losick Richard , Beckwith Jon
Published: November 2003

FtsI, also known as penicillin‐binding protein 3, is a transpeptidase required for the synthesis of peptidoglycan in the division septum of the bacterium, Escherichia coli. FtsI has been estimated to be present at about 100 molecules per cell, well below the detection limit of immunoelectron microscopy. Here, we confirm the low abundance of FtsI and use immunofluorescence microscopy, a highly sensitive technique, to show that FtsI is localized to the division site during the later stages of cell growth. FtsI was also sometimes observed at the cell pole; polar localization was not anticipated and its significance is not known. We conclude (i) that immunofluorescence microscopy can be used to localize proteins whose abundance is as low as approximately 100 molecules per cell; and (ii) that spatial and temporal regulation of FtsI activity in septum formation is achieved, at least in part, by timed localization of the protein to the division site.
Bacterial copper‐ and zinc‐cofactored superoxide dismutase contributes to the pathogenesis of systemic salmonellosis
Authors: Farrant Jayne L. , Sansone Assunta , Canvin James R. , Pallen Mark J. , Langford Paul R. , Wallis Timothy S. , Dougan Gordon , Kroll J. Simon
Published: November 2003

Copper/zinc‐cofactored superoxide dismutase ([Cu,Zn]‐SOD) has been found in the periplasm of many bacterial species but its biological function is unknown. Here we report the cloning and characterization of sodC, encoding [Cu,Zn]‐SOD, from Salmonella typhimurium. The predicted protein sequence shows only 58% identity to Escherichia coli SodC, and from this its chromosomal location and its immediate proximity to a phage gene, sodC, in Salmonella is speculated to have been acquired by bacteriophage‐mediated horizontal transfer from an unknown donor. A sodC mutant of S. typhimurium was unimpaired on aerobic growth in rich medium but showed enhanced sensitivity in vitro to the microbicidal action of superoxide. S. typhimurium, S. choleraesuis and S. dublin sodC mutants showed reduced lethality in a mouse model of oral infection and persisted in significantly lower numbers in livers and spleens after intraperitoneal infection, suggesting that [Cu,Zn]‐SOD plays a role in pathogenicity, protecting Salmonella against oxygen radical‐mediated host defences. There was, however, no observable difference compared with wild type in the interaction of sodC mutants with porcine pleural, mouse peritoneal or J774 macrophages in vitro, perhaps reflecting the hierarchical capacity of different macrophage lines to kill Salmonella, the most efficient overwhelming the proposed protective effect of periplasmic SOD.
Clonal descent and microevolution of Neisseria meningitidis during 30 years of epidemic spread
Authors: Morelli Giovanna , Malorny Burkhard , Müller Kerstin , Seiler Andrea , Wang Jian‐Fu , Del Valle Jesus , Achtman Mark
Published: November 2003

Serogroup A meningococci of subgroups III, IV‐1 and IV‐2 are probably descended from a common ancestor that existed in the nineteenth century. The 10.5 kb sequences spanning five distinct chromosomal loci, encoding cell‐surface antigens, a secreted protease or housekeeping genes and intergenic regions, were almost identical in strains of those subgroups isolated in 1966, 1966 and 1917 respectively. During the subsequent two to three decades, all of these loci varied as a result of mutation, translocation or import of DNA from unrelated neisseriae. Thus, microevolution occurs frequently in naturally transformable bacteria. Many variants were isolated only once or within a single geographical location and disappeared thereafter. Other variants achieved genetic fixation within months or a few years. The speed with which sequence variation is either eliminated or fixed may reflect sequential bottlenecks associated with epidemic spread and contrasts with the results of phylogenetic analyses from bacteria that do not cause epidemics.
Phase variation in tcpH modulates expression of the ToxR regulon in Vibrio cholerae
Authors: Carroll Patricia A. , Tashima Karen T. , Rogers Marc B. , DiRita Victor J. , Calderwood Stephen B.
Published: November 2003

We evaluated a spontaneous mutant of Vibrio cholerae, which was avirulent in an infant mouse and had reduced expression of cholera toxin and TcpA in response to environmental signals. The toxR, toxS and toxT genes in the mutant were normal, but transcription of toxT was absent. A plasmid expressing wild‐type tcpP and tcpH complemented the mutant. The mutation resulted from a frameshift in a string of nine G residues within tcpH; similar slipped‐strand mutations in tcpH arose at a frequency of 10−4 during overnight growth and in the majority of colonies by the end of 5 days of growth in ToxR‐inducing conditions. Transcription of tcpPH was regulated by temperature and pH independently of ToxR or ToxT. These results suggest that TcpH couples environmental signals (temperature and pH) to expression of the ToxR regulon, and provide a model for phase variation in the co‐ordinate expression of cholera virulence factors.
Intracellular targeting of exoenzyme S of Pseudomonas aeruginosa via type III‐dependent translocation induces phagocytosis resistance, cytotoxicity and disruption of actin microfilaments
Authors: Frithz‐Lindsten Elisabet , Du Yidong , Rosqvist Roland , Forsberg Åke
Published: November 2003

Exoenzyme S (ExoS) is an ADP‐ribosyltransferase secreted by the opportunistic pathogen Pseudomonas aeruginosa. The amino‐terminal half of ExoS exhibits homology to the YopE cytotoxin of pathogenic Yersinia. Recently, YopE was found to be translocated into the host cell by a bacteria–cell contact‐dependent mechanism involving the ysc‐encoded type III secretion system. By using an approach in which exoS was expressed in different strains of Yersinia, including secretion and translocation mutants, we could demonstrate that ExoS was secreted and translocated into HeLa cells by a similar mechanism to that described previously for YopE. Similarly to YopE, the presence of ExoS in the host cell elicited a cytotoxic response, correlating with disruption of the actin microfilament structure. A similar cytotoxic response was also induced by a mutated form of ExoS with a more than 2000‐fold reduced ADP‐ribosyltransferase activity. However, the enzymatically active ExoS elicited a more definite rounding up of the HeLa cells, which also correlated with decreased viability of the cells after prolonged infection compared with cells infected with strains expressing mutated ExoS or YopE. This suggests that ExoS can act through two different mechanisms on the host cell. The expression of ExoS by Yersinia also mediated an anti‐phagocytic effect on macrophages. In addition, we present evidence that extracellularly located P. aeruginosa is able to target ExoS into eukaryotic cells. Taken together, our data suggest that P. aeruginosa, by analogy with Yersinia, targets virulence proteins into the eukaryotic cytosol via a type III secretion‐dependent mechanism as part of an anti‐phagocytic strategy.
Phage display selection of peptides against enzyme I of the phosphoenolpyruvate–sugar phosphotransferase system (PTS)
Authors: Mukhija Seema , Erni Bernhard
Published: November 2003

The bacterial phosphoenolpyruvate–sugar phosphotransferase system (PTS) mediates the uptake and phosphorylation of carbohydrates and is involved in signal transduction. In response to the availability of carbohydrates it modulates catabolite repression, intermediate metabolism, gene expression and chemotaxis. It is ubiquitous in bacteria but does not occur in animals and plants. Uniqueness and pleiotropic function make the PTS a target for new antibacterial drugs. Enzyme I is the first component of the divergent protein phosphorylation cascade of the PTS. It transfers phosphoryl groups from phosphoenolpyruvate to the general phosphoryl carrier protein HPr. Six 15‐mer, nine 10‐mer and nine 6‐mer peptides that inhibit enzyme I were selected from phage display libraries. Of these, 16 were synthesized and characterized. The majority of the peptides contain a histidine with an adjacent arginine. Two peptides were found to contain cysteines but no histidine. All peptides are rich in basic residues and lack acidic amino acids. The peptides inhibit the phosphotransferase system in vitro with IC50 of between 10 μM and 2 mM. Some, but not all, of the peptides inhibit cell growth in the agar diffusion test by an as yet undefined mechanism. All peptides are phosphorylated by enzyme I, and some are regenerated by slow autocatalytic hydrolysis of the phospho–peptide bond.
Dephosphorylation of the phosphoprotein PII in Synechococcus PCC 7942: identification of an ATP and 2‐oxoglutarate‐regulated phosphatase activity
Authors: Irmler Angelika , Sanner Silvia , Dierks Henning , Forchhammer Karl
Published: November 2003

The phosphorylation state of the putative signal transduction protein PII from the cyanobacterium Synechococcus sp. strain PCC 7942 depends on the cellular state of nitrogen and carbon assimilation. In this study, dephosphorylation of phosphorylated PII protein (PII‐P) was investigated both in vivo and in vitro. The in vivo studies implied that PII‐P dephosphorylation is regulated by inhibitory metabolites involved in the glutamine synthetase–glutamate synthase pathway of ammonium assimilation. An in vitro assay for PII‐P dephosphorylation was established that revealed a Mg2+‐dependent PII‐P phosphatase activity. PII‐P phosphatase and PII kinase activities could be separated biochemically. A partially purified PII‐P phosphatase preparation also catalysed the dephosphorylation of phosphoserine/phosphothreonine residues on other proteins in a Mg2+‐dependent manner. However, only dephosphorylation of PII‐P was regulated by synergistic inhibition by ATP and 2‐oxoglutarate. As the same metabolites stimulate the PII kinase activity, it appears that the phosphorylation state of PII is determined by ATP and 2‐oxoglutarate‐dependent reciprocal reactivity of PII towards its phosphatase and kinase.
Chaperone‐mediated reduction of RepA dimerization is associated with RepA conformational change
Authors: Dibbens Justin A. , Muraiso Kanae , Chattoraj Dhruba K.
Published: November 2003

RepA, the initiator protein of plasmid P1, binds to multiple sites (iterons) in the origin. The binding normally requires participation of chaperones, DnaJ, DnaK and GrpE. When purified, RepA appears dimeric and is inactive in iteron binding. On reaction with chaperones, a species active in iteron binding is formed and found to be monomeric. To test whether the chaperones can reduce dimerization, RepA was used to replace the dimerization domain of the λ repressor. The hybrid protein repressed the λ operator efficiently, indicating that RepA can dimerize in vivo. A further increase in repressor activity was seen in dnaJ mutant cells. These results are consistent with a chaperone‐mediated reduction of RepA dimerization. We also found that RepA mutants defective in dimerization still depend on DnaJ for iteron binding. Conversely, RepA mutants that no longer require chaperones for iteron binding remain dimerization proficient. These results indicate that the chaperone dependence of RepA activity is not solely owing to RepA dimerization. Our results are most simply explained by a chaperone‐mediated conformational change in RepA protomer that activates iteron binding. This conformational change also results in reduced RepA dimerization.
Capsular polysaccharide synthesis in Streptococcus pneumoniae serotype 14: molecular analysis of the complete cps locus and identification of genes encoding glycosyltransferases required for the biosynthesis of the tetrasaccharide subunit
Authors: Kolkman Marc A. B. , Wakarchuk Warren , Nuijten Piet J. M. , Van Der Zeijst Bernard A. M.
Published: November 2003

We have reported previously on seven genes (cps14B–H ) of Streptococcus pneumoniae serotype 14, which are part of the type 14 capsular polysaccharide synthesis (cps14 ) locus. This study describes the cloning and sequencing of the remaining part of the cps14 locus. The entire cps14 gene cluster consists of 12 open reading genes (cps14A to cps14L ), which appear to be arranged as a single transcriptional unit. The flanking regions of the cps14 locus contain vestiges of insertion elements. Moreover, a 115‐bp‐long repeated DNA element, which is also present in several other intergenic regions on the pneumococcal chromosome, has been identified upstream of cps14AAll 12 open reading frames (ORFs) were inactivated by the insertion of a tetracycline resistance cassette. The cps14A to cps14J and cps14L mutants were unencapsulated, whereas only a limited amount of capsular polysaccharide was expressed by a cps14K insertion mutant. Comparison with DNA and protein sequences available in databases allowed us to predict functions for four out of the five new cps14 gene products. The biosynthetic function of Cps14I was determined experimentally by analysis of intermediates in the synthesis of the type 14 tetrasaccharide subunit, catalysed by membrane preparations of Escherichia coli expressing pneumococcal glycosyltransferases. The cps14I gene encodes the β‐1,3‐N‐acetylglucosaminyltransferase activity necessary for the addition of the third sugar in the synthesis of the type 14 repeating unit. The activity encoded by cps14J was established using a synthetic glycosyltransferase acceptor: cps14J encodes a β‐1,4‐galactosyltransferase, which requires β‐linked GlcNAc as an acceptor. Thus, Cps14J is responsible for the addition of the last (fourth) sugar in the synthesis of the type 14 subunit.
The finM promoter and the traM promoter are the principal promoters of the traM gene of the antibiotic resistance plasmid R100
Authors: Stockwell David T. , Dempsey Walter B.
Published: November 2003

finP multicopy repression and traJ multicopy derepression indicate that the ratio of sense to antisense transcripts is important in the regulation of R100 conjugation. The extension of R100 traM transcripts into traJ shows that promoters in traM can affect this ratio, making the regulation of traM transcription important in the regulation of R100 conjugation. Since R100 traM, traY and traI proteins bind to the traM promoter region, we examined traM transcription in R100‐1 traM, traY and traI mutants and compared it with traM transcription in both R100‐1 and R100. We verified that the traM and finM promoters provide virtually all the transcripts originating in the R100‐1 traM gene. When either is deleted, as in VAR22 or VAR30, the remaining promoter is highly active. We show here that traY positively regulates R100‐1 traM transcription, as has been found for F. We found that traI did not regulate R100‐1 traM transcription. The measured activity of the native R100 traM promoter was 12% of that in R100‐1, whereas the native R100 finM promoter was 45% of that in R100‐1. These data and data from the R100‐1 traY and traI mutants show that the activities of the two promoters varied independently.
The role of ribosomal RNAs in macrolide resistance
Authors: Sander Peter , Prammananan Therdsak , Meier Albrecht , Frischkorn Klaus , Böttger Erik C.
Published: November 2003

Macrolides are bacteriostatic antibiotics which interfere with the peptidyltransfer function of the ribosome. We have investigated the molecular mechanisms underlying macrolide resistance in Mycobacterium smegmatis, an eubacterium carrying two rRNA operons. Surprisingly, drug resistance was associated not with alterations in ribosomal proteins, but with a single point mutation in the peptidyltransferase region of one of the two 23S RNA genes, i.e. A2058→G or A2059→G. This mutation resulted in a heterozygous organism with a mutated and a wild‐type rRNA operon respectively. Reverse transcriptase sequencing indicated the expression of both wild‐type and mutated rRNAs. The mutated operon was introduced into genetically engineered rrn− strains of M. smegmatis carrying a single functional rRNA operon and into parental M. smegmatis with two chromosomal rRNA operons, using gene transfer as well as gene replacement techniques. The results obtained demonstrate the dominant nature of resistance. As exemplified in our results on macrolide resistance, a complete set of genetic tools is now available, which allows questions of dominance vs. recessivity and gene dosage effects in eubacterial ribosomal nucleic acids to be addressed experimentally in vivo.
Regulation of the yeast SPS19 gene encoding peroxisomal 2,4‐dienoyl‐CoA reductase by the transcription factors Pip2p and Oaf1p: β‐oxidation is dispensable for Saccharomyces cerevisiae sporulation in acetate medium
Authors: Gurvitz Aner , Rottensteiner Hanspeter , Hiltunen J. Kalervo , Binder Maximilian , Dawes Ian W. , Ruis Helmut , Hamilton Barbara
Published: November 2003

The yeast SPS19 gene encoding the peroxisomally targeted 2,4‐dienoyl‐CoA reductase shares its promoter region (291 bp) with the sporulation‐specific gene SPS18. SPS19 is induced during sporulation in diploids but to a lesser extent than SPS18; under oleate induction conditions, SPS19, but not SPS18, is transcribed via an oleate response element (ORE) independently of ploidy or sporulation. The SPS19 ORE is the binding target of the Pip2p and Oaf1p transcription factors, and an SPS19–lacZ reporter gene, which is highly expressed in oleate‐induced cells, is not activated in haploids devoid of either protein. We examined the expression of CYC1–lacZ reporter constructs carrying the SPS19 and CTA1 OREs in diploids propagated under sporulation conditions and have shown that OREs are not sufficient for heterologous expression during yeast development. In addition, diploids deleted at either PIP2 or OAF1 demonstrated abundant ascosporogenesis, indicating that these genes are not essential for sporulation. A Δpex6 strain lacking peroxisomal structures and one devoid of fatty acyl‐CoA oxidase (Δpox1), the first step in fungal β‐oxidation, were both proficient for sporulation and, hence, β‐oxidation and the peroxisomal compartment containing it are dispensable for meiotic development.
Specific phosphatidylethanolamine dependence of Serratia marcescens cytotoxin activity
Authors: Hertle Ralf , Brutsche Sandra , Groeger Wolfram , Hobbie Silke , Koch Walter , Könninger Ulrich , Braun Volkmar
Published: November 2003

The cytolytic and haemolytic activity of Serratia marcescens is determined by the ShlA protein, which is secreted across the outer membrane with the aid of the ShlB protein. In the absence of ShlB, inactive ShlA* remains in the periplasm of Escherichia coli transformed with an shlA‐encoding plasmid, which indicates that ShlB converts ShlA* to active ShlA. ShlA* in a periplasmic extract and partially purified ShlA* were activated in vitro by partially purified ShlB. When both proteins were highly purified, ShlA* was only activated by ShlB when phosphatidylethanolamine (PE) or phosphatidylserine was added to the assay, while phosphatidylglycerol contributed little to ShlA* activation. Lyso‐PE, cardiolipin, phosphatidylcholine, phosphatidic acid, lipopolysaccharide and various detergents could not substitute for PE. Although radioactively labelled PE was so tightly associated with ShlA that it remained bound to ShlA after heating and SDS–PAGE, it was not covalently linked to ShlA as PE could be removed by thin‐layer chromatography with organic solvents. The number of PE molecules associated per molecule of ShlA was 3.9 ± 2.2. Active ShlA was inactivated by treatment with phospholipase A2, which indicated that PE is also required for ShlA activity. ShlA‐255 (containing the 255 N‐terminal amino acids of ShlA) reversibly complemented ShlA* to active ShlA and was inactivated by phospholipase A2, which demonstrated that PE binds to the N‐terminal portion of ShlA; this region has previously been found to be involved in ShlA secretion and activation. Electrospray mass spectroscopy of ShlA‐255 determined a molar mass that corresponded to that of unmodified ShlA‐255. An E. coli mutant that synthesized only minute amounts of PE did not secrete ShlA but contained residual cell‐bound haemolytic activity. Since PE binds strongly to ShlA* in the absence of ShlB without converting ShlA* to haemolytic ShlA, ShlB presumably imposes a conformation on ShlA that brings PE into a position to mediate interaction of the hydrophilic haemolysin with the lipid bilayer of the eukaryotic membrane.
The carboxy‐terminal C2‐like domain of the α‐toxin from Clostridium perfringens mediates calcium‐dependent membrane recognition
Authors: Guillouard Isabelle , Alzari Pedro M. , Saliou Bernard , Cole Stewart T.
Published: November 2003

The lethal, cytolytic α‐toxin (phospholipase C) of Clostridium perfringens consists of two distinct modules: the larger N‐terminal domain catalyses phospholipid hydrolysis, and its activity is potentiated by a smaller C‐terminal domain. Calcium ions are essential for the binding of α‐toxin to lipid films. Sixteen α‐toxin variants with single amino acid substitutions in the C‐terminal region were obtained using site‐directed mutagenesis and T7 expression technology. Five of these variants showed reduced phospholipase C activity and were considerably less active than native α‐toxin under calcium‐limiting conditions. Replacement of Thr‐272 by Pro diminished phospholipase C activity, severely affected haemolysis and platelet aggregation and perturbed a surface‐exposed conformational epitope. The results of sequence comparisons and molecular modelling indicate that the C‐terminal region probably belongs to the growing family of C2β‐barrel domains, which are often involved in membrane interactions, and that the functionally important substitutions are clustered at one extremity of the domain. The combined findings suggest that the C‐terminal region of α‐toxin mediates interactions with membrane phospholipids in a calcium‐dependent manner. Mutations to this domain may account for the natural lack of toxicity of the α‐toxin homologue, phospholipase C of Clostridium bifermentans.