bluuebunny/crossref_metadata_2025 huggingface dataset:
dnaC mutations suppress defects in DNA replication‐ and recombination‐associated functions in priB and priC double mutants in Escherichia coli K‐12
Authors: Sandler Steven J. , Marians Kenneth J. , Zavitz Kenton H. , Coutu Jaime , Parent Michelle A. , Clark Alvin J.
Published: March 2003

PriA, PriB and PriC were originally discovered as proteins essential for the ΦX174 in vitro DNA replication system. Recent studies have shown that PriA mutants are poorly viable, have high basal levels of SOS expression (SOSH), are recombination deficient (Rec−), sensitive to UV irradiation (UVS) and sensitive to rich media. These data suggest that priA's role may be more complex than previously thought and may involve both DNA replication and homologous recombination. Based on the ΦX174 system, mutations in priB and priC should cause phenotypes like those seen in priA2::kan mutants. To test this, mutations in priB and priC were constructed. We found that, contrary to the ΦX174 model, del(priB)302 and priC303::kan mutants have almost wild‐type phenotypes. Most unexpectedly, we then found that the priBC double mutant had very poor viability and/or a slow growth rate (even less than a priA2::kan mutant). This suggests that priB and priC have a redundant and important role in Escherichia coli. The priA2::kan suppressor, dnaC809, partially suppressed the poor viability/slow growth phenotype of the priBC double mutant. The resulting triple mutant (priBC dnaC809 ) had small colony size, recombination deficiency and levels of SOS expression similar to a priA2::kan mutant. The priBC dnaC809 mutant, however, was moderately UVR and had good viability, unlike a priA2::kan mutant. Additional mutations in the triple mutant were selected to suppress the slow growth phenotype. One suppressor restored all phenotypes tested to nearly wild‐type levels. This mutation was identified as dnaC820 (K178N) [mapping just downstream of dnaC809 (E176G)]. Experiments suggest that dnaC820 makes dnaC809 suppression of priA and or priBC mutants priB and or priC independent. A model is proposed for the roles of these proteins in terms of restarting collapsed replication forks from recombinational intermediates.
Mutual suppression of mukB and seqA phenotypes might arise from their opposing influences on the Escherichia coli nucleoid structure
Authors: Weitao Tao , Nordström Kurt , Dasgupta Santanu
Published: March 2003

A strain of Escherichia coli in which both the seqA and mukB genes were inactivated displayed partial suppressions of their individual phenotypes. Temperature sensitivity, anucleate cell production and poor nucleoid folding seen in the mukB strain were suppressed by the seqA null mutation, whereas filamentation, asymmetric septation and compact folding of the nucleoids observed in the seqA strain were suppressed by inactivation of the mukB gene function. However, the asynchronous initiation of chromosome replication in the seqA strain was not reversed in the mukBseqA double mutant. Membrane‐associated nucleoids were isolated from the wild‐type, mukB, seqA and mukBseqA strains and their sedimentation rates were compared under identical conditions. Whereas the mukB mutation caused unfolding of the nucleoid, the seqA mutation led to a more compact packaging of the chromosome. The mukBseqA double mutant regained the wild‐type nucleoid organization as revealed from its rate of sedimentation. Microscopic appearances of the nucleoids were consistent with the sedimentation profiles. The mukB mutant was oversensitive to novobiocin and this susceptibility was suppressed in the mukBseqA strain, suggesting possible roles of MukB and SeqA in maintaining chromosome topology. The mutual phenotypic suppression of mukB and seqA alleles thus suggests that these genes have opposing influences on the organization of the bacterial nucleoid.
Subcellular localization of plasmids containing the oriC region of the Escherichia coli chromosome, with or without the sopABC partitioning system
Authors: Niki Hironori , Hiraga Sota
Published: March 2003

Fluorescence in situ hybridization (FISH) analysis has revealed the subcellular localization of specific chromosomal segments and plasmid molecules during the cell division cycle in Escherichia coli: the replication origin (oriC) segments on the chromosome are localized at nucleoid borders, and actively partitioning mini‐F plasmid molecules are localized at the 1/4 and 3/4 positions of the cell. In contrast, mini‐F plasmid molecules lacking the sopABC segment are randomly localized in cytoplasmic areas at cell poles. In this study, we analysed the subcellular localization of an oriC plasmid that contains the minimum E. coli chromosomal replication origin and its flanking regions. These oriC plasmid molecules were mainly localized in cytosolic areas at cell poles. On the other hand, oriC plasmid DNA molecules carrying the sopABC segment of F plasmid were localized at cell quarter sites, as were actively partitioning mini‐F plasmid DNA molecules. Therefore, we conclude that oriC itself and its flanking regions are not sufficient for positioning the replication origin domain of the E. coli chromosome within the cell.
Analysis of the function of Escherichia coli poly(A) polymerase I in RNA metabolism
Authors: Mohanty Bijoy K. , Kushner Sidney R.
Published: March 2003

To help understand the role of polyadenylation in Escherichia coli RNA metabolism, we constructed an IPTG‐inducible pcnB [poly(A) polymerase I, PAP I] containing plasmid that permitted us to vary poly(A) levels without affecting cell growth or viability. Increased polyadenylation led to a decrease in the half‐life of total pulse‐labelled RNA along with decreased half‐lives of the rpsO, trxA, lpp and ompA transcripts. In contrast, the transcripts for rne (RNase E) and pnp (polynucleotide phosphorylase, PNPase), enzymes involved in mRNA decay, were stabilized. rnb (RNase II) and rnc (RNase III) transcript levels were unaffected in the presence of increased polyadenylation. Long‐term overproduction of PAP I led to slower growth and irreversible cell death. Differential display analysis showed that new RNA species were being polyadenylated after PAP I induction, including the mature 3′‐terminus of 23S rRNA, a site that was not tailed in wild‐type cells. Quantitative reverse transcriptase–polymerase chain reaction (RT–PCR) demonstrated an almost 20‐fold variation in the level of polyadenylation among three different transcripts and that PAP I accounted for between 94% and 98.6% of their poly(A) tails. Cloning and sequencing of cDNAs derived from lpp, 23S and 16S rRNA revealed that, during exponential growth, C and U residues were polymerized into poly(A) tails in a transcript‐dependent manner.
The Tat protein export pathway
Authors: Berks Ben C. , Sargent Frank , Palmer Tracy
Published: March 2003

The Tat (twin‐arginine translocation) system is a bacterial protein export pathway with the remarkable ability to transport folded proteins across the cytoplasmic membrane. Preproteins are directed to the Tat pathway by signal peptides that bear a characteristic sequence motif, which includes consecutive arginine residues. Here, we review recent progress on the characterization of the Tat system and critically discuss the structure and operation of this major new bacterial protein export pathway.
The transfer region of IncI1 plasmid R64: similarities between R64 tra and Legionella icm/dot genes
Authors: Komano Teruya , Yoshida Tetsu , Narahara Koji , Furuya Nobuhisa
Published: March 2003

The entire nucleotide sequence of the transfer region of IncI1 plasmid R64 was determined together with previously reported sequences. Twenty‐two transfer genes, traE–Y and nuc, were newly identified in the present study. The protein products of 17 genes were detected by maxicell experiments or by the T7 RNA polymerase expression system. Mutagenesis experiments indicated that 16 genes were indispensable for R64 transfer both in liquid and on surfaces. In summary, the R64 transfer region located within an ≈ 54 kb DNA segment was shown to encode the most complex transfer system so far studied. It contains at least 49 genes and may produce 58 different proteins as a result of shufflon DNA rearrangement and overlapping genes. Among the 49 genes, 23 tra, trb and nik genes have been shown to be indispensable for R64 conjugal transfer in liquid and on surfaces. Twelve additional pil genes are required only for liquid matings. The amino acid sequences of 10 R64 tra/trb products share similarity with those of the icm/dot products of Legionella pneumophila that are responsible for its virulence, suggesting that the R64 transfer and L. pneumophila icm/dot systems have evolved from a common ancestral genetic system.
Multiple N‐acetyl neuraminic acid synthetase (neuB) genes in Campylobacter jejuni: identification and characterization of the gene involved in sialylation of lipo‐oligosaccharide
Authors: Linton Dennis , Karlyshev Andrey V. , Hitchen Paul G. , Morris Howard R. , Dell Anne , Gregson Norman A. , Wren Brendan W.
Published: March 2003

N‐acetyl neuraminic acid (NANA) is a common constituent of Campylobacter jejuni lipo‐oligosaccharide (LOS). Such structures often mimic human gangliosides and are thought to be involved in the triggering of Guillain–Barré syndrome (GBS) and Miller–Fisher syndrome (MFS) following C. jejuni infection. Analysis of the C. jejuni NCTC 11168 genome sequence identified three putative NANA synthetase genes termed neuB1, neuB2 and neuB3. The NANA synthetase activity of all three C. jejuni neuB gene products was confirmed by complementation experiments in an Escherichia coli neuB‐deficient strain. Isogenic mutants were created in all three neuB genes, and for one such mutant (neuB1) LOS was shown to have increased mobility. C. jejuni NCTC 11168 wild‐type LOS bound cholera toxin, indicating the presence of NANA in a LOS structure mimicking the ganglioside GM1. This property was lost in the neuB1 mutant. Gas chromatography–mass spectrometry and fast atom bombardment–mass spectrometry analysis of LOS from wild‐type and the neuB1 mutant strain demonstrated the lack of NANA in the latter. Expression of the neuB1 gene in E. coli confirmed that NeuB1 was capable of in vitro NANA biosynthesis through condensation of N‐acetyl‐d‐mannosamine and phosphoenolpyruvate. Southern analysis demonstrated that the neuB1 gene was confined to strains of C. jejuni with LOS containing a single NANA residue. Mutagenesis of neuB2 and neuB3 did not affect LOS, but neuB3 mutants were aflagellate and non‐motile. No phenotype was evident for neuB2 mutants in strain NCTC 11168, but for strain G1 the flagellin protein from the neuB2 mutant showed an apparent reduction in molecular size relative to the wild type. Thus, the neuB genes of C. jejuni appear to be involved in the biosynthesis of at least two distinct surface structures: LOS and flagella.
Low levels of Ypt protein prenylation cause vesicle polarization defects and thermosensitive growth that can be suppressed by genes involved in cell wall maintenance
Authors: Bialek‐Wyrzykowska Urszula , Bauer Bettina E. , Wagner Wolfgang , Kohlwein Sepp D. , Schweyen Rudolf J. , Ragnini Antonella
Published: March 2003

The Rab/Ypt small G proteins are essential for intracellular vesicle trafficking in mammals and yeast. The vesicle‐docking process requires that Ypt proteins are located in the vesicle membrane. C‐terminal geranylgeranyl anchors mediate the membrane attachment of these proteins. The Rab escort protein (REP) is essential for the recognition of Rab/Ypt small G proteins by geranylgeranyltransferase II (GGTase II) and for their delivery to acceptor membranes. What effect an alteration in the levels of prenylated Rab/Ypt proteins has on vesicle transport or other cellular processes is so far unknown. Here, we report the characterization of a yeast REP mutant, mrs6‐2, in which reduced prenylation of Ypt proteins occurs even at the permissive temperature. A shift to the restrictive temperature does not alter exponential growth during the first 3 h. The amount of Sec4p, but not Ypt1p, bound to vesicle membranes is reduced 2.5 h after the shift compared with wild‐type or mrs6‐2 cells incubated at 25°C. In addition, vesicles fail to be polarized towards the bud and small budded binucleate cells accumulate at this time point. Growth in 1 M sorbitol or overexpression of MLC1, encoding a myosin light chain able to bind the unconventional type V myosin Myo2, or of genes involved in cell wall maintenance, such as SLG1, GFA1 and LRE1, suppresses mrs6‐2 thermosensitivity. Our data suggest that, at least at high temperature, a critical minimal level of Ypt protein prenylation is required for maintaining vesicle polarization.
Modulation of host immune responses, induction of apoptosis and inhibition of NF‐κB activation by the Bordetella type III secretion system
Authors: Yuk Ming Huam , Harvill Eric T. , Cotter Peggy A. , Miller Jeff F.
Published: March 2003

Bordetella bronchiseptica establishes respiratory tract infections in laboratory animals with high efficiency. Colonization persists for the life of the animal and infection is usually asymptomatic in immunocompetent hosts. We hypothesize that this reflects a balance between immunostimulatory events associated with infection and immunomodulatory events mediated by the bacteria. We have identified 15 loci that are part of a type III secretion apparatus in B. bronchiseptica and three secreted proteins. The functions of the type III secretion system were investigated by comparing the phenotypes of wild‐type bacteria with two strains that are defective in type III secretion using in vivo and in vitro infection models. Type III secretion mutants were defective in long‐term colonization of the trachea in immunocompetent mice. The mutants also elicited higher titres of anti‐Bordetella antibodies upon infection compared with wild‐type bacteria. Type III secretion mutants also showed increased lethal virulence in immunodeficient SCID‐beige mice. These observations suggest that type III‐secreted products of B. bronchiseptica interact with components of both innate and adaptive immune systems of the host. B. bronchiseptica induced apoptosis in macrophages in vitro and inflammatory cells in vivo and type III secretion was required for this process. Infection of an epithelial cell line with high numbers of wild type, but not type III deficient B. bronchiseptica resulted in rapid aggregation of NF‐κB into large complexes in the cytoplasm. NF‐κB aggregation was dependent on type III secretion and aggregated NF‐κB did not respond to TNFα activation, suggesting B. bronchiseptica may modulate host immunity by inactivating NF‐κB. Based on these in vivo and in vitro results, we hypothesize that the Bordetella type III secretion system functions to modulate host immune responses during infection.
Maturation and secretion of the non‐typable Haemophilus influenzae HMW1 adhesin: roles of the N‐terminal and C‐terminal domains
Authors: Grass Susan , St. Geme Joseph W.
Published: March 2003

Non‐typable Haemophilus influenzae is a common cause of human disease and initiates infection by colonizing the upper respiratory tract. The non‐typable H. influenzae HMW1 and HMW2 adhesins mediate attachment to human epithelial cells, an essential step in the process of colonization. HMW1 and HMW2 have an unusual N‐terminus and undergo cleavage of a 441‐amino‐acid N‐terminal fragment during the course of their maturation. Following translocation across the outer membrane, they remain loosely associated with the bacterial surface, except for a small amount that is released extracellularly. In the present study, we localized the signal sequence to the first 68 amino acids, which are characterized by a highly charged region from amino acids 1–48, followed by a more typical signal peptide with a predicted leader peptidase cleavage site after the amino acid at position 68. Additional experiments established that the SecA ATPase and the SecE translocase are essential for normal export and demonstrated that maturation involves cleavage first between residues 68 and 69, via leader peptidase, and next between residues 441 and 442. Site‐directed mutagenesis revealed that HMW1 processing, secretion and extracellular release are dependent on amino acids in the region between residues 150 and 166 and suggested that this region interacts with the HMW1B outer membrane translocator. Deletion of the C‐terminal end of HMW1 resulted in augmented extracellular release and elimination of HMW1‐mediated adherence, arguing that the C‐terminus may serve to tether the adhesin to the bacterial surface. These observations suggest that the HMW proteins are secreted by a variant form of the general secretory pathway and provide insight into the mechanisms of secretion of a growing family of Gram‐negative bacterial exoproteins.
Formate protects stationary‐phase Escherichia coli and Salmonella cells from killing by a cationic antimicrobial peptide
Authors: Barker Helen C. , Kinsella Niamh , Jaspe Almudena , Friedrich Thorsten , O’Connor C. David
Published: March 2003

For a sustained infection, enteric bacterial pathogens must evade, resist or tolerate a variety of antimicrobial host defence peptides and proteins. We report here that specific organic acids protect stationary‐phase Escherichia coli and Salmonella cells from killing by a potent antimicrobial peptide derived from the human bactericidal/permeability‐increasing protein (BPI). BPI‐derived peptide P2 rapidly halted oxygen consumption by stationary‐phase cells preincubated with glucose, pyruvate or malate and caused a 109‐fold drop in cell viability within 90 min of addition. In marked contrast, O2 consumption and viability were not significantly affected in stationary‐phase cells preincubated with formate or succinate. Experiments with fdhH, fdoG, fdnG, selC and sdhO mutants indicate that protection by formate and succinate requires their oxidation by the Fdh‐N formate dehydrogenase and succinate dehydrogenase respectively. Protection was also dependent on the BipA GTPase but did not require the RpoS sigma factor. We conclude that the primary lesion caused by this cationic peptide is not gross permeabilization of the bacterial cytoplasmic membrane but may involve specific disruption of the respiratory chain. Because P2 shares sequence similarity with a range of other antimicrobial peptides, its cytotoxic mechanism has broader significance. Additionally, protective quantities of formate are secreted by E. coli and Salmonella during growth suggesting that such compounds are important determinants of bacterial survival in the host.
Acid resistance of Helicobacter pylori depends on the UreI membrane protein and an inner membrane proton barrier
Authors: Rektorschek Marina , Buhmann Anita , Weeks David , Schwan Dorothee , Bensch Klaus W. , Eskandari Sepehr , Scott David , Sachs George , Melchers Klaus
Published: March 2003

ureI encodes an inner membrane protein of Helicobacter pylori. The role of the bacterial inner membrane and UreI in acid protection and regulation of cytoplasmic urease activity in the gastric microorganism was studied. The irreversible inhibition of urease when the organism was exposed to a protonophore (3,3′,4′,5‐tetrachlorsalicylanide; TCS) at acidic pH showed that the inner membrane protected urease from acid. Isogenic ureI knockout mutants of several H. pylori strains were constructed by replacing the ureI gene of the urease gene cluster with a promoterless kanamycin resistance marker gene (kanR). Mutants carrying the modified ureAB‐kanR‐EFGH operon all showed wild‐type levels of urease activity at neutral pH in vitro. The mutants resisted media of pH > 4.0 but not of pH < 4.0. Whereas wild‐type bacteria showed high levels of urease activity below pH 4.0, this ability was not retained in the ureI mutants, resulting in inhibition of metabolism and cell death. Gene complementation experiments with plasmid‐derived H. pylori ureI restored wild‐type properties. The activation of urease activity found in structurally intact but permeabilized bacteria treated with 0.01% detergent (polyoxy‐ethylene‐8‐laurylether; C12E8), suggested a membrane‐limited access of urea to internal urease at neutral pH. Measurement of 14C‐urea uptake into Xenopus oocytes injected with ureI cRNA showed acid activation of uptake only in injected oocytes. Acceleration of urea uptake by UreI therefore mediates the increase of intracellular urease activity seen under acidic conditions. This increase of urea permeability is essential for H. pylori survival in environments below pH 4.0. ureI‐independent urease activity may be sufficient for maintenance of bacterial viability above pH 4.0.
Differential RNA elongation controls the variant surface glycoprotein gene expression sites of Trypanosoma brucei
Authors: Vanhamme Luc , Poelvoorde Philippe , Pays Annette , Tebabi Patricia , Van Xong Hoang , Pays Etienne
Published: March 2003

The protozoan parasite Trypanosoma brucei develops antigenic variation to escape the immune response of its host. To this end, the trypanosome genome contains multiple telomeric expression sites competent for transcription of variant surface glycoprotein genes, but as a rule only a single antigen is expressed at any time. We used reverse transcription‐PCR (RT‐PCR) to analyse transcription of different segments of the expression sites in different variant clones of two independent strains of T. brucei. The results indicated that RNA polymerase is installed and active at the beginning of many, if not all, expression sites simultaneously, but that a progressive arrest of RNA elongation occurs in all but one site. This defect is linked to inefficient RNA processing and RNA release from the nucleus. Therefore, functional transcription in the active site appears to depend on the selective recruitment of a RNA elongation/processing machinery.
Hyperactive forms of the Pdr1p transcription factor fail to respond to positive regulation by the Hsp70 protein Pdr13p
Authors: Hallstrom Timothy C. , Moye‐Rowley W. Scott
Published: March 2003

Multidrug resistance in Saccharomyces cerevisiae is commonly associated with the overproduction of ATP‐binding cassette transporter proteins such as Pdr5p or Yor1p. The Cys6‐Zn(II)2 cluster‐containing transcription factors Pdr1p and Pdr3p are key regulators of expression of these pleiotropic drug resistance (PDR) loci. Previous experiments have demonstrated that the Hsp70 protein encoded by the PDR13 gene is a positive regulator of Pdr1p function. We have examined the mechanism underlying the control of Pdr1p by Pdr13p. Expression of deletion, insertion and amino acid substitution mutant variants of Pdr1p suggest that the centre region of the transcription factor is the target for Pdr13p‐mediated positive regulation. Immunological and fusion protein analyses demonstrate that Pdr13p is located in the cytoplasm, while Pdr1p is found in the nucleus. Biochemical fractionation experiments indicate that Pdr13p is associated with a high‐molecular‐weight complex and suggest the association of some fraction of Pdr13p with ribosomes.
Effects of local transcription and H‐NS on inversion of the fim switch of Escherichia coli
Authors: O′gara James P. , Dorman Charles J.
Published: March 2003

The fim switch of Escherichia coli is responsible for phase‐variable expression of type 1 fimbriae. Switching in the ON‐to‐OFF and OFF‐to‐ON directions is promoted by the FimB recombinase, while the FimE recombinase directs switching predominantly in the ON‐to‐OFF direction. The effects of local promoter activity and the H‐NS nucleoid‐associated protein on inversion of the switch were assessed. In contrast to FimB‐mediated inversion, inversion of the switch by the FimE recombinase was unaffected by the H‐NS status of the cell. Transcription towards the switch from within a translationally inactivated fimE gene was found to bias the switch strongly in the OFF direction, creating a FimE+‐like phenotype in the absence of the FimE protein. This biasing was H‐NS dependent and was also contingent on transcription from within the switch. These data show that local transcription and a nucleoid‐associated protein both contribute to the modulation of a site‐specific recombination event on the bacterial chromosome.
The Bacillus subtilis cell division protein FtsL localizes to sites of septation and interacts with DivIC
Authors: Sievers Jörg , Errington Jeff
Published: March 2003

FtsL is a small bitopic membrane protein required for vegetative cell division and sporulation in Bacillus subtilis. We investigated its localization by fluorescence microscopy using a green fluorescent protein (GFP) fusion. GFP–FtsL was localized at mid‐cell in vegetative cells and at the asymmetric septum in sporulating cells. We also show that FtsL forms a ring‐like structure at the division site and that it remains localized at mid‐cell during the whole septation process. By yeast two‐hybrid analysis and non‐denaturing polyacrylamide gel electrophoresis (PAGE) with purified proteins, FtsL was found to interact with another membrane‐bound division protein, the FtsL‐like DivIC protein.
Host adaptation and the emergence of infectious disease: the Salmonella paradigm
Authors: Kingsley Robert A. , Bäumler Andreas J.
Published: March 2003

The recent emergence of food‐borne pathogens, such as Salmonella enterica serotype Enteritidis (S. enteritidis) and Escherichia coli O157:H7, has generated increasing interest in how infectious diseases can invade, persist and spread within new host populations. To alter their host range pathogens require adaptations, which ensure their circulation in a new animal population. Adaptations for circulation in different populations of vertebrate hosts seem to have been acquired multiple times within the genus Salmonella because extant Salmonella serotypes differ greatly with regard to host range. In this article, mechanisms involved in host adaptation are deduced by considering the influence of the host immune response on circulation of Salmonella serotypes within populations of vertebrate animals. This approach contributes to the identification of genes involved in host adaptation and provides new insights into the emergence of food‐borne pathogens.
Identification of a major facilitator protein from Escherichia coli involved in efflux of metabolites of the cysteine pathway
Authors: Daßler Tobias , Maier Thomas , Winterhalter Christoph , Böck August
Published: March 2003

A chromosomal fragment has been identified in a gene bank from Escherichia coli, which augmented the yield of cysteine in an industrial production strain. Subcloning and genetic analysis showed that an open reading frame coding for a product of 299 amino acids (Orf299) was responsible. Orf299 was synthesized in the T7 polymerase/promoter system and exhibited the properties of an integral membrane protein. Mutational interruption of orf299 did not cause a distinct phenotype; however, transformants overexpressing orf299 had lost the ability to grow in minimal medium unless it was supplemented with a source of reduced sulphur compounds, and they excreted considerable amounts of cysteine and O‐acetyl‐l‐serine, especially in the presence of thiosulphate. Most of the cysteine was found to be masked in 2‐methyl‐2,4‐thiazolidinedicarboxylic acid. N‐acetyl‐l‐serine was also present in the medium, but it is open to question whether it represents a primary excretion product. Measurement of the induction status of the cysteine regulon by means of a cysK′–′lacZ gene fusion demonstrated that the regulon is not induced upon growth in the presence of a poor sulphur source and that the introduction of a constitutive cysB allele alleviates this deficiency. The results indicate that orf299 codes for an export pump for different metabolites of the cysteine pathway. Its relation to other efflux systems and the physiological role are discussed.
Interacting interfaces of the P4 antirepressor E and the P2 immunity repressor C
Authors: Eriksson Sara K. Renberg , Liu Tao , Haggård‐Ljungquist Elisabeth
Published: March 2003

Antirepressors have been identified as proteins interacting with transcriptional repressors leading to expression of the repressed genes. The defective satellite phage/plasmid P4 has the capacity to derepress the unrelated prophage P2 after infection, thereby getting access to the late functions of the helper that are required for P4 lytic growth. The derepression of prophage P2 is mediated by the P4 E protein that function as an antirepressor by binding to the P2 immunity repressor C. A P2 mutant, sos, has been isolated that is insensitive to the action of the P4 E protein. In the present study, we show that sos is a point mutation in the P2 immunity repressor gene C and that it makes P4 E unable to turn the transcriptional switch of P2 from the lysogenic state to the lytic mode in a two plasmid reporter system. Furthermore, the interaction between C and E, when analysed in the yeast two‐hybrid system, is blocked by the sos mutation. An analysis of C mutants indicates that the dimerization function of C is located in the C‐terminal part of the protein and the dimerization defective mutants are unable to bind to their operator DNA. The sos mutation does not affect the capacity of the protein to dimerize. Using the yeast two‐hybrid system, compensatory E mutants have been isolated that can interact with Sos, but they are unable to turn the transcriptional switch controlled by the Sos repressor. However, one point mutation in the E protein is shown to be unable to turn the transcriptional switch controlled by the wild‐type C repressor.
Functional specificity of the replication fork‐arrest complexes of Bacillus subtilis and Escherichia coli: significant specificity for Tus–Ter functioning in E. coli
Authors: Andersen P. A. , Griffiths A. A. , Duggin I. G. , Wake R. G.
Published: March 2003

The Escherichia coli replication terminator TerB was inserted in its two alternate orientations into a Bacillus subtilis fork‐arrest assay plasmid. After transferring these new plasmids into B. subtilis, which could overproduce the E. coli terminator protein Tus, it was shown that the E. coli Tus–TerB complex could cause polar replication fork arrest, albeit at a very low level, in B. subtilis. A new B. subtilis–E. coli shuttle plasmid was designed to allow the insertion of either the TerI (B. subtilis) or TerB (E. coli) terminator at the same site and in the active orientation in relation to the approaching replication fork generated in either organism. Fork‐arrest assays for both terminator‐containing plasmids replicating in both organisms which also produced saturating levels of either the B. subtilis terminator protein (RTP) or Tus were performed. The efficiency of the Tus–TerB complex in causing fork arrest was much higher in E. coli than in B. subtilis. The efficiency of the B. subtilis RTP–TerI complex was higher in B. subtilis than in E. coli, but the effect was significantly less. Evidently a specificity feature in E. coli operates to enhance appreciably the fork‐arrest efficiency of a Tus–Ter complex. The specificity effect is of less significance for an RTP–Ter complex functioning in B. subtilis.
A chimeric ribozyme in Clostridium difficile combines features of group I introns and insertion elements
Authors: Braun Veit , Mehlig Markus , Moos Michael , Rupnik Maja , Kalt Bettina , Mahony David E. , Von Eichel‐Streiber Christoph
Published: March 2003

CdISt1, a DNA insertion of 1975 bp, was identified within tcdA‐C34, the enterotoxin gene of the Clostridium difficile isolate C34. Located in the catalytic domain A1‐C34, CdISt1 combines features of two genetic elements. Within the first 434 nt structures characteristic for group I introns were found; encoding the two transposase‐like proteins tlpA and tlpB nucleotides 435–1975 represent the remainder of a IS605‐like insertion element. We show that the entire CdISt1 is accurately spliced from tcdA‐C34 primary transcripts and that purified TcdA‐C34 toxin is of regular size and catalytic activity. A search for CdISt1‐related sequences demonstrates that the element is widespread in toxinogenic and non‐toxinogenic C. difficile strains, indicating the mobility of CdISt1. In strain C34, we characterize 10 CdISt1 variants; all are highly homologous to CdISt1 (> 93% identity), integrated in bacterial open reading frames (ORFs), show the typical composite structure of CdISt1 and are precisely spliced from their primary transcripts. CdISt1‐like chimeric ribozymes appear to combine the invasiveness of an insertion element with the splicing ability of a group I intron, rendering transposition harmless for the interrupted gene.
Functional mapping of the Yersinia enterocolitica adhesin YadA. Identification of eight NSVAIG – S motifs in the amino‐terminal half of the protein involved in collagen binding
Authors: Tahir Yasmin El , Kuusela Pentti , Skurnik Mikael
Published: March 2003

The virulence plasmid‐encoded YadA of Yersinia enterocolitica serotype O:3 is a 430‐amino‐acid outer membrane protein, synthesized with a 25‐amino‐acid signal peptide. YadA forms homotrimeric surface structures that function as adhesin between bacteria and collagen as well as other host proteins. The structure–function relationships of YadA were studied, and the collagen‐binding determinants of YadA were located to its amino‐terminal half. Collagen did not bind to any of the overlapping 16‐mer YadA peptides, indicating that the collagen binding site of YadA is conformational. Epitope mapping of YadA identified 12 linear antigenic epitopes altogether. Seven epitopes were uniquely recognized by an anti‐YadA antiserum able to inhibit collagen binding. Four of these epitopes shared a motif NSVAIG–S that is repeated eight times within the N‐terminal half of YadA. Site‐directed mutagenesis showed that these motifs are absolutely required for YadA‐mediated collagen binding, revealing a novel type of collagen‐binding mechanism.
Characterization of the O‐antigen gene clusters of Yersinia pseudotuberculosis and the cryptic O‐antigen gene cluster of Yersinia pestis shows that the plague bacillus is most closely related to and has evolved from Y. pseudotuberculosis serotype O:1b
Authors: Skurnik Mikael , Peippo Anne , Ervelä Elise
Published: March 2003

One of the most virulent and feared bacterial pathogens is Yersinia pestis, the aetiologic agent of bubonic plague. Characterization of the O‐antigen gene clusters of 21 serotypes of Yersinia pseudotuberculosis and the cryptic O‐antigen gene cluster of Y. pestis showed that the plague bacillus is most closely related to and has evolved from Y. pseudotuberculosis serotype O:1b. The nucleotide sequences of both gene clusters (about 20.5 kb each) were determined and compared to identify the differences that caused the silencing of the Y. pestis gene cluster. At the nucleotide sequence level, the loci were 98.9% identical and, of the 17 biosynthetic genes identified from the O:1b gene cluster, five were inactivated in the Y. pestis cluster, four by insertions or deletions of one nucleotide and one by a deletion of 62 nucleotides. Apparently, the expression of the O‐antigen is not beneficial for the virulence or to the lifestyle of Y. pestis and, therefore, as one step in the evolution of Y. pestis, the O‐antigen gene cluster was inactivated.
The outer membrane protein, Antigen 43, mediates cell‐to‐cell interactions within Escherichia coli biofilms
Authors: Danese Paul N. , Pratt Leslie A. , Dove Simon L. , Kolter Roberto
Published: March 2003

Transcription of the agn43 locus, which specifies an outer membrane protein of Escherichia coli, is regulated in a phase‐variable fashion by the OxyR–DNA binding protein and Dam methylase. Despite its well‐characterized regulation, the function of Ag43 has remained elusive until now. Previous studies indicated that Ag43 mediates autoaggregation of certain strains of E. coli in liquid culture. Given this phenotype, we examined the role of Ag43 in biofilm formation. Here, we report that Ag43 contributes to E. coli biofilm formation in glucose‐minimal medium, but not in Luria–Bertani broth. In addition, we show that flagellar‐mediated motility is required for biofilm formation in both rich and minimal environments. Altogether, our results suggest that E. coli uses both common and specific gene sets for the development of biofilms under various growth conditions.
The Myxococcus xanthus socE and csgA genes are regulated by the stringent response
Authors: Crawford Eugene W. , Shimkets Lawrence J.
Published: March 2003

Disruption of the Myxococcus xanthus socE gene bypasses the requirement for the cell contact‐dependent C‐signalling system mediated by CsgA and restores fruiting body morphogenesis and spore differentiation. The socE gene has been identified by genetic complementation, cloned and sequenced. SocE is highly basic, unique and is predicted to be a soluble protein with a molecular size of 53.6 kDa. The socE and csgA genes have opposite transcription patterns during the M. xanthus life cycle. socE expression is high in growing cells and declines during the early stages of development. Expression of csgA is low in vegetative cells and increases during development. socE transcription is negatively regulated by the stringent response, the major amino acid‐sensing pathway in M. xanthus. A relA null mutation, which eliminates the stringent response, prevents the decline in socE expression normally observed at the onset of development. CsgA is positively regulated by the stringent response and is negatively regulated by socE. A relA mutation virtually eliminates developmental csgA expression. Expression of socE in Escherichia coli leads to a rapid loss of viability in relA− cells during stationary phase, suggesting a relationship with the stringent response.
The spvB gene‐product of the Salmonella enterica virulence plasmid is a mono(ADP‐ribosyl)transferase
Authors: Otto Helge , Tezcan‐Merdol Dilek , Girisch Roman , Haag Friedrich , Rhen Mikael , Koch‐Nolte Friedrich
Published: March 2003

A number of well‐known bacterial toxins ADP‐ribosylate and thereby inactivate target proteins in their animal hosts. Recently, several vertebrate ecto‐enzymes (ART1–ART7) with activities similar to bacterial toxins have also been cloned. We show here that psiblast, a position‐specific‐iterative database search program, faithfully connects all known vertebrate ecto‐mono(ADP‐ribosyl)transferases (mADPRTs) with most of the known bacterial mADPRTs. Intriguingly, no matches were found in the available public genome sequences of archaeabacteria, the yeast Saccharomyces cerevisiae or the nematode Caenorhabditis elegans. Significant new matches detected by psiblast from the public sequence data bases included only one open reading frame (ORF) of previously unknown function: the spvB gene contained in the virulence plasmids of Salmonella enterica. Structure predictions of SpvB indicated that it is composed of a C‐terminal ADP‐ribosyltransferase domain fused via a poly proline stretch to a N‐domain resembling the N‐domain of the secretory toxin TcaC from nematode‐infecting enterobacteria. We produced the predicted catalytic domain of SpvB as a recombinant fusion protein and demonstrate that it, indeed, acts as an ADP‐ribosyltransferase. Our findings underscore the power of the psiblast program for the discovery of new family members in genome databases. Moreover, they open a new avenue of investigation regarding salmonella pathogenesis.
Surface expression of O‐specific lipopolysaccharide in Escherichia coli requires the function of the TolA protein
Authors: Gaspar Joe A. , Thomas John A. , Marolda Cristina L. , Valvano Miguel A.
Published: March 2003

We investigated the involvement of Tol proteins in the surface expression of lipopolysaccharide (LPS). tolQ, ‐R, ‐A and ‐B mutants of Escherichia coli K‐12, which do not form a complete LPS‐containing O antigen, were transformed with the O7+ cosmid pJHCV32. The tolA and tolQ mutants showed reduced O7 LPS expression compared with the respective isogenic parent strains. No changes in O7 LPS expression were found in the other tol mutants. The O7‐deficient phenotype in the tolQ and tolA mutants was complemented with a plasmid encoding the tolQRA operon, but not with a similar plasmid containing a frameshift mutation inactivating tolA. Therefore, the reduction in O7 LPS was attributed to the lack of a functional tolA gene, caused either by a direct mutation of this gene or by a polar effect on tolA gene expression exerted by the tolQ mutation. Reduced surface expression of O7 LPS was not caused by changes in lipid A‐core structure or downregulation of the O7 LPS promoter. However, an abnormal accumulation of radiolabelled mannose was detected in the plasma membrane. As mannose is a sugar unique to the O7 subunit, this result suggested the presence of accumulated O7 LPS biosynthesis intermediates. Attempts to construct a tolA mutant in the E. coli O7 wild‐type strain VW187 were unsuccessful, suggesting that this mutation is lethal. In contrast, a polar tolQ mutation affecting tolA expression in VW187 caused slow growth rate and serum sensitivity in addition to reduced O7 LPS production. VW187 tolQ cells showed an elongated morphology and became permeable to the membrane‐impermeable dye propidium iodide. All these phenotypes were corrected upon complementation with cloned tol genes but were not restored by complementation with the tolQRA operon containing the frameshift mutation in tolA. Our results demonstrate that the TolA protein plays a critical role in the surface expression of O antigen subunits by an as yet uncharacterized involvement in the processing of O antigen.
Occurrence of three PII‐like signal transmitter proteins in the diazotrophic proteobacterium Azoarcus sp. BH72
Authors: Martin Dietmar E. , Hurek Thomas , Reinhold‐Hurek Barbara
Published: March 2003

PII‐like signal transmitter proteins are involved in the regulation of ammonium assimilation and nitrogen fixation. We report the identification of three PII‐like proteins in the diazotrophic, endophytic proteobacterium Azoarcus sp. BH72, encoded by glnB (monocistronically transcribed) or in the glnKamtB and glnYamtY operons. Phylogenetic analysis revealed that glnB, glnK and glnY represent distinct lineages within the Proteobacteria. A combined approach of two‐dimensional gel electrophoresis, Western blotting with paralogue‐specific antibodies, N‐terminal sequencing and marker exchange mutagenesis allowed us to analyse PII protein expression of Azoarcus sp. BH72 in vivo. GlnK and GlnB were present on all nitrogen sources. Knock‐out mutant analysis revealed that GlnB was the only detectable PII protein in a glnK− background, whereas GlnY was only present in a glnK/glnB− double mutant. Nitrogen limitation enhanced transcript abundance of glnK strongly, glnY moderately and glnB not at all in wild‐type, glnB−/glnK− or glnK− backgrounds respectively. Phenotypic characterization of knock‐out mutants revealed that, unlike in other Proteobacteria, neither glnK nor glnB were essential for nitrogen fixation. As the growth of a double mutant was drastically impaired only on minimal media, both proteins are probably involved in the control of ammonium and nitrate assimilation. The PII‐like proteins differed from each other in details of N‐sensing. They were covalently modified by uridylylation upon nitrogen limitation, as shown by mass spectrometry; however, the modification patterns in relation to the supplied nitrogen source differed. The novel paralogue GlnY was unusual, as it only occurred in the uridylylated state in vivo and thus lacked a deuridylylation response to nitrogen excess.
The interaction domains of the DnaA and DnaB replication proteins of Escherichia coli
Authors: Seitz Harald , Weigel Christoph , Messer Walter
Published: March 2003

The initiation of chromosome replication in Escherichia coli requires the recruitment of the replicative helicase DnaB from the DnaBC complex to the unwound region within the replication origin oriC, supported by the oriC‐bound initiator protein DnaA. We defined physical contacts between DnaA and DnaB that involve residues 24–86 and 130–148 of DnaA and residues 154–210 and 1–156 of DnaB respectively. We propose that contacts between DnaA and DnaB occur via two interaction sites on each of the proteins. Interaction domain 24–86 of DnaA overlaps with its N‐terminal homo‐oligomerization domain (residues 1–86). Interaction domain 154–210 of DnaB overlaps or is contiguous with the domains known to interact with plasmid initiator proteins. Loading of the DnaBC helicase in vivo can only be performed by DnaA derivatives containing (in addition to residues 24–86 and the DNA‐binding domain 4) a structurally intact domain 3. Nucleotide binding by domain 3 is, however, not required. The parts of DnaA required for replication of pSC101 were clearly different from those used for helicase loading. Domains 1 and 4 of DnaA, but not domain 3, were found to be involved in the maintenance of plasmid pSC101.
FlbT, the post‐transcriptional regulator of flagellin synthesis in Caulobacter crescentus, interacts with the 5′ untranslated region of flagellin mRNA
Authors: Anderson Paul E. , Gober James W.
Published: March 2003

Flagellar gene expression‘ in Caulobacter crescentus is regulated by a complex trans‐acting hierarchy, in which the assembly of early structural proteins is required for the expression of later structural proteins. The flagellins that comprise the filament are regulated at both the transcriptional and the post‐transcriptional levels. Post‐transcriptional regulation is sensitive to the assembly of the flagellar basal body and hook structures. In mutant strains lacking these structures, flagellin genes are transcribed, but not translated. Mutations in the flagellar regulatory gene, flbT, restore flagellin translation in the absence of flagellar assembly. In this report, we investigate the mechanism of FlbT‐mediated post‐transcriptional regulation. We show that FlbT is associated with the 5′ untranslated region (UTR) of fljK (25 kDa flagellin) mRNA and that this association requires a predicted loop structure in the transcript. Mutations within this loop abolished FlbT association and resulted in increased mRNA stability, indicating that FlbT promotes the degradation of flagellin mRNA by associating with the 5′ UTR. We also assayed the effects on gene expression using mutant transcripts fused to lacZ. Interestingly, the mutant transcript that failed to associate with FlbT in vitro was still repressed in mutants defective in flagellum assembly, suggesting that other factors in addition to FlbT couple assembly to translation.
The putative l‐lactate dehydrogenase from Methanococcus jannaschii is an NADPH‐dependent l‐malate dehydrogenase
Authors: Madern Dominique
Published: March 2003

The enyme encoded by Methanococcus jannaschii open reading frame (ORF) 0490 was purified and characterized. It was shown to be an NADPH‐dependent [lactate dehydrogenase (LDH)‐like]l‐malate dehydrogenase (MalDH) and not an l‐lactate dehydrogenase, as had been suggested previously on the basis of amino acid sequence similarity. The results show the importance of biochemical data in the assignment of ORF function in genomic sequences and have implications for the phylogenetic distribution of members of the MalDH/LDH enzyme superfamilies within the prokaryotic kingdom.