bluuebunny/crossref_metadata_2025 huggingface dataset:
Vibrio spp. secrete proaerolysin as a folded dimer without the need for disulphide bond formation
Authors: Hardie Kim R. , Schulze Angela , Parker Michael W. , Buckley J. Thomas
Published: March 2004

Proaerolysin is an extracellular dimeric protein that is secreted across the inner and outer membranes of Aeromonas spp. in separate steps. To investigate the role of protein folding in the second step, one or more cysteine residues were introduced and the mutant proaerolysins were expressed in Aeromonas hydrophila and Aeromonas salmonicida, as well as Vibrio cholerae. Replacing Met‐41 with Cys resulted in expression of a protein that could form a dimer in which the monomers were linked together by a disulphide bridge. A double mutant was also made, in which Gly‐202 and Ile‐445 were replaced with cysteine in order to allow the formation of an intrachain disulphide bridge when the molecule was correctly folded. The M41C covalent dimer and G202C/I445C proaerolysin with the new intrachain bridge were both easily detected inside the bacteria, and they later appeared in the culture supernatants. Small amounts of incorrectly folded proaerolysin were also observed in the cells, but they were not secreted. We conclude that proaerolysin folds and dimerizes before being released from the cell, and that correct folding is a requirement for secretion to occur. The proton ionophore CCCP reduced release of the folded proteins. Unoxidized protein was secreted by cells grown in β‐mercaptoethanol and by a dsbA mutant of V. cholerae, indicating that disulphide bond formation may not be essential for release.
Transcriptional regulation of repressor synthesis in mycobacteriophage L5
Authors: Nesbit Chadd E. , Levin Margaret E. , Donnelly‐Wu Mary K. , Hatfull Graham F.
Published: March 2004

Mycobacteriophage L5 is a temperate phage of the mycobacteria that forms stable lysogens in Mycobacterium smegmatis. Lysogeny is maintained by the putative repressor, the gene 71 product, which also mediates immunity to superinfection. We show here that there are three promoters located upstream of gene 71 which are active in an L5 lysogen but which do not require any phage‐encoded proteins. In early lytic growth, gene 71 is also transcribed from a promoter, Pleft, located at the right end of the genome and which appears to be a target of gp71 regulation. A model is given for the regulation of L5 life cycles.
Pilus biogenesis and epithelial cell adherence of Neisseria gonorrhoeae pilC double knock‐out mutants
Authors: Rudel Thomas , Boxberger Hans‐Jürgen , Meyer Thomas F.
Published: March 2004

The phase‐variable PilC proteins of pathogenic Neisseria species have recently been implicated in both assembly and cellular adherence functions of the type 4 pili of these pathogens. We describe here the cloning of full‐length pilC1 and pilC2 genes and the complete sequencing of the pilC2 gene of Neisseria gonorrhoeae MS11. Sequential inactivation of both genes by gene replacement in piliated (P+) variants of N. gonorrhoeae MS11 led initially to a non‐piliated (P−) phenotype; however, spontaneous P+ variants could be derived from some pilC1,2 double mutants which produced morphologically intact pili. Purified pili from pilC1,2 mutants revealed no detectable PilC protein. Instead, a novel protein about 70 kDa in size appeared in the pili preparations of P+ mutants; this protein exhibited no immunological cross‐reactivity with PilC1 or PilC2. We propose that this novel factor replaces the function of PilC in pilus biogenesis. Using isogenic N. gonorrhoeae strains which produce identical PilE (pilin) proteins we demonstrate that pili associated with the 70 kDa protein do not confer gonococcal adherence to human epithelial cells, in contrast to pili assembled in the presence of PilC1 or PilC2.
Uptake and nuclear transport of Neisseria IgA1 protease‐associated α‐proteins in human cells
Authors: Pohlner Johannes , Langenberg Uwe , Wölk Uwe , Beck Susanne C. , Meyer Thomas F.
Published: March 2004

Pathogenic Neisseria species, the causative agents of gonorrhoea and bacterial meningitis, encode a family of polymorphic exo‐proteins which are autoproteolytically processed into several distinct extracellular components, including an IgA1 protease and an α‐protein. IgA1 protease, a putative virulence determinant, is a sequence‐specific endopeptidase known to cleave human IgA1, but additional target proteins have been postulated. The physical linkage of IgA1 protease and a‐protein suggests a functional relationship of both precursor components. Previous work has shown that α‐protein is essential neither for extracellular transport nor for the proteolytic activity of IgA1 protease. Intriguingly, α‐proteins carry amino acid sequences reminiscent of nuclear location signals of viral and eukaryotic proteins. Here we demonstrate the functionality of these nuclear location signal sequences in transfected eukaryotic cells. Chimeric α‐proteins show nuclear transport and selectively associate with nucleolar structures. More importantly, native purified α‐proteins are capable of entering certain human primary cells from the exterior via an endocytotic route and accumulate in the nuclei. The neisserial α‐proteins share several features with eukaryotic transcription factors, such as the formation of dimers via a heptad repeat sequence. We propose a role for a‐proteins in the regulation of host‐cell functions. As the α‐proteins are covalently connected with IgA1 protease they may also serve as carriers for the IgA1 protease into human cells where additional proteolytic targets may exist. Neisseria meningitidis, which locally colonizes the nasopharyngeal mucosa of many human individuals without apparently causing symptoms, secretes this nucleus‐targeted factor in large quantities.
Transcriptional activation of the Escherichia coli bgl operon: negative regulation by DNA structural elements near the promoter
Authors: Singh Jatinder , Mukerji Mitali , Mahadevan S.
Published: March 2004

The bgl operon of Escherichia coli is transcriptionally inactive in wild‐type cells. DNA insertion sequences (IS) constitute a major class of spontaneous mutations that activate the cryptic bgl promoter. In an attempt to study the molecular mechanism of activation mediated by insertion sequences, transcription of the bgl promoter was carried out in vitro. Stimulation of transcription is observed when a plasmid containing an insertionally activated bgl promoter is used as a template in the absence of proteins other than RNA polymerase. Deletions that remove sequences upstream of the bgl promoter, and insertion of a 1.2 kb DNA fragment encoding resistance to kanamycin, activate the promoter. Point mutations within a region of dyad symmetry upstream of the promoter, which has the potential to extrude into a cruciform structure under torsional stress, also lead to activation. Introduction of a sequence with dyad symmetry, upstream of an activated bgl promoter carrying a deletion of upstream sequences, results in a fourfold reduction in transcription. These results suggest that the cryptic nature of the bgl promoter is because of the presence of DNA structural elements near the promoter that negatively affect transcription.
Characterization of AGT1 encoding a general α‐glucoside transporter from Saccharomyces
Authors: Han Eun‐Kyoung , Cotty Francis , Sottas Chantal , Jiang Hua , Michels Corinne A.
Published: March 2004

Molecular genetic analysis is used to characterize the AGT1 gene encoding an α‐glucoside transporter. AGT1 is found in many Saccharomyces cerevisiae laboratory strains and maps to a naturally occurring, partially functional allele of the MAL1 locus. Agt1p is a highly hydrophobic, postulated integral membrane protein. It is 57% identical to Mal61p, the maltose permease encoded at MAL6, and is also a member of the 12 transmembrane domain superfamily of sugar transporters. Like Mal61p, Agt1p is a high‐affinity, maltose/proton symporter, but Mal61p is capable of transporting only maltose and turanose, while Agt1p transports these two α‐glucosides as well as several others including isomaltose, α‐methylglucoside, maltotriose, palatinose, trehalose and melezitose. AGT1 expression is maltose inducible and induction is mediated by the Mal‐activator. The sequence of the upstream region of AGT1 is identical to that of the maltose‐inducible MAL61 gene over a 469 bp region containing the UASMAL but the 315 bp sequence immediately upstream of AGT1 shows no significant homology to the sequence immediately upstream of MAL61. The evolutionary origin of the MAL1 allele to which AGT1 maps and the relationship of AGT1 to other α‐glucoside fermentation genes is discussed.
Unusual regulatory mechanism for a Streptomyces multidrug resistance gene, ptr, involving three homologous protein‐binding sites overlapping the promoter region
Authors: Salah‐Bey Khadidja , Thompson Charles J.
Published: March 2004

A promoter controlling expression of the pristinamycin multidrug resistance gene (ptr), originally isolated from Streptomyces pristinaespiralis, is inducible by many toxic compounds in various Streptomyces species. Studies of ptr promoter control were carried out in the heterologous host, Streptomyces lividans. In S. lividans, a regulatory protein or a protein complex (Pip), identified by its ability to bind to the ptr promoter in gel‐retardation experiments, was induced by pristinamycin I (PI). In situ copper‐phenanthroline foot‐printing analysis identified three (A, B, and C) similar Pip‐binding sites having the sequence GTACA(C/G)CGTA(C/T). These sites overlapped with functionally important regions of the promoter: the ‘A’ site overlapped with the −35 hexamer, ‘B’ overlapped with the −10 hexamer and ‘C’ was located between the transcription start site and the Shine—Dalgarno sequence. A GT—AG dinucleotide mutation was introduced at positions 8–9 of the consensus sequence to generate seven variant promoters: three mutated in one of the three sites, three mutated in two sites, and one mutated in all three sites. Whereas these promoters had reduced antibiotic (PI)‐induced activity, their levels of expression in the absence of PI was higher. This suggested an unusual regulatory mechanism in which Pip could act either as an activator or repressor. Gel shift experiments revealed Pip or its homologues in many other Streptomyces species, suggesting that it is widely employed in the regulation of antibiotic resistance genes and perhaps secondary metabolism.
The recA gene of Lactococcus lactis: characterization and involvement in oxidative and thermal stress
Authors: Duwat Patrick , Ehrlich S. Dusko , Gruss Alexandra
Published: March 2004

The role of recA in Lactococcus lactis, a microaerophilic fermenting organism, was examined by constructing a recA‐disrupted strain. This single alteration had a surprisingly pleiotropic effect. In addition to its roles in homologous recombination and DNA repair, recA is also involved in responses to oxygen and heat stresses. We found that oxygen stress induced by aeration causes reductions in growth and stationary‐phase survival of the recA strain. Toxicity is a consequence of hydroxyl radical production via the Fenton Reaction and is alleviated by catalase or Ferrozine addition. These results suggest that oxygen radicals are not efficiently eliminated and accumulate in lactococcal cultures, and that RecA is needed to deal with the damage they incur. Unexpectedly, thermal stress arrested growth of the recA strain. Immunological data indicate that the recA mutant is deficient in heat‐shock proteins DnaK, GroEL, and GrpE. Poor growth at elevated temperature is therefore due to a diminished heat‐shock response in the recA strain. In contrast, levels of a novel heat‐shock protein, HflB, are elevated. In Escherichia coli, HflB downregulates the heat‐shock response by promoting degradation of the transcription factor σ32. We propose that recA regulates the heat‐shock response via HflB. This work provides the first evidence showing that two major pathways of stress response, induced by heat shock and DNA damage, are interactive.
Single amino acid substitutions in the N‐terminus of Vibrio cholerae TcpA affect colonization, autoagglutination, and serum resistance
Authors: Chiang Su L. , Taylor Ronald K. , Koomey Michael , Mekalanos John J.
Published: March 2004

The toxin‐coregulated pilus (TCP) of Vibrio cholerae O1 is required for successful infection of the host. TcpA, the structural subunit of TCP, belongs to the type IV family of pilins, which includes the PilE pilin of Neisseria gonorrhoeae. Recently, single amino acid changes in the N‐terminus of PilE were found to abolish autoagglutination in gonococci. As type IV pilins demonstrate some similarities in function and amino acid sequence, site‐directed mutagenesis and allelic exchange were used to create corresponding mutations in TcpA. All four mutant strains demonstrated autoagglutination defects, and all were highly defective for colonization in the infant mouse model. These results support the previously proposed correlation between autoagglutination and colonization. Finally, all four mutants are serum sensitive, indicating that TcpA plays a role in serum resistance, a phenotype previously attributed to TcpC. As the mutations have similar effects in N. gonorrhoeae and V. cholerae, our results support the idea that type IV pilins have similar functions in a variety of pathogenic bacteria.
Adhesion properties of mutants of Staphylococcus aureus defective in fibronectin‐binding proteins and studies on the expression of fnb genes
Authors: Greene C. , McDevitt D. , Francois P. , Vaudaux P.E. , Lew D.P. , Poster T.J.
Published: March 2004

Staphylococcus aureus 8325‐4 has the potential to express two distinct cell wall‐associated fibronectin‐binding proteins called FnBPA and FnBPB. In order to test if both proteins are expressed in S. aureus and if both are required for promoting bacterial adhesion to fibronectin‐coated surfaces, insertion mutations were isolated in each gene. A DNA fragment encoding tetracycline resistance was inserted into fnbA and a fragment encoding erythromycin resistance was inserted into fnbB. A double fnbA fnbB mutant was also constructed. The fnbA and fnbB single mutants showed no significant reduction in their adhesion to polymethylmethacrylate coverslips that had been coated in vitro with fibronectin. However, the double mutant was completely defective in adhesion. Monospecific antibodies directed against the non‐conserved N‐terminal regions of both proteins confirmed the lack of expression of FnBPs in the mutant strains. Wild‐type fnbA and fnbB genes cloned seperately on a multicopy plasmid were each able to restore fully the adhesion‐defective phenotype of the 8325‐4 fnbA fnbB mutant. This demonstrates that both fnb genes are expressed in S. aureus and that both contribute to the ability of strain 8325‐4 to adhere to fibronectin‐coated surfaces. The double mutant was also defective in adhesion to coverslips that had been removed from tissue cages implanted subcutaneously in guinea‐pigs, which suggests that fibronectin is important in promoting attachment of S. aureus to biomaterial in vivo.
Molecular genetics and transport analysis of the copper‐resistance determinant (pco) from Escherichia coli plasmid pRJ1004
Authors: Brown Nigel L. , Barrett Siobhan R. , Camakaris James , Lee Barry T.O. , Rouch Duncan A.
Published: March 2004

The copper‐resistance determinant (pco) of Escherichia coli plasmid pRJ1004 was cloned and sequenced. Tn1000 transposon mutagenesis identified four complementation groups, mutations in any of which eliminated copper resistance. DNA sequence analysis showed that the four complementation groups contained six open reading frames, designated pcoABCDRS. The protein product sequences derived from the nucleotide sequence show close homology between this copper‐resistance system and the cop system of a plasmid pPT23D of Pseudomonas syringae pv. tomato. The PcoR and PcoS protein sequences show homology to the family of two‐component sensor/responder phosphokinase regulatory systems. A seventh reading frame (pcoE) was identified from DNA sequence data, and lies downstream of a copper‐regulated promoter. Transport assays with 64Cu(II) showed that the resistant cells containing the plasmid had reduced copper accumulation during the log phase of growth, while increased accumulation had previously been observed during stationary phase. Chromosomal mutants defective in cellular copper management were obtained and characterized. In two of these mutants pco resistance was rendered totally inactive, whilst in another two mutants pco complemented the defective genes. These data indicate that plasmid‐borne copper resistance in E. coli is linked with chromosomal systems for copper management.
A cell‐surface polysaccharide that facilitates rapid population migration by differentiated swarm cells of Proteus mirabilis
Authors: Gygi Daniel , Rahman M. Mahbubur , Lai Hsin‐Chih , Carlson Russell , Guard‐Petter Jean , Hughes Colin
Published: March 2004

Swarming by Proteus mirabilis is characterized by cycles of rapid population migration across surfaces, following differentiation of typical vegetative rods into long, hyperflagellated, virulent swarm cells. A swarm‐defective TnphoA insertion mutant was isolated that was not defective in cell motility, differentiation or control of the migration cycle, but was specifically impaired in the ability to undergo surface translocation as a multicellular mass. The mutation, previously shown to compromise urinary tract virulence, was located within a 1112 bp gene that restored normal swarming of the mutant when expressed in trans. The gene encoded a 40.6 kDa protein that is related to putative sugar transferases required for lipopolysaccharide (LPS) core modification in Shigella and Salmonella. The immediately distal open reading frame encoded a protein that is related to dehydrogenases involved in the synthesis of LPS O‐side‐chains, enterobacterial common antigen and extracellular polysaccharide (PS). Gel electrophoresis and electron microscopy showed that the mutant still made LPS but it had lost the ability to assemble a surface (capsular) PS, which gas‐liquid chromatography and mass spectrometry indicated to be an acidic type II molecule rich in galacturonic acid and galactosamine. We suggest that this surface PS facilitates translocation of differentiated cell populations by reducing surface friction.
Lethality of rep recB and rep recC double mutants of Escherichia coli
Authors: Uzest Marilyne , Ehrlich S. Dusko , Michel Béédicte
Published: March 2004

A rep mutation in combination with a recB or a recC mutation renders Escherichia coli non‐viable. This conclusion is based on the following lines of evidence: (i) double mutants cannot be constructed by P1 transduction; (ii) induction of the λ Gam protein, which inactivates most of the RecBCD activities, is lethal in rep mutants; (iii) rep recBts recCts mutants are not viable at high temperature. The reasons for a requirement for the RecBCD enzyme in rep strains were investigated. Initiation of chromosome replication, elongation and chromosomal segregation do not seem impaired in the rep recBts recCts mutant at the non‐permissive temperature. The viability of other rep derivatives was tested. rep recA recD triple mutants are not viable, whereas rep recD and rep recA double mutants are. Inactivation of both exoV activity and recBC‐dependent homologous recombination is therefore responsible for the non‐viability of rep recBC strains. However, sbcA and sbcB mutations, which render recBC mutants recombination proficient, do not restore viability of rep recBC mutants, indicating that recombination via the RecF or the RecE pathways cannot functionally replace RecBCD‐mediated recombination. The specific requirement for RecBCD suggests the occurrence of double‐strand DNA breaks in rep strains. Additional arguments in favour of the presence of DNA lesions in rep mutants are as follows: (i) expression of SOS repair functions delays lethality of rep derivatives after inactivation of RecBCD; (ii) sensitivity of rep strains to ultraviolet light is increased by partial inactivation of RecBCD. A model for the recovery of cells from double‐strand breaks in rep mutants is discussed.
Four genes, two ends, and a res region are involved in transposition of Tn5053: a paradigm for a novel family of transposons carrying either a mer operon or an integron
Authors: Kholodii G.Ya. , Mindlin S.Z. , Bass I.A. , Yurieva O.V. , Minakhina S.V. , Nikiforov V.G.
Published: March 2004

The complete nucleotide sequence of an 8447 bp‐long mercury‐resistance transposon (Tn5053) has been determined. Tn5053 is composed of two modules: (i) the mercury‐resistance module and (ii) the transposition module. The mercury‐resistance module carries a mer operon, merRTPFAD, and appears to be a single‐ended relic of a transposon closely related to the classical mercury‐resistance transposons Tn21 and Tn501. The transposition module of Tn5053 is bounded by 25 bp terminal inverted repeats and contains four genes involved in transposition, i.e. tniA, tniB, tniQ, and tniR. Transposition of Tn5053 occurs via cointegrate formation mediated by the products of the tniABQ genes, followed by site‐specific cointegrate resolution. This is catalysed by the product of the tniR gene at the res region, which is located upstream of tniR. The same pathway of transposition is used by Tn402 (Tn5090) which carries the integron of R751. Transposition genes of Tn5053 and Tn402 are interchangeable. Sequence analysis suggests that Tn5053 and Tn402 are representatives of a new family of transposable elements, which fall into a recently recognized superfamily of transposons including retroviruses, insertion sequences of the IS3 family, and transposons Tn552 and Tn7. We suggest that the tni genes were involved in the dissemination of integrons.
Meningococcal pilin: a glycoprotein substituted with digalactosyl 2,4‐diacetamido‐2,4,6‐trideoxyhexose
Authors: Stimson Elaine , Virji Mumtaz , Makepeace Katherine , Dell Anne , Morris Howard R. , Payne Gail , Saunders Jon R. , Jennings Michael P. , Barker Stephanie , Panico Maria , Blench Ian , Moxon E. Richard
Published: March 2004

Neisseria meningitidis pili are filamentous protein structures that are essential adhesins in capsulate bacteria. Pili of adhesion variants of meningococcal strain C311 contain glycosyl residues on pilin (PilE), their major structural subunit. Despite the presence of three potential N‐linked glycosylation sites, none appears to be occupied in these pilins. Instead, a novel O‐linked trisaccharide substituent, not previously found as a constituent of glycoproteins, is present within a peptide spanning amino acid residues 45 to 73 of the PilE molecule. This structure contains a terminal 1‐4‐linked digalactose moiety covalently linked to a 2,4‐diacetamido‐2,4,6‐trideoxyhexose sugar which is directly attached to pilin. Pilins derived from galactose epimerase (galE) mutants lack the digalactosyl moiety, but retain the diacetamidotrideoxyhexose substitution. Both parental (#3) pilins and those derived from a hyper‐adherent variant (#16) contained identical sugar substitutions in this region of pilin, and galE mutants of #3 were similar to the parental phenotype in their adherence to host cells. These studies have confirmed our previous observations that meningococcal pili are glycosylated and provided the first structural evidence for the presence of covalently linked carbohydrate on pili. In addition, they have revealed a completely novel protein/saccharide linkage.
Regulation of insecticidal crystal protein production in Bacillus thuringiensis
Authors: Baum James A. , Malvar Thomas
Published: March 2004

The production of insecticidal crystal proteins (ICPs) in Bacillus thuringiensis normally coincides with sporulation, resulting in the appearance of parasporal crystalline inclusions within the mother cell. In most instances, the temporal and spatial regulation of ICP gene expression is determined at the transcriptional level by mother‐cell‐specific sigma factors that share homology with σE and σK from Bacillus subtilis. The crylll ICP genes are a notable exception; these genes are transcribed from σA‐like promoters during vegetative growth, are induced or derepressed at the onset of stationary phase, and are overexpressed in sporulation mutants of B. thuringiensis blocked in the phosphorylation of Spo0A, a key regulator of sporulation initiation. Transcription alone, however, cannot account for the impressive ability of this bacterium to accumulate insecticidal proteins. A variety of post‐transcriptional and post‐translational mechanisms also contribute to the efficient production of ICPs in B. thuringiensis, thus making this bacterium a cost‐effective biological control agent.
Genetic organization, nucleotide sequence and regulation of expression of genes encoding phenol hydroxylase and catechol 1,2‐dioxygenase in Acinetobacter calcoaceticus NCIB8250
Authors: Ehrt Sabine , Schirmer Falck , Hillen Wolfgang
Published: March 2004

We have mutated Acinetobacter calcoaceticus NCIB8250 to growth deficiency on phenol as sole carbon source and isolated genes with similarity to phenol hydroxylase and catechol 1,2‐dioxygenase by complementation. Sequence analysis reveals the presence of six open reading frames (ORFs) with similarities to a Pseudomonas multicomponent phenol hydroxylase which are followed by an ORF with similarity to catA from A. calcoaceticus ADP1. Transformation of these genes to ADP1 confers the ability to grow at the expense of phenol as sole carbon source. Primer extension analysis indicates phenol‐inducible transcription from an RpoN‐dependent promoter sharing sequence similarity with the σ54 consensus promoter sequence, except that the −12 box is GG instead of GC. A catA::lacZ transcriptional fusion shows the same induction profile for β‐galactosidase expression as transcription from the σ54‐dependent promoter. This result suggests that catA is cotranscribed in the same operon with the phenol hydroxylase‐encoding genes and is consistent with the fact that no apparent additional promoter is found for catA by sequence analysis or primer extension. Catechol 1,2‐dioxygenase activity is induced in NCIB8250 by benzoate, whereas β‐galactosidase expression from the catA::lacZ fusion is not. This observation leads to the hypothesis that two differentially regulated catA genes should be present in that strain.
Identification of the minimal replicon of plasmid pMEA300 of the methylotrophic actinomycete Amycolatopsis methanolica
Authors: Vrijbloed J.W. , Jelínková M. , Hessels G.I. , Dijkhuizen L.
Published: March 2004

The actinomycete Amycolatopsis methanolica contains a 13.3 kb plasmid (pMEA300), capable of enhancing the spontaneous mutation frequency of its host. Depending on the growth medium pMEA300 is not only maintained as an integrated element but can additionally be present as a multicopy, autonomously replicating plasmid. The minimal replicon of pMEA300 was identified. Two unlinked DNA fragments of 2.6 kb and 0.8 kb were required for pMEA300 maintenance. Sequence analysis of the 2.6 kb fragment revealed at least two open reading frames, orfA and orfB, encoding putative proteins of 170 amino acids (18 373 Da) and 416 amino acids (45 260 Da), respectively. No clear similarities were found between the deduced amino acid sequences of the putative orfA and orfB products of pMEA300 and replication proteins identified for various Streptomyces plasmids. The pMEA300 proteins of A. methanolica thus may represent unfamiliar types. The 0.8 kb fragment contained a single complete open reading frame (korA), encoding a protein of 118 amino acids (12 917 Da). The putative KorA protein of pMEA300 shows sequence similarity with various other Streptomyces plasmid‐encoded Kor proteins which may belong to the GntR family of transcriptional repressor proteins. The data provide preliminary evidence for the possible involvement of a kil—kor system in autonomous replication of pMEA300.
Differential expression of two siderophore‐dependent iron‐acquisition pathways in Erwinia chrysanthemi 3937: characterization of a novel ferrisiderophore permease of the ABC transporter family
Authors: Mahé Bruno , Masclaux Céline , Rauscher Lise , Enard Corine , Expert Dominique
Published: March 2004

In planta expression of a high‐affinity iron‐uptake system involving the siderophore chrysobactin in Erwinia chrysanthemi 3937 contributes greatly to invasive growth of this pathogen on its natural host, African violets. A previous study reported that global regulation by iron in this strain was mediated at the transcriptional level via the cbr locus which, when inactivated by insertional mutation, prevents the chrysobactin system from being tightly repressed by FeCl3‐ Herein, we report the nucleotide sequence of this locus and the functional analysis of its encoded products. Sequence analysis of a 4.8 kb genomic segment of a plasmid encompassing the cbr locus and characterization of the cognate translated products made it possible to uncover a system exhibiting similarity with prokaryotic transporters implicated in the transport of iron complexes. Accordingly, the CbrA product was shown to be the periplasmic component of a permease complex also including two integral membrane proteins, CbrB and CbrC, and the ATP‐binding unit CbrD. This system allowed internalization of Fe(III) when supplied to bacterial cells as 59FeCl3 or 59Fe dicitrate, via complexation to a second siderophore recently detected in strain 3937. Most notably, we demonstrate that this second siderophore‐mediated iron‐acquisition system is operational in bacterial cells grown in the presence of FeCl3. The regulatory effect of cbr was further assessed on a lacZ chrysobactin operon fusion indicating that the transcriptional control exerted by cbr on expression of the chrysobactin system is of homeostatic nature. In conclusion, E. chrysanthemi provides an interesting model in which iron acquisition involves an inductive process resulting in differential expression of two siderophore‐mediated pathways in relation to external iron accessibility.
Insertion of inverted Ter sites into the terminus region of the Escherichia coli chromosome delays completion of DNA replication and disrupts the cell cycle
Authors: Sharma Bela , Hill Thomas M.
Published: March 2004

To investigate the co‐ordination between DNA replication and cell division, we have disrupted the DNA replication cycle of Escherichia coli by inserting inverted Ter sites into the terminus region to delay completion of the chromosome. The inverted Ter sites (designated InvTer::spcr) were initially inserted into the chromosome of a Δtus strain to allow unrestrained chromosomal replication. We then introduced a functional tus gene by transforming the InvTer::spcr strain with a plasmid carrying the tus gene under control of an arabinose‐inducible promoter. In the presence of 0.2% arabinose, the cells formed long filaments, suggesting that activation of the inverted Ter sites by Tus arrested DNA replication and delayed the onset of cell division. Induction of sfiA, a gene in the SOS regulon, was observed following arrest of DNA replication; however, when a sfiB114 allele was introduced into InvTer::spcr strain, long filaments were still formed, suggesting that the sfi‐independent pathway also caused filamentation. Either recA::camr or lexA3 alleles suppressed filamentation when introduced in the InvTer strain. Interestingly, in both the recA::camr and lexA3 mutants, virtually all cells had a nucleoid, suggesting that cell division was proceeding even though DNA replication was not complete. These results suggest that DNA replication and cell division are uncoupled when recA is inactivated or when genes repressed by LexA cannot be induced.
Expression of flagella and motility by Shigella
Authors: Girón Jorge A.
Published: March 2004

Since the discovery of Shigella as the aetiologic agent of acute dysentery almost 100 years ago, this organism has been described as a non‐motile and non‐flagellated organism that invades the human colonic mucosa. In this study, the production of flagella by prototypic strains of all four Shigella species and, moreover, by fresh clinical isolates was demonstrated by electron microscopy. The flagellum of Shigella (flash) is ∼10 µm long and 12–14 nm in diameter and is typically seen emanating from one pole of the bacterium. Flash is composed of a putative structural polypeptide subunit of 33–38 kDa that shares immunological similarities with Escherichia coli, Salmonella spp., and Proteus mirabilis flagellins, and with the recently described recombinant Shigella flagellins (FliCSS and FliCSF) expressed in E. coli K‐12. A fliCSS ‐specific oligo probe hybridized with all four Shigella species, while a fliCSF probe hybridized with all Shigella flexneri and Shigella dysenteriae strains, but not with all Shigella sonnei or Shigella boydii strains, indicating genetic divergence among their flagellin genes. Shigella exhibits motility in low‐concentration motility agar under physiological growth conditions. The expression of flash and motility appears to be strictly regulated by unidentified genetic and environmental factors. These heretofore undescribed features may allow the bacteria to circumvent the natural intestinal mucosal defences leading to bacterial colonization and disease. The motility of shigellae may represent an evolutionary adaptation important for bacterial survival.
NPI1, an essential yeast gene involved in induced degradation of Gap1 and Fur4 permeases, encodes the Rsp5 ubiquitin—protein ligase
Authors: Hein Claudine , Springael Jean‐Yves , Volland Christiane , Haguenauer‐Tsapis Rosine , André Bruno
Published: March 2004

When yeast cells growing on a poor nitrogen source are supplied with NH4+ ions, several nitrogen permeases including the general amino acid permease (Gap1p) are rapidly and completely inactivated. This report shows that inactivation by NH4+ of the Gap1 permease is accompanied by its degradation. A functional NPI1 gene product is required for both inactivation and degradation of Gap1p. Molecular analysis of the NPI1 gene showed that it is identical to RSP5. The RSP5 product is a ubiquitin—protein ligase (E3 enzyme) whose physiological function was, however, unknown. Its C‐terminal region is very similar to that of other members of the E6‐AP‐like family of ubiquitin‐protein ligases. Its N‐terminal region contains a single C2 domain that may be a Ca2+‐dependent phospholipid interaction motif, followed by several copies of a recently identified domain called WW(P). The Npi1/Rsp5 protein has a homologue both in humans and in mice, the latter being involved in brain development. Stress‐induced degradation of the uracil permease (Fur4p), a process in which ubiquitin is probably involved, was also found to require a functional NPI1/RSP5 product. Chromosomal deletion of NPI1/RSP5 showed that this gene is essential for cell viability. In the viable np1/rsp5 strain, expression of NPI1/RSP5 is reduced as a result of insertion of a Ty1 element in its 5′ region. Our results show that the Npi1/Rsp5 ubiquitin‐protein ligase participates in induced degradation of at least two permeases, Gap1p and Fur4p, and probably also other proteins.
Tissue‐specific glycogen branching isoenzymes in a multicellular prokaryote, Streptomyces coelicolor A3(2)
Authors: Bruton Celia J. , Plaskitt Kitty A. , Chater Keith F.
Published: March 2004

In the overtly differentiated colonies of Streptomyces coelicolor A3(2), discrete phases of glycogen synthesis are found at the vegetative/aerial mycelium boundary (phase I) and in the immature spore chains at aerial hyphal tips (phase II). We have characterized two S. coelicolor glgB genes encoding glycogen branching enzyme, which are well separated in the genome. Disruption of glgBI led to the formation of abnormal polyglucan deposits at phase I, with phase II remaining normal, whereas disruption of glgBII interfered specifically with phase II deposits, and not with those of phase I. Thus, each branching enzyme isoform is involved in a different phase of glycogen synthesis. This situation contrasts with that in simple bacteria, which typically have a single set of enzymes for glycogen metabolism, and more closely resembles that in plants.
Coupling of Escherichia coli hns mRNA levels to DNA synthesis by autoregulation: implications for growth phase control
Authors: Free Andrew , Dorman Charles J.
Published: March 2004

The H‐NS protein of enteric bacteria is one of the major proteins of the bacterial nucleoid and seems to play an important role in nucleoid structure. Transcription of the hns gene encoding the H‐NS protein appears to be negatively regulated by H‐NS itself both in vitro and in vivo. We have examined the role of this mode of regulation in wild‐type cells in vivo. We find that hns transcription is down‐regulated when DNA synthesis is blocked in growing cells, in a manner that is dependent upon continuing H‐NS protein synthesis. These data suggest that hns autoregulation serves to match de novo H‐NS synthesis to the demands of DNA synthesis and may maintain a relatively constant H‐NS:DNA ratio. It has previously been suggested that hns transcription is activated as cells enter stationary phase, which would require a complete relaxation of autoregulatory control given that DNA synthesis decreases at this time. However, we show here that levels of hns mRNA in fact decline at the onset of stationary phase in a manner fully consistent with the autoregulation model. We also fail to detect any significant accumulation of the H‐NS protein in stationary phase.
Identification of a methyl‐accepting chemotaxis protein in Rhodobacter sphaeroides
Authors: Ward Mandy J. , Harrison David M. , Ebner Martin J. , Armitage Judith P.
Published: March 2004

Analysis of the DNA sequence directly upstream of the chemotaxis operon of Rhodobacter sphaeroides identified a single gene whose product has strong similarity to the methyl‐accepting chemotaxis proteins (MCPs) found in enteric bacteria. The deduced protein had a highly conserved signalling sequence and only one very hydrophobic region at the N‐terminus, in contrast to enteric MCPs. A possible cytoplasmic location of the majority of the protein was supported by Western blotting. The mcpA gene was insertionally inactivated and the resulting phenotype examined using swarm plate assays. The mutant lacking McpA lost chemotaxis to a wide range of attractant stimuli but only under aerobic conditions; it retained almost normal chemotaxis under anaerobic/photosynthetic conditions. The identification of a sensory protein which is active only under one set of growth conditions suggests that R. sphaeroides probably has several MCPs, which co‐ordinately respond to changes in environmental conditions. Southern hybridization at relaxed stringency to the conserved sequence of the R. sphaeroides and Caulobacter crescentus mcp genes identified three possible additional mcp genes.
Cation‐selectivity of the l‐glutamate transporters of Escherichia coli, Bacillus stearothermophilus and Bacillus caldotenax: dependence on the environment in which the proteins are expressed
Authors: Tolner Berend , Ubbink‐Kok Trees , Poolmann Bert , Konings Wil N.
Published: March 2004

l‐Glutamate transport by the H+‐glutamate and Na+‐glutamate symport proteins of Escherichia coli K‐12 (GltPEc and GltSEc, respectively) and the Na+‐H+‐glutamate symport proteins of Bacillus stearothermophilus (GltTBs) and Bacillus caldotenax (GltTBc) was studied in membrane vesicles derived from cells in which the proteins were either homologously or heterologously expressed. Substrate and inhibitor specificity studies indicate that GltPEc, GltTBs and GltTBc fall into the same group of transporters, whereas GltSEc is distinctly different from the others. Also, the cation specificity of GltSEc is different; GltSEc transported l‐glutamate with (at least) two Na+, whereas GltPEc, GltTBs and GltTBc catalysed an electrogenic symport of l‐glutamate with ≥two H+, i.e. when the proteins were expressed in E. coli Surprisingly studies in membrane vesicles of B. stearothermophilus and B. caldotenax indicated a Na+‐H+‐l‐glutamate symport for both GltTBs and GltTBc. The Na+ dependency of the GltT transporters in the Bacillus strains increased with temperature. These observations suggest that the conformation of the transport proteins in the E. coli and the Bacillus membranes differs, which influences the coupling ion selectivity.
Cell‐surface‐bound Yersinia translocate the protein tyrosine phosphatase YopH by a polarized mechanism into the target cell
Authors: Persson Cathrine , Nordfelth Roland , Holmström Anna , H»kansson Sebastian , Rosqvist Roland , Wolf‐Watz Hans
Published: March 2004

YopH is translocated by cell‐surface‐bound bacteria through the plasma membrane to the cytosol of the HeLa cell. The transfer mechanism is contact dependent and polarizes the translocation to only occur at the contact zone between the bacterium and the target cell. More than 99% of the PTPase activity is associated with the HeLa cells. In contrast to the wild‐type strain, the yopBD mutant cannot deliver YopH to the cytosol. Instead YopH is deposited in localized areas in the proximity of cell‐associated bacteria. A yopN mutant secretes 40% of the total amount of YopH to the culture medium, suggesting a critical role of YopN in regulation of the polarized translocation. Evidence for a region in YopH important for its translocation through the plasma membrane of the target cell but not for secretion from the pathogen is provided.
The regulator of streptomycin gene expression, StrR, of Streptomyces griseus is a DNA binding activator protein with multiple recognition sites
Authors: Retzlaff Lutz , Distler Jürgen
Published: March 2004

In Streptomyces griseus the expression of at least one streptomycin biosynthetic gene, strB1, is dependent on the pathway‐specific activator protein StrR. We show here that StrR is a DNA‐binding protein which specifically interacts with the strB1 promoter fragment. Footprinting experiments demonstrate that the StrR protein binds to an inverted repeat located upstream of the strB1 promoter. Further StrR‐binding sites having the consensus sequence GTTCGActG(N)11CagTcGAAc were identified in the str—sts gene clusters of S. griseus and Streptomyces glaucescens by sequence comparison, gel retardation, and footprinting studies. The genetic and biochemical evidence strongly supports the model of the StrR protein activating the expression of streptomycin biosynthetic genes by interacting with multiple binding sites within the str—sts gene clusters of S. griseus and S. glaucescens.
Transcriptional regulation of ferric citrate transport in Escherichia coli K‐12. Fecl belongs to a new subfamily of σ70‐type factors that respond to extracytoplasmic stimuli
Authors: Angerer Annemarie , Enz Sabine , Ochs Martina , Braun Volkmar
Published: March 2004

Transcription of the ferric citrate transport system of Escherichia coli K‐12 is repressed by Fe2+‐Fur and activated by ferric citrate. Ferric citrate does not have to enter the cytoplasm; it initiates a signal transduction mechanism by binding to the outer membrane receptor FecA. Presumably, a conformational change is transmitted in a TonB‐dependent manner to the FecR protein. FecR activates Fecl, and Fecl activates transcription of the fecABCDE transport genes. In this communication, Fecl was isolated after cloning fecl downstream of an ideal ribosome‐binding site. Overexpressed Fecl formed inclusion bodies which were solubilized and purified in active form using a mild detergent. Fecl, in conjunction with RNA polymerase core enzyme, directed transcription from the fecA promoter in an in vitro run‐off transcription assay. Furthermore, Fecl retarded the electrophoretic mobility of a specific 75 bp DNA fragment located upstream of fecA. An in vivo competition experiment between the fecA promoters of wild‐type and mutant strains identified the nucleotide positions 2747, 2749, 2751 and 2753, located within the 75 bp fragment, as important for Fecl‐induced transcription. Mobility band shift of fecA promoter DNA caused by cell lysates required growth of cells in the presence of ferric citrate and expression of FecA, Fecl and FecR. These data support the previous assignment of Fecl, based on sequence homologies, to a new subfamily of eubacterial RNA polymerase σ70 factors that respond to extra‐cytoplasmic stimuli and regulate extracytoplasmic functions.
Regulatory characteristics and promoter analysis of csiE, a stationary phase‐inducible gene under the control of σS and the cAMP—CRP complex in Escherichia coli
Authors: Marschall Christoph , Hengge‐Aronis Regine
Published: March 2004

Sigma‐S and the cAMP—CRP complex are global regulatory factors involved in stationary‐phase induction of large groups of genes in Escherichia coli. csiE, a gene located at 57.25 min (co‐ordinate 2674) of the physical map of the E. coli chromosome, is under the control of both of these factors. Sigma‐S plays a positive, though not absolutely essential, role in the expression of csiE. Regulation by cAMP—CRP has both positive and negative elements, with the latter being dependent on the presence of σS, whose expression is negatively influenced by cAMP—CRP. csiE has a single transcriptional start site located 33 bp upstream of the initiation codon. By a 5′‐deletion approach, we show that 72 bp upstream of the csiE transcriptional start site are sufficient for regulation by σS and cAMP—CRP. A deletion upstream of nucleotide −38 with respect to the start site eliminates positive cAMP—CRP control and makes the remaining expression fully dependent on σS. Our results indicate that transcription at the csiE promoter can be initiated in vivo by σS‐containing RNA polymerase alone as well as by σ70‐containing RNA polymerase in conjunction with cAMP—CRP or a cAMP—CRP‐dependent secondary regulator. The promoter region of poxB, the structural gene for pyruvate oxidase, which is also under the control of σS and cAMP—CRP, is very similar to the corresponding region of csiE, suggesting a similar regulatory mechanism also for poxB.
Molecular handles on adaptive mutation
Authors: Rosenberg Susan M. , Harris Reuben S. , Torkelson Joel
Published: March 2004

In one experimental system, several handles on the molecular mechanism of apparent adaptive mutation have emerged. The system is reversion of a lac frame‐shift mutation in Escherichia coli. The molecular handles include a requirement for homologous recombination; the implication of DNA double‐strand breaks as a molecular intermediate; a unique sequence spectrum of −1 deletions in mononucleotide repeats which implies polymerase errors, and also implies a failure of post‐synthesis mismatch repair on those errors; and the involvement of sexual functions at some stage of the process. These molecular handles are revealing an unexpected new mechanism of mutagenesis.
Transcription antitermination: the λ paradigm updated
Authors: Friedman David I. , Court Donald L.
Published: March 2004

Coliphage λ employs systems of transcription termination and antitermination to regulate gene expression. Early gene expression is regulated by the phage‐encoded N protein working with a series of Escherichia coli proteins, Nus, at RNA sites, NUT, to modify RNA polymerase to a termination‐resistant form. Expression of λ late genes is regulated by the phage‐encoded Q antitermination protein. Q, which appears to use only one host factor, acts at a DNA site, qut, to modify RNA polymerase to a termination‐resistant form. This review focuses on recent studies which show that: (i) N can mediate antitermination in vitro, independent of Nus proteins, (ii) Early genes in another lambdoid phage HK022 are also regulated by antitermination, where only an RNA signal appears necessary and sufficient to create a termination‐resistant RNA polymerase. (iii) A part of the qut signal appears to be read from the non‐template DNA strand. (iv) A host‐encoded inhibitor of N antitermination appears to act through the NUT site as well as with the α subunit of RNA polymerase, and is antagonized by NusB protein.