bluuebunny/crossref_metadata_2025 huggingface dataset:
FruA, a putative transcription factor essential for the development of Myxococcus xanthus
Authors: Ogawa Mari , Fujitani Shoji , Mao Xiaohua , Inouye Sumiko , Komano Teruya
Published: November 2003

A new developmental gene, fruA, of Myxococcus xanthus was cloned using a one‐step cloning vector, TnV. DNA sequencing of the wild‐type allele of the fruA gene indicated that the fruA gene encodes a protein of 229 amino acid residues with a calculated molecular weight of 24 672. The deduced amino acid sequence of FruA protein showed similarity to those of many bacterial regulatory proteins carrying a DNA‐binding helix‐turn‐helix motif. The transcription‐initiation site of the fruA gene was determined by a primer‐extension experiment. Development of M. xanthus cells with a disrupted fruA gene stopped at the stage of mound formation. Although cells were able to aggregate to form mounds, myxospores were not formed. By Northern and Western blot analysis, it was found that the fruA expression was not detected during vegetative growth but initiated at around 6 h and reached the highest level at 12 h after the onset of development. Expression of the fruA gene was dependent on the expression of asg, bsg, csg, dsg, and esg genes, indicating that a series of intercellular signalling is necessary for the expression of the fruA gene. The effects of the fruA mutation on ‐galactosidase expression of various developmentally regulated genes fused with the lacZ gene were analysed; three developmental lacZ fusions (Ω4469, Ω4273 and Ω4500) were either poorly induced or not induced at all, while three other lacZ fusions (Ω4408, Ω4521 and Ω4455) expressed at the early stage of development were normally induced but were unable to be repressed at a later stage of development as in the wild‐type strain. Interestingly, in the fruA mutant, tps (the gene for protein S) was not activated. From these results together with analysis of the amino acid sequence of FruA, we propose that FruA is a putative transcription factor required for the development of M. xanthus.
Structural relationship between a bacterial developmental protein and eukaryotic PP2C protein phosphatases
Authors: Adler Elliot , Donella‐Deana Arianna , Arigoni Fabrizio , Pinna Lorenzo A. , Stragier Patrick
Published: November 2003

Bacillus subtilis SpoIIE is a Ser protein phosphatase whose action on the phosphoprotein SpoIIAA triggers the cell type‐specific activation of a sporulation transcription factor. Here we report that SpoIIE displays sequence similarity to the PP2C family of eukaryotic Ser/Thr protein phosphatases, and that residues common to these proteins are required for the function of both SpoIIE and TPD1, a yeast PP2C. These findings suggest that SpoIIE and the PP2C protein phosphatases are structurally related, and reveal a striking formal similarity between the SpoIIAA regulatory circuit and that of mammalian mitochondrial pyruvate dehydrogenase. This similarity may reflect an evolutionarily conserved mechanism of biological regulation based on the interplay of His protein kinase‐like Ser kinases and PP2C‐like protein phosphatases.
Stationary‐phase regulation of the Saccharomyces cerevisiaeSOD2 gene is dependent on additive effects of HAP2/3/4/5‐ and STRE‐binding elements
Authors: Flattery‐O′Brien Jacinta A. , Grant Chris M. , Dawes Ian W.
Published: November 2003

SOD2, encoding manganese superoxide dismutase (MnSOD), is essential for stationary‐phase survival of yeast cells. In addition, stationary‐phase cells are more resistant to oxidative stress than exponential‐phase cells. The use of a SOD2::lacZ fusion construct in this study shows that transcription of SOD2 increases 6.5‐fold as cells enter stationary phase in rich, glucose medium. The increase in SOD2 expression appears to be due to two phenomena — the switch to a non‐fermentable carbon source and nutrient limitation. Analysis of SOD2 transcription in mutant Saccharomyces cerevisiae strains showed that the gene was negatively regulated by intracellular cAMP levels which decrease as cells enter stationary phase. Mutation of ‘stress‐responsive’ (STRE) elements in the SOD2 promoter which respond to cAMP levels resulted in the loss of cAMP‐dependent expression but only partially reduced the increase in expression as cells entered stationary phase. A putative Yap1p‐binding site was found to be inactive and mutation of YAP1 had no effect on the STRE‐mediated expression. To fully eliminate the stationary‐phase response, it was necessary to mutate a HAP2/3/4/5 complex binding site in addition to the STRE elements. It is postulated that the effects of the STRE sites and the HAP2/3/4/5 complex binding site are additive.
A branch in the ToxR regulatory cascade of Vibrio cholerae revealed by characterization of toxT mutant strains
Authors: Champion Gretchen A. , Neely Melody N. , Brennan Molly A. , DiRita Victor J.
Published: November 2003

Co‐ordinate expression of genes associated with pathogenicity in Vibrio cholerae requires two transcription activators, ToxR and ToxT. Work carried out to date suggests that ToxR activates transcription of the toxT gene and that ToxT directly activates transcription of several genes whose products play a role in colonization or CT production by V. cholerae. Previous work also suggests that ToxR can directly activate transcription of the CT operon (ctxAB) independently of ToxT, thereby implying a degree of complexity in control of the ctxAB operon not found with other genes of the ToxR regulon. We tested the regulatory cascade model of virulence gene expression by constructing strains of classical and El Tor V. cholerae deleted for the coding sequence for the putative DNA‐binding domain of toxT. Phenotypic analysis of these strains suggests that V. cholerae has ToxT‐dependent and ToxT‐independent branches of its virulence regulon. The results also raise questions about the precise role for ToxR in activation of ctxAB transcription.
Integration host factor stimulates both FimB‐ and FimE‐mediated site‐specific DNA inversion that controls phase variation of type 1 fimbriae expression in Escherichia coli
Authors: Blomfield Ian C. , Kulasekara Don H. , Eisenstein Barry I.
Published: November 2003

The site‐specific DNA inversion that controls phase variation of type 1 fimbriation in E. coli is catalysed by two recombinases, FimB and FimE. Efficient inversion by either recombinase also requires the leucine‐responsive regulatory protein (Lrp). In addition, FimB recombination is stimulated by the integration host factor (IHF). The effect of IHF on FimE inversion has not previously been reported. Here it is shown that IHF stimulates FimE recombination; in strain MG1655, mutants containing lesions in either the α (ihfA) or β (ihfB) subunits of IHF show a marked decrease in both FimB‐ (100‐fold) and FimE (15 000‐fold)‐promoted switching. IHF is shown to bind with high affinity to sites both adjacent to (site I) and within (site II) the fim invertible element. Furthermore, mutations in site I or site II that lower the affinity of IHF binding in vitro were found to lower the frequency of FimE and/or FimB recombination in vivo. Although site I and site II mutations in combination have an effect on FimB‐promoted switching comparable to that of IHF knockout mutations (100‐fold), the cis site mutations have a much less marked effect (100‐fold) on FimE‐promoted switching.
Promoter‐independent cold‐shock induction of cspA and its derepression at 37°C by mRNA stabilization
Authors: Fang Li , Jiang Weining , Bae Weonhye , Inouye Masayori
Published: November 2003

The gene for CspA, the major cold‐shock protein of Escherichia coli is known to be dramatically induced upon temperature downshift. Here, we report that three‐base substitutions around the Shine–Dalgarno sequence in the 159‐base 5′‐untranslated region of the cspA mRNA stabilizes the mRNA 150‐fold, resulting in constitutive expression of cspA at 37°C. This stabilization was found to be at least partially due to resistance against RNase E degradation. The cold‐shock induction of cspA was also achieved by exchanging its promoter with the non‐cold‐shock lpp promoter. The results presented indicate that the cspA gene is efficiently transcribed even at 37°C. However, the translation of the cspA mRNA is blocked because of its extreme instability at 37°C. The presented results also demonstrate that the cspA gene is constitutively transcribed at all temperatures; however, its expression at 37°C is prevented by destabilizing its mRNA.
Regulation of O‐antigen chain length is required for Shigella flexneri virulence
Authors: Van Den Bosch Luisa , Manning Paul A. , Morona Renato
Published: November 2003

It is shown that Shigella flexneri maintains genetic control over the modal chain length of the O‐antigen polysaccharide chains of its lipopolysaccharide (LPS) molecules because such a distribution is required for virulence. The effect of altering O‐antigen chain length on S. flexneri virulence was investigated by inserting a kanamycin (Km)‐resistance cassette into the rol gene (controlling the modal O‐antigen chain length distribution), and into the rfbD gene, whose product is needed for synthesis of dTDP‐rhamnose (the precursor of rhamnose in the O‐antigen). The mutations had the expected effect on LPS structure. The rol::Km mutation was impaired in the ability to elicit keratoconjunctivitis, as determined by the Serény test. The rol::Km and rfbD::Km mutations prevented plaque formation on HeLa cells, but neither mutation affected the ability of S. flexneri to invade and replicate in HeLa cells. Microscopy of bacteria‐infected HeLa cells stained with fluorescein isothiocyanate (FITC)‐phalloidin demonstrated that both the rol::Km and rfbD::Km mutants were defective in F‐actin tail formation: the latter mutant showed distorted F‐actin tails. Plasma‐membrane protrusions were occasionally observed. Investigation of the location of IcsA (required for F‐actin tail formation) on the cell surface by immunofluorescence and immunogold electron microscopy showed that while most rol mutant bacteria produced little or no cell‐surface IcsA, 10% resembled the parental bacterial cell (which had IcsA at one cell pole; the rfbD mutant had IcsA located over its entire cell surface although it was more concentrated at one end of the cell). That the O‐antigen chains of the rol::Km mutant did not mask the IcsA protein was demonstrated by using the endorhamnosidase activity of Sf6c phage to digest the O‐antigen chains, and comparing untreated and Sf6c‐treated cells by immunofluorescence with anti‐IcsA serum.
Bacteriophage T12 of Streptococcus pyogenes integrates into the gene encoding a serine tRNA
Authors: McShan W. Michael , Tang Y.‐F. , Ferretti Joseph J.
Published: November 2003

The region of temperate bacteriophage T12 responsible for integration into the chromosome of Streptococcus pyogenes has been identified. The integrase gene (int) and the phage attachment site (attP) are found immediately upstream of the gene for speA, the latter of which is known to be responsible for the production of erythrogenic toxin A (also known as pyrogenic exotoxin A). The integrase gene has a coding capacity for a protein of 41 457 Da, and the C‐terminus of the deduced protein is similar to other conserved C‐terminal regions typical of phage integrases. Upstream of int is a second open reading frame, which is capable of encoding an acidic protein of 72 amino acids (8744 Da); the position of this region in relation to int suggests it to be the phage excisionase gene (xis). The arms flanking the integrated prophage (attL and attR) were identified, allowing determination of the sequences of the phage (attP) and bacterial (attB) attachment sites. A fragment containing the integrase gene and attP was cloned into a streptococcal suicide vector; when introduced into S. pyogenes by electrotransformation, this plasmid stably integrated into the bacterial chromosome at attB. The insertion site for the phage into the S. pyogenes chromosome was found to be in the anticodon loop of a putative type II gene for a serine tRNA. attP and attB share a region of identity that is 96 bp in length; this region of identity corresponds to the 3′ end of the tRNA gene such that the coding sequence remains intact after integration of the prophage. The symmetry of the core region of att may set this region apart from previously described phage attachment sites (Campbell, 1992), and may play a role in the biology of this medically important bacteriophage.
Repetitive sequences found in the chromosome of the myxobacterium Nannocystisexedens are similar to msDNA: a possible retrotransposition event in bacteria
Authors: Lampson Bert C. , Rice Scott A.
Published: November 2003

The first reverse transcriptase (RT) to be found in a prokaryotic cell is encoded by an element called a retron which resides in the chromosome of many different bacteria. In addition, all retrons code for a functionally obscure RNA–DNA satellite molecule called msDNA. msDNA is synthesized from an RNA template by the retron‐encoded RT. An unusual retron element is described here from the myxobacterium Nannocystis exedens. This retron does not appear to have a typical RT gene in close proximity (1 kb) to the gene msd (which encodes the DNA strand of msDNA). The gene msr (which encodes the RNA strand of msDNA) appears to be duplicated and flanks both sides of the msd gene. Also discovered throughout the chromosome of this bacterium is a set of repeated sequences related to msDNA. These repeat sequences match only part of the sequence of msDNA and may have become incorporated into the chromosome of this bacterium by reverse transcription.
Interspecies recombination, and phylogenetic distortions, within the glutamine synthetase and shikimate dehydrogenase genes of Neisseria meningitidis and commensal Neisseria species
Authors: Zhou Jiaji , Bowler Lucas D. , Spratt Brian G.
Published: November 2003

Visual inspection showed clear evidence of a history of intraspecies recombinational exchanges within the neighbouring meningococcal shikimate dehydrogenase (aroE ) and glutamine synthetase (glnA) genes, which was supported by the non‐congruence of the trees constructed from the sequences of these genes from different meningococcal strains, and by statistical tests for mosaic structure. Many examples were also found of highly localized interspecies recombinational exchanges between the meningococcal aroE and glnA genes and those of commensal Neisseria species. These exchanges appear to have inflated the sequence variation at these loci, and have resulted in major distortions of the phylogenetic trees constructed from the sequences of the aroE and glnA genes of human pathogenic and commensal Neisseria species. Statistical tests for sequence mosaicism, and for anomalies within the Neisseria species trees, strongly supported the view that frequent interspecies recombination has occurred within aroE and glnA. The high levels of sequence variation, and intra‐ and interspecies recombination, within aroE and glnA did not appear to be due to a ‘hitch‐hiking’ effect caused by positive selection for variation at a neighbouring gene. Our results suggest that interspecies recombinational exchanges with commensal Neisseria occur frequently in some meningococcal ‘housekeeping’ genes as they can be observed readily even when there appears to be no obvious selection for the recombinant phenotypes.
Bidirectional regulation of two DNA‐damage‐inducible genes, MAG1 and DDI1, from Saccharomyces cerevisiae
Authors: Liu Yule , Xiao Wei
Published: November 2003

MAG1 encodes a Saccharomyces cerevisiae 3‐methyladenine DNA glycosylase that initiates a base‐excision‐repair pathway and protects yeast cells from killing by methylating agents such as methyl methanesulphonate (MMS). In the promoter region of the MAG1 gene, there is an 8 bp GC‐rich direct repeat (DR). Here we report that the DR sequence functions as an upstream activating site (UAS) that upregulates the expression of MAG1 as well as another DNA‐damage‐inducible gene, DDI1, which is transcribed divergently from MAG1. Deletions, or point mutations, within this repeat completely abolished DNA‐damage induction and resulted in a reduced basal‐level expression for both MAG1 and DDI1 genes. Furthermore, yeast cells carrying the MAG1 gene with the DR deletion displayed an increased sensitivity to MMS compared with wild‐type cells. The DR sequence alone can activate transcription of a CYC1 minimal promoter and confer a partial DNA‐damage responsiveness. Electrophoretic mobility‐shift assays indicate that the DR function is not due to interaction with the yeast RPA. Like MAG1the DDI1 gene is also controlled by an upstream repressing site (URS) located 5′ to the direct repeat. Based on this and previous studies, a model is proposed whereby the constitutive expression of MAG1 and DDI1 is controlled by two functionally opposite regulatory elements, UAS and URS, probably through an antagonistic mechanism, whereas the damage‐induced expression appears to be regulated by mechanisms of derepression at the URS as well as activation at the UAS.
The ftsH gene of Bacillus subtilis is involved in major cellular processes such as sporulation, stress adaptation and secretion
Authors: Deuerling Elke , Mogk Axel , Richter Carolin , Purucker Martina , Schumann Wolfgang
Published: November 2003

The ftsH gene of Bacillus subtilis has been identified as a general stress gene which is transiently induced after thermal or osmotic upshift. The FtsH protein exhibits 70.1% homology to FtsH of Escherichia coli which constitutes an essential ATP‐ and Zn2+‐dependent protease anchored in the cytoplasmic membrane via two N‐terminal transmembrane domains. This paper describes the isolation and functional characterization of an ftsH null mutant which was obtained by integration of a cat‐cassette near the 5′ end of ftsH, thereby preventing the synthesis of FtsH protein. In contrast to the situation in E. coli, ftsH is dispensable in B. subtilis but results in a pleiotropic phenotype. While the mutant cells grew mostly as large filaments under physiological conditions, they turned out to be extremely sensitive to heat and salt stress. Although ftsH is necessary for adaptation to heat, it is not involved in the regulation of the heat‐shock response. The induction profiles of representative genes of the CIRCE and sigma‐B regulon and class III heat‐shock genes lon and clpC were identical in the wild type and the ftsH null mutant. Furthermore, the ftsH knockout strain was unable to sporulate, and this failure was probably due to the absence of Spo0A protein which is essential for entry into the sporulation programme. In addition, secretion of bulk exoproteins was severely impaired in the ftsH null mutant after entry into stationary phase. The α‐amylase and subtilisin activity in the supernatant was specifically tested. Whereas the activity of α‐amylase increased after entry into stationary phase in both the wild type and the ftsH mutant strain, that of subtilisin encoded by aprE was prevented at the level of transcription in the mutant. Most of these results can be explained by the failure to synthesize appropriate amounts of Spo0A protein in the ftsH null mutant and point to ftsH as a developmental checkpoint
The Yersinia Yop virulon: a bacterial system for subverting eukaryotic cells
Authors: Cornelis Guy R. , Wolf‐Watz Hans
Published: November 2003

The Yop virulon enables Yersinia spp. (Y. pestis, Y. pseudotuberculosis and Y. enterocolitica) to survive and multiply in the lymphoid tissues of their host. It is an integrated system allowing extracellular bacteria to communicate with the host cell's cytosol by the injection of effector proteins. It is composed of the following four elements. (i) A contact or type III secretion system called Ysc, which is devoted to the secretion of Yop proteins. This secretion apparatus, comprising some 22 proteins recognizes the Yops by a short N‐terminal signal that is not cleaved off during secretion. (ii) A system designed to deliver bacterial proteins into eukaryotic target cells. This system is made of YopB, YopD and possibly other Yops such as LcrV. (iii) A control element (YopN). (iv) A set of effector Yop proteins designed to disarm these cells or disrupt their communications (YopE, YopH, YpkA/YopO, and YopM). The whole virulon is encoded by a 70 kb plasmid designated pYV. Transcription of the genes is controlled both by temperature and by contact with a eukaryotic cell.
The Bordetella pertussis sigma subunit of RNA polymerase confers enhanced expression of fha in Escherichia coli
Authors: Steffen Pierre , Goyard Sophie , Ullmann Agnes
Published: November 2003

We have cloned the rpoD gene coding for the major sigma factor of Bordetella pertussis. The deduced amino acid sequence reveals a protein of 733 residues which has extensive amino acid homology with the principal σ factors of a number of divergent prokaryotes. It is larger than most σ factors identified to date, having a molecular mass of 81.3 kDa. We have designated this factor sigma 80. In a heterologous complementation assay, B. pertussisrpoD was able to complement the Escherichia coli rpoD temperature‐sensitive mutant UQ285. Furthermore, B. pertussisrpoD conferred better specificity to the E. coli RNA polymerase, allowing increased expression of the B. pertussis virulence‐associated fha promoter, but could not activate the ptx and cya promoters in the E. coli UQ285 strains carrying the B. pertussis bvg locus. We discuss the implications of these results on the mechanisms involved in the activation of virulence‐associated promoters.
Transcription activation at Class II CAP‐dependent promoters
Authors: Busby Steve , Ebright Richard H.
Published: November 2003

Transcription activation at Class II CAP‐dependent promoters provides a paradigm for understanding how a single activator molecule can make multiple interactions with the transcription machinery, with each interaction being responsible for a specific mechanistic consequence. At Class II CAP‐dependent promoters, the DNA target site for CAP is centred near position −42, overlapping and replacing the −35 determinant for binding of RNA polymerase (RNAP). Transcription activation requires two distinct mechanistic components. The first component is ‘anti‐inhibition,’ overcoming an inhibitory effect of the RNAP α subunit C‐terminal domain (αCTD). This component involves direct contact between amino acids 156–164 (activating region 1) of the upstream subunit of the CAP dimer and a target in αCTD. The second component is ‘direct activation’, facilitating isomerization of the RNAP–promoter closed complex to the transcriptionally competent open complex. This component involves direct contact between amino acids 19, 21 and 101 (activating region 2) of the downstream subunit of the CAP dimer and a target in the RNAP α subunit N‐terminal domain (αNTD).
Sulfadoxine resistance in the human malaria parasite Plasmodium falciparum is determined by mutations in dihydropteroate synthetase and an additional factor associated with folate utilization
Authors: Wang Ping , Read Martin , Sims Paul F. G. , Hyde John E.
Published: November 2003

Sulfadoxine/pyrimethamine (Fansidar) is widely used in Africa for treating chloroquine‐resistant falciparum malaria. To clarify how parasite resistance to this combination arises, various lines of Plasmodium falciparum were used to investigate the role of naturally occurring mutations in the target enzyme, dihydropteroate synthetase (DHPS), in the parasite response to sulfadoxine inhibition. An improved drug assay was employed to identify a clear correlation between sulfadoxine‐resistance levels and the number of DHPS mutations. Moreover, tight linkage was observed between DHPS mutations and high‐level resistance in the 16 progeny of a genetic cross between sulfadoxine‐sensitive (HB3) and sulfadoxine‐resistant (Dd2) parents. However, we also demonstrate a profound influence of exogenous folate on IC50 values, which, under physiological conditions, may have a major role in determining resistance levels. Importantly, this phenotype does not segregate with dhps genotypes in the cross, but shows complete linkage to the two alleles of the dihydrofolate reductase (dhfr) gene inherited from the parental lines. However, in unrelated lines, this folate effect correlates less well with DHFR sequence, indicating that the gene responsible may be closely linked to dhfr, rather than dhfr itself. These results have major implications for the acquisition of Fansidar resistance by malaria parasites.
In Saccharomyces cerevisiae a short amino acid sequence facilitates excretion in the growth medium of periplasmic proteins
Authors: Venturini Marina , Morrione Andrea , Pisarra Patrizia , Martegani Enzo , Vanoni Marco
Published: November 2003

In Saccharomyces cerevisiae the cell wall is a barrier to excretion of proteins in the growth medium. Although small proteins are more easily released than bigger ones, other factors besides molecular sieving may play a role in partitioning of periplasmic proteins. By using several complementary approaches including enzyme‐activity assays, quantitative immunoblotting on subcellular fractions and growth media, as well as a novel approach involving the use of flow cytometry and specific antibodies, we show that residues 1–8 of mature glucoamylase greatly enhance excretion of both glucoamylase and β‐galactosidase in vivo and facilitate extraction of periplasmic proteins in vitro. Immunological data obtained by flow cytometry on whole cells indicate that this amino acid sequence increases the fraction of enzyme reaching the outer cell‐wall layers. This amino acid sequence may define a novel type of topogenic sequence, facilitating the crossing of the yeast cell wall in vivo and facilitating extraction of periplasmic proteins by non‐disruptive means in vitro.
Isolation and properties of enhancer‐bypass mutants of sigma 54
Authors: Syed Adeela , Gralla Jay D.
Published: November 2003

The N‐terminal activation domain of Escherichia coli sigma 54 was randomly mutated to provide a library of changes that might allow the required enhancer function to be bypassed. Five clones harbouring mutant sigma factors were obtained that exhibited this property in that they enhanced growth under nitrogen‐limiting conditions in cells lacking NtrC. DNA sequence analysis located all mutations to four leucines in a small region between amino acids 25 and 31. No mutant sigma factors retained the hydrophobic character of the leucine residues. Mutant sigma factors were shown to transcribe in vitro without the need for enhancer binding activator or ATP hydrolysis, confirming the in vivo phenotype. These and other data suggest that a very small set of leucines is critical for keeping polymerase function in check, allowing high responsiveness to physiological induction via enhancer proteins such as NtrC.
CheY‐mediated modulation of Campylobacter jejuni virulence
Authors: Yao Ruijin , Burr Donald H. , Guerry Patricia
Published: November 2003

Four motile, non‐adherent and non‐invasive mutants of Campylobacter jejuni 81‐176 generated by a site‐specific insertional mutagenesis scheme were characterized at the molecular level and all contained a duplication of the same region of the chromosome. When this region was cloned from wild‐type 81‐176 and transferred into 81‐176 on a shuttle plasmid, the same non‐invasive phenotype as the original mutants was observed, suggesting that the region contained a repressor of adherence and invasion. The smallest piece of DNA identified which was capable of repressing adherence and invasion was a 0.8 kb fragment encoding the cheY gene of C. jejuni. To confirm further that CheY was responsible for the observed non‐adherent and non‐invasive phenotypes, the cheY gene was inserted into the arylsulfatase gene of 81‐176 to generate a strain with two chromosomal copies of cheY. This diploid strain displayed the same non‐adherent and non‐invasive phenotype as the original mutants. Insertional inactivation of the cheY gene in 81‐176 resulted in an approx. threefold increase in adherence and invasion in vitro, but this strain was unable to colonize or cause disease in animals. The diploid cheY strain, although able to colonize mice, was attenuated in a ferret disease model.
SopA, the outer membrane protease responsible for polar localization of IcsA in Shigella flexneri
Authors: Egile Coumaran , D’Hauteville Hélène , Parsot Claude , Sansonetti Philippe J.
Published: November 2003

The spreading ability of Shigella flexneri, a facultative intracellular Gram‐negative bacterium, within the host‐cell cytoplasm is the result of directional assembly and accumulation of actin filaments at one pole of the bacterium. IcsA/VirG, the 120 kDa outer membrane protein that is required for intracellular motility, is located at the pole of the bacterium where actin polymerization occurs. Bacteria growing in laboratory media and within infected cells release a certain proportion of the surface‐exposed IcsA after proteolytic cleavage. In this study, we report the characterization of the sopA gene which is located on the virulence plasmid and encodes the protein responsible for the cleavage of IcsA. The deduced amino acid sequence of SopA exhibits 60% identity with those of the OmpT and OmpP outer membrane proteases of Escherichia coli. The construction and phenotypic characterization of a sopA mutant demonstrated that SopA is required for exclusive polar localization of IcsA on the bacterial surface and proper expression of the motility phenotype in infected cells.
Cross‐talk to the genes for Bacillus anthracis capsule synthesis by atxA, the gene encoding the frans‐activator of anthrax toxin synthesis
Authors: Uchida Ikuo , Makino Sou‐ichi , Sekizaki Tsutomu , Terakado Nobuyuki
Published: November 2003

The two major virulence factors of Bacillus anthracis are the tripartite toxin and the polyglutamate capsule, which are encoded by genes on the large plasmids, pX01 and pX02, respectively. The genes atxA, located on pX01, and acpA, located on pX02, encode positive frans‐acting proteins that are involved in bicarbonate‐mediated regulation of toxin and capsule production, respectively. A derivative strain cured of pX01 produced less capsular substance than the parent strain harbouring both pX01 and pX02, and electroporation of the strain cured of pX01 with a plasmid containing the cloned atxA gene resulted in an increased level of capsule production. An acpA‐null mutant was complemented by not only acpA but also the atxA gene. The cap region, which is essential for encapsulation, contains three genes capB, capC, and cap A, arranged in that order. The atxA gene stimulated capsule synthesis from the cloned cap region. Transcriptional analysis of cap by RNA slot‐blot hybridization and primer‐extension analysis revealed that atxA activated expression of cap in trans at the transcriptional level. These results indicate that cross‐talk occurs, in which the pX01‐located gene, atxA, activates transcription of the cap region genes located on pX02. We identified two major apparent transcriptional start sites, designated P1 and P2, located at positions 731 bp and 625 bp, respectively, upstream of the translation‐initiation codon of capB. Transcription initiated from P1 and P2 was activated by both atxA and acpA, and activation appeared to be stimulated by bicarbonate. Deletion analysis of the upstream region of the cap
Pathogenicity islands of virulent bacteria: structure, function and impact on microbial evolution
Authors: Hacker J. , Blum‐Oehler G. , Mühldorfer I. , Tschäpe H.
Published: November 2003

Virulence genes of pathogenic bacteria, which code for toxins, adhesins, invasins or other virulence factors, may be located on transmissible genetic elements such as transposons, plasmids or bacteriophages. In addition, such genes may be part of particular regions on the bacterial chromosome, termed‘pathogenicity islands’(Pais). Pathogenicity islands are found in Gram‐negative as well as in Gram‐positive bacteria. They are present in the genome of pathogenic strains of a given species but absent or only rarely present in those of non‐pathogenic variants of the same or related species. They comprise large DNA regions (up to 200 kb of DNA) and often carry more than one virulence gene, the G+C contents of which often differ from those of the remaining bacterial genome. In most cases, Pais are flanked by specific DNA sequences, such as direct repeats or insertion sequence (IS) elements. In addition, Pais of certain bacteria (e.g. uropathogenic Escherichia coli, Yersinia spp., Helicobacter pylori) have the tendency to delete with high frequencies or may undergo duplications and amplifications. Pais are often associated with tRNA loci, which may represent target sites for the chromosomal integration of these elements. Bacteriophage attachment sites and cryptic genes on Pais, which are homologous to phage integrase genes, plasmid origins of replication or IS elements, indicate that these particular genetic elements were previously able to spread among bacterial populations by horizontal gene transfer, a process known to contribute to microbial evolution.
Double‐glycine‐type leader peptides direct secretion of bacteriocins by ABC transporters: colicin V secretion in Lactococcus lactis
Authors: van Belkum Marco J. , Worobo Randy W. , Stiles Michael E.
Published: November 2003

Many non‐lantibiotic bacteriocins of lactic acid bacteria are produced as precursors which have N‐terminal leader peptides that share similarities in amino acid sequence and contain a conserved processing site of two glycine residues in positions ‐1 and ‐2. A dedicated ATP‐binding cassette (ABC) transporter is responsible for the proteolytic cleavage of the leader peptides and subsequent translocation of the bacteriocins across the cytoplasmic membrane. To investigate the role that these leader peptides play in the recognition of the precursor by the ABC transporters, the leader peptides of leucocin A, lactococcin A or colicin V were fused to divergicin A, a bacteriocin from Carnobacterlum divergens that is secreted via the cell's general secretion pathway. Production of divergicin was monitored when these fusion constructs were introduced into Leuconostoc gelidum, Lactococcus lactis and Escherichia coli, which carry the secretion apparatus for leucocin A, lactococcins A and B, and colicin V, respectively. The different leader peptides directed the production of divergicin in the homologous hosts. In some cases production of divergicin was also observed when the leader peptides were used in heterologous hosts. For ABC‐transporter‐dependent secretion in E. coli the outer membrane protein TolC was required. Using this strategy, colicin V was produced in L. lactis by fusing this bacteriocin behind the leader peptide of leucocin A.
Molecular characterization of the Aspergillus nidulans treA gene encoding an acid trehalase required for growth on trehalose
Authors: D'Enfert Christophe , Fontaine Thierry
Published: November 2003

Aspergillus nidulans conidiospores contain high levels of the non‐reducing disaccharide trehalose. We show that upon induction of conidiospore germination, the trehalose pool is rapidly degraded and a glycerol pool is transiently accumulated. A trehalase with an acidic pH optimum was purified from conidiospores. Characterization of the treA gene encoding this trehalase shows that it is homologous to Saccharomyces cerevisiae vacuolar acid trehalase, the product of the ATH1 gene, and to two related proteins of unknown function identified in Mycobacterium tuberculosis and Mycobacterium leprae. A. nidulans mutants that lack acid trehalase activity were constructed by gene replacement at the treA locus. Analysis of these mutants suggests that the treA gene product is localized in the conidiospore wall, is required for growth on trehalose as a carbon source, and is not involved in the mobilization of the intracellular pool of trehalose. Therefore, it is proposed that a cytoplasmic regulatory trehalase is controlling this latter process.
The gene encoding the major proline transporter of Aspergillus nidulans is upregulated during conidiospore germination and in response to proline induction and amino acid starvation
Authors: Tazebay Uygar Halis , Sophianopoulou Vicky , Scazzocchio Claudio , Diallinas George
Published: November 2003

In Aspergillus nidulans a highly specific l‐proline transporter is encoded by the prnB gene which is tightly linked to all other genes involved in proline catabolism. In mycelia, the expression of the prn structural genes is finely co‐regulated in response to proline induction and nitrogen/carbon catabolite repression. In this study we establish that prnB expression is also activated during germination of conidiospores. This activation persists until the development of 6 h‐old mycelia and it is independent of proline induction mediated by the pathway‐specific prnA gene product. We then show that, in mycelia, prnB transcription is activated in response to proline or histidine starvation. This process has two components: a prnA‐dependent and a prnA‐independent component. A cis‐acting element that conforms to the consensus target of the GCN4/CPC1 transcriptional activators mediating amino acid biosynthesis activation in other fungi is involved in the activation of prnB transcription in response to amino acid starvation. We also show that the stimulation of prnB expression in germinating conidiospores is not due exclusively to transient internal amino acid starvation occurring during the transition from conidiospore to mycelium. This is the first report that an amino acid transporter gene is upregulated during development and in response to amino acid starvation and specific amino acid induction.
Functional analysis of ssaJ and the ssaK/U operon, 13 genes encoding components of the type III secretion apparatus of Salmonella Pathogenicity Island 2
Authors: Hensel Michael , Shea Jacqueline E. , Raupach Bärbel , Monack Denise , Falkow Stanley , Gleeson Colin , Kubo Toru , Holden David W.
Published: November 2003

We have investigated the structure and transcriptional organization of 13 genes of Salmonella Pathogenicity Island 2 (SPI2) that encode components of the second type III secretion apparatus of Salmonella typhimurium. ssaK, L, M, V, N, O, P, Q, R, S, T, U constitute one operon of 10 kb. ssaJ lies upstream of ssaK and is the terminal gene of another operon. The deduced products of ssaJ, ssaK, ssaV, ssaN, ssaO, ssaQ, ssaR, ssaS, ssaT, and ssaU show greatest similarity to the Yersinia spp. genes yscJ, yscL, lcrD, yscN, yscO, yscQ, yscR, yscS, yscT, and yscU, respectively. The products of the ssaL, ssaM and ssaP genes do not have significant similarity to products of other type III secretion systems, and might be important for the specific function of the SPI2 type III secretion system. Bacterial strains carrying different ssa mutations display minor alterations in terms of serum sensitivity when compared with the wild‐type strain, but none are defective in replication within macrophage‐like RAW 264.7 cells. However, some of the ssa mutant strains invade HEp2 cells less efficiently and are less cytotoxic to RAW 264.7 macrophages than the wild‐type strain. We show that the invasion defect is correlated with a lack of SipC in culture supernatants of these mutant strains. SipC is a product of the SPI1 type III secretion system of S. typhimurium, and is important for epithelial cell invasion. Therefore, mutations in SPI2 can affect the SPI1 secretion system, which raises the possibility of an interaction between the two type III secretion systems.
Analysis of the interaction between the bacterial superantigen streptococcal pyrogenic exotoxin A (SpeA) and the human T‐cell receptor
Authors: Kline J. Bradford , Collins Carleen M.
Published: November 2003

Streptococcus pyogenes that produces the bacterial superantigen streptococcal pyrogenic exotoxin A (SpeA) is associated with outbreaks of streptococcal toxic shock syndrome (STSS) in the United States and Europe. SpeA stimulates Vβ2.1, 12.2, 14.1, and 15.1‐positive T cells, and the lymphokine production from the activated T cells is believed to result in the symptoms associated with STSS. The T‐cell receptor (TCR)–SpeA interaction is crucial for superantigenic activity, and studies were undertaken to determine regions of both SpeA and the TCR involved in the formation of MHC/SpeA/TCR complexes. Previously, recombinant toxins encoded by speA alleles 1, 2, and 3 as well as toxins resulting from 19 distinct point mutations in speA1 were generated. Here, these 22 toxin forms were incubated with human peripheral blood mono‐ nuclear cells (PBMCs), and the percentages of T‐cell blasts bearing Vβ chains 2.1, 12.2, and 14.1 were quantified by flow cytometry. The analysis indicates that the residues of SpeA needed for a productive TCR interaction differ for each Vβ chain examined. An amino acid substitution at only one site significantly affected the toxin’s ability to stimulate Vβ2.1‐expressing T cells, three individual amino acid substitutions resulted in significant loss of ability to stimulate Vβ12.2‐expressing T cells, and substitution at 13 individual sites significantly affected the ability to stimulate Vβ14.1‐expressing T cells. To elucidate the regions of the Vβ chains that interacted with SpeA, synthetic peptides representative of the human Vβ12.2 complementary‐determining regions (CDRs) 1, 2, and 4 were used to block the SpeA‐mediated proliferation of human PBMCs. The CDR1, CDR2 and CDR4 peptides were each able to block proliferation, with the activity of CDR1 > CDR2 > CDR4. Combinations of CDR1 peptide with CDR2 or CDR4 peptides allosterically enhanced the ability of each to block proliferation, suggesting SpeA has distinct binding sites for the CDR loops.
Host‐cell cyclophilin is important for the intracellular replication of Leishmania major
Authors: Hoerauf A. , Rascher Ch. , Bang R. , Pahl A. , Solbach W. , Brune K. , Röllinghoff M. , Bang H.
Published: November 2003

The antiparasitic effects of cyclosporin A were examined in leishmanial infection by analysing the role of CsA‐binding proteins (cyclophilins) in the host–parasite interaction. We hypothesized that the leishmanicidal effects of CsA on Leishmania major infected macrophages might be mediated through a cyclophilin of either the parasite or the host cell. Two cyclophilins (20 and 22 kDa) were purified from L. major parasites and N‐terminally sequenced. Although enzyme activity of these cyclophilins was inhibited by CsA, pretreatment of L. major parasites with CsA did not result in reduction of a subsequent macrophage infection, arguing against a role of L. major cyclophilins as infectivity potentiators. However, host‐cell cyclophilin A (CypA) was found to be critically involved in the intracellular replication of L. major parasites in murine macrophages. An antisense oligonucleotide to murine CypA was constructed and added to cultures of peritoneal macrophages prior to infection with L. major parasites. This treatment strongly reduced the expression of CypA in macrophages and resulted in the inhibition of the intracellular replication of L. major amastigotes. These data indicate that interaction of amastigotes with host‐cell cyclophilin is an important part of the intracellular replication machinery of L. major and define, for the first time, a direct involvement of a cyclophilin in the survival strategies of an intracellular parasite.
Dynamic bacterial genome organization
Authors: Kolstø Anne‐Brit
Published: November 2003

Recently completed projects of sequencing chromosomal fragments and entire chromosomes, as well as physical mapping of genomes, have opened novel inroads to the understanding of the biology of bacterial genomes. From these studies one may draw some conclusions. (i) The organization of orthologous genes on the bacterial chromosome is not conserved during evolution. (ii) The bacterial genome is more complex and also more flexible than hitherto thought. Genetic elements are sometimes part of the chromosome, while at other times they are independent elements or parts of alternative replicons (e.g. large plasmids). Such replicons, carrying essential genes, now seem to deserve the designation ‘secondary chromosomes’. A study of the regulation of replication and segregation of these essential genetic elements will be of great interest.
Effect of mutations in Shigella flexneri chromosomal and plasmid‐encoded lipopolysaccharide genes on invasion and serum resistance
Authors: Hong Mei , Payne Shelley M.
Published: November 2003

This study shows that both length and distribution of lipopolysaccharide (LPS) are important for Shigella flexneri invasion and virulence. Mutants were generated in the chromosomal LPS synthesis genes rfa, rfb, and rol, and in a plasmid‐encoded O‐antigen chain‐length regulator, cldpHS‐2. LPS analysis showed that mutations in rfb genes and in a candidate rfaL gene either eliminated the entire O‐antigen side chains or produced chains of greatly reduced length. Mutation in a previously unidentified gene, rfaX, affected the LPS core region and resulted in reduced amounts of O‐antigen. Mutants defective in cldpHS‐2 or rol had different distributions of O‐antigen chain lengths. The results of tissue‐culture cell invasion and plaque assays, the Serény test, and serum‐sensitivity assay suggested roles for the different LPS synthesis genes in bacterial survival and virulence; rfaL, rfaX and rfb loci are required for serum resistance and intercellular spread, but not for invasion; cldpHS‐2 is required for resistance to serum killing and for full inflammation in the Serény test, but not for invasion or intercellular spread, while rol is required for normal invasiveness and plaque formation, but not for serum resistance. Thus, O‐antigen synthesis and chain‐length regulation genes encoded on both the chromosome and the small plasmid pHS‐2 play important roles in S. flexneri invasion and virulence.
Role of the transcriptional activator RocR in the arginine‐degradation pathway of Bacillus subtilis
Authors: Gardan Rozenn , Rapoport Georges , Débarbouillé Michel
Published: November 2003

In Bacillus subtilis, genes involved in arginine and ornithine catabolism constitute two operons, rocABC and rocDEF. Inducible expression of these two operons is SigL‐dependent and requires the transcriptional activator RocR. RocR is a member of the NtrC/NifA family of regulators. To study the molecular mechanisms leading to the activation of RocR, we constructed a series of mutants affected in various steps of arginine catabolism. Results obtained using these mutants strongly suggest that the true inducer is ornithine or citrulline. Constitutive mutants of rocR containing either missense mutations, frameshift mutations resulting from deletions, or in‐frame deletions leading to the synthesis of N‐terminal truncated RocR polypeptides were obtained. Analysis of these mutants indicates that the N‐terminal part of RocR is an intramolecular repressor domain. AhrC is a second positive regulatory protein of the rocABC and rocDEF operons. Two missense mutations modifying the N‐terminal domain of RocR led to high constitutive expression of the Roc regulon in the absence of AhrC. Constitutive RocR proteins still require the presence of UAS1 and therefore probably bending of the DNA region located between the UAS1 and the promoter, suggesting that AhrC is not involved in DNA bending which facilitates interaction between RocR and σ54‐RNA polymerase. We suggest that the positive role of AhrC involves protein–protein interaction with RocR.