bluuebunny/crossref_metadata_2025 huggingface dataset:
Three disparately regulated genes for σ32‐like transcription factors in Bradyrhizobium japonicum
Authors: Narberhaus Franz , Krummenacher Philipp , Fischer Hans‐Martin , Hennecke Hauke
Published: November 2003

Bradyrhizobium japonicum possesses a subclass of heat‐shock genes whose members are transcribed from a σ32 consensus promoter. Having identified previously one gene (rpoH1) encoding a σ32‐like RNA polymerase transcription factor, we report here the characterization of two additional rpoH‐like genes (rpoH2 and rpoH3). B. japonicum thus represents the first example of an organism possessing an rpoH multigene family. All three rpoH genes encode functional proteins that are able to initiate transcription from the Escherichia coli groE promoter. Each rpoH gene is apparently regulated by a different mechanism. Although both rpoH1 and rpoH2 are transcribed from σ70‐type promoters, transcription of the rpoH1 operon was found to be heat inducible by an unknown mechanism, whereas the level of rpoH2 mRNA decreased after heat shock. At extreme temperatures (48°C), rpoH2 was transcribed from a second promoter that resembled the E. coliσE‐type promoter. The rpoH3 gene was found to be associated with two upstream genes, ragA and ragB, coding for a classical two‐component regulatory system. Transcription initiated from a promoter that mapped in front of the putative response regulator gene ragA, suggesting that ragA, ragB and rpoH3 are organized in an operon. The ragA promoter was similar to a σ32 consensus promoter. The three B. japonicum rpoH genes also varied in their significance to support growth of the organism. While the rpoH2 gene could not be eliminated by mutation, knock‐out mutants of rpoH1 and/or rpoH3 were readily obtained and shown to be indistinguishable from the wild type under aerobic growth conditions or during root‐nodule symbiosis. We conclude that rpoH2 is essential for the synthesis of cellular proteins under physiological growth conditions, whereas rpoH1, and probably also rpoH3, are involved in their synthesis during the stress response.
H‐NS: a modulator of environmentally regulated gene expression
Authors: Atlung Tove , Ingmer Hanne
Published: November 2003

H‐NS is a small chromatin‐associated protein found in enterobacteria. H‐NS has affinity for all types of nucleic acids but binds preferentially to intrinsically curved DNA. The major role of H‐NS is to modulate the expression of a large number of genes, mostly by negatively affecting transcription. Many of the H‐NS‐modulated genes are regulated by environmental signals, and expression of most of these genes is positively regulated by specific transcription factors. Therefore one of the purposes of H‐NS could be to repress expression of some genes under conditions characteristic of a non‐intestinal environment, but allow expression of specific genes in response to certain stimuli in the intestinal environment. The hns gene is autoregulated. In vivo the H‐NS to DNA ratio is fairly constant except during cold shock, when it increases three‐ to fourfold. In this review we propose that only the preferential binding to intrinsically curved DNA plays a role under normal growth conditions, and we discuss the different mechanisms by which H‐NS might affect gene expression and how H‐NS could be involved in the response to different stress situations. Finally, we summarize the evolutionary and functional relationship between H‐NS and the homologous StpA.
YopK of Yersinia pseudotuberculosis controls translocation of Yop effectors across the eukaryotic cell membrane
Authors: Holmström Anna , Pettersson Jonas , Rosqvist Roland , Håkansson Sebastian , Tafazoli Farideh , Fällman Maria , Magnusson Karl‐Eric , Wolf‐Watz Hans , Forsberg Åke
Published: November 2003

Introduction of anti‐host factors into eukaryotic cells by extracellular bacteria is a strategy evolved by several Gram‐negative pathogens. In these pathogens, the transport of virulence proteins across the bacterial membranes is governed by closely related type III secretion systems. For pathogenic Yersinia, the protein transport across the eukaryotic cell membrane occurs by a polarized mechanism requiring two secreted proteins, YopB and YopD. YopB was recently shown to induce the formation of a pore in the eukaryotic cell membrane, and through this pore, translocation of Yop effectors is believed to occur (Håkansson et al., 1996b). We have previously shown that YopK of Yersinia pseudotuberculosis is required for the development of a systemic infection in mice. Here, we have analysed the role of YopK in the virulence process in more detail. A yopK‐mutant strain was found to induce a more rapid YopE‐mediated cytotoxic response in HeLa cells as well as in MDCK‐1 cells compared to the wild‐type strain. We found that this was the result of a cell‐contact‐dependent increase in translocation of YopE into HeLa cells. In contrast, overexpression of YopK resulted in impaired translocation. In addition, we found that YopK also influenced the YopB‐dependent lytic effect on sheep erythrocytes as well as on HeLa cells. A yopK‐mutant strain showed a higher lytic activity and the induced pore was larger compared to the corresponding wild‐type strain, whereas a strain overexpressing YopK reduced the lytic activity and the apparent pore size was smaller. The secreted YopK protein was found not to be translocated but, similar to YopB, localized to cell‐associated bacteria during infection of HeLa cells. Based on these results, we propose a model where YopK controls the translocation of Yop effectors into eukaryotic cells.
Intercontinental spread of promiscuous mercury‐resistance transposons in environmental bacteria
Authors: Yurieva Olga , Kholodii Gennady , Minakhin Leonid , Gorlenko Zhosephine , Kalyaeva Eza , Mindlin Sofia , Nikiforov Vadim
Published: November 2003

We demonstrate that horizontal spread of mer operons similar to worldwide spread of antibiotic‐resistance genes in medically important bacteria occurred in bacteria found in ores, soils and waters. The spread was mediated by different transposons and plasmids. Some of the spreading transposons were damaged in different ways but this did not prevent their further spread. Certain transposons are mosaics composed of segments belonging to distinct sequence types. These mosaics arose as a result of homologous and site‐specific recombination. Our data suggest that the mercury‐resistance operons of Gram‐negative environmental bacteria can be considered as a worldwide population composed of a relatively small number of distinct recombining clones shared, at least partially, by environmental and clinical bacteria.
Regulation of the Caulobacter flagellar gene hierarchy; not just for motility
Authors: Wu Jianguo , Newton Austin
Published: November 2003

The Caulobacter crescentus flagellum serves not only as a motility apparatus, but also as a key landmark in the differentiation of this asymmetrically dividing bacterium. A distinctive aspect of flagellum biosynthesis is the periodic expression of the flagellar genes during the cell cycle in a sequence corresponding to the order of gene product assembly into the growing flagellum. This program of gene expression is achieved in part by the organization of flagellar genes into a four‐tiered regulatory hierarchy that controls their expression at both the transcriptional and post‐transcriptional levels. Because of the close interconnection of the developmental program to the asymmetric cell‐division cycle in C. crescentus, studies of flagellar gene regulation and motility have also begun to reveal basic mechanisms responsible for control of the cell cycle itself. Here, we review recent work on regulation of the flagellar gene hierarchy in C. crescentus and consider regulatory mechanisms that are distinct from those described in Escherichia coli and Salmonella typhimurium.
The global activator GacA of Pseudomonas aeruginosa PAO positively controls the production of the autoinducer N‐butyryl‐homoserine lactone and the formation of the virulence factors pyocyanin, cyanide, and lipase
Authors: Reimmann Cornelia , Beyeler Markus , Latifi Amel , Winteler Harald , Foglino Maryline , Lazdunski Andrée , Haas Dieter
Published: November 2003

The global activator GacA, a highly conserved response regulator in Gram‐negative bacteria, is required for the production of exoenzymes and secondary metabolites in Pseudomonas spp. The gacA gene of Pseudomonas aeruginosa PAO1 was isolated and its role in cell‐density‐dependent gene expression was characterized. Mutational inactivation of gacA resulted in delayed and reduced formation of the cell‐density signal N‐butyryl‐l‐homoserine lactone (BHL), of the cognate transcriptional activator RhlR (VsmR), and of the transcriptional activator LasR, which is known to positively regulate RhlR expression. Amplification of gacA on a multicopy plasmid caused precocious and enhanced production of BHL, RhlR and LasR. In parallel, the gacA gene dosage markedly influenced the BHL/RhlR‐dependent formation of the cytotoxic compounds pyocyanin and cyanide and the exoenzyme lipase. However, the concentrations of another known cell‐density signal of P. aeruginosa, N‐oxododecanoyl‐l‐homoserine lactone, did not always match BHL concentrations. A model accounting for these observations places GacA function upstream of LasR and RhlR in the complex, cell‐density‐dependent signal‐transduction pathway regulating several exoproducts and virulence factors of P. aeruginosa via BHL.
HIP1 propagates in cyanobacterial DNA via nucleotide substitutions but promotes excision at similar frequencies in Escherichia coli and Synechococcus PCC 7942
Authors: Robinson Pamela J. , Cranenburgh Rocky M. , Head Ian M. , Robinson Nigel J.
Published: November 2003

The sequence 5′‐GCGATCGC‐ 3′, designated HIP1, for highly iterated palindrome, was first identified at the borders of a gene‐deletion event and subsequently shown to constitute up to 2.5% of the DNA in some cyanobacteria. It is now reported that HIP1 is polyphyletic, occurring in several distinct cyanobacterial lineages and not defining a clade. HIP1 does not introduce gaps into sequence alignments. It aligns with partial HIP1 sites in related sequences showing that it propagates by nucleotide substitutions rather than insertion. Constructs have been created to determine the frequencies at which deletion events occur between palindromes located within the selectable marker neoDeletion between HIP1 sites was more frequent in Synechococcus PCC 7942 than deletion between control palindromes, 5′‐CCGATCGG‐3′, designated PAL0. However, this is not due to a recombinase that recognises HIP1 and is peculiar to cyanobacteria because similar deletion frequencies were detected in Escherichia coli. Furthermore, the frequency of deletion of DNA flanked asymmetrically by one HIP1 site and one PAL0 site was less than the frequency of deletion of DNA flanked symmetrically by identical copies of either palindrome. This is consistent with deletion by copy‐choice.
Cloning and functional characterization of Neisseria gonorrhoeae tonB, exbB and exbD genes
Authors: Biswas Gour D. , Anderson James E. , Sparling P. Frederick
Published: November 2003

Neisseria gonorrhoeae is able to utilize iron (Fe) from a variety of sources including transferrin (TF) and lactoferrin (LF). To gain insight into the molecular mechanisms used by gonococci to scavenge Fe from TF and LF, we cloned a 3.5 kb segment of wild‐type DNA that repaired the defect in tlu mutants, which are unable to take up Fe from either TF or LF despite exhibiting apparently normal ligand binding to the receptor. Nucleotide sequence determination identified three open reading frames (ORFs), designated ORF1, ORF2, and ORF3, which were arranged in tandem. The deduced amino acid sequence of the 852 bp ORF1 encoded a 28 kDa protein that exhibited 26–32% identity with TonB proteins of nine other bacteria. The 663 bp ORF2 predicted a 24 kDa protein and the 435 bp long ORF3 predicted a 15 kDa protein. These predicted protein sequences exhibited 32–38% and 24–36% identity, respectively, with ExbB and ExbD proteins of three other bacteria. Thus, the sequence comparison identified the ORF1, ORF2 and ORF3 as gonococcal homologues of the E. coli tonB, exbB and exbD genes. An insertional mutation in the tonB homologue resulted in the failure of gonococci to grow with TF, LF or human haemoglobin (HB) as sole Fe sources and in the inability to take up 55Fe from TF and LF. The tonB mutation did not prevent the utilization of Fe from citrate (CT) or haemin (HM). Binding of TF, LF and HB to whole cells in a solid‐phase binding assay was largely unaffected by the tonB mutation. We conclude that the pathways for utilization of Fe bound to TF, LF and HB but not to HM or CT were dependent on the TonB system.
Mycobacterial recA is cotranscribed with a potential regulatory gene called recX
Authors: Papavinasasundaram K. G. , Movahedzadeh Farahnaz , Keer Jacquie T. , Stoker Neil G. , Colston M. Joseph , Davis Elaine O.
Published: November 2003

The recA gene of Mycobacterium smegmatis has been cloned and sequenced. The amino acid sequence of the RecA protein is highly homologous to other RecA proteins. Three other potential open reading frames were identified. One of these showed extensive homology to a protein, HypB, involved in the incorporation of nickel into hydrogenases. Another, found downstream of and overlapping recA, was similar to a gene, recX, which has been proposed to play a regulatory role related to recA function. The homology between the M. smegmatis sequence and that of Mycobacterium tuberculosis extended upstream of the recA coding region for 140 bp including a motif identical to the Cheo‐box consensus sequence which has been shown to bind LexA. In addition, the transcriptional start sites were found to be identical to those identified previously for M. tuberculosis. Transcriptional fusions to the reporter gene chloramphenicol acetyltransferase (CAT) revealed that recA was DNA‐damage inducible and that expression required sequences at some distance from the mapped transcriptional start sites. Although a motif with only one mismatch to the Cheo box was found in the intergenic region between orf1 and orf2 these open reading frames were not DNA‐damage inducible, nor was this motif required for regulation of recA expression. Gel retardation assays revealed that the reason for this was that LexA did not bind to this sequence containing a mismatch. Reverse transcription/polymerase chain reaction analysis of M. smegmatis RNA demonstrated that recA and orf3 (recX ) are within the same trancriptional unit.
Energy requirement for pullulanase secretion by the main terminal branch of the general secretory pathway
Authors: Possot Odile M. , Letellier Lucienne , Pugsley Anthony P.
Published: November 2003

The energy requirement for the second step in pullulanase secretion by the general secretory pathway was studied in Escherichia coli. In order to uncouple the two steps in the secretion pathway (across the cytoplasmic and outer membranes, respectively) and to facilitate kinetic analysis of secretion, a variant form of pullulanase lacking its N‐terminal fatty acid membrane anchor was used. The transport of the periplasmic secretion intermediate form of this protein across the outer membrane was not inhibited by concentrations of sodium arsenate in excess of those required to reduce ATP levels to ≤10% of their normal value. Pullulanase secretion was inhibited by the protonophore carbonyl cyanide m‐chlorophenyl hydrazone at concentrations which were similar to those reported by others to be required to prevent solute uptake or the export and processing of preproteins across the cytoplasmic membrane, but which were in excess of those required to fully dissipate the proton‐motive force and to reduce lactose uptake to a significant extent.
The C‐terminal domain of the secretin PulD contains the binding site for its cognate chaperone, PulS, and confers PulS dependence on pIVf1 function
Authors: Daefler Simon , Guilvout Ingrid , Hardie Kim R. , Pugsley Anthony P. , Russel Marjorie
Published: November 2003

Related outer membrane proteins, termed secretins, participate in the secretion of macromolecules across the outer membrane of many Gram‐negative bacteria. In the pullulanase‐secretion system, PulS, an outer membrane‐associated lipoprotein, is required both for the integrity and the proper outer membrane localization of the PulD secretin. Here we show that the PulS‐binding site is located within the C‐terminal 65 residues of PulD. Addition of this domain to the filamentous phage secretin, pIV, or to the unrelated maltose‐binding protein rendered both proteins dependent on PulS for stability. A chimeric protein composed of bacteriophage f1 pIV and the C‐terminal domain of PulD required properly localized PulS to support phage assembly. An in vivo complex formed between the pIV‐PulD65 chimera and PulS was detected by co‐immunoprecipitation and by affinity chromatography.
Ffh and FtsY in a Mycoplasma mycoides signal‐recognition particle pathway: SRP RNA and M domain of Ffh are not required for stimulation of GTPase activity in vitro
Authors: Macao Bertil , Luirink Joen , Samuelsson Tore
Published: November 2003

Mycoplasma mycoides contains a signal‐recognition particle (SRP) composed of an RNA molecule and an SRP54 homologue (Ffh). We have now identified a mycoplasma homologue to the α subunit of the mammalian SRP receptor and Escherichia coli FtsY. The protein (MmFtsY) was expressed in E. coli and purified to homogeneity. MmFtsY has a weak intrinsic GTPase activity but GTP hydrolysis was markedly stimulated when it was combined with mycoplasma Ffh (MmFfh) and SRP RNA. Also, in the absence of SRP RNA GTPase activity was significantly enhanced. Furthermore, GTP hydrolysis was stimulated when MmFtsY was combined with the N‐terminal GTPase domain (N+G) of MmFfh. These findings indicate that basic features of the GTPase activation mechanism are independent of the C‐terminal M domain of the MmFfh protein. We propose that the activation is mediated to a large extent by contacts between the GTPase domains of the mycoplasma Ffh and FtsY proteins and that the contribution of the M domain and SRP RNA in the activation mechanism is mainly for modifying the conformation of the MmFfh GTPase domain.
The cioAB genes from Pseudomonas aeruginosa code for a novel cyanide‐insensitive terminal oxidase related to the cytochrome bd quinol oxidases
Authors: Cunningham Louise , Pitt Melinda , Williams Huw D.
Published: November 2003

The structural genes for the cyanide‐insensitive terminal oxidase (CIO) of Pseudomonas aeruginosa were sequenced. The locus comprised two open reading frames, cioA and cioB, coding for gene products of 488 and 335 amino acid residues with predicted molecular masses of 54 241 and 37 016 Da respectively. These genes were encoded by a 2.7 kb transcript and probably comprise an operon. Upstream of a major transcriptional start site is a −10 promoter region and, approximately at nucleotides −50 and +13, there are sequences homologous to the binding site of the transcriptional regulator Anr. The deduced amino acid sequences of CioA and CioB are homologous to the cytochrome bd quinol oxidases of Escherichia coli and Azotobacter vinelandii. However, no cytochrome d‐like signals were found in wild‐type P. aeruginosa strains. An atypical cytochrome d‐like signal was seen under low‐aeration growth conditions but only in strains in which the cioAB genes were present on a high‐copy‐number plasmid. The appearance of these cytochrome d‐like signals was not paralleled by a concomitant increase in CIO activity. These data support the hypothesis that the CIO of P. aeruginosa does not contain haem d. This raises the possibility that there is a family of bacterial quinol oxidases related to the cytochrome bd of E. coli that can differ in their haem composition from the E. coli paradigm.
Characterization of the negative elements involved in silencing the bgl operon of Escherichia coli: possible roles for DNA gyrase, H‐NS, and CRP–cAMP in regulation
Authors: Mukerji Mitali , Mahadevan S.
Published: November 2003

The bgl operon of Escherichia coli is rendered cryptic and uninducible in wild‐type cells by the presence of DNA structural elements that negatively regulate transcription. We have carried out a detailed analysis of the sequences implicated in negative regulation. Fine‐structure deletion analysis of the upstream sequences showed the presence of at least two elements involved in silencing the promoter. Chemical probing of genomic DNA in vivo showed that a region of dyad symmetry, present upstream of the promoter, is hypersensitive to KMnO4. The hypersensitive region detected corresponds to the potential cruciform structure implicated earlier in negative regulation. Enhancement of transcription from the wild‐type promoter, observed in the presence of the gyrase inhibitor novobiocin, was absent in a mutant that carried point mutations in the inverted repeat. This observation suggests that the activation seen in a gyrase mutant is mediated by destabilization of the cruciform because of reduced supercoiling. Deletion of sequences downstream of the potential cruciform also resulted in an increase in transcription, indicating the presence of a second regulatory element. Measurement of transcription from the bgl promoter carrying the deletion, in a strain that has a mutation in the hns gene, indicated that this region is likely to be involved in binding to H‐NS or a protein regulated by H‐NS, which acts as a non‐specific repressor. We also provide evidence which suggests that transcriptional activation by mutations at the cAMP receptor protein (CRP)‐binding site is mediated partly by antagonization of the negative effect of H‐NS by CRP–cAMP as a result of its increased affinity for the mutant site.
Sequence requirements for the termination of rolling‐circle replication of plasmid pT181
Authors: Zhao Adam C. , Khan Saleem A.
Published: November 2003

Most small multicopy plasmids of Gram‐positive bacteria and many in Gram‐negative bacteria replicate by a rolling‐circle (RC) mechanism. The replication initiator proteins encoded by the RC plasmids and single‐stranded bacteriophages of Escherichia coli have origin‐specific nicking‐closing activities that are required for the initiation and termination of RC replication. We have investigated the sequence requirements for termination of RC replication of plasmid pT181. The initiator nick site is located in the loop of a hairpin region (IRII) within the pT181 origin of replication. By mutational analysis, we have found that several nucleotides within the stem of IRII which are critical for the initiation activity are dispensable for termination of replication. We also demonstrate that nucleotides in the right arm of IRII, but not the left arm, are absolutely required for termination of RC replication. We have also identified specific nucleotides in IRII that are critical for its termination activity. The sequence of the right arm of the hairpin must be located downstream of the initiator nick site for termination, suggesting that termination requires a specific orientation of the initiator protein at the origin.
Analysis of the architecture of the transcription factor σN (σ54) and its domains by circular dichroism
Authors: Missailidis S. , Cannon W. V. , Drake A. , Wang X. Y. , Buck M.
Published: November 2003

Enhancer‐dependent transcription in bacteria requires the alternative transcription factor σN (σ54), which forms an RNA polymerase holoenzyme that binds promoters as a transcriptionally inactive complex. We have examined the structure of σN by circular dichroism (CD) analysis. The σN protein and its domains are well structured in the absence of the core RNA polymerase subunits or promoter DNA. Denaturation of σN by temperature as followed by changes in CD shows a concomitant loss of secondary and tertiary structures with a melting temperature of 36°C. The secondary structure displays a two‐state melting curve with a second Tm of 85°C. The amino‐terminal Region I activation domain together with the acidic Region II does not contribute to the two‐state melting. In marked contrast, the integrity of the C‐terminal DNA‐binding domain is required for the two‐state melting. Measurements of pKb also demonstrated that a C‐terminal part of σN, but not regions I or I + II, is required for the structural integrity of σN at high pH. Measurements of pKa suggested that α‐helical structures are important in σN for the establishment of tertiary structural elements. The tertiary structure near ultraviolet CD signals of σN do not require regions I or I + II but were strongly diminished by C‐terminal truncation of σN. Promoter DNA binding resulted in aconformational change in σN, permitting the determination of a binding constant. A typical B‐DNA conformation was adopted by the promoter DNA. Implications for the modular domain organization of σN, the function of C‐terminal sequences, and domain communication and its role in activation of transcription are discussed.
Inactivation of the replication‐termination system affects the replication mode and causes unstable maintenance of plasmid R1
Authors: Krabbe Margareta , Zabielski Jan , Bernander Rolf , Nordström Kurt
Published: November 2003

Two so‐called Ter sites, which bind the Escherichia coli Tus protein, are located near the replication origin of plasmid R1. Inactivation of the tus gene caused a large decrease in the stability of maintenance of the R1 mini‐derivative pOU47 despite the presence of a functional partition system on the plasmid. Deletion of the right Ter site caused a drop in stability similar to that observed after inactivation of the tus gene. Substitution of 2 bp required for Tus binding also caused unstable plasmid maintenance, whereas no effects on stability were observed when the left Ter site was deleted. Inactivation of the tus gene was coupled to an increased occurrence of multimeric plasmid forms as shown by gel electrophoresis of pOU47 DNA. Inactivation of the recA gene did not increase plasmid stability, suggesting that the multimerization was not mediated by RecA. Plasmid DNA was isolated from the tus strain carrying plasmid pOU47 and from a wild‐type strain carrying pOU47 in which the right Ter site had been inactivated; in both cases, electron microscopy revealed the presence of multimers as well as rolling‐circle structures with double‐stranded tails. Thus, the right Ter site in plasmid R1 appears to stabilize the plasmid by preventing multimerization and shifts from theta to rolling‐circle replication.
HlyX, the FNR homologue of Actinobacillus pleuropneumoniae, is a [4Fe–4S]‐containing oxygen‐responsive transcription regulator that anaerobically activates FNR‐dependent Class I promoters via an enhanced AR1 contact
Authors: Green Jeffrey , Baldwin Mandy L.
Published: November 2003

The hlyX gene of the pig pathogen Actinobacillus pleuropneumoniae encodes HlyX, a homologue of FNR, the anaerobic transcription regulator of Escherichia coli. The hlyX gene complements the anaerobic respiratory deficiencies of E. coli fnr mutants but also induces the expression of an otherwise latent haemolysin. Therefore, FNR and HlyX have distinct but overlapping regulons. The hlyX gene has been overexpressed as a gst ::hlyX fusion and the HlyX protein purified. Similar to FNR, HlyX can acquire a [4Fe–4S] cluster, which promotes binding to the FNR box (Kd of 20–30 nM) under anaerobic conditions. Expression of hlyX in E. coli induced the anaerobic production of at least five polypeptides, including the yfiD gene product, which were not induced by fnr. Analysis of the yfiD promoter region revealed the presence of two FNR boxes situated at −61.5 and −114.5. Consistent with this observation, expression from the semi‐synthetic Class I promoter FF+20pmelR was efficiently activated by HlyX but not by FNR. The weaker level of FNR‐mediated activation of Class I promoters suggests that there is a poorer activating contact (activating region 1 (AR1) equivalent) between FNR and RNA polymerase at these promoters and that HlyX possesses an additional or improved AR1. The AR1 of HlyX is partially characterized by a surface‐exposed region around amino acid A187, which confers the altered specificity and provides an explanation for the existence of distinct but overlapping HlyX and FNR regulons.
The Bacillus subtilis DivIVA protein targets to the division septum and controls the site specificity of cell division
Authors: Edwards David H. , Errington Jeffery
Published: November 2003

The Bacillus subtilisdivIVA gene, first defined by a mutation giving rise to anucleate minicells, has been cloned and characterized. Depletion of DivIVA leads to inhibition of the initiation of cell division. The residual divisions that do occur are abnormally placed and sometimes misorientated relative to the long axis of the cell. The DivIVA phenotype can be suppressed by disruption of the MinCD division inhibitor, suggesting that DivIVA controls the topological specificity of MinCD action and thus septum positioning. A DivIVA–GFP fusion targets to new and used sites of cell division, consistent with it having a direct role in topological specification.
Normal processing of AP sites in Apn1‐deficient Saccharomyces cerevisiae is restored by Escherichia coli genes expressing either exonuclease III or endonuclease III
Authors: Masson Jean‐Yves , Ramotar Dindial
Published: November 2003

Escherichia coli exonuclease III and endonuclease III are two distinct DNA‐repair enzymes that can cleave apurinic/apyrimidinic (AP) sites by different mechanisms. While the AP endonuclease activity of exonuclease III generates a 3′‐hydroxyl group at AP sites, the AP lyase activity of endonuclease III produces a 3′‐α,β unsaturated aldehyde that prevents DNA‐repair synthesis. Saccharomyces cerevisiae Apn1 is the major AP endonuclease/3′‐diesterase that also produces a 3′‐hydroxyl group at the AP site, but it is unrelated to either exonuclease III or endonuclease III. apn1 deletion mutants are unable to repair AP sites generated by the alkylating agent methyl methane sulphonate and display a spontaneous mutator phenotype. This work shows that either exonuclease III or endonuclease III can functionally replace yeast Apn1 in the repair of AP sites. Two conclusions can be derived from these findings. The first of these conclusions is that yeast cells can complete the repair of AP sites even though they are cleaved by AP lyase. This implies that AP lyase can contribute significantly to the repair of AP sites and that yeast cells have the ability to process the α,β unsaturated aldehyde produced by endonuclease III. The second of these conclusions is that unrepaired AP sites are strictly the cause of the high spontaneous mutation rate in the apn1 deletion mutant.
Characterization of BpH3, an H‐NS‐like protein in Bordetella pertussis
Authors: Goyard Sophie , Bertin Philippe
Published: November 2003

This study describes the characterization of BpH3, a Bordetella pertussis DNA‐binding protein. Sequence analysis reveals significant homology with the H‐NS sequence of Escherichia coli and Haemophilus influenzae, particularly in the C‐terminal part of the proteins. Our results provide evidence that H‐NS and BpH3 display functional homology. First, expression of BpH3 in an hns mutant results in restoration of motility, an H‐NS‐dependent phenotype. This effect is dependent on the level of BpH3 expression and results from transcriptional activation of the flagellar master operon. Second, the high level of β‐glucosidase associated with hns mutations is reversed to the low wild‐type level in the presence of BpH3. Third, BpH3 is able, like H‐NS, to preferentially bind in vitro to curved DNA fragments, such as flhDC and bla promoter regions. Our results are the first demonstration that proteins homologous to H‐NS exist in bacteria phylogenetically distant from H. influenzae and enterobacteria.
Iron‐regulated haemolysin gene from Edwardsiella tarda
Authors: Hirono Ikuo , Tange Nahoko , Aoki Takashi
Published: November 2003

We have cloned and sequenced the haemolysin gene locus from Edwardsiella tarda (ETH). This region encoded two open reading frames, designated ethA and ethB. ethA is the haemolysin gene consisting of 4782 bp encoding a product of 165.3 kDa and ethB is an activation/secretion protein gene of 1677 bp that encodes a product of 61.9 kDa. There were two putative ferric uptake regulator (Fur) binding sites on the 5′ upstream region of the ethB gene overlapping the promoter region and ribosome‐binding site. The haemolysin produced by the cloned gene was secreted by Escherichia coli. The deduced amino acid sequences of the ethA and ethB genes were found to be homologous to those of the haemolysin and activation/secretion proteins of Haemophilus ducreyi, Proteus mirabilis, and Serratia marcescens. E. coli carrying the ethA gene but not the ethB gene completely lost haemolytic activity, although the ethA gene was transcribed. The protein expressed by E. coli carrying a recombinant plasmid which encoded the ethA gene had haemagglutination activity. The EthB protein was necessary for activation of EthA protein (haemolysin). The ethA and ethB genes were very prevalent in haemolytic E. tarda strains isolated from diseased fish. Transcription of the ethB gene was regulated by iron. The ethA and ethB genes were transcribed independently.
Sulphation of Rhizobium sp. NGR234 Nod factors is dependent on noeE, a new host‐specificity gene
Authors: Hanin M. , Jabbouri S. , Quesada‐Vincens D. , Freiberg C. , Perret X. , Promé J.‐C. , Broughton W. J. , Fellay R.
Published: November 2003

Rhizobia secrete specific lipo‐chitooligosaccharide signals (LCOs) called Nod factors that are required for infection and nodulation of legumes. In Rhizobium sp. NGR234, the reducing N‐acetyl‐d‐glucosamine of LCOs is substituted at C6 with 2‐O‐methyl‐l‐fucose which can be acetylated or sulphated. We identified a flavonoid‐inducible locus on the symbiotic plasmid pNGR234a that contains a new nodulation gene, noeEwhich is required for the sulphation of NGR234 Nod factors (NodNGR). noeE was identified by conjugation into the closely related Rhizobium fredii strain USDA257, which produces fucosylated but non‐sulphated Nod factors (NodUSDA). R. fredii transconjugants producing sulphated LCOs acquire the capacity to nodulate Calopogonium caeruleum. Furthermore, mutation of noeE (NGRΔnoeE ) abolishes the production of sulphated LCOs and prevents nodulation of Pachyrhizus tuberosus. The sulphotransferase activity linked to NoeE is specific for fucose. In contrast, the sulphotransferase NodH of Rhizobium meliloti seems to be less specific than NoeE, because its introduction into NGRΔnoeE leads to the production of a mixture of LCOs that are sulphated on C6 of the reducing terminus and sulphated on the 2‐O‐methylfucose residue. Together, these findings show that noeE is a host‐specificity gene which probably encodes a fucose‐specific sulphotransferase.
DNase I footprinting, DNA bending and in vitro transcription analyses of ClcR and CatR interactions with the clcABD promoter: evidence of a conserved transcriptional activation mechanism
Authors: McFall Sally M. , Klem Thomas J. , Fujita Nobuyuki , Ishihama Akira , Chakrabarty A. M.
Published: November 2003

In Pseudomonas putida, benzoate and 3‐chlorobenzoate are converted to catechol and 3‐chlorocatechol, respectively, which are then catabolized to tricarboxylic acid cycle intermediates via the catBCA and clcABD pathways. The catBCA and clcABD operons are regulated by homologous transcriptional activators CatR and ClcR. Previous studies have demonstrated that in addition to sequence similarities, CatR and ClcR share functional similarities which allow catR to complement clcR. In this study, we demonstrate that CatR activates the clcABD promoter in vitro without inducer, but more transcript is produced when inducer is added. DNase I footprinting and DNA‐bending analyses demonstrate that CatR binds to and bends the clcABD promoter to the same angle as does ClcR plus its inducer, 2‐chloromuconate. This implies that CatR binds to the clc promoter in its active conformation. Transcription of the clcABD promoter by the α‐subunit truncation mutant (α‐235) of RNA polymerase was sharply reduced, indicating that the α‐subunit C‐terminal domain is important. However, a small amount of transcript was produced under these conditions, indicating that other contact sites on the RNA polymerase may play a role in activation.
Studies of the repressor (BlaI) of β‐lactamase synthesis in Staphylococcus aureus
Authors: Gregory P. D. , Lewis R. A. , Curnock S. P. , Dyke K. G. H.
Published: November 2003

Purified BlaI, the putative repressor of the β‐lactamase operon in Staphylococcus aureus, binds specifically to two regions of dyad symmetry (operators) located in the blaZ–blaR1 intergenic region. BlaI binds with similar affinity to the two regions and to the related sequence upstream of the mec gene found in methicillin‐resistant strains of S. aureus, providing physical evidence for the cross‐talk previously observed between these systems. A change from a lysine in the N‐terminus of BlaI to an alanine or deletion of the C‐terminal 23 amino acids severely reduces its DNA‐binding ability, demonstrating the functional importance of both the N‐ and C‐termini. An operator DNA–protein complex observed with crude cell lysates from repressed cells, indistinguishable from that observed with purified BlaI, was eliminated by induction of the β‐lactamase operon. Furthermore, BlaI is proteolytically cleaved in response to the addition of inducer in a blaR1‐dependent manner, providing primary evidence for the molecular basis of induction. Thus, BlaI is shown to be the repressor of the β‐lactamase system.
Paracoccus denitrificans CcmG is a periplasmic protein–disulphide oxidoreductase required for c‐ and aa3‐type cytochrome biogenesis; evidence for a reductase role in vivo
Authors: Page M. Dudley , Ferguson Stuart J.
Published: November 2003

Cloning and sequencing of the Paracoccus denitrificans ccmG gene indicates that it codes for a periplasmic protein–disulphide oxidoreductase; the presence of the sequence Cys‐Pro‐Pro‐Cys at the CcmG active site suggests that it may act in vivo to reduce disulphide bonds rather than to form them. A CcmG–PhoA fusion confirmed the periplasmic location. Disruption of the ccmG gene resulted in not only the expected phenotype of pleiotropic deficiency in c‐type cytochromes, but also loss of spectroscopically detectable cytochrome aa3, cytochrome c oxidase and ascorbate/TMPD oxidase activities; there was also an enhanced sensitivity to growth inhibition by some component of rich media and by oxidized thiol compounds. Dithiothreitol promoted the growth of the ccmG mutant on rich media and substantially restored spectroscopically detectable cytochrome aa3 and cytochrome c oxidase activity, although it did not restore c‐type cytochrome biogenesis. Assembly of the disulphide‐bridged proteins methanol dehydrogenase and Escherichia coli alkaline phosphatase was unaffected in the ccmG mutant. It is proposed that P. denitrificans CcmG acts in vivo to reduce protein–disulphide bonds in certain protein substrates including c‐type cytochrome polypeptides and/or polypeptides involved in c‐type cytochrome biogenesis.
ftsW is an essential cell‐division gene in Escherichia coli
Authors: Boyle David S. , Khattar Medhat M. , Addinall Stephen G. , Lutkenhaus Joe , Donachie William D.
Published: November 2003

In the absence of exogenous promoters, plasmid‐mediated complementation of the temperature‐sensitive ftsW201 allele requires the presence of the full coding sequence of ftsW plus upstream DNA encompassing the C‐terminus of mraY and the full coding sequence of murD. We used molecular and genetic techniques to introduce an insertional inactivation into the chromosomal copy of ftsW, in the presence of the plasmid‐borne wild‐type ftsW gene under the control of PBAD. In the absence of arabinose, the ftsW‐null strain is not viable, and a shift from arabinose‐ to glucose‐containing liquid medium resulted in a block in division, followed by cell lysis. Immunofluorescence microscopy revealed that in ftsW‐null filaments, the FtsZ ring is absent in 50–60% of filaments, whilst between one and three Z‐rings per filament can be detected in the remainder of the population, with the majority of these containing only one Z‐ring per filament. We also demonstrated that the expression of only ftsWS (the smaller of two ftsW open reading frames) from PBAD is sufficient for complementation of the ftsW‐null allele. We conclude that FtsW is an essential cell‐division protein in Escherichia coli, and that it plays a role in the stabilization of the FtsZ ring during cell division.
Cloning, sequence and mutagenesis of the structural gene of Pseudomonas aeruginosa CysB, which can activate algD transcription
Authors: Delic‐Attree I. , Toussaint B. , Garin J. , Vignais P. M.
Published: November 2003

Pseudomonas aeruginosa strains infecting patients with cystic fibrosis (CF) acquire a mucoid phenotype due to overproduction of alginate. The key enzyme in alginate synthesis is AlgD, whose promoter is transcriptionally active in mucoid strains and under the control of several trans‐acting factors, including the integration host factor (IHF). The algD promoter (palgD ) contains two IHF‐binding sites (ihf1 and ihf2 ). Study of IHF binding to ihf2 of palgD, by electrophoretic mobility‐shift assays, led to the discovery of a protein of 36 kDa (p36) able to bind downstream from ihf2, to the 3′ region of palgD. The gene encoding p36 was isolated from the mucoid strain CHA of P. aeruginosa and sequenced. It can encode a 324‐amino‐acid protein, which shares a high degree of sequence identity (63%) with CysB from Escherichia coli and from Salmonella typhimurium, a transcriptional factor of the LysR superfamily. Furthermore, both p36 and S. typhimurium CysB bind the same site of palgD ; p36 was therefore termed CysB and its structural gene was called cysB. Next to cysB, on the opposite DNA strand, cysH was capable of encoding a protein sharing 26% identity with CysH (PAPS reductase) of E. coli and an even greater identity (54%) with the nucleotide‐deduced protein from Arabidopsis. A CysB‐deficient mutant of CHA, constructed by insertional inactivation of cysB, was a cysteine auxotroph and was unable to form a specific complex with palgD in vitro. Activity of palgD in the cysB mutant, in CHA and in the non‐mucoid strain PAO was assessed by the use of a transcriptional algD–xylE fusion. Cells of PAO and of the cysB mutant grown in minimal media in the presence of 0.3 M NaCl exhibited a palgD activity, which was 10% or less that of the mucoid strain CHA. Thus, P. aeruginosa CysB can act as an activator of algD expression.
Mutations in a dispensable region of the UaY transcription factor of Aspergillus nidulans differentially affect the expression of structural genes
Authors: Oestreicher Nathalie , Scazzocchio Claudio , Suárez Teresa
Published: November 2003

The uaY gene encodes a transcriptional activator mediating uric acid induction of at least nine genes of the purine‐utilization pathway. In this article, we characterize a loss‐of‐function mutation, uaY205, as a 16 bp deletion that results in premature translation termination, and substitutes the C‐terminal 63 amino acids for 13 amino acid residues. Reversion analysis demonstrates that the C‐terminal 63 amino acid residues are unnecessary for UaY function, and that the loss‐of‐function phenotype resulting from the uaY205 mutation is caused by the new amino acid sequence present in the mutant protein. Revertants in two different frames (wild type and +1) restore function but show subtle differences in the expression of genes controlled by the UaY protein. Two strains showing elevated expression of genes under UaY control were shown to carry, in addition to a mutation leading to the recovery of the wild‐type open reading frame, mutations in unlinked genes. Using crude extracts of Aspergillus nidulans, we have been able to detect, for the first time, in transcription factors of this class, specific retardation of a promoter probe. The binding activity is at least partially dependent on the presence of inducer. The gel shift experiments show that the novel inhibitory sequence present in the UaY205 protein can act either by affecting the stability of the protein, or via an inter‐ or intramolecular interaction impairing the specific DNA‐binding activity.
Regulation of the Escherichia coli K5 capsule gene cluster by transcription antitermination
Authors: Stevens Mark P. , Clarke Bradley R. , Roberts Ian S.
Published: November 2003

The expression of the Escherichia coli K5 (group II) capsular polysaccharide requires the rfaH gene. By reverse transcriptase‐polymerase chain reaction (RT‐PCR) it was possible to demonstrate that RfaH increases the transcription of region 2 genes by readthrough transcription from the region 3 promoter. A mutation in the rfaH gene reduced this readthrough transcription from the region 3 promoter by 10‐fold as measured by quantitative RT‐PCR. The region 3 promoter was mapped to 741 bp 5′ of the initiation codon of the kpsM gene. Deletion and insertion mutagenesis of the JUMPstart sequence, which is 28 bp 5′ of kpsM and is conserved upstream of RfaH‐regulated operons and other polysaccharide biosynthesis genes, confirmed that this sequence was required for expression of the K5 antigen and for the antitermination activity of RfaH. The JUMPstart sequence could cause RfaH‐dependent antitermination at other Rho‐dependent terminators, suggesting that the JUMPstart sequence may function in a manner analogous to a λnut site. On the basis of these results we propose a model by which RfaH regulates expression of E. coli group II capsule gene clusters by allowing readthrough transcription to proceed from region 3 into region 2 and that sequences within the JUMPstart sequence are essential for this process.
Quorum sensing by peptide pheromones and two‐component signal‐transduction systems in Gram‐positive bacteria
Authors: Kleerebezem Michiel , Quadri Luis E. N. , Kuipers Oscar P. , De Vos Willem M.
Published: November 2003

Cell‐density‐dependent gene expression appears to be widely spread in bacteria. This quorum‐sensing phenomenon has been well established in Gram‐negative bacteria, where N‐acyl homoserine lactones are the diffusible communication molecules that modulate cell‐density‐dependent phenotypes. Similarly, a variety of processes are known to be regulated in a cell‐density‐ or growth‐phase‐dependent manner in Gram‐positive bacteria. Examples of such quorum‐sensing modes in Gram‐positive bacteria are the development of genetic competence in Bacillus subtilis and Streptococcus pneumoniae, the virulence response in Staphylococcus aureus, and the production of antimicrobial peptides by several species of Gram‐positive bacteria including lactic acid bacteria. Cell‐density‐dependent regulatory modes in these systems appear to follow a common theme, in which the signal molecule is a post‐translationally processed peptide that is secreted by a dedicated ATP‐binding‐cassette exporter. This secreted peptide pheromone functions as the input signal for a specific sensor component of a two‐component signal‐transduction system. Moreover, genetic linkage of the common elements involved results in autoregulation of peptide‐pheromone production.