bluuebunny/crossref_metadata_2025 huggingface dataset:
Co‐ordinate regulation of Salmonella typhimurium invasion genes by environmental and regulatory factors is mediated by control of hilA expression
Authors: Bajaj Vivek , Lucas Robin L. , Hwang Clara , Lee Catherine A.
Published: November 2003

During infection of their hosts, salmonellae enter intestinal epithelial cells. It has been proposed that when Salmonellatyphimurium is present in the intestinal lumen, several environmental and regulatory conditions modulate the expression of invasion factors required for bacterial entry into host cells. We report here that the expression of six different S. typhimurium invasion genes encoded on SPI1 (Salmonella pathogenicity island 1) is co‐ordinately regulated by oxygen, osmolarity, pH, PhoPQ, and HilA. HilA is a transcriptional activator of the OmpR/ToxR family that is also encoded on SPI1. We have found that HilA plays a central role in the co‐ordinate regulation of invasion genes by environmental and regulatory conditions. HilA can activate the expression of two invasion gene–lacZY fusions on reporter plasmids in Escherichia coli, suggesting that HilA acts directly at invasion‐gene promoters in S. typhimurium. We have found that the regulation of invasion genes by oxygen, osmolarity, pH, and PhoPQ is indirect and is mediated by regulation of hilA expression by these environmental and regulatory factors. We hypothesize that the complex and co‐ordinate regulation of invasion genes by HilA is an important feature of salmonella pathogenesis and allows salmonellae to enter intestinal epithelial cells.
Expression of the Bacillus subtilis gabP gene is regulated independently in response to nitrogen and amino acid availability
Authors: Ferson Amy E. , Wray, Jr Lewis V. , Fisher Susan H.
Published: November 2003

Expression from the Bacillus subtilis nrg‐21 locus increases 26‐fold during nitrogen‐limited growth. The DNA corresponding to this locus was cloned and sequenced. The nucleotide sequence revealed a gene that could encode a protein with sequence similarity to the Escherichia coliγ‐aminobutyric acid (GABA) permease. A transposon insertion in this locus eliminated the uptake of GABA and severely inhibited the utilization of GABA as a nitrogen source. Primer extension analysis revealed that the B. subtilis gabP gene was transcribed from two overlapping promoters. Transcription from the P1 promoter was repressed during growth in the presence of amino acids. The product of the codY gene proved to be required for this repression. Transcription from the P2 promoter increased during nitrogen‐limited growth and was dependent upon the product of the tnrA gene. Deletion analysis revealed that activation of the P2 promoter during nitrogen‐limited growth requires a nucleotide sequence located upstream of its −35 region. Regulation of gabP expression by the CodY and TnrA regulatory systems, which respond to different physiological signals, allows for a wide range of gabP expression during growth on various nitrogen sources.
Effect of UVM induction on mutation fixation at non‐pairing and mispairing DNA lesions
Authors: Sayeedur Rahman M. , Dunman Paul M. , Wang Ge , Murphy Holly S. , Zafri Humayun M.
Published: November 2003

Mutation fixation at an ethenocytosine (εC) residue borne on transfected M13 single‐stranded DNA is significantly enhanced in response to pretreatment of Escherichia coli cells with UV, alkylating agents or hydrogen peroxide, a phenomenon that we have called UVM for UV modulation of mutagenesis. The UVM response does not require the E. coli SOS or adaptive responses, and is observed in cells defective for oxyR, an oxidative DNA damage‐responsive regulatory gene. UVM may represent either a novel DNA‐repair phenomenon, or an unrecognized feature of DNA replication in damaged cells that affects a specific class of non‐coding DNA lesions. To explore the range of DNA lesions subject to the UVM effect, we have examined mutation fixation at 3,N 4‐ethenocytosine and 1,N 6‐ethenoadenine, as well as at O6‐methylguanine (O6mG). M13 viral single‐stranded DNA constructs bearing a single mutagenic lesion at a specific site were transfected into cells pretreated with UV or 1‐methyl‐3‐nitro‐1‐nitrosoguanidine (MNNG). Survival of transfected viral DNA was measured as transfection efficiency, and mutagenesis at the lesion site was analysed by a quantitative multiplex sequence analysis technology. The results suggest that the UVM effect modulates mutagenesis at the two etheno lesions, but does not appear to significantly affect mutagenesis at O6mG. Because the modulation of mutagenesis is observed in cells incapable of the SOS response, these data are consistent with the notion that UVM may represent a previously unrecognized DNA damage‐inducible response that affects the fidelity of DNA replication at certain mutagenic lesions in Escherichia coli.
Stationary‐phase induction of GLR1 expression is mediated by the yAP‐1 transcriptional regulatory protein in the yeast Saccharomyces cerevisiae
Authors: Grant Chris M. , MacIver Fiona H. , Dawes Ian W.
Published: November 2003

Glutathione (GSH) is an abundant low‐molecular‐mass thiol which has been implicated in numerous cellular processes including protection against cytotoxic agents such as xenobiotics, carcinogens and free radicals. Utilization of GSH results in its conversion to the oxidized form (GSSG), and it is recycled to GSH by the action of glutathione reductase (GLR) using the reducing power of NADPH. We show that GLR activity is increased by three‐ to fourfold during stationary‐phase growth compared to exponential phase growth, and that a yeast strain deleted for GLR1, encoding glutathione reductase, shows an elevated sensitivity to H2O2 challenge during stationary phase. These data indicate an increased requirement for GSH as the cell arrests growth and enters stationary phase. The stationary‐phase increase in GLR activity is entirely dependent upon the action of the yAP‐1 transcriptional regulatory protein, previously implicated in regulating GLR activity in response to oxidative stress. Thus, both oxidant‐ and growth phase‐mediated control of GLR1 expression are regulated by the same transcriptional control mechanism. In addition, strains lacking GLR or yAP‐1 do not accumulate GSSG during stationary‐phase growth, indicating that the cell possesses alternative means of preventing an accumulation of GSSG during stationary phase.
The Aspergillus nigerGCN4 homologue, cpcA, is transcriptionally regulated and encodes an unusual leucine zipper
Authors: Wanke Christoph , Eckert Sabine , Albrecht Gerd , Van Hartingsveldt Wim , Punt Peter J. , Van Den Hondel Cees A.M.J.J. , Braus Gerhard H.
Published: November 2003

The general control transcriptional regulator gene cpcA of Aspergillus niger was cloned by complementation of a Saccharomyces cerevisiaeΔgcn4 mutant strain. The encoded protein conferred resistance to amino acid analogues when expressed in yeast. Disruption of cpcA in A. niger resulted in a strain which is sensitive towards 3‐aminotriazole and fails to respond to amino acid starvation. cpcA encodes a transcript of ≈2400 nucleotides in length that includes a 5′ leader region of 900 nucleotides. The 5′ leader region contains two small open reading frames, suggesting translational control of gene expression. Steady‐state mRNA levels of cpcA increase by a factor of three upon amino acid starvation. The coding region of cpcA is interrupted by a 57 bp intron and the deduced amino acid sequence displays an ≈30% overall identity to yeast GCN4p and Neurospora crassa cpc‐1p. Critical amino acid residues of the transcriptional activation domains of GCN4p are conserved in cpcAp. The basic DNA‐binding domain shows up to 70% amino acid sequence identity to other basic zipper (bZIP)‐type transcriptional activators. cpcAp binds specifically to a GCN4p recognition element in gel retardation experiments. The C‐terminal dimerization domain encodes a leucine zipper with only a single leucine residue.
Vitamin B12 repression of the btuB gene in Salmonella typhimurium is mediated via a translational control which requires leader and coding sequences
Authors: Ravnum S. , Andersson D. I.
Published: November 2003

Expression of the Salmonella typhimurium btuB gene, which encodes an outer membrane protein required for vitamin B12 uptake, is repressed by the presence of external vitamin B12. We have, by means of a mutational analysis, investigated which btuB sequences are required for repression. Analysis of btuB::lacZ transcriptional and translational fusions of various lengths showed that the control was exerted mainly at the translational level and required both coding and leader sequences in the btuB transcript. Regulatory mutants with a B12 non‐repressible phenotype were isolated and the mutations were shown to be located at several sites within the btuB leader. Analysis of constructs carrying site‐directed point mutations, which either destabilized or restabilized a putative RNA hairpin that sequesters the btuB ribosomal binding site, demonstrated that this hairpin was essential for normal repression. Comparison of the S. typhimurium btuB gene with the previously characterized S. typhimurium cbiA and Escherichia coli btuB genes reveals significant similarities as well as differences in the cis‐acting sequences required for repression.
Disruption of hspR, the repressor gene of the dnaK operon in Streptomyces albus G
Authors: Grandvalet Cosette , Servant Pascale , Mazodier Philippe
Published: November 2003

hspR is the distal gene of the Streptomyces albus dnaK operon. It encodes a protein similar to GlnR, the repressor of the Bacillus subtilis glutamine synthetase gene. Transcriptional analysis showed that disruption of hspR led to constitutive high‐level expression of the dnaK operon. SDS–PAGE analysis revealed overproduction and accumulation of the chaperone DnaK at low temperature. HSP94, a heat‐inducible protein cross‐reacting with anti‐ClpB antibodies, was also shown to be constitutively overexpressed at low temperature in the hspR mutant. Those features were lost when the mutant was complemented in trans by an intact copy of hspR. The hspR mutant was impaired in its growth on solid rich medium: colonies grow slowly at 30°C. However, formation of aerial mycelium and sporulation was not prevented. In liquid culture growth curves of the mutant and the wild type were similar. The kinetics of groEL gene induction were not modified by the hspR null mutation, indicating that HspR was not directly involved in the control of groEL transcription. Thus, in contrast with B. subtilis and other Gram‐positive bacteria, transcription of StreptomycesdnaK and groEL operons is not controlled by the same regulator.
Molecular and chemical characterization of the lipopolysaccharide O‐antigen and its role in the virulence of Yersinia enterocolitica serotype O:8
Authors: Zhang Lijuan , Radziejewska‐Lebrecht Joanna , Krajewska‐Pietrasik Danuta , Toivanen Paavo , Skurnik Mikael
Published: November 2003

The Y. enterocolitica O:8 (YeO8) O‐antigen repeat units consist of five sugar residues: N‐acetyl‐d‐galactosamine (GalNAc), d‐galactose (Gal), d‐mannose (Man), l‐fucose (Fuc), and 6‐deoxy‐d‐gulose (6d‐Gul). The nucleotide sequence of the O‐antigen gene cluster of the YeO8 strain 8081‐c was determined. Altogether, 18 open reading frames (ORFs) were identified and shown to be essential for O‐antigen biosynthesis. We previously characterized the 3′‐end of the O‐antigen gene cluster and identified four genes: two for GDP‐Man biosynthesis, one for UDP‐Gal biosynthesis, and one for O‐antigen polymerase. Based on sequence similarity, Tn5‐insertion phenotypes and chemical analysis, the 14 new genes were assigned the following functions: four genes are involved in the biosynthesis of CDP‐6d‐Gul and two in GDP‐Fuc biosynthesis. Five gene products were assigned sugar transferase functions and one gene product was similar to Wzx, the O‐antigen flippase. Two genes remained unassigned. By genetic complementation we also showed that YeO8 O‐antigen biosynthesis was dependent on N‐acetyl‐glucosaminyl:undecaprenylphosphate transferase (GlcNAc transferase), the WecA (formerly known as Rfe) protein. Data obtained from chemical‐composition analysis suggest that in addition to being GlcNAc transferase, WecA may also function as a GalNAc transferase. Using a restriction‐deficient derivative of Y. enterocolitica O:8 strain 8081, a rough mutant, designated 8081‐R2, was isolated. 8081‐R2 was complemented in trans with a cloned O‐antigen gene cluster restoring surface O‐antigen expression. The virulence of the wild‐type strain and that of the complemented strain were significantly higher (approx. 100‐fold) than that of the rough mutant in an orally infected mouse model, showing that YeO8 O‐antigen is a virulence factor.
Promoter strength influences phase variation of neisserial opa genes
Authors: Belland Robert J. , Morrison S. G. , Carlson John H. , Hogan Daniel M.
Published: November 2003

The opa multigene family of Neisseria gonorrhoeae encodes 11 related outer‐membrane proteins which phase vary in vitro and in vivo. Illegitimate recombination within direct pentameric DNA repeats, encoding the signal‐peptide region of pre‐Opas, leads to switches in expression states. Despite the conserved nature of the variation mechanism, specific genes are expressed at high frequencies in the transition from Opa– to Opa+. The genes which are expressed at elevated frequencies differ from the rest of the family with respect to promoter structure, based on sequence comparisons between the opa genes of strain MS11mk. We have analysed transcription of the opa gene family of N. gonorrhoeae MS11mk, focussing on the different promoters found among the 11 genes to determine whether increased levels of expression are associated with increased phase‐variation rates. Primer extension and Northern blotting was used to assess the levels of transcription of three representative opa genes (opaA, B and C) in ‘on’ and ‘off’ states. Full‐length opa mRNA was detected primarily in strains expressing the homologous gene. Truncated opa mRNA was constitutively expressed from all opa genes regardless of their expression state. Quantitative comparisons in N. gonorrhoeae were complicated by the simultaneous expression of all 11 genes and the cross‐reactivity of mRNA probes. Expression levels from the individual promoters were therefore assessed by creating transcriptional and translational lacZ fusions to each of the representative opa promoters which lacked the DNA repeats responsible for variation. The expression levels were compared to the phase‐variation rates of translational opa::phoA fusions containing the same promoters in addition to the corresponding coding repeat regions. A strong correlation was found between expression levels from the different promoters and the variation rates at which ‘on’ variants appeared from an ‘off’ population (i.e. opaA > opaB > opaC). These results provide an explanation for the favoured expression of specific Opa proteins and indicate that expression of opa genes may be regulated at the level of transcription.
A conserved helicase motif of the AddA subunit of the Bacillus subtilis ATP‐dependent nuclease (AddAB) is essential for DNA repair and recombination
Authors: Kooistra Jan , Haijema Bert Jan , Hesseling‐Meinders Anne , Venema Gerard
Published: November 2003

Various mutations were introduced in a conserved helicase domain (motif VI) of the AddA subunit of the Bacillus subtilis ATP‐dependent nuclease (AddAB) by site‐directed mutagenesis. These mutations affected the helicase activity and the ATP‐dependent exonuclease activity on double‐stranded DNA (dsDNA) as the substrate to various degrees, but had hardly any effect on the exonuclease activity on single‐stranded DNA (ssDNA), suggesting that exonuclease activity on dsDNA of the enzyme requires unwinding of the DNA. This idea was supported by the finding that, initially, the rate and extent of unwinding of the DNA were higher than those of its degradation to acid‐soluble products by the exonucleolytic activity. The effects of the mutations on DNA repair and recombination correlated strongly with their effects on helicase activity. Taken together, these results suggest that motif VI is essential for the helicase activity, and that this activity is required for DNA repair and recombination.
Isolation and analysis of a gene encoding α‐glucuronidase, an enzyme with a novel primary structure involved in the breakdown of xylan
Authors: Ruile Peter , Winterhalter Christoph , Liebl Wolfgang
Published: November 2003

This is the first report describing the analysis of a gene encoding an α‐glucuronidase, an enzyme essential for the complete breakdown of substituted xylans. A DNA fragment that carries the gene for α‐glucuronidase was isolated from chromosomal DNA of the hyperthermophilic bacterium Thermotoga maritima MSB8. The α‐glucuronidase gene (aguA) was identified and characterized with the aid of nucleotide sequence analysis, deletion experiments and expression studies in Escherichia coli, and the start of the coding region was defined by amino‐terminal sequencing of the purified recombinant enzyme. The aguA gene encodes a 674‐amino‐acid, largely hydrophilic polypeptide with a calculated molecular mass of 78 593 Da. The α‐glucuronidase of T. maritima has a novel primary structure with no significant similarity to any other known amino acid sequence. The recombinant enzyme was purified to homogeneity as judged by SDS–PAGE. Gel filtration analysis at low salt concentrations revealed a high apparent molecular mass (<630kDa) for the recombinant enzyme, but the oligomeric structure changed upon variation of the ionic strength or the pH, yielding hexameric and/or dimeric forms which were also enzymatically active. The enzyme hydrolysed 2‐O‐(4‐O‐methyl‐α‐d‐glucopyranosyluronic acid)‐d‐xylobiose (MeGlcAX2) to xylobiose and 4‐O‐methylglucuronic acid. The Km for MeGlcAX2 was 0.95mM. The pH optimum was 6.3. Maximum activity was measured at 85°C, about 25°C or more above the values reported for all other α‐glucuronidases known to date. When incubated at 55–75°C, the enzyme suffered partial inactivation, but thereafter the residual activity remained nearly constant for several days.
Identification and analysis of genes from Streptomyces pristinaespiralis encoding enzymes involved in the biosynthesis of the 4‐dimethylamino‐l‐phenylalanine precursor of pristinamycin I
Authors: Blanc Véronique , Gil Patricia , Bamas‐Jacques Nathalie , Lorenzon Sophie , Zagorec Monique , Schleuniger Josiane , Bisch Didier , Blanche Francis , Debussche Laurent , Crouzet Joe¨l , Thibaut Denis
Published: November 2003

Four pap genes (papA, papB, papC, papM ) were found by sequencing near to snbA, a Streptomyces pristinaespiralis gene which was previously shown to encode one of the pristinamycin I (PI) synthetases. Analysis of the homologies observed from the deduced amino acid sequences suggested that these four genes could be involved in the biosynthesis of the PI precursor 4‐dimethylamino‐l‐phenylalanine (DMPAPA). This was first verified when disruption of papA in S. pristinaespiralis led to a PI− phenotype, which was reversed by the addition of DMPAPA into the culture medium. Further confirmation was obtained when papM was overexpressed in Escherichia coli and the corresponding protein purified to homogeneity. It catalysed the two successive N‐methylation steps of 4‐amino‐l‐phenylalanine leading to DMPAPA via 4‐methylamino‐l‐phenylalanine. These results allowed us to assign a function to each of the four pap genes and to propose a biosynthetic pathway for DMPAPA.
The leader peptide is essential for the post‐translational modification of the DNA‐gyrase inhibitor microcin B17
Authors: Madison Lara L. , Vivas Eugenio I. , Li Yue‐Ming , Walsh Christopher T. , Kolter Roberto
Published: November 2003

Microcin B17 (MccB17) is a ribosomally encoded DNA‐gyrase inhibitor. Ribosomally encoded antibiotics are derived from precursors containing an N‐terminal leader, which is removed during maturation, and a C‐terminal structural peptide. PreMccB17, the translational product of mcbA, is modified into proMccB17 by the action of three enzymes, McbB, McbC, and McbD. A chromosomally encoded peptidase then converts proMccB17 into MccB17. The role of McbB, McbC, and McbD is to convert glycine, cysteine, and serine residues present in preMccB17 into four thiazole and four oxazole rings. Using a modification‐specific antibody rather than antimicrobial activity, we show that the 26‐amino‐acid N‐terminal leader of preMccB17 is essential for the conversion of preMccB17 into proMccB17. Neither a preMccB17 peptide lacking the leader nor a preMccB17–β‐galactosidase fusion lacking the leader are post‐translationally modified.
Plasmid replication and partition in Escherichiacoli: is the cell membrane the key?
Authors: Firshein William , Kim Peter
Published: November 2003

The DNA–membrane complex has been the subject of intensive investigation for over 35 years as the possible site for DNA replication in the prokaryotic cell and the site through which newly synthesized chromosomes are segregated into daughter cells. However, the molecular mechanisms which control these phenomena are, for the most part, poorly understood despite genetic, biochemical, and morphologic evidence in favour of their existence. This is probably due to the transient nature and non‐covalent interactions that occur between DNA and the membrane. In addition, there is a paucity of knowledge concerning the nature of the membrane receptors for DNA and whether the membrane plays simply a structural or metabolic role in the two processes. Plasmids can provide important insights into the role of the membrane in replication and partitioning because the plasmid life cycle is relatively simple, with replication occurring during the cell cycle and partitioning during cell division. The replicon model of Jacob et al. (1963, Cold Spring Harbor Symp Quant Biol 28: 329–348) still represents a good conceptual framework (with modifications) to explain how plasmid replication and partitioning are linked by the membrane. In its simplest form, the model focuses on specific membrane binding sites (possibly along the equator of the cell) for plasmid (or bacterial) replication, with the membrane acting as a motive force to separate the newly synthesized replicons and their attached sites into daughter cells. Indeed, proteins involved in both plasmid replication and partitioning have been found in membrane fractions and some plasmids require membrane binding for initiation and an active partitioning. We propose that several factors are critical for both plasmid DNA replication and partitioning. One factor is the extent of negative supercoiling (brought about by an interplay of various topoisomerases, but most importantly by DNA gyrase). Supercoiling is known to be critical for initiation of DNA replication but may also be important for the formation of a partition complex in contact with the cell membrane. Another factor is the presence of specific subdomains of the membrane which can interact specifically with origin DNA and possibly other regions involved in partitioning. Such domains may be induced transiently or be present at all times during the cell cycle.
Characterization of a mitogen‐activated protein (MAP) kinase from Plasmodium falciparum
Authors: Graeser Ralph , Ku¨ry Patrick , Franklin Richard M. , Kappes Barbara
Published: November 2003

A mitogen‐activated protein (MAP) kinase gene, PfMAP, from Plasmodium falciparum was recently identified. We expressed this gene in Escherichia coli to test whether it encodes a functional MAP kinase. Recombinant PfMAP kinase autophosphorylates on both the tyrosine and threonine residues within the TXY motif, and readily phosphorylates myelin basic protein as exogenous substrate. This identifies the PfMAP gene product as a true member of the growing family of MAP kinases. Wild‐type PfMAP kinase expressed in COS‐7 (SV40 transformed African green monkey kidney) cells seemed to induce apoptosis in these cells. Western blots and immunoprecipitations indicated that the kinase is expressed during the growth of the parasite in the red blood cell as three major forms: truncated forms with apparent molecular masses of 40 kDa and 80 kDa, and as a protein of ≈150 kDa. The 40 kDa form is present throughout the intraerythrocytic development, whereas the two larger forms are only detected in mature parasites. The 40 kDa and 80 kDa forms are tyrosine phosphorylated, indicating that they represent the active forms of the PfMAP kinase. The total PfMAP kinase activity constantly increases with the maturation of the parasite.
SpoIVB has two distinct functions during spore formation in Bacillus subtilis
Authors: Oke Valerie , Shchepetov Mikhail , Cutting Simon
Published: November 2003

The Bacillus subtilis SpoIVB protein is a critical component of the intercompartmental signal‐transduction pathway that activates the sigma factor, σK, in the mother cell of the sporulating cell. SpoIVB, synthesized in the forespore chamber, must act across two layers of phospholipid membrane to facilitate proteolytic processing of inactive pro‐σK to active σK. We have used a genetic approach to dissect SpoIVB function and found that this protein has two distinct developmental functions. One function is that of intercompartmental signalling of pro‐σK processing. The other role is essential to spore formation and is illustrated by mutations of SpoIVB which allow cell–cell signalling of pro‐σK processing but prevent the formation of viable spores. Using localized and site‐specific mutagenesis we have identified a functional domain of SpoIVB that is involved in its non‐signalling role.
Plasmid stability: comments on the dimer catastrophe hypothesis
Authors: Boe L. , Tolker‐Nielsen T.
Published: November 2003

Using a derivative of the plasmid pBR322 we have tested the dimer catastrophe hypothesis of plasmid instability. Most of the theory was confirmed by our observations, but our data suggest that some of the quantitative aspects need modification. In a recF strain of Escherichia coli we estimated the difference in loss rate between the plasmid in the monomeric and the dimeric state to be a factor of 13–14 and the difference in the loss rate between the plasmid in the monomeric and the trimeric state to be a factor of 14–50. We were able to confirm that plasmid oligomers were heterogeneously distributed within a rec+ population, but we were unable to detect any pronounced difference in the level of growth inhibition exerted by the plasmid when in the monomeric, dimeric, or trimeric state. This leaves open the question as to whether runaway plasmid multimerization was prevented (i) by a small correlation between the inhibition of growth and the ‘multimeric status’ of the plasmid, (ii) by intramolecular homologous recombination, or (iii) whether the process of runaway multimerization is too slow to be recognized within the duration of the experiments, i.e. 200 generations of growth.
Increase in negative supercoiling of plasmid DNA in Escherichia coli exposed to cold shock
Authors: Mizushima Tohru , Kataoka Kazuhiro , Ogata Yasuyuki , Inoue Ryu‐ichi , Sekimizu Kazuhisa
Published: November 2003

Negative supercoiling of plasmid DNA in Escherichia coli cells can decrease transiently when exposed to heat shock. The effect of cold shock on DNA supercoiling was examined, and analysis by agarose gel electrophoresis in the presence of chloroquine revealed that negative supercoiling of plasmid DNA in cells increased when cells were exposed to cold shock. This increase was transient and was nil when the cells were pretreated with nalidixic acid, an inhibitor of DNA gyrase. In a mutant deficient in expression of HU protein, the increase in negative supercoiling of DNA by cold shock is less apparent than in wild‐type cells. It is proposed that DNA gyrase and HU protein have a role in the DNA supercoiling reaction seen with cold shock.
Integration host factor is a transcriptional cofactor of pilE in Neisseria gonorrhoeae
Authors: Hill Stuart A. , Samuels D. Scott , Carlson John H. , Wilson Jeanne , Hogan Dan , Lubke Lori , Belland Robert J.
Published: November 2003

Integration host factor (IHF) is a small, heterodimeric DNA‐binding protein with pleiotropic function. IHF was purified to apparent homogeneity from Neisseria gonorrhoeae. Gel‐retardation assays demonstrated binding of IHF to the pilE promoter region. The IHF‐binding site was identified by DNase I protection assays and mapped proximal to three previously defined pilE promoters. Removal of the putative IHF‐binding domain from pilE promoter DNA negated retardation of the DNA fragment when assessed by gel‐shift analysis. Kleinschmidt electron microscopy showed pronounced kinking of pilE promoter DNA following incubation with IHF. Isogenic N. gonorrhoeae strains were constructed that contained either a wild‐type pilE locus or a deleted pilE locus where the IHF‐binding domain was removed. Primer‐extension analysis and Northern blotting of total gonococcal RNA showed that in the absence of IHF binding at the pilE promoter, transcription was reduced 10‐fold. Together, these data indicate that IHF is a transcriptional co‐activator of pilE.
Towards a structural biology of bacterial conjugation
Authors: Silverman Philip M.
Published: November 2003

Horizontal DNA transfer among bacteria (conjugation) requires the formation of secure intercellular contacts before DNA transfer can occur. The formation of such contacts among Gram‐negative bacteria is mediated by conjugative pili. Like other pili, conjugative pili are filaments extending from the surface of donor cells. They are composed, in so far as is known, entirely of conjugative pilin subunits. Here, we review the structure of F‐pilin, the subunit of which F‐pili are composed. We emphasize recent studies suggesting a specific domain organization for F‐pilin and present a model of how these domains might be arranged in filament subunits.
Molecular characterization of hpuAB, the haemoglobin–haptoglobin‐utilization operon of Neisseria meningitidis
Authors: Lewis Lisa A. , Gray Elizabeth , Wang Ying‐Ping , Roe Bruce A. , Dyer David W.
Published: November 2003

We previously identified HpuB, an 85 kDa Fe‐repressible protein required for utilization of Fe from, and binding to, haemoglobin and the haemoglobin–haptoglobin complex. The gene for hpuB was cloned from Neisseria meningitidis strain DNM2 and the predicted amino acid sequence indicates that HpuB is an outer membrane receptor belonging to the TonB family of high‐affinity transport proteins. A second open reading frame, predicted to encode a 34.8 kDa lipoprotein, was discovered 5′ to hpuB, and was designated hpuA. HpuA was identified in a total‐membrane‐protein preparation by construction of a mutant lacking HpuA. Acylation of HpuA was confirmed by [3H]‐palmitic acid labelling of meningococci. Consensus promoter sequences were not apparent 5′ to hpuB. The hpuA insertion mutation exerted a polar effect, abolishing expression of hpuB, suggesting that hpuA and hpuB are co‐transcribed. The 3.5 kb polycistronic hpuAB mRNA was identified and shown to be transcriptionally repressed by iron. The transcriptional start site was identified 33 nucleotides 5′ to the hpuA translational start site, appropriately positioned around consensus promoter and ferric uptake regulator (Fur)‐box sequences. The structure of this operon suggests that HpuA–HpuB is a two‐component receptor analogous to the bipartite transferrin receptor TbpB–TbpA.
PilC of Neisseria meningitidis is involved in class II pilus formation and restores pilus assembly, natural transformation competence and adherence to epithelial cells in PilC‐deficient gonococci
Authors: Ryll Roland R. , Rudel Thomas , Scheuerpflug Ina , Barten Roland , Meyer Thomas F.
Published: November 2003

Type 4 pili produced by the pathogenic Neisseria species constitute primary determinants for the adherence to host tissues. In addition to the major pilin subunit (PilE), neisserial pili contain the variable PilC proteins represented by two variant gene copies in most pathogenic Neisseria isolates. Based upon structural differences in the conserved regions of PilE, two pilus classes can be distinguished in Neisseria meningitidis. For class I pili found in both Neisseria gonorrhoeae and N. meningitidis, PilC proteins have been implicated in pilus assembly, natural transformation competence and adherence to epithelial cells. In this study, we used primers specific for the pilC2 gene of N. gonorrhoeae strain MS11 to amplify, by the polymerase chain reaction, and clone a homologous pilC gene from N. meningitidis strain A1493 which produces class II pili. This gene was sequenced and the deduced amino acid sequence showed 75.4% and 73.8% identity with the gonococcal PilC1 and PilC2, respectively. These values match the identity value of 74.1% calculated for the two N. gonorrhoeae MS11 PilC proteins, indicating a horizontal relationship between the N. gonorrhoeae and N. meningitidispilC genes. We provide evidence that PilC functions in meningococcal class II pilus assembly and adherence. Furthermore, expression of the cloned N. meningitidis pilC gene in a gonococcal pilC1,2 mutant restores pilus assembly, adherence to ME‐180 epithelial cells, and transformation competence to the wild‐type level. Thus, PilC proteins exhibit indistinguishable functions in the context of class I and class II pili.
Construction and analysis of a recombination‐deficient (radA) mutant of Haloferax volcanii
Authors: Woods Wayne G. , Dyall‐Smith Michael L.
Published: November 2003

By deleting the radA open reading frame of an extreme halophile, Haloferax volcanii, we created and characterized a recombination‐deficient archaeon. This strain, Hf. volcanii DS52, has no detectable DNA recombination, is more sensitive to DNA damage by UV light and ethylmethane sulfonate, and has a slower growth rate than the wild type. These characteristics are similar to those observed in recombination mutants of Eukarya and Bacteria, and show that the radA gene belongs in the recA/RAD51 family by function as well as sequence homology. In addition, strain DS52 was not transformable by plasmids pWL102 or pUBP2 (which contain pHV2 and pHH1 replicons, respectively), although it was readily transformed by plasmids containing a pHK2 replicon, indicating a role for radA in the maintenance or replication of some halobacterial plasmids. Despite its slower growth rate, Hf. volcanii DS52 was still easy to culture and transform, and should be suitable for use in studies where a recombination‐deficient background is desired.
Region‐specific meiotic recombination in Schizosaccharomyces pombe: the rec11 gene
Authors: Li Ywan Feng , Numata Masayuki , Wahls Wayne P. , Smith Gerald R.
Published: November 2003

Mutations in the rec11 gene of Schizosaccharomyces pombe reduce meiotic recombinant frequencies by as much as a factor of 300 on chromosome III but less than a factor of 4 in the intervals tested on chromosomes I and II. To gain insight into the function of this region‐ (or chromosome‐) specific activator of recombination, we have cloned and sequenced the rec11 gene. Meiotic crosses with rec11 disruption mutations placed the rec11 gene 6 cM from ade6 on chromosome III. Transcripts of rec11 accumulated transiently at 2–3 h after induction of meiosis in a pat1‐114 (Ts) mutant. Reverse transcriptase/polymerase chain reaction (RT‐PCR) analysis of these transcripts revealed eight introns. The spliced RNA is predicted to encode a polypeptide of 923 amino acids with only very limited homology to reported proteins. The transient accumulation of rec11 transcripts and the phenotype of rec11 mutations suggest that the novel rec11 gene product acts early in meiosis to activate recombination preferentially on chromosome III.
The penicillin sensory transducer, BlaR, involved in the inducibility of β‐lactamase synthesis in Bacillus licheniformis is embedded in the plasma membrane via a four‐α‐helix bundle
Authors: Hardt Karin , Joris Bernard , Lepage Sophie , Brasseur Robert , Lampen J. Oliver , Frère Jean‐Marie , Fink Anthony L. , Ghuysen Jean‐Marie
Published: November 2003

Prediction studies, conformational analyses and membrane‐topology mapping lead to the conclusion that the penicillin sensory transducer, BlaR, involved in the inducibility of β‐lactamase synthesis in Bacillus licheniformis, is embedded in the plasma membrane bilayer via four transmembrane segments TM1–TM4 that form a four‐α‐helix bundle. The extracellular 262‐amino‐acid‐residue polypeptide, S340–R601, that is fused at the carboxy end of TM4, possesses the amino acid sequence signature of a penicilloyl serine transferase. It probably functions as penicillin sensor. As an independent entity, this polypeptide behaves as a high‐affinity penicillin‐binding protein. As a component of the full‐size BlaR, it adopts a different conformation presumably because of interactions with the extracellular 63‐amino‐acid‐residue P53–S115 loop that connects TM2 and TM3. Reception of the penicillin‐induced signal requires a precise conformation of the sensor but it does not involve penicilloylation of the serine residue S402 of motif STYK. Signal transmission through the plasma membrane by the four‐α‐helix bundle may proceed in a way comparable to that of the aspartate receptor, Tar. Signal emission in the cytosol by the intracellular 189‐amino‐acid‐residue Y134–K322 loop that connects TM3 and TM4, may proceed via the activation of a putative metallopeptidase.
FnrP and NNR of Paracoccus denitrificans are both members of the FNR family of transcriptional activators but have distinct roles in respiratory adaptation in response to oxygen limitation
Authors: Van Spanning Rob J. M. , De Boer Anthonius P. N. , Reijnders Willem N. M. , Westerhoff Hans V. , Stouthamer Adriaan H. , Van Der Oost John
Published: November 2003

The Paracoccus denitrificansfnrP gene encoding a homologue of the Escherichia coli FNR protein was localized upstream of the gene cluster that encodes the high‐affinity cbb3‐type oxidase. FnrP harbours the invariant cysteine residues that are supposed to be the ligands of the redox‐sensitive [4Fe–4S] cluster in FNR. NNR, another FNR‐like transcriptional regulator in P. denitrificans, does not. Analysis of FnrP and NNR single and double mutants revealed that the two regulators each exert exclusive control on the expression of a discrete set of target genes. In FnrP mutants, the expression of cytochrome c peroxidase was blocked, that of membrane‐bound nitrate reductase and the cbb3‐type oxidase was significantly reduced, whilst the activity of the bb3‐type quinol oxidase was increased. The amounts of the nitrite and nitric oxide reductases in these FnrP mutants were the same as in the wild type. NNR mutants, on the other hand, were disturbed exclusively in the concentrations of nitrite reductase and nitric oxide reductase. An FnrP.NNR double mutant combined the phenotypes of the single mutant strains. In all three mutants, the concentrations and/or activities of the aa3‐type oxidase, cytochrome c550, cytochrome c552, and nitrous oxide reductase equalled those in the wild type. As the FNR boxes in front of the FnrP‐ and NNR‐regulated genes are highly similar to or even identical to each other, the absence of cross‐talk between the regulation by FnrP and NNR implies that as yet unidentified factors are important in the control. It is proposed that the redox state of an intracellular redox couple other than the oxygen/water couple is one of the factors that modulates the activity of FnrP.
Transcriptional activation by FNR and CRP: reciprocity of binding‐site recognition
Authors: Sawers Gary , Kaiser Manuela , Sirko Agnieszka , Freundlich Martin
Published: November 2003

Anaerobic expression of the focA pfl operon is dependent on the transcription factors ArcA and FNR and transcription is directed by multiple, anaerobically regulated promoters. A FNR‐binding site is centred at −41.5 bp relative to the P6 promoter, inactivation of which severely impairs anaerobic expression of the complete operon. Mutations were introduced into this binding site to create a consensus recognition site for the cAMP‐receptor protein, CRP (CC‐site), and one that was recognised by both CRP and FNR (CF‐site). Transcription directed by these mutant binding sites in vivo in different promoter constructs was analysed by primer extension and by constructing lacZ operon fusions. With a derivative including only the P6 promoter and the CF‐binding site, transcription was shown to be independent of oxygen and was activated by CRP or FNR. In agreement with previous findings, FNR only activated transcription anaerobically. In a construct including the CC‐binding site transcription was strong, CRP dependent and initiated at the identical site to the wild‐type promoter. Transcription activation from the CC‐site was exquisitely sensitive to low cAMP concentration. Surprisingly, in a crp mutant, anaerobically inducible, FNR‐dependent transcription directed by the CC‐site was detected, indicating that FNR can recognise a consensus CRP‐binding site in vivo. A strain unable to synthesise CRP or FNR exhibited no transcription from the P6 promoter. Essentially the same results were observed in a series of constructs that also included the promoter P7 and its regulatory sequences. Evidence is also presented which demonstrates that CRP activates transcription from the natural FNR‐binding site of the P6 promoter. In vitro DNA‐binding studies showed that CRP specifically interacted with the FNR‐binding site, protecting exactly the same sequence as that protected by the FNR protein. Interaction of CRP with the natural FNR‐binding site was reduced greater than 50‐fold compared to its interaction with the mutant CC‐binding site. Although we could not demonstrate that FNR interacted with the CC‐binding site in vitro, it did bind to the CF‐site giving the same protection as observed with the wild‐type FNR‐binding site. FNR also activated transcription from the CF‐site in vitro, giving further support to the idea that a single functional DNA half‐site is sufficient to direct binding and transcription activation by a dimeric transcription factor.
A gene involved in both protein secretion during growth and starvation‐induced development encodes a subunit of the NADH:ubiquinone oxidoreductase in Myxococcus xanthus
Authors: Laval‐Favre Karine , Letouvet‐Pawlak Barbara , Friedrich Thorsten , Alexandre Joe¨l , Guespin‐Michel Janine F.
Published: November 2003

The secretion of numerous proteins during vegetative growth of Myxococcus xanthus, and the multicellular development cycle induced upon starvation of these bacteria, are partially interrelated in so far as mutants impaired in extracellular protein production are unable to undergo development. We have cloned and sequenced a gene in which a Tn5 insertion leads to a decrease in the production of most, if not all, extracellular proteins, and prevents development and sporulation. The deduced protein is homologous to the putative ubiquinone‐binding subunit of bacterial and mitochondrial NADH:ubiquinone oxidoreductases (complex I). This is the first example of the presence of this complex in a bacterium from subclass δ of the proteobacteria. This gene is expressed during growth and during early development. As its disruption by Tn5 does not impair growth of the mutant strain, we assume the presence of a second alternative NADH oxidoreductase, and suggest that the phenotypic alterations caused by the mutation are due to a decrease in the proton‐motive force.
Different structures of selected and unselected araB‐lacZ fusions
Authors: Maenhaut‐Michel Genevieve , Blake Catherine E. , Leach David R. F. , Shapiro James A.
Published: November 2003

Formation of araB‐lacZ coding‐sequence fusions is a key adaptive mutation system. Eighty‐four independent araB‐lacZ fusions were sequenced. All fusions carried rearranged MuR linker sequences between the araB and lacZ domains indicating that they arose from the standard intermediate of the well‐characterized Mu DNA rearrangement process, the strand transfer complex (STC). Five non‐standard araB‐lacZ fusions isolated after indirect sib selection had novel structures containing back‐to‐back inverted MuR linkers. The observation that different isolation procedures gave rise to standard and non‐standard fusions indicates that cellular physiology can influence late steps in the multi‐step biochemical sequence leading to araB‐lacZ fusions. Each araB‐lacZ fusion contained two novel DNA junctions. The MuR‐lacZ junctions showed‘hot‐spotting’according to established rules for Mu target selection. The araB‐MuR and MuR‐MuR junctions all involved exchanges at regions of short sequence homology. More extensive homology between MuR and araB sequences indicates potential STC isomerization into a resolvable four‐way structure analogous to a Holliday junction. These results highlight the molecular complexity of araB‐lacZ fusion formation, which may be thought of as a multi‐step cell biological process rather than a unitary biochemical reaction.
The Bacillus subtilis chromatin‐associated protein Hbsu is involved in DNA repair and recombination
Authors: Fernández Silvia , Rojo Fernando , Alonso Juan C.
Published: November 2003

The Bacillus subtilis hbs gene encodes an essential chromatin‐associated protein termed Hbsu. Hbsu, the counterpart of the Escherichia coli HU protein, binds DNA in a non‐specific way but has a clear preference for bent, kinked or altered DNA sequences. To investigate the role of Hbsu in DNA repair and DNA recombination we have constructed a series of site‐directed mutants in the hbs gene and used these mutant genes to substitute the wild‐type chromosomal hbs gene. The hbs47 mutation, which codes for a mutant protein in which residue Phe‐47 has been replaced by Trp, does not cause any discernible phenotype. Additional substitution of residue Arg‐55 by Ala (hbs4755 mutation) rendered cells deficient in DNA repair, homologous recombination and (i protein‐mediated site‐specific recombination. We have also tested the effect on DNA repair of the hbs4755 mutation in combination with mutations in different functions of homologous DNA recombination (recA, recF, recG, recti and addAB). The hbs4755 mutation did not modify the sensitivity of recH and addAB cells to the DNA‐damaging agents methylmethane sulphonate (MMS) or 4‐nitroquinoline‐1‐oxide (4NQO), and it only marginally affected recF and recG cells. The hbs4755 mutation blocked intermolecular recombination in recH cells and markedly reduced it (20‐ to 50‐fold) in recF and recG cells, but had no effect on addAB cells. Taken together, these data indicate that the Hbsu protein is required for DNA repair and for homologous DNA recombination.
Genetic evidence of separate repressor and activator activities of the XylR regulator of the TOL plasmid, pWWO, of Pseudomonas putida
Authors: Bertoni Giovanni , Perez‐Martfn Jose , de Lorenzo Victor
Published: November 2003

The XylR protein encoded by pWWO, the TOL (toluene biodegradation) plasmid of Pseudomonas putida, activates at a distance the transcription of Pu and Ps, which are the two σ54‐dependent promoters of the plasmid, but it also downregulates its own σ70‐promoter, Pr, which divergently overlaps the upstream activating sites of Ps. All regulatory elements that control Pr activity have been faithfully reproduced in Escherichia coli, and the basis of the autoregulation of XylR transcription has been examined by monitoring the activity in vivo of different combinations of mutant proteins and promoters in rpoN+ and rpoN‐ genetic backgrounds. By using PsIPr regions bearing deleted or offset binding sites for XylR and the σ54‐containing RNA polymerase, we could show that formation of a nucleoprotein complex involving the polymerase bound to the divergent promoter Ps is not required for downregulation of Pr. Mutant XylR proteins, G268N and A311V (mutated within the NTP‐binding region of XylR) or R453H (affected in multi‐merization), which are unable to activate (‐dependent transcription from Ps, were indistinguishable from the wild‐type XylR in their ability to repress a reporter Pr‐lacZ fusion. Autoregulation of XylR is therefore due exclusively to the binding of the protein to its target sites at the Pr promoter. This allows one to define sensu stricto XylR as a transcriptional repressor, independently of its activator role in other promoters.