bluuebunny/crossref_metadata_2025 huggingface dataset:
Parallel induction strategies for cat‐86: separating chloramphenicol induction from protein synthesis inhibition
Authors: Rogers Elizabeth J. , Ambulos Nicholas P. , Gu Zhiping , Lovett Paul S.
Published: October 2006

Induction of cat‐86 translation results from the stalling of a ribosome at a discrete location in the leader region of the transcript. Stalling destabilizes an adjacent region of secondary structure that sequesters the cat‐86 ribosome binding site, thereby activating cat‐86 translation. Two well characterized antibiotics, chloramphenicol and erythromycin, induce cat‐86 by stalling a ribosome at the appropriate leader site. Here we demonstrate differences between the two antibiotics with respect to induction. First, induction by chloramphenicol is dependent on nucleotides in the leader sequence that are different from those necessary for erythromycin induction. Second, variants of Bacillus subtilis that are chloramphenicol resistant because of chromosome mutations permit cat‐86 induction by chloramphenicol, whereas erythromycin‐resistance host mutations block or greatly reduce cat‐86 induction by erythromycin. Third, selected strains of B. subtilis bearing alterations in proteins of the 50S ribosomal subunit interfere with cat‐86 induction by chloramphenicol, yet these strains are chloramphenicol sensitive. Lastly, induction by chloramphenicol is not reversed by removal of the antibiotic whereas erythromycin induction is reversible. The data indicate that chloramphenicol induction results from an effect of the drug that is not identical to its role as a general inhibitor of ribosome elongation. Induction by erythromycin, on the other hand, could not be distinguished from its antibiotic activity.
The presence of a novel type of surface polysaccharide in Rhizobium meliloti requires a new fatty acid synthase‐like gene cluster involved in symbiotic nodule development
Authors: Petrovics György , Putnoky Peter , Reuhs Bradley , Kim John , Thorp Tina A. , Noel K. Dale , Carlson Russell W. , Kondorosi Adam
Published: October 2006

Bacterial exopolysaccharide (EPS) and lipopolysaccharide (LPS) molecules have been shown to play important roles in plant‐bacterium interactions. Here we have demonstrated that the fix‐23 loci, which compensate for exo mutations during symbiotic nodule development, are involved in the production of a novel polysaccharide that is rich in 3‐deoxy‐D manno‐2‐octulosonic acid (Kdo) but is not the classical LPS. This molecule is likely to be a surface antigen since antiserum to whole Rhizobium meliloti cells reacts strongly with it, and since mutations in fix‐23 result in an inability to produce this polysaccharide and to bind bacteriophage 16‐3. It is likely that this Kdo‐rich polysaccharide is analogous to certain Escherichia coli K‐antigens which are anchored to the membrane via a phospholipid moiety. DNA sequence analysis of one gene cluster of this region revealed that the predicted protein products of six genes exhibit a high degree of homology and similar organization to those of the rat fatty acid synthase multifunctional enzyme domains.
Expression of the ferric enterobactin receptor (PfeA) of Pseudomonas aeruginosa: involvement of a two‐component regulatory system
Authors: Dean Charles R. , Poole Keith
Published: October 2006

Expression of the ferric enterobactin receptor in Pseudomonas aeruginosa is inducible by enterobactin and requires sequences upstream of the structural gene (pfeA). Nucleotide sequencing of a 2.5 kilo‐base pair (kb) region of DNA immediately upstream of pfeA revealed two open reading frames (ORFs), pfeR and pfeS, which appeared to comprise an operon. The predicted products of pfeR and pfeS (molecular weight 26 796 and 50 597, respectively) exhibited a high degree of homology to response‐regulator and sensor components, respectively, of the superfamily of prokaryotic environmentally responsive protein pairs. Consistent with an apparent role in regulating expression of pfeA in response to enterobactin, introduction of pfeR/pfeS into P. aeruginosa on a high‐copy‐number vector enhanced enterobactin‐dependent expression of pfeA. Furthermore, a pfeR mutant obtained by in vitro mutagenesis and gene replacement failed to express PfeA despite the presence of enterobactin in the culture medium. Analysis of the hydropathy profiles of PfeR and PfeS supported a cytoplasmic location for PfeR and a cytoplasmic membrane location for PfeS.
Isolation and analysis of a linear plasmid‐located gene of Borrelia burgdorferi B29 encoding a 27 kDa surface lipoprotein (P27) and its overexpression in Escherichia coli
Authors: Reindl Markus , Redl Bernhard , Stöffler Georg
Published: October 2006

Using an antiserum of a patient with cutaneous manifestations of Lyme borreliosis we have isolated the gene encoding a 27 kDa protein antigen (P27) of Borrelia burgdorferi B29 from a λ‐gt11 expression library. Nucleotide sequence analysis revealed that it is a basic protein of 248 amino acids with a typical prokaryotic leader sequence of 17 amino acid residues at the N‐terminus of the proposed translation product. Biochemical investigations showed that P27 is a surface‐exposed lipoprotein. From pulsed‐field gel electrophoresis and subsequent Southern blot analysis it is evident that the p27 gene is located on a linear plasmid of a size of approximately 55 kb. It was over‐expressed in Escherichia coli and the purified recombinant protein was used for biochemical and serological studies. Northern and Western blot analysis demonstrated that p27 is expressed in the European B. burgdorferi strain B29, but not in the American strain B31.
Preparation and analysis of isogenic mutants in the transferrin receptor protein genes, tbpA and tbpB, from Neisseria meningitidis
Authors: Irwin Sean W. , Averil Nuzhat , Cheng Cindy Y. , Schryvers Anthony B.
Published: October 2006

Isogenic mutants were constructed in the tbpA and tbpB genes from Neisseria meningitidis strain B16B6, which code for the transferrin receptor proteins, Tbp1 and Tbp2. Insertion mutants of the tbpA and tbpB genes were obtained by shuttle mutagenesis and by in vitro cassette mutagenesis, respectively. The Isogenic mutants were verified by Southern blot and Western blot analysis. Isogenic mutants deficient in Tbp1 or Tbp2 demonstrated a reduced transferrin binding activity in intact cells and total membranes but were incapable of utilizing transferrin iron for growth. Tbp1 could be isolated by affinity methods from the mutant lacking Tbp2 but isolation of Tbp2 from the mutant lacking Tbp1 required the presence of exogenous Tbp1.
The region of human transferrin involved in binding to bacterial transferrin receptors is iocalized in the C‐lobe
Authors: Alcantara J. , Yu R. H. , Schryvers A. B.
Published: October 2006

Iron‐saturated human transferrin was digested with either chymotrypsin or trypsin to produce C‐lobe and N‐lobe protein fragments. Individual protein fragments were purified by a combination of gel filtration and Concanavalin A affinity chromatographic procedures. The C‐lobe and N‐lobe fragments of human transferrin were then used in binding assays to assess their ability in binding to the bacterial transferrin receptors. Competitive binding assays demonstrated that the C‐lobe fragment of human transferrin binds as well as intact human transferrin to bacterial transterrin receptors from Neisseria meningitidis, Neisseria gonorrhoeae and Haemophlius influenzae. Using isogenic mutants of N. meningitidis deficient in either of the transferrin‐binding proteins (Tbps), we demonstrated that both transferrin‐binding proteins were able to bind to the C‐lobe fragment of human transferrin.
Identification of an operon involved in the assimilatory nitrate‐reducing system of Azotobacter vineiandii
Authors: Ramos Francisco , Blanco Gonzalo , Gutiérrez Juan C. , Luque Francisco , Tortolero Maria
Published: October 2006

A number of mutants lacking nitrate reductase (Nas−) or nitrite reductase (Nis−) activities have been isolated and characterized. An operon including two new genes (nasA and nasB) has been defined and cloned from an Azotobacter vinelandii gene bank. nasA encodes for nitrite reductase apoenzyme, whereas nasB is specific for nitrate reductase activity. Nitrate reductase exerts a regulatory effect on nasAB.
Lactococcus lactis: high‐level expression of tetanus toxin fragment C and protection against lethal challenge
Authors: Wells Jeremy M. , Wilson Peter W. , Norton Pamela M. , Gasson Michael J. , Le Page Richard W. F.
Published: October 2006

To determine if the food‐grade bacterium Lactococcus lactis holds promise as a vaccine antigen delivery vector we have investigated whether this bacterium can be made to produce high levels of a heterologous protein antigen. A regulated expression system has been developed which may be generally suitable for the expression of foreign antigens (and other proteins) In L. lactis. The system utilizes the fast‐acting T7 RNA polymerase to transcribe target genes, and provides the first example of the successful use of this polymerase in a Gram‐positive bacterium. When the performance of the expression system was characterized using tetanus toxin fragment C (TTFC) up to 22% of soluble cell protein was routinely obtained as TTFC. Mice immunized subcutaneously with L. lactis expressing TTFC were protected from lethal challenge with tetanus toxin. These results show for the first time that L. lactis is able to express substantial quantities of a heterologous protein antigen and that this organism can present this antigen to the Immune system in an immunogenic form.
Yeast DNA recombination and repair proteins Rad 1 and Radio constitute a complex in vivo mediated by localized hydrophobic domains
Authors: Bardwell A. Jane , Bardwell Lee , Johnson Douglas K. , Friedberg Errol C.
Published: October 2006

The Saccharomyces cerevisiae Rad 1 and Rad 10 proteins are required for damage‐specific incision during nucleotide excision repair and also for certain mitotic recombination events between repeated sequences. Previously we have demonstrated that Rad1 and Rad10 form a specific complex in vitro. Using the ‘two‐hybrid’ genetic assay system we now report that Rad1 and Rad10 proteins are subunits of a specific complex in the cell nucleus. The Rad10‐binding domain of Rad1 protein maps to a localized region between amino acids 809–997. The Rad1 ‐binding domain of Radio protein maps between amino acids 90–210. These domains are evolutionarily conserved and are hydrophobic in character. Although significant homology exists between Rad10 and the human‐DNA‐repair protein Ercc1 in this region, we were unable to detect any interaction between Ercc1 and Rad1 proteins. We conclude that Rad1 and Rad10 operate in DNA repair and mitotic recombination as a constitutive complex.
Regulation of epidermin biosynthetic genes by EpiQ
Authors: Peschel Andreas , Augustin Johannes , Kupke Thomas , Stevanovic Stefan , Götz Friedrich
Published: October 2006

We investigated the role of epiQ in the biosynthesis of the lantibiotic epidermin. epiQ was essential for epidermin production. It was shown that EpiQ controls epidermin production by transcriptionally activating the epiA promoter, used for transcription of most of the epidermin biosynthetic genes. Additional copies of epiQ increased epidermin production in the epidermin‐producing wild‐type strain Staphylococcus epidermidis Tü3298. The epiA promoter region was characterized by primer extension analysis. Two inverted repeats, putative operator sites for EpiQ binding, are located upstream of the −35 region and one is localized downstream of the‐10 region. Crude protein extracts from S. epidermidis Tü3298 and epiQ expressing Escherichia coli cells led to gel mobility shifts of a DNA fragment bearing the inverted repeat which is located immediately upstream of the ‐35 region. DNA fragments bearing the other two inverted repeats were not shifted. The epiQ gene product could be detected by overexpression in the E. coli T7 system using antiserum raised against synthetic pep‐tides of EpiQ. Furthermore, EpiQ, like other DNA‐binding proteins, was shown to bind strongly to heparin sepharose.
Site‐specific insertion of gene cassettes into integrons
Authors: Collis Christina M. , Grammaticopoulos Georgia , Briton Jayne , Stokes H.W. , Hall Ruth M.
Published: October 2006

Site‐specific insertion of gene cassettes into the insert region of integrons has been demonstrated. Insertion was only observed if the integron DNA integrase was expressed in the recipient cell and if the cassette DNA was ligated prior to transformation. The essential ligation products were resistant to treatment with exonuclease III, indicating that they were closed circular molecules. Insertion of cassettes into integron fragments containing either no insert (one recombination site), or one gene cassette (two recombination sites), was demonstrated. In the latter case, insertion occurred predominantly at the core site located 5′ to the resident cassette, which corresponds to the only site available when no insert is present in the recipient. When DNA molecules including two gene cassettes were used, insertion of only one of the gene cassettes was generally observed, suggesting that resolution of the circular molecule to generate two independent circular cassettes occurred more rapidly than insertion into the recipient integron.
Iron and oxygen regulation of Escherichia coli MnSOD expression: competition between the global regulators Fur and ArcA for binding to DNA
Authors: Tardat Brigitte , Touati Danièle
Published: October 2006

Manganese superoxide dismutase (the sod A gene product) in Escherichia coli, is negatively regulated by two global regulators, ArcA (aerobic respiration control) and Fur (ferric uptake regulation), coupling its expression to aerobic metabolism and the intracellular iron pool. Footprinting analyses were carried out on the sodA promoter region with purified Fur protein and with ArcA protein overproduced in crude extracts. ArcA is able to bind in vitro in the absence of the in vivo triggering signal. The binding occurs in one step and study of contacts within the operator sequence reveals binding on one side of the double helix. The DNA protection extends to a much larger region (about 65 bp) than would be expected for a 27 kDa protein, suggesting polymerization. Both Fur and ArcA footprints encompass the –35 and –10 promoter region and there is considerable overlap of their binding sequences, in agreement with in vivo results suggesting that either regulator alone can block sodA transcription. Furthermore, competition experiments show that Fur and ArcA binding to the sodA promoter are mutually exclusive and that ArcA can easily displace Fur, but not vice versa. The biological significance of this in vitro behaviour is discussed.
Spatial expression and autoregulation of hetR, a gene involved in the control of heterocyst development in Anabaena
Authors: Black Todd A. , Cai Yuping , Wolk C. Peter
Published: October 2006

The spatially patterned differentiation of hetero‐cysts in the filamentous cyanobacterium Anabaena requires a functional hetR gene. Transcriptional fusions to luxAB show that hetR is transcribed at a low level throughout the filament when Anabaena is grown with combined nitrogen, and that induction of the gene begins within 2 h following nitrogen deprivation. By 3.5 h, induction is localized to spaced foci. By 6h, there is an overall induction of at least threefold in whole cultures, reflecting at least a 20‐fold increase within spatially separated cells. The induction requires the presence of a functional hetR gene, indicating that hetR is autoregulatory. Full induction of a heterocyst structural gene, hepA, also requires a functional hetR locus.
comF, a Bacillus subtilis late competence locus, encodes a protein similar to ATP‐dependent RNA/DNA helicases
Authors: Londoño‐Vallejo J. A. , Dubnau D.
Published: October 2006

We have sequenced and genetically characterized comF, a Bacillus subtilis competence locus, previously identified by Tn917 transposon insertion mutagenesis. Expression of the locus, in which three open reading frames (ORFs) were found, is driven by a single σ;A‐like promoter in front of comFORF1 and is dependent on early regulatory competence genes and only expressed in competence medium. The predicted amino acid sequences of two of the ORFs showed similarities to known proteins in the Gen‐Bank and SwissProt databases: ComFORF1 is similar to an extensive family of ATP‐dependent RNA/DNA helicases with closer similarity to the DEAD protein subfamily and to the PriA protein in Escherichia coli. The latter is a DNA translocase/helicase required for primosome assembly at the replication fork of phage φX174. ComFORF3 is 22% identical to Com101, a protein required for genetic competence in Haemophilus influenzae, a naturally competent Gram‐negative bacterium. In‐frame comFORF1 deletions were 1000‐fold deficient in transformability compared to the wild type, whereas disruptions of the other two ORFs were only five‐ to 10‐fold lower. These observations allow us to hypothesize that the ComFORF1 late gene product plays an essential role during the binding and uptake events involved in Bacillus subtilis transformation.
Identification of nodSUIJ genes in Nod locus 1 of Azorhizobium caulinodans: evidence that nodS encodes a methyltransferase involved in Nod factor modification
Authors: Geelen Danny , Mergaert Peter , Geremia Roberto A. , Goormachtig Sofie , Van Montagu Marc , Holsters Marcelle
Published: October 2006

The Azorhizobium caulinodans strain ORS571 nodu‐lation genes nodSUIJ were located downstream from nodABC. Complementation data and transcriptional analysis suggest that nodABCSUIJ form a single operon. Mutants with Tn5 insertions in the genes nodS, nodU, and nodJ were delayed in nodulation of Sesbania rostrata roots and stems. The NodS amino acid sequences of ORS571, Bradyrhizobium japonicum, and Rhizobium sp. strain NGR234, contain a consensus with similarity to 5‐adenosylmethionine (SAM)‐utilizing methyltransferases. A naringenin‐inducible nodS‐dependent protein of approximately 25kDa could be cross‐linked to radiolabelled SAM. By applying L‐[methyl‐3H]‐methionine in vivo. Nod factors of ORS571, known to be N‐methylated, could be labelled in wild type and nodU mutants but not in nodS mutants. Therefore, we propose that NodS is a SAM‐utilizing methyltransferase involved in Nod factor synthesis.
Co‐dependent positive regulation of the ansBF promoter of Escherichia coli by CRP and the FNR protein: a molecular analysis
Authors: Jennings Michael P. , Beacham Ifor R.
Published: October 2006

Transcription of the ansB gene, encoding l‐asparaginase II, is positively regulated by cAMP receptor protein (CRP) and by the product of the fnr gene, the FNR protein. These global regulatory proteins mediate the expression of ansB in Escherichia coli in response to carbon source and to anaerobiosis, respectively, and are required concurrently for optimal ansB expression. The mechanism whereby CRP and FNR interact co‐operatively with the ansB promoter to achieve transcription has not previously been established. We have utilized an ansB‘‐’1lacZ fusion, in conjunction with deletion analysis and site‐directed mutagenesis, to identify two sites which interact with these regulatory proteins In the ansB promoter. The first is an FNR site, centred 41.5 bp upstream of the major transcriptional start site. The second site, located 28 bp upstream of the FNR site, is the site of CRP regulation. This site is homologous to both the CRP and FNR binding‐site consensus sequences and may respond to both CRP and FNR. The concurrent requirement for CRP and FNR for optimal expression of ansB may be explained if, first, essentially no transcription occurs unless the FNR is bound at the downstream site, and, second, the level of transcription when FNR alone is present is enhanced when CRP binds at the upstream site.
Cloning and characterization of the psaE gene of the cyanobacterium Synechococcus sp. PCC 7002: characterization of a psaE mutant and overproduction of the protein in Escherichia coli
Authors: Zhao J. , Snyder W. B. , Mühlenhoff U. , Rhiel E. , Warren P. V. , Golbeck J. H. , Bryant D. A.
Published: October 2006

The psaE gene, encoding a 7.5 kDa peripheral protein of the photosystem I complex, has been cloned and characterized from the cyanobacterium Synechococcus sp. PCC 7002. The gene is transcribed as an abundant monocistronic transcript of approximately 325 nt. The PsaE protein has been overproduced in Escherichia coli, purified to homogeneity, and used to raise polyclonal antibodies. Mutant strains, in which the psaE gene was insertionally inactivated by interposon mutagenesis, were constructed and characterized. Although the PS I complexes of these strains were similar to those of the wild type, the strains grew more slowly under conditions which favour cyclic electron transport and could not grow at all under photoheterotrophic conditions. The results suggest that PsaE plays a role in cyclic electron transport in cyanobacteria.
Molecular analysis of two ScrR repressors and of a ScrR–FruR hybrid repressor for sucrose and D‐fructose specific regulons from enteric bacteria
Authors: Jahrels K. , Lengeler J. W.
Published: October 2006

The scr regulon of pUR400 and the chromosomally encoded scr regulon of Klebsiella pneumoniae KAY2026 are both negatively controlled by a specific repressor (ScrR). As deduced from the nucleotide sequences, both scrR genes encode polypeptides of 334 residues (85.5% identical base pairs, 91.3% identical amino acids), containing an N‐terminal helix‐turn‐helix motif. Comparison with other regulatory proteins revealed 30.6% identical amino acids to FruR, 27.0% to Lacl and 28.1% to GaIR. Six scrRs super‐repressor mutations define the inducer‐binding domain. The scr operator sequences were identified by in vivo titration tests of the sucrose repressor and by in vitro electrophoretic mobility shift assays. D‐fructose, an intracellular product of sucrose transport and hydrolysis, and D‐fructose 1‐phosphate were shown to be molecular inducers of both scr regulons. An active ScrR–FruR hybrid repressor protein was constructed with the N‐terminal part of the sucrose repressor of K. pneumoniae and the C‐terminal part of the fructose repressor of Salmonella typhimurium, LT2. Gel retardation assays showed that the hybrid protein bound to scr‐specific operators, and that D‐fructose 1‐phosphate, the inducer for FruR, was the only inducer. In vivo, neither the operators of the fru operon nor of the pps, operon, the natural targets for FruR, were recognized, but the scr operators were. These data and the data obtained from the super‐repressor alleles confirm previous models on the binding of repressors of the Lacl family to their operators.
A family of IS1031 elements in the genome of Acetobacter xylinum: nucleotide sequences and strain distribution
Authors: Coucheron Dag H.
Published: October 2006

An insertion sequence (here called IS 1031A) from Acetobacter xylinum ATCC 23769 has recently been isolated. This study describes the complete nucleotide sequence of IS 1031A as well as the sequences of two novel iso‐IS 1031 elements, IS1031C and IS1031D, from A. xylinum ATCC 23769. The three ISs are all exactly 930 bp long, have imperfect terminal inverted repeats of 24 bp for IS1031A and 21 bp for IS1031C and IS1031D, are flanked by three base pair direct repeats, and contain an open reading frame encoding a putative basic protein of 278 amino acids. Because of nucleotide substitutions, IS1031C and IS1031D differ from IS 1031A by 12.9% while IS1031C differs from IS1031D by only 0.6%. Hybridization analyses of total DNA from nine A. xylinum strains showed that all strains contained IS 1031‐like elements varying in copy number from three to at least 16. None of three Acetobacter aceti strains examined contained IS1031‐like elements. Taken together, the results suggest that A. xylinum contains a family of IS 1031 elements with considerably diversified nucleotide sequences.
IS 10 mRNA stability and steady state levels in Escherichia coli: indirect effects of translation and role of rne function
Authors: Jain Chaitanya , Kleckner Nancy
Published: October 2006

Translation of the IS 10 transposase gene is known to be very infrequent. We have identified mutations whose genetic properties suggest that they act directly to increase or decrease the intrinsic level of translation initiation. Also, we have analysed in detail the effects of these mutations on IS 10 mRNA using one particular IS 10 derivative. In this case, increases or decreases in translation are accompanied by increases or decreases in both the steady state level and the half‐life of transposase mRNA; effects on steady state levels are much more dramatic than effects on message half‐life. At wild‐type levels of translation initiation, the rate‐limiting step in physical decay of full length IS 10 message for a particular IS 10 derivative is shown to be rne‐dependent endonucleolytic cleavage; 3′ exonucleases appear to play a secondary role, degrading primary cleavage products. Analysis of interplay between translation mutations and rne function, together with the above observations, suggests that translation stabilizes messages in a general way against rne‐dependent endonucleolytic cleavage, and that significant protection may be conferred by one or a few ribosomes. However, dramatic effects of translation on steady state message levels are still observed in an rne mutant and involve the 3′ end of the transcript; we propose that these additional effects reflect translation‐mediated stimulation of transcript release.
Preferential cis action of IS 10 transposase depends upon its mode of synthesis
Authors: Jain Chaitanya , Kleckner Nancy
Published: October 2006

A number of bacterial DNA‐binding proteins, including IS element transposases, act preferentially in cis. We show below that the degree of preferential cis action by IS 10 transposase depends upon its mode of synthesis at steps subsequent to transcription initiation. Cis preference is increased several fold by mutations that decrease translation initiation, by the presence of IS 10‐specific antisense RNA and by plasmids that increase the level of cellular RNases. Conversely, cis preference is decreased by mutations that increase translation initiation; in some cases, cis preference is nearly abolished. Mutations that alter the rate of transcription initiation have no effect. In light of other observations, we suggest that cis preference is strongly dependent upon the rate at which transcripts are released from their templates and/or the half‐life of the transposase message. These observations provide further evidence that inefficient translation plays multiple roles in the biology of IS 10.
The int genes of bacteriophages P22 and λ are regulated by different mechanisms
Authors: Wulff Daniel L. , Ho Yen Sen , Powers Susan , Rosenberg Martin
Published: October 2006

Bacteriophage P22 and λ are related bacteriophages with similar gene organizations. In λ the cII‐dependent PI promoter is responsible for λint gene expression. The only apparent counterpart to PI in P22 is oriented in the opposite direction, and cannot transcribe the P22 int gene. We show that this promoter, called Pal, is active both in vivo and in vitro, and is dependent upon the P22 cII‐like gene, called c1. We have also determined the DNA sequence of a 3.3 kb segment that closes the gap between previously reported sequences to give a continuous sequence between the P22 pL promoter and the int gene. The newly determined sequence is densely packed with genes from the pL direction, and the proteins predicted by the sequence show excellent correlation with the proteins mapped by Youderian and Susskind in 1980. However, the sequence contains no apparent genes in the opposite (pal) direction, and no additional binding motifs for the P22 c1 protein. We conclude that int gene expression in P22 is regulated by a different mechanism than in λ.
Hc1‐mediated effects on DNA structure: a potential regulator of chlamydial development
Authors: Barry Clifton E. , Brickman Timothy J. , Hackstadt Ted
Published: October 2006

Chlamydiae are obligate intracellular bacteria which undergo a unique developmental cycle, alternating between non‐replicative elementary bodies (EBs) and replicatlve reticulate bodies (RBs). The transition from RB to EB is characterized by condensation of the chromosome into a dense nucleoid structure. The chlamydial histone homologue Hc1 is sufficient to induce formation of a similar structure in Escherichia coli. High‐level Hc1 expression in E. coli is self‐limiting and down‐regulates transcription, translation, and replication at concentrations similar to those observed in chlamydial elementary bodies. Expression of Hc1 at sub‐structural levels may have specific regulatory functions through its interaction with chromosomal DNA, In E. coli this is reflected in a dramatic shift in the pattern of gene expression. The differential expression of the outer membrane porin proteins OmpC and OmpF and analysis of lacZ fusions with promoter regions sensitive to supercoiling suggests that low‐level Hc1 expression results in a net relaxation of chromosomal DNA. Topological analysis of plasm id DNA from both E. coli and Chlamydia trachomatis supports a decrease in superhelicity preceding nucleoid formation. In vitro analysis of purified Hc1–DNA interactions supports preferential binding based upon DNA conformation. These results suggest a dual role in which Hc1‐mediated changes in gene expression may precede metabolic inactivity.
Pleiotropic effects of the rpoC10 mutation affecting the RNA polymerase β′ subunit of Escherichia coli on factor‐dependent transcription termination and antitermination
Authors: Ito Koichi , Nakamura Yoshikazu
Published: October 2006

Escherichia coli RNA polymerase is composed of four different subunits, 2α, β, β′ and σ;. Among these subunits, the role of β′ is poorly understood. The rpoC10 mutation affecting β′ has been isolated as a suppressor mutation of the temperature‐sensitive nusA11 mutant. DNA sequence analysis revealed that the rpoC10 mutant is a substitution of Lys for Glu‐402. This increased positive charge appears to compensate for the increased negative charge present in the nusA11 protein (Asp for Gly‐181). In vivo measurements of reporter gene expression have revealed that rpoC10 restores ρ‐dependent termination but fails to restore ρ‐independent termination in nusA.11 Moreover, the rpoC10 mutation, in combination with any nusA mutation, inhibited λ Q‐mediated antitermination without affecting N antitermination and severely restricted λ phage development. The inhibition of Q function and λ growth could be compensated for by overproducing Q. These results suggest that the RNA polymerase β′ subunit plays a crucial rote in factor‐dependent transcription termination and antitermination.
Processing and methylation of PulG, a pilin‐like component of the general secretory pathway of Klebsiella oxytoca
Authors: Pugsley Anthony P.
Published: October 2006

The signal sequence of the Klebsiella oxytoca pulG gene product, which is required for extracellular secretion of the enzyme pullulanase, is similar in many respects to the corresponding segment of the precursors of type IV (me‐Phe) pilins. The significance of this similarity is confirmed by the observation that the pulO gene product processes prePulG at the consensus type IV prepilin peptidase cleavage site at the amino‐terminal end of the PulG signal sequence. Like most type IV pilins, processed PuiG was found to have a methylated amino‐terminal phenylaianine residue. Site‐directed mutagenesis was used to replace amino acids in prePulG that correspond to residues shown by others to be essential for processing, methylation and assembly of type IV pilins. The glycine residue on the amino‐terminal side of the prePulG cleavage site is absolutely required for processing and for pullulanase secretion. The glutamate residue at position 11 (+5) is also required for pullulanase secretion but not for processing or methylation. This result contrasts with that reported for corresponding variants of Pseudomonas aeruginosa type IV prepilin, which were processed but only inefficiently IV‐methylated. Cleavage of prePulG and pullulanase secretion were both unaffected by replacement of the phenylalanine residue on the car‐boxy‐terminal side of the cleavage site by leucine, isoleucine or valine, by a conservative substitution within the hydrophobic core of the prePulG signal sequence, or by a glutamine to proline substitution within the processed segment. However, replacement of the same glutamine residue by arginine abolished secretion without affecting either processing or methylation.
Genetic organization and sequence of the rfb gene cluster of Yersinia enterocolitica serotype O:3: similarities to the dTDP‐L‐rhamnose biosynthesis pathway of Salmonella and to the bacterial polysaccharide transport systems
Authors: Zhang Lijuan , Al‐Hendy Ayman , Toivanen Paavo , Skumik Mikael
Published: October 2006

The Yersinia enterocolitica O:3 lipopotysaccharide O‐antigen is a homopotymer of 6‐deoxy‐L‐altrose. The cloned rfb region was sequenced, and 10 open reading frames were identified. Transposon mutagenesis, deletion analysis and transcomplementatton experiments showed that eight of the genes, organized into two operons, rfbABC and rfbDEFGH, are essential for 0‐antigen synthesis. Functional tandem promoters were identified upstream of both operons. Of the deduced polypeptides RfbA, RfbF and RfbG were similar to Salmonella proteins involved in the dTDP‐l‐rhamnose biosynthesis. Rhamnose and 6‐deoxy‐l‐altrose are C3‐epimers suggesting that analogous pathways function in their biosynthesis. RfbD and RfbE were similar to capsular polysaccharide export proteins, e.g. KpsM and KpsT of Escherichia coli. This and transposon mutagenesis showed that RfbD and RfbE function as O‐antigen exporters.
Cloning, expression and sequencing of Helicobacter felis urease genes
Authors: Ferrero Richard L. , Labigne Agnès
Published: October 2006

Urease genes from Helicobacter felis were cloned and expressed in Escherichia coli cells. A genomic bank of Sau3A‐digested H. felis chromosomal DNA was created using a cosmid vector. Cosmid clones were screened for urease activity following subculture on a nitrogen‐limiting medium. Subcloning of DNA from an urease‐positive cosmid cione led to the construction of plLL205 (9.5 kb) which conferred a urease activity of 1.2±0.5 μmole urea min‐1 mg‐1 bacterial protein to E. coli HB101 bacteria grown on a nitrogen‐limiting medium. Random mutagenesis using a MiniTn3‐Km transposable element permitted the identification of three DNA regions on plLL205 which were necessary for the expression of an urease‐positive phenotype in E. coii clones. To localize the putative structural genes of H. felis on plLL205, extracts of clones harbouring the mutated copies of the plasmid were analysed by Western blotting with anti‐H. felis rabbit serum. One mutant cione did not synthesize the putative UreB subunit of H. felis urease and it was postulated that the transposable element had disrupted the corresponding structural gene. By sequencing the DNA region adjacent to the transposon insertion site two open reading frames, designated ureA and ureB, were identified. The polypeptides encoded by these genes had caicuiated moiecuiar masses of 26 074 and 61 663 Da, respectively, and shared 73.5% and 88.2% identity with the corresponding gene products of Helicobacter pylori urease.
A pleiotropic reduced virulence (Rvi−) mutant of Erwinia carotovora subspecies atroseptica is defective in flagella assembly proteins that are conserved in plant and animal bacterial pathogens
Authors: Mulholland Vincent , Hinton Jay C. D. , Sidebotham Julie , Toth Ian K. , Hyman Lisabeth J. , Perombelon Michel C. M. , Reeves Philip J. , Salmond George P. C.
Published: October 2006

Erwinia carotovora subsp. atroseptica was mutage‐nized and assayed for virulence in planta. Those mutants which exhibited reduced virulence (Rvi‐) were assayed for growth rate, auxotrophy and extracellular enzyme secretion and seven mutants were found to be wild type for all of these phenotypes. When screened for other phenotypes, two were found to be non‐motile. One mutant was complemented for motility by a heterologous gene library. A 2.7kb XmaIII‐Clal complementing fragment was sequenced and the gene products were found to have similarity to flagella biosynthesis gene products from several bacteria. Further similarity was found to a pathogenicity protein from the plant pathogen Xanthomonas campestris pv. glycines and to the Spa pathogenicity proteins of the human pathogen Shigella fiexneri, which are involved in the surface presentation of antigens. These studies highlight the emergence of common themes in the molecular strategies employed by both plant and animal bacterial pathogens for the targeting of proteins involved in the elaboration of disease.
Generation of isogenic K54 capsule‐deficient Escherichia coli strains through TnphoA‐mediated gene disruption
Authors: Russo Thomas A. , Guenther Jane E. , Wenderoth Suzanne , Frank Michael M.
Published: October 2006

Transposon muta genesis, using IS50L::phoA(Tn‐phoA), was performed in a K54/O4/H5 blood isolate of Escherichia coli (CP9), to generate a library of random mutants. Five hundred and twenty‐six independent CP9 TnphoA mutants were isolated with active gene fusions to alkaline phosphatase. From this mutant library, eight capsule‐deficient strains were detected and were found to have a single copy of TnphoA. Sixteen additional capsule deficient mutants with TnphoA inserts were subsequently obtained that did not possess active PhoA fusions. In conjunction with the initial eight capsule‐deficient isolates we have defined genes on three different XbaI fragments as being involved in capsule production. Generalized transduction with the bacteriophage T4 established that these insertions were responsible for the loss of capsule and that they are linked. These capsule‐deficient strains can be used to assess the pathogenic role of the K54 capsular polysaccharide.
Sequence and properties of mecA, a negative regulator of genetic competence in Bacillus subtilis
Authors: Kong Liyun , Siranosian Kathryn Jaacks , Grossman Alan D. , Dubnau David
Published: October 2006

The development of competence in Bacillus subtilis is regulated by growth conditions and several regulatory genes. In complex media competence development is poor, and there is little or no expression of late competence genes, mec mutations permit competence development and late competence gene expression in complex media, and bypass the requirements for many of the competence regulatory genes. In this paper we describe the cloning and characterization of mecA. The mecA gene product acts negatively in the development of competence. Null mutations in mecA allowed expression of a late competence gene comG, under conditions where it is not normally expressed, including in complex media and in cells mutant for several competence regulatory genes. Overexpression of MecA from a multicopy plasmid resulted in inhibition of comG transcription. The DNA sequence of mecA was determined and the predicted gene product showed no significant similarity to any protein in the database. Expression of a mecA–lacZ translational fusion was constitutive during growth and did not vary significantly in the different media tested. The rote of mec A in competence development and other stationary phase phenomena is discussed.
Isolation and characterization of hydroxylamine‐induced mutations in the Erwinia herbicola ice nucleation gene that selectively reduce warm temperature ice nucleation activity
Authors: Gurian‐Sherman Douglas , Lindow Steven E. , Panopoulos Nickolas J.
Published: October 2006

Cells of ice nucleation active bacterial species catalyse ice formation over the temperature range of ‐2 to ‐12°C. Current models of ice nucleus structure associate the size of ice nucleation protein aggregates with the temperature at which they catalyse ice formation. To better define the structural features of ice nucleation proteins responsible for the functional heterogeneity of ice nuclei within a genetically homogeneous collection of cells we used in vitro chemical mutagenesis to isolate mutants with reduced ability to nucleate ice at warm assay temperatures but which retain normal or near normal nucleation activity at cold temperatures (WIND, i.e. warm ice nucleus‐deficient mutants). Nearly half of the mutants obtained after hydroxylamine mutagenesis of the iceE gene from Erwinia herbicola had this phenotype. The phenotypes and location of lesions on the genetic map of iceE were determined for a number of mutants. All WIND mutations were restricted to the portion of iceE encoding the repetitive region of the poty peptide. DNA sequencing of two WIND mutants revealed single nucleotide substitutions changing a conserved serine or glycine residue to phenylalanine and serine, respectively. The implications of these findings in structure/function models for the ice nucleation protein are discussed.
Function of the rfb gene cluster and the rfe gene in the synthesis of O antigen by Shigella dysenteriae 1
Authors: Klena J. D. , Schnaitman C. A.
Published: October 2006

A plasmid that included both an 8.9 kb chromosomal DNA insert containing genes from the rfb cluster of Shigella dysenteriae 1 and a smaller insert containing the rfp gene from a S. dysenteriae 1 multicopy plasmid resulted in efficient expression of O antigen in an rfb‐deleted strain of Escherichia coli K‐12. Eight genes were identified in the rfb fragment: the rfbB‐CAD cluster which encodes dTDP‐rhamnose synthesis, rfbX which encodes a hydrophobic protein involved in assembly of the O antigen, rfc which encodes the O antigen polymerase, and two sugar transferase genes. The production of an O antigen also required the E. coli K‐12 rfe gene, which is known to encode a transferase which adds N‐acetyl‐glucosamine phosphate to the carrier lipid unde‐caprenol phosphate. Thus Rfe protein appears to function as an analogue of the Salmonella RfbP protein to provide the first sugar of the O unit. Functional analysis of the other genes was facilitated by the fact that partial O units of one, two or three sugars were efficiently transferred to the lipopolysaccharide core. This analysis indicated that the plasmid‐encoded Rfp protein is the transferase that adds the second sugar of the O unit while the two rfb transferases add the distal sugars to make an O antigen whose structure is (Rha–Rha–Gal–GlcNAc)n. The use of the rfe gene product as the transferase that adds the first sugar of an O unit is a novel mechanism which may be used for the synthesis of other enteric O antigens.
Nitrate regulation of anaerobic respiratory gene expression in Escherichia coli
Authors: Stewart Valley
Published: October 2006

Synthesis of most anaerobic respiratory pathways is subject to dual regulation by anaerobiosis and nitrate. Anaerobic induction is mediated by the FNR protein. Dual interacting two‐component regulatory systems mediate nitrate induction and repression. The response regulator protein NARL binds DNA to control nitrate induction and repression of genes encoding nitrate respiration enzymes and alternate anaerobic respiratory enzymes, respectively. The homologous protein NARP controls nitrite induction of at least two operons. Nitrate and nitrite signalling are both mediated by the homologous sensor proteins NARX and NARQ. Recent mutational analyses have defined a heptamer sequence necessary for specific DNA binding by the NARL protein. These heptamers are located at different positions in the control regions of different operons. The NARL protein‐binding sites in the narG (nitrate reductase) and narK (nitrate‐nitrite antiporter) operon control regions are located approximately 200 bp upstream of the transcription initiation site. The integration host factor (IHF) greatly stimulates nitrate induction of these operons, indicating that a specific DNA loop brings NARL protein, bound at the upstream region, into the proximity of the promoter for transcription activation. Other NARL protein‐dependent operons do not require IHF for nitrate induction, and the arrangement of NARL heptamer sequences in these control regions is quite different. This complexity of signal transduction pathways, coupled with the diversity of control region architecture, combine to provide many interesting areas for future investigation. An additional challenge is to determine how or if the FNR and NARL proteins interact to mediate dual positive control of transcription initiation.
The FtsZ protein of Bacillus subtilis is localized at the division site and has GTPase activity that is dependent upon FtsZ concentration
Authors: Wang Xunde , Lutkenhaus Joe
Published: October 2006

The ftsZ gene is essential for cell division in both Escherichia coli and Bacillus subtilis. In E. coli FtsZ forms a cytokinetic ring at the division site whose formation is under cell‐cycle control. In addition, the FtsZ from E. coli has a GTPase activity that shows an unusual lag in vitro. In this study we show that FtsZ in Bacillus subtilis forms a ring that is at the tip of the invaginating septum. The FtsZ ring is dynamic since it is formed as division is initiated, changes diameter during septation, and disperses upon completion of septation. In vitro the purified FtsZ from B. subtilis exhibits a GTPase activity without a demonstrable lag, but the GTPase activity is markedly dependent upon the FtsZ concentration, suggesting that the FtsZ protein must oligomerize to express the GTPase activity.
The genes of the glutamine synthetase adenylylation cascade are not regulated by nitrogen in Escherichia coli
Authors: van Heeswijk Wally C. , Rabenberg Maarten , Westerhoff Hans V. , Kahn Daniel
Published: October 2006

Regulation of glutamine‐synthetase (GS) activity in enteric bacteria involves a complex cascade of events. In response to nitrogen limitation, a transferase catalyses the uridylylation of the PII protein, which in turn stimulates deadenylylation of GS. Deadenylylated GS is the more active form of the enzyme. Here we characterize in detail the genes from Escherichia coli encoding uridylyl‐transferase (gInD), the PII protein (glnB), and adenylyl‐transferase (glnE). glnD is transcribed from its own promoter, glnE is cotranscribed with another gene, orfXE, whereas glnB is partly cotranscribed with a gene encoding a homologue of the transcription activator NtrC. All three gln regulatory genes were constitutively expressed at a tow level, i.e. their expression was independent of the nitrogen status and the RNA polymerase sigma factor σ;54. We conclude that the functioning of the GS adenylylation cascade is regulated by modulation of the activities of uridylyl‐transferase and adenylyl‐transferase, rather than by changes in the expression of their genes.