bluuebunny/crossref_metadata_2025 huggingface dataset:
The Rhizobium leguminosarum FnrN protein is functionally similar to Escherichia coli Fnr and promotes heterologous oxygen‐dependent activation of transcription
Authors: Schlüter A. , Patschkowski T. , Unden G. , Priefer U. B.
Published: October 2006

An open reading frame from Rhizobium leguminosarum bv. viciae strain VF39, previously identified and found to be similar to Escherichia coli fnr and Rhizobium meliloti fixK (orf240, thereafter called fnrN), was further analysed. Analysis of the expression of an fnrN‐lacZ transcriptional fusion revealed that fnrN is preferentially expressed under oxygen limitation. Using R. meliloti fixN‐lacZ fusions it was shown that the fnrN gene product only mediates transcriptional activation under microaerobiosis, indicating that the FnrN protein responds, directly or indirectly, to oxygen. Plasmids which expressed fnrN under the control of an E. coli promoter were able to complement an E. coli fnr mutant with respect to anaerobic growth on nitrate but not fumarate, and to promote anaerobic but not aerobic activation of the Fnr‐dependent E. coli genes narGHJI, nirB and fdnGHI coding for nitrate reductase, NADH‐dependent nitrite reductase and formate dehydrogenase‐N, respectively. Fumarate and DMSO reductase activities were not induced by FnrN. The E. coli fnr gene substituted for fnrN in oxygen‐regulated transcription of nirB‐ and fixN‐lacZ fusions in R leguminosarum. The results indicate that Fnr and FnrN are functionally very similar and share a common mode of oxygen‐dependent transcriptional activation. From hybridization studies, it appeared that fnrN‐like genes are present in a number of different R. leguminosarum strains.
Genetic analysis of the immunity region of phage‐plasmid P4
Authors: Ghisotti Daniela , Chiaramonte Raffaelia , Forti Francesca , Zangrossi Sandro , Sironi Gianpiero , Deho Gianni
Published: October 2006

In the prophage P4, expression of the early genes is prevented by premature termination of transcription from the constitutive promoter Ple. In order to identify the region coding for the immunity determinant, we cloned several fragments of P4 DNA and tested their ability to confer immunity to P4 superinfection. A 357 bp long fragment (P4 8418‐8774) is sufficient to confer immunity to an infecting P4 phage and to complement the immunity‐defective P4 cl405 mutant, both in the presence and in the absence of the helper phage P2.

The immunity region covers PLE and the cl locus. We were unable to obtain evidence of translation of the region, thus we suggest that P4 immunity is not elicited by a protein but by a transcript (or transcripts) encoded by the region downstream of the promoter PLE, The promoter PLE appears to be necessary for the expression of P4 immunity: fragments in which the PLE region is deleted did not complement P4 cl405 for lysogenization, although they still interfered with P4 growth. Two complementary sequences downstream of PLE(seqA and seqB) at the 5’and 3’ends of the immunity region play an essential role in the control of P4 immunity.
A plasmid‐encoded type IV fimbrial gene of enteropathogenic Escherichia coli associated with localized adherence
Authors: Donnenberg M. S. , Girón J. A. , Nataro J. P. , Kaper J. B.
Published: October 2006

Enteropathogenic Escherichia coli (EPEC) form adherent microcolonies on the surface of tissue culture cells in a pattern termed localized adherence. Localized adherence requires the presence of a large EPEC adherence factor (EAF) plasmid. Recently a bundle‐forming pilus has been described in EPEC possessing the EAF plasmid. An analysis of 22 non‐invasive EPEC TnphoA mutants revealed that seven have insertions in the EAF plasmid and are incapable of localized adherence. We report here the mapping of the TnphoA insertions in these mutants. The nucleotide sequence of the gene interrupted in these TnphoA mutants (bfpA) was determined and found to correspond to the N‐terminal amino acid sequence of the major structural protein of the bundle‐forming pilus. The bfpA gene bears sequence similarities to members of the type IV fimbrial gene family and encodes a potential site for processing by a prepilin peptidase. A plasmid containing bfpA as the only open reading frame directs the synthesis of a protein recognized by antiserum raised against the bundle‐forming pilus. TnphoA mutants at this locus are unable to synthesize BfpA, but synthesis is restored by introduction of a plasmid containing the cloned gene. The minimum fragment of DNA required to restore localized adherence is considerably greater than that required to restore BfpA synthesis. BfpA expression, as assessed by alkaline phophatase activity in bfpA::TnphoA mutants, is affected by temperature and growth medium. These studies describe an EPEC plasmid‐encoded fimbrial gene, a candidate for the elusive EPEC adherence factor responsible for localized adherence.
Interaction of two variable proteins (PilE and PilC) required for pilus‐mediated adherence of Neisseria gonorrhoeae to human epithelial cells
Authors: Rudel Thomas , van Putten Jos P. M. , Gibbs Carol P. , Haas Rainer , Meyer Thomas F.
Published: October 2006

Pili confer the initial ability of Neisseria gonorrhoeae to bind to epithelial cells. Pilin (PilE), the major pilus subunit, and a minor protein termed PilC, reportedly essential for pilus biogenesis, undergo intra‐strain phase and structural variation. We demonstrate here that at least two different adherence properties are associated with the gonococcal pili: one is specific for erythrocytes, which is virtually unaffected by PilE variation, and another is specific for epithelial cells, and is modulated in response to the variation of PilE. Based on this finding, mutants of a recA ‐ strain were selected that had lost the ability to bind to human cornea epithelial cells (A‐) but retained the ability to form pili (P+) and to agglutinate human erythrocytes (H+). The adherence‐negative mutants failed to produce detectable levels of PMC1 or PilC2 proteins, representing pilC phase variants generated in the absence of RecA. The A‐pilC phase variants were indistinguishable from their A+parents and spontaneous A+ revertants with regard to the amount of PilE produced and its electrophoretic mobility, the degrees of piliation and haemagglutination, and the pilE nucleotide sequence. These data demonstrate a central role for PilC in pilus‐mediated adherence of N. gonorrhoeae to human epithelial cells and further indicate that neither PNC1 nor PilC2 is obligatory for the assembly of gonococcal pili.
Human Menkes X‐chromosome disease and the staphylococcal cadmium‐resistance ATPase: a remarkable similarity in protein sequences
Authors: Silver Simon , Nucifora Giuseppina , Phung Le T.
Published: October 2006

A search with the proposed amino acid translation product from the new ‘candidate gene’ for human Menkes disease against protein sequence libraries showed a remarkable similarity to that for the cadmium efflux ATPase from Staphylococcus aureus resistance plasmids. The Menkes sequence appears closer to the CadA Cd2+ sequence than to P‐type ATPases from animal sources. Menkes syndrome is an X‐chromosome invariably fatal disease that results from abberant copper metabolism. The gene that is defective in Menkes patients, i.e. the Menkes candidate gene, encodes a P‐type ATPase, whose properties satisfactorily explain the phenotype of the disease. P‐type ATPases are all cation pumps, either for uptake (e.g. the bacterial Kdp K+ ATPase), for efflux (e.g. the muscle sarcoplasmic reticulum Ca2+ ATPase), or for cation exchange (e.g. the animal cell Na+/K+ ATPase). These enzymes have a conserved aspartate residue that is transiently phosphorylated from ATP during the transport cycle, hence the name ‘P‐type’ ATPase. The Menkes sequence shares with the staphylococcal CadA ATPase those regions common to all P‐type ATPases and also an N‐terminal dithiol region that was proposed to be a ‘metal‐binding motif’. There are one or two copies of this motif in the available CadA sequences and six copies in the Menkes sequence.
Analysis in Neisseria meningitidis and other Neisseria species of genes homologous to the FKBP immunophilin family
Authors: McAllister Carl F. , Stephens David S.
Published: October 2006

The immunophilin family of FK506‐binding proteins (FKBPs), involved in eukaryotic protein folding and cell regulation, have recently been found to have prokaryotic homologues. Genes with sequences homologous to those encoding human FKBPs were examined in Neisseria species. An FKBP DNA sequence was present, as shown by the polymerase chain reaction and Southern blotting experiments, in the chromosome of Neisseria meningitidis (14 strains) and in all 11 different commensal Neisseria spp. studied, but was not found in Neisseria gonorrhoeae (11 strains tested) or in Moraxella catarrhalis. The nucleotide and predicted protein sequences of the FKBP‐encoding domain from five of the meningococcal strains were highly conserved (e.g. ≥97% homologous). The meningococcal nucleotide sequence was ≥93% homologous and the consensus meningococcal protein sequence was ≥97% homologous to FKBP sequences found in seven different commensal Neisseria spp. The meningococcal nucleotide and predicted protein sequences were ≥59% homologous to the conserved C‐terminus of the human FKBP gene family.

The FKBP nucleotide sequence was present as a single copy in the chromosome of commensal Neisseria spp. and in most strains of N. meningitidis. The FKBP gene was linked to the silent pilin locus, pilS, in class II‐piliated meningococcal strains. In meningococcal strains expressing class I pili, the FKBP gene was linked to one of several pilS loci but not the pilE locus present in these strains. FKBP genes found in commensal Neisseria spp. were not linked to known pilin loci.
Clustering and co‐ordinated activation of carotenoid genes in Myxococcus xanthus by blue light
Authors: Ruiz‐Vázquez Rosa , Fontes Marta , Murillo Francisco J.
Published: October 2006

Blue light activates carotenoid production in the non‐photosynthetic, Gram‐negative bacterium Myxococcus xanthus. Light is known to stimulate the expression of two unlinked genes for carotenoid synthesis, carB and carC, through a mechanism in which the regulatory genes carA, carQ and carR take part. Genes carQ and carR are linked together at a separate locus, whereas carA is linked to carB. We have introduced Tn5 at various sites between carA and carB. Chemical analyses of the mutant strains demonstrate the presence in this region of a cluster of genes for carotenoid synthesis. Gene expression analysis strongly argues for most (or all) of the genes in the cluster being transcribed from a single, light‐inducible promoter under the control of genes carA, carQ and carR.
Transcriptional regulation of Salmonella enterica virulence plasmid genes in cultured macrophages
Authors: Rhen Mikael , Riikonen Petri , Taira Suvi
Published: October 2006

The plasmid‐carried spv genes promote virulence of salmonellae in mice by allowing bacterial growth in the reticuloendothelial tissue. When the bacteria are cultivated under normal laboratory conditions the spv genes appear dormant. This communication explores the transcriptional regulation of spv genes within murine macrophage‐like J774‐A.1 cells utilizing a new reporter system. Transcriptional fusions were constructed between promoter elements of the Salmonella enterica var. Typhimurium spv genes and the KS71A fimbrial gene cluster. The expression of KS71A fimbriae In fusion‐carrying Escherichia coli strains was found to be under the control of the transcriptional activator gene spvR. In strains overproducing SpvR, KS71A fimbriae were assembled on the bacterial cell surface and could be detected by bacterial agglutination or immunofluorescence of intact bacteria; the reporter activity was quantified by estimating the percentage of fluorescent bacteria and by immunoblotting of cell lysates. The activity of the reporters, when transformed into the parent Typhimurium TML R66, was low and revealed less than 0.3% fimbriated cells under in vitro culture conditions. A 15–30‐fold increase in fimbriation was observed when the bacteria were cultivated within J774‐A.1 cells. No such increase occurred when the reporter fusions were transformed into TML R66 cured of the virulence plasmid. Insertional inactivation of the spvR gene of the virulence plasmid in Typhimurium TML R66 also abolished induction, whereas corresponding inactivation of spvA or spvB did not reduce induction. No increase in reporter activity was obtained in Typhimurium of line Q1, which is naturally avirulent for mice, although the strain was provided with virulence plasmid pEX102 of line TML R66. We conclude that the intracellular environment of J774‐A.1 cells induces the spv genes and that this induction requires gene functions of both the bacterial chromosome and the virulence plasmid.
Retrotransfer of IncP piasmid R751 from Escherichia coli maxicells: evidence for the genetic sufficiency of self‐transferable plasmids for bacterial conjugation
Authors: Heinemann Jack A. , Ankenbauer Robert G.
Published: October 2006

Gene transfer between organisms is a prime contributor to evolution. Bacterial conjugation is probably the most important mechanism by which genes are spread among prokaryotes and perhaps also contributes to eukaryotic evolution. Conjugation is mediated by plasmids. The mechanism of conjugation remains ill‐understood despite progress in the identification, mapping and sequencing of genes required for plasmid transmission. All conjugation‐specific genes (those required only for DNA transfer and establishment) identified to date map to plasmids. We found that IncP plasmids could enter and subsequently convert maxicells, which are trapped in a metabolic state that prevents de novo expression of chromosomal genes, into conjugative donors. This suggests that IncP plasmids encode not only necessary functions but indeed all functions specific to DNA transmission. Thus, like viruses, plasmids can convert non‐viable cells into gene vectors.
Identification and characterization of a siderophore regulatory gene (pfrA) of Pseudomonas putida WCS358: homology to the alginate regulatory gene aigQ of Pseudomonas aeruginosa
Authors: Venturi Vittorio , Ottevanger Clemens , Leong John , Weisbeek Peter J.
Published: October 2006

Genes encoding biosynthesis of pseudobactin 358 (a microbial iron transport agent) and its cognate outer membrane receptor protein. PupA, are transcribed only under iron limitation in plant growth‐promoting Pseudomonas putida WCS358. Two cosmid clones were identified from a gene bank of WCS358 DNA which could independently and in an iron‐dependent manner activate transcription from a WCS358 siderophore gene promoter in heterologous Pseudomonas strain A225. The functional region of one of the clones was localized by subcloning, transposon Tn3Gus mutagenesis, and DNA sequencing. Genomic transposon insertion mutants in the functional region lost the capacity to activate a siderophore gene promoter fusion transcriptionally; furthermore, these mutants no longer produced pseudobactin 358. The activating region consisted of a single gene designated pfrA (Pseudomonasferric regulator). The pfrA gene codes for a single polypeptide, PfrA, of approximately 18kDa, which has 58% identity to AlgQ (also known as AlgR2), a positive regulator involved in transcriptionally regulating alginate biosynthesis in Pseudomonas aeruginosa. Cross‐complementation studies between the pfrA gene of P. putida and the algQ gene of P. aeruginosa revealed that pfrA can restore mucoidy (alginate production) in an algQ mutant and that algQ could poorly complement a pfrA genomic mutant. It is concluded that PfrA is involved in the positive regulation of siderophore biosynthetic genes in response to iron limitation; furthermore, pfrA and algQ appeared to be interchangeable between P. putida and P. aeruginosa.
The influence of DNA topology on the environmental regulation of a pH‐regulated locus in Salmonella typhimurium
Authors: Karem Kevin , Foster John W.
Published: October 2006

Salmonella typhimurium is exposed to major shifts in H+ concentration both in its natural and pathogenic environments. The organism undergoes extensive changes in gene expression in response to these pH fluctuations. A current question of regulatory biology is how a change in external pH selectively modulates transcription. We have analysed the expression of one such pH‐regulated locus, aniG, and found it is controlled by several additional environmental conditions including osmolarity and oxygen. For factors such as osmolarity and anaerobiosis, an environmentally triggered change in DNA supercoiling has been suggested as a means for controlling gene expression. Thus, environmentally induced changes in DNA topology were explored as a possible common means for establishing the multiple controls on aniG. The involvement of DNA supercoiling in the genetic response of S. typhimurium to external pH has not previously been defined. This report establishes that alkaline environments lower the linking number of reporter plasmids when compared to acidic environments. A consistent pattern was then established whereby conditions or mutations leading to either increased or decreased negative supercoiling were associated with altered expression of aniG. A similar relationship was observed for another environmentally regulated locus, proU. The DNA topology effects on aniG expression were dependent on the presence of EarA, the negative regulator of aniG. These data can be explained by a model in which repressor‐operator interactions are very sensitive to changes in operator conformation. These environmentally induced topological influences on operator DNA structure contribute to the magnitude of pH control exerted upon aniG.
The major heat‐modifiable outer membrane protein CD is highly conserved among strains of Branhamella catarrhalis
Authors: Murphy Timothy F. , Kirkham Charmaine , Lesse Alan J.
Published: October 2006

The outer membrane of Branhamella catarrhalis contains a major, heat‐modifiable outer membrane protein called CD which has epitopes on the surface of the intact bacterium. The gene encoding CD was cloned and expressed in Escherichia coli. The protein migrates in gels as a doublet, indicating that CD is encoded by single gene whose gene product has two stable conformations. The nucleotide sequence of the gene encoding CD was determined and shows homology with the OprF outer membrane protein of Pseudomonas species. The CD protein contains a proline‐rich region, which appears to account for its aberrant migration in gels. Restriction fragment‐length analysis of 30 isolates of B. catarrhalis with oligonucleotide probes corresponding to sequences in the CD gene produced identical patterns in Southern blot assays. The major heat‐modifiable outer membrane protein CD shares homology with the OprF protein and is highly conserved among strains of B. catarrhalis.
Characterization of comE, a late competence operon of Bacillus subtilis required for the binding and uptake of transforming DNA
Authors: Hahn Jeanette , Inamine Gordon , Kozlov Yura , Dubnau David
Published: October 2006

The binding and transport of DNA by competent Bacillus subtilis requires the assembly of a specialized apparatus. We present here the characterization of comE, an operon under competence control that is required for both DNA binding to the competent ceil surface, and for uptake. comE contains three open reading frames (0RF1‐3) read in the forward direction, preceded by a long untranslated leader sequence and an apparent EσA promoter. A minor promoter also is responsible for transcription of ORF2 and ‐3. A transcript containing a single ORF is produced in the reverse direction. The reverse ORF overlaps ORF1 and the untranslated comE leader. The comE transcript is present at a very low level during growth and at an elevated level in stationary‐phase cells. Converseiy, the reverse transcript is present during exponential growth and disappears during stationary phase. The reverse ORF resembles prokaryotic and eukaryotic pyrroiine‐5‐carboxylate reductases, while ORF2 is similar to several dCMP deaminases. 0RF1 and 0RF3 are predicted to be integral membrane proteins. The latter is specifically required for DNA uptake but not for binding.
Integration host factor binds to a unique class of complex repetitive extragenic DNA sequences in Escherichia coli
Authors: Oppenheim Amos B. , Rudd Kenneth E. , Mendelson Itai , Teff Dinah
Published: October 2006

Interspersed repeated DNA sequences are characteristic features of both prokaryotic and eukaryotic genomes. REP sequences are defined as conserved repetitive extragenic palindromic sequences and are found in Escherichia coli, Salmonella typhimurium and other closely related enteric bacteria. These REP sequences may participate in the folding of the bacterial chromosome. In this work we describe a unique class of 28 conserved complex REP clusters, about 100bp long, in which two inverted REPs are separated by a singular integration host factor (IHF) recognition sequence. We term these sequences RIP (for repetitive IHF‐binding palindromic) elements and demonstrate that IHF binds to them specifically. It is estimated that there are about 70 RIP elements in E. coli. Our analysis shows that the RIP elements are evenly distributed around the bacterial chromosome. The possible function of the RIP element is discussed.
nif gene expression studies in Rhodobacter capsulatus: nfrC‐independent repression by high ammonium concentrations
Authors: Hübner Philipp , Masepohl Bernd , Klipp Werner , Bickle Thomas A.
Published: October 2006

The expression of nif genes in Rhodobacter capsulatus depends on the two regulatory genes, rpoN and nifA, encoding a nif‐specific alternative sigma factor of RNA polymerase and a nif‐specific transcriptional activator, respectively. The expression of the rpoN gene itself is also RPON/NIFA dependent. In order to better characterize the regulation of nif gene induction, chromosomal nifH‐, rpoN‐, nifAl‐ and nifA2‐ lacZ fusions were constructed and the expression of these different nif‐lacZ fusions was determined under photoheterotrophic conditions at different starting ammonium concentrations. The two nifA genes were found to be induced first, followed by nifH and finally by rpoN upon weak, medium and strong nitrogen starvation, respectively. This induction profile and the correlation between the expression of the different nif genes suggested that nifAl expression is the limiting factor for nif gene induction. This hypothesis was tested by construction of different nifA1 overexpressing mutants. Contrary to the current model of n/Y gene expression in R. capsulatus, which predicted constitutive nif gene expression in such mutants, a strong repression of nifH and rpoW was found at high ammonium concentration. The low nifH expression under these conditions is unaffected by nifA2 and is not increased in a ntrC mutant, ruling out any role of NTRC as a mediator of this repression. This finding implies an additional, so far unidentified, regulation by fixed nitrogen in R capsulatus. Changing the expression level of rpoN indicated that low levels of RPON are already sufficient for full nifH induction. The nifA1 and rpoN expression mutants were also tested for diazotrophic growth. Similar generation times were determined for the mutants and for the wild type, but diazotrophic growth of the nifA1 overexpressing ntrC mutant RCM14 did not start until after a prolonged lag phase of two to three days.
Isogenic P‐fimbrial deletion mutants of pyelonephritogenic Escherichia coli: the role of α Gal(1–4)β Gal binding in virulence of a wild‐type strain
Authors: Mobley Harry L.T. , Jarvis Karen G. , Elwood John P. , Whittle Donna I. , Lockatell C. Virginia , Russell Robert G. , Johnson David E. , Donnenberg Michael S. , Warren John W.
Published: October 2006

Escherichia coli strains causing acute pyelonephritis often express multiple fimbrial types and haemolysin, which may contribute to their ability to adhere to, and interact with, kidney epithelial cells. Strain CFT073, a pap+, sfa+, pil+, hly+ pyelonephritis strain, previously established as virulent in the CBA mouse model of ascending urinary tract infection and cytotoxic for cultured human renal epithelial cells, was selected for construction of isogenic strains. From a gene bank of this strain, two distinct copies of the pap operon were isolated. The two P‐fimbrial determinants were sub‐cloned into pCVD442, a positive selection suicide vector containing the sacB gene of Bacillus subtilis. Deletion mutations were introduced into each of the two constructs, within papEFG of one operon and papDEFG of the other. Suicide vectors carrying pap deletions were mobilized from E. coli SM10 lambda pir into CFT073 (NalR) and cointegrates were passaged on non‐selective medium. The first pap mutation was identified by screening a Southern blot of DNA from sucrose‐resistant colonies using a papEFG probe. This mutant retained the MRHA+ phenotype since a second functional copy of pap was still present. A double pap‐deletion mutant, UPEC76, confirmed by Southern blotting, was unable to agglutinate human type O erythrocytes or α Gal(1–4)β Gal‐coated latex beads.

CBA mice (N =100) were challenged transurethrally with 105, 106, 107, or 109 cfu of strains CFT073 or UPEC76. After one week, quantitative cultures of urine, bladder, and kidney were done and histologic changes were examined. No substantive differences in organism concentration or histological findings between parent and mutant were detected in urine, bladder, or kidney at any challenge concentration. We conclude that adherence by P fimbriae of uro‐pathogenic E. coli strain CFT073 plays only a subtle role in the development of acute pyelonephritis in the CBA mouse model.
Proteolytic cleavage at arginine residues within the hydrophilic disulphide loop of the Escherichia coli Shiga‐like toxin I A subunit is not essential for cytotoxicity
Authors: Burgess Beverley J. , Roberts Lynne M.
Published: October 2006

Bschehchia coli Shiga‐like toxin I is a type II ribosome‐inactivating protein composed of an A subunit with RNA‐specific N‐glycosidase activity, non‐covalently associated with a pentamer of B subunits possessing affinity for galabiose‐containing glycolipids. The A subunit contains a single intrachain disulphide bond encompassing a hydrophilic sequence containing two trypsin‐sensitive arginine residues. By analogy with other bacterial toxins it has been proposed that proteolytic nicking, deemed essential for a cytotoxic effect, occurs within this disulphide‐bonded loop to generate the A1 and A2 fragments. Reduced A1 is then believed to translocate an internal membrane to inactivate protein synthesis in the cytosol. In this report, the disulphide‐loop arginines of the SLT I A subunit were mutated to block the specific proteolysis presumed to occur. However, the mutant generated remained an effective toxin having similar catalytic activity to wild‐type toxin and only a marginally reduced cytotoxicity towards cultured cells. We conclude that the disulphide‐loop arginine residues are not the unique and essential processing sites previously assumed, but that processing may occur at alterNatlve accessible sites to compensate for loss of target sites within the loop.
The response of the picoplanktonic marine cyanobacterium Synechococcus species WH7803 to phosphate starvation involves a protein homologous to the periplasmic phosphate‐binding protein of Escherichia coli
Authors: Scanlan David J. , Mann Nicholas H. , Carr Noel G.
Published: October 2006

During phosphate‐limited growth the marine phycoerythrin‐containing picoplanktonic cyanobacterium Synechococcus sp. WH7803 synthesizes novel polypeptides, including two abundant species of 100 kDa and 32kDa. The 32kDa polypeptide was localized to the cell wall, although in a related strain, Synechococcus sp. WH8103, it could be detected in both the cell wall fraction and the periplasm. The gene (designated pstS) encoding this polypeptide was cloned and shown to be present in a single copy. The deduced amino acid sequence indicated a polypeptide consisting of 326 amino acids with a calculated Mr of 33763. Comparison of this sequence with that obtained by microsequencing the N‐terminus of the 32kDa polypeptide showed that the mature protein was synthesized as a precursor, the first 24 amino acid residues being cleaved between two alanine residues at positions 24 and 25. The amino acid sequence of the mature polypeptide showed 35% identity and 52% similarity to the periplasmic phosphate‐binding protein (PstS) from Escherichia coli, including three regions of much stronger homology which, by comparison with E. coli PstS, are directly involved in phosphate binding. Northern blot analysis revealed a pstS transcript of 1.2 kb in RNA extracted from cells grown in Pi‐replete conditions and one of 1.4 kb in considerably increased abundance under Pi‐depleted conditions. Homologues of the pstS gene were detected in other marine phycoerythrin‐containing Synechococcus strains, but not in freshwater or halotolerant species.
Bacillus subtilis CtaA and CtaB function in haem A biosynthesis
Authors: Svensson Birgitta , Lübben Mathias , Hederstedt Lars
Published: October 2006

Haem A, a prosthetic group of many respiratory oxidases, is probably synthesized from haem B (protohaem IX) in a pathway in which haem O is an intermediate. Possible roles of the Bacillus subtilis ctaA and CtaB gene products in haem O and haem A synthesis were studied. Escherichia coli does not contain haem A. The CtaA gene on plasmids in E. coli resulted in haem A accumulation in membranes. The presence of CtaB together with ctaA increased the amount of haem A found in E. coli. Haem O was not detected in wild‐type B. subtilis strains. A previously isolated B. subtilis CtaA deletion mutant was found to contain haem B and haem O, but not haem A. B. subtilis ctaB deletion mutants were constructed and found to tack both haem A and haem O. The results with E. coli and B. subtilis strongly suggest that the B. subtilis CtaA protein functions in haem A synthesis. It is tentatively suggested that it functions in the oxygeNatlon/oxidation of the methyl side group of carbon 8 of haem O. B. subtilis CtaB, which is homologous to Saccharomyces cerevisiae COX10 and E. coli CyoE, also has a role in haem A synthesis and seems to be required for both cytochrome a and cytochrome o synthesis.
Expression of meningococcal epitopes in LamB of Escherichia coli and the stimulation of serosubtype‐specific antibody responses
Authors: McCarvil J. , McKenna A. J. , Grief C. , Hoy C. S. , Sesardic D. , Maiden M. C. J. , Feavers I. M.
Published: October 2006

The class 1 outer membrane protein (OMP), a major variable surface antigen of Neisseria meningitidis, is a component of novel meningococcal vaccines currently in field trials. Serological variants of the protein are also used to serosubtype meningococci. Most of the amino acid changes that give rise to antigenic variants of the protein occur in two variable regions (VR1 and VR2) that are thought to form loops on the cell surface. The polymerase chain reaction (PCR) was used to amplify the nucleotide sequences encoding VR1 and VR2 from the chromosomal DNA of N. meningitidis strain M1080. These were cloned in frame into the lamB gene of the Escherichia coli expression vector pAJC264. Whole‐cell enzyme‐linked immunosorbent assays (ELISAs), using monoclonal antibodies, and SDS PAGE confirmed that, upon induction, strains of E. coli carrying these constructs expressed hybrid LamB proteins containing the N. meningitidis surface loops. These strains were used to immunize rabbits and the resultant polyclonal antisera reacted specifically with the class 1 OMP of reference strain M1080 (P1.7). Immunogold labelling of meningococcal cells and whole‐cell dot‐blot analyses with these antisera showed that the variable epitopes were exposed on the cell surface and confirmed that this approach could be used to obtain serosubtype‐specific antisera. The binding profiles of the antisera were determined from their reactions with overlapping synthetic peptides and their reactivity compared with that of relevant serosubtype‐specific monoclonal antibodies. This approach was used successfully to raise antisera against two other class 1 OMP VR2s. A fourth antiserum raised against a VR2, including the P1.1 epitope, was not subtype specific.
DNA rearrangements and phenotypic switching in prokaryotes
Authors: Dybvig K.
Published: October 2006

Microorganisms have numerous strategies for coping with environmental changes. In many systems, a single cell has the capacity to generate a seemingly infinite array of phenotypic variants in just a few generations of growth. The resulting heterogeneous population is well equipped for sudden environmental change; even if only a few cells in the population possess a phenotype needed for survival, these cells have the capacity to regenerate a similarly diverse population. Phenotypic switching in these systems usually results from high‐frequency DNA rearrangements which are the subject of this review.
Topoisomerase mutations affect the relative abundance of many Escherichia coli proteins
Authors: Steck Todd R. , Franco Robert J. , Wang Jian‐Ying , Drlica Karl
Published: October 2006

The relative abundance of 88 proteins was measured in extracts from three strains of Escherichia coli K‐12 that are isogenic except for the topA and gyrB genes. Mutations in these genes slightly raise or lower, respectively, steady‐state DNA supercoiling levels but have little effect on growth rate. Altered protein abundances were observed in the mutant strains relative to wild type. Many proteins exhibited minimum abundance at wild‐type supercoiling levels, and other proteins exhibited maximal abundance at relaxed levels. A smaller number showed maximal abundance at elevated levels of supercoiling. These data suggest that small, non‐lethal changes in DNA supercoiling can have widespread effects on patterns of gene expression.
A novel strategy for the isolation of luxl homologues: evidence for the widespread distribution of a LuxR:Luxl superfamily in enteric bacteria
Authors: Swift S. , Winson M. K. , Chan P. F. , Bainton N. J. , Birdsall M. , Reeves P. J. , Rees C. E. D. , Chhabra S. R. , Hill P. J. , Throup J. P. , Bycroft B. W. , Salmond G. P. C. , Williams P. , Stewart G. S. A. B.
Published: October 2006

The pheromone N‐(3‐oxohexanoyl)‐L‐homoserine lactone (OHHL) regulates expression of bioluminescence in the marine bacterium Vibrio fischeri, the production of carbapenem antibiotic in Erwinia carotovora and exoenzymes in both E. carotovora and Pseudomonas aeruginosa. A characteristic feature of this regulatory mechanism in V. fischeri is that it is cell density‐dependent, reflecting the need to accumulate sufficient pheromone to trigger the induction of gene expression. Using a lux plasmid‐based bioluminescent sensor for OHHL, pheromone production by E. carotovora, Enterobacter agglomerans, Hafnia alvei, Rahnella aquatilis and Serratia marcescens has been demonstrated and shown also to be cell density‐dependent. Production of OHHL implies the presence in these bacteria of a gene equivalent to luxl. Chromosomal banks from all five enteric bacteria have yielded clones capable of eliciting OHHL production when expressed in Escherichia coli. The luxl homologue from both E. carotovora (carl) and E. agglomerans (eagl) were characterized at the DNA sequence level and the deduced protein sequences have only 25% identity with the V. fischeri Luxl. Despite this, carl, eagl and luxl are shown to be biologically equivalent. An insertion mutant of eagl demonstrates that this gene is essential for OHHL production in E. agglomerans.
The rpsL gene and streptomycin resistance in single and multiple drug‐resistant strains of Mycobacterium tuberculosis
Authors: Nair Jaygopal , Rouse David A. , Bai Gill‐Han , Morris Sheldon L.
Published: October 2006

The recent emergence of indolent and rapidly fatal drug‐resistant strains of Mycobacterium tuberculosis has renewed interest in defining the molecular mechanisms of drug resistance in the tubercle bacilli. In this report, we have examined the mechanism of resistance to streptomycin (Sm) in M. tuberculosis through the cloning and nucleotide sequence analysis of the gene encoding the ribosomal SR protein (rpsL gene) from streptomycin‐resistant strains and their streptomycin‐sensitive parental strains. We have demonstrated that five singly SmRM. tuberculosis strains and an SmR isolate that has reduced sensitivity to multiple antibiotics have identical point mutations at codon 43 of the rpsL gene. Mutations at this same site confer SmR in Escherichia coli. In contrast, two other multiple drug‐resistant M. tuberculosis strains that are resistant to Sm have rpsL genes that have the same nucleotide sequence as their drug‐sensitive parent strains, suggesting that different resistance mechanisms are involved in these strains.
Cloning of Bordetella bronchiseptica urease genes and analysis of colonization by a urease‐negative mutant strain in a guinea‐pig model
Authors: Monack D. M. , Falkow S.
Published: October 2006

The genes encoding urease were cloned from Bordetella bronchiseptica and the 5.2 kb of DNA essential for expression analysed in a T7 RNA polymerase transcription‐translation system. At least four poly‐peptides with predicted molecular weights of 69 000, 26 000, 12 200 and 11 000 were found. Partial DNA sequence of the gene encoding the 69 000 Da polypeptide revealed high amino acid identity to the α‐subunit of Proteus mirabilis urease, UreC and jack bean urease. A stable, unmarked deletion was constructed in this gene to create a urease‐negative mutant of B. bronchiseptica. To assess colonization in a guinea‐pig model, the urease‐negative strain was inoculated with the urease‐positive parental strain in a mixed infection. The urease‐negative strain out competed the urease‐positive strain in the trachea, lungs and caecum. We demonstrate that urease is not essential for B. bronchiseptica colonization of the guinea‐pig respiratory and digestive tracts.
Correlation of gene transcription with the time of initiation of chromosome replication in Escherichia coli
Authors: Theisen Patrick W. , Grimwade Julia E. , Leonard Alan C. , Bogan Joseph A. , Helmstetter Charles E.
Published: October 2006

Transcriptional levels of the Escherichia coli mioC and gidA genes, which flank the chromosomal origin of replication (oriC) and the dnaA gene, were correlated with the time of initiation of chromosome replication. The transcripts were measured either in dnaC2(ts) mutants that had been aligned for initiation of chromosome replication by a temperature shift or in synchronous cultures of cells obtained using the baby machine technique. In both types of experiments, mioC transcription was inhibited prior to initiation of chromosome replication and resumed several minutes after initiation. Conversely, gidA and dnaA transcription were both inhibited after initiation of replication, coincident with the period of hemimethylation of oriC DNA. It is proposed that mioC transcription prevents initiation of chromosome replication, and must terminate before replication can begin. It is further proposed that the eclipse period between rounds of replication, i.e. the minimum interval between successive initiations, encompasses the time required to methylate GATC sequences in newly replicated oriC plus the time required to terminate mioC transcription. Conversely, the active transcription of gidA and dnaA prior to initiation is consistent with their positive effects on initiation, and their shutdown after initiation could serve to limit premature reinitiation.
A proline‐rich protein, verprolin, involved in cytoskeletal organization and cellular growth in the yeast Saccharomyces cerevisiae
Authors: Donnelly Sean F. H. , Pocklington Michael J. , Pallotta Dominick , Orr Elisha
Published: October 2006

A gene (VRP1) encoding a novel proline‐rich protein (verprolin) has been isolated from the yeast Saccharomyces cerevisiae as a result of its hybridization to a chick vinculin cDNA probe. The deduced protein sequence contains 24% proline residues present as proline‐rich motifs throughout the verprolin sequence. Several of these motifs resemble recently identified sequences shown to bind Src homology 3 (SH3) domains in vitro. Replacement of the wild‐type VRP1 allele with a mutant allele results in strains that grow slower than wild‐type strains and are temperature sensitive. The vrp1 mutants are impaired in both cell shape and size and display aberrant chitin and actin localization. We propose that verprolin is involved in the maintenance of the yeast actin cytoskeleton, through interactions with other proteins, possibly containing SH3 domains.
Restoration of attachment, virulence and nodulation of Agrobacterium tumefaciens chvB mutants by rhicadhesin
Authors: Swart Saskia , Smit Gerrit , Lugtenberg Ben J.J. , Kijne Jan W.
Published: October 2006

In contrast to wild‐type Agrobacterium tumefaciens strains, β‐1,2‐glucan‐deficient chvB mutants were found to be unable to attach to pea root hair tips. The mutants appeared to produce rhicadhesin, the protein that mediates the first step in attachment of Rhizobiaceae cells to plant root hairs, but the protein was inactive. Both attachment to root hairs and virulence of the ChvB mutants could be restored by treatment of the plants with active rhicadhesin, whereas treatment of plants with β‐1,2‐glucan had no effect on attachment or virulence. Moreover, nodulation ability of a chvB mutant carrying a Sym plasmid could be restored by pretreatment of the host plant with rhicadhesin. Apparently the attachment‐minus and avirulence phenotype of chvB mutants is caused by lack of active rhicadhesin, rather than directly being caused by a deficiency in β‐1,2‐glucan synthesis. The results strongly suggest that rhicadhesin is essential for attachment and virulence of A. tumefaciens cells. They also indicate that the mechanisms of binding of Agrobacterium and Rhizobium bacteria to plant target cells are similar, despite differences between these target cells.
Suppression of temperature‐sensitive assembly mutants of heat‐labile enterotoxin B subunits
Authors: Sandkvist Maria , Bagdasarian Michael
Published: October 2006

Deletions or substitutions of amino acids at the carboxyl‐terminus of the heat‐labile enterotoxin B sub‐unit (EtxB) affect its assembly into pentamers in a temperature‐dependent manner. At 42°C, the mutations prevent the B subunits from achieving their final pentameric structure resulting in membrane association of the monomers. However, mutant B subunits produced at 30°C assemble, in the periplasm, into pentamers that remain stable when transferred to 42°C, indicating that the mutant pentamers are stable under conditions where their formation is inhibited. The mutant pentamers are, similarly to wild‐type pentamers, SDS‐resistant and stable, in vitro, at temperatures up to 65°C. This suggests that although the C‐terminal amino acids are part of the subunit interface, they appear not to contribute significantly to the stability of the final pentameric complex, but are instead essential for the formation or stabilization of an assembly intermediate in the pentamerization process. Single second site mutations suppress the assembly defect of mutant EtxB191.5, which carries substitutions at its C‐terminus. The Thr→IIe replacement at position 75 in the α2‐helix probably restores the van der Waals contact between residues 75 and 101, which had been greatly reduced by the Met→Leu substitution at position 101 in the β6‐strand of EtxB191.5. Interaction between the α2‐helix and β6‐strand which contains the c‐terminus probably stabilizes a conformation essential for assembly and is therefore required for the formation of pentamers.
pH‐dependent permeabilization of the plasma membrane of mammalian cells by anthrax protective antigen
Authors: Milne Jill C. , Collier R. John
Published: October 2006

Protective antigen (PA) of anthrax toxin forms ion‐conductive channels in planar lipid bilayers and liposomes under acidic pH conditions. We show here that PA has a similar permeabilizing action on the plasma membranes of CHO‐K1 and three other mammalian cell lines (J774A.1, RAW264.7 and Vero). Changes in membrane permeability were evaluated by measuring the efflux of the K+ analogue, 86Rb+, from prelabelled cells, and the influx of 22Na+. The permeabilizing activity of PA was limited to a proteolytically activated form (PAN) and was dependent on acidic pH for membrane insertion (optimal at pH 5.0), but not for sustained ion flux. The flux was reduced in the presence of several known channel blockers: tetrabutyl‐, tetrapentyl‐, and tetrahexylammonium bromides. PAN facilitated the membrane translocation of anthrax edema factor under the same conditions that induced changes in membrane permeability to ions. These results indicate that PAN permeabilizes cellular membranes under conditions that are believed to prevail in the endosomal compartment of toxin‐sensitive cells; and they provide a basis for more detailed studies of the relationship between channel formation and translocation of toxin effector moieties in vivo.
The XbaI‐BlnI‐CeuI genomic cleavage map of Salmonella enteritidis shows an inversion relative to Salmonella typhimurium LT2
Authors: Liu Shu‐Lin , Hessel Andrew , Sanderson Kenneth E.
Published: October 2006

We have established the genomic cleavage map of Salmonella enteritidis strain SSU7998 using pulsed‐field gel electrophoresis. The chromosome of 4600kb was analysed by XbaI (16 fragments), I‐CeuI (7 fragments) and BlnI (12 fragments); the genome also contains a plasmid of 60 kb. Cleavage sites of I‐CeuI, in the large subunit ribosomal RNA gene, are conserved from Salmonella typhimurium and Escherichia coli K‐12, and the XbaI and BinI sites in glt‐tRNA are also conserved, but other sites are less conserved. Transposon Tn10, located at 60 different positions in the chromosome of S. typhimurium, was transduced by bacteriophage P22 into S. enteritidis and the insertion mapped using the XbaI and BlnI sites on Tn10. Gene order in S. enteritidis is identical to S. typhimurium LT2 and similar to E. coli K‐12 except for an inversion of 815 kb, which covers the terminus region including T1 and T2. Endpoints are in the NDZs, or non‐divisible zones, in which inversion endpoints were not detected in experiments in E. coli K‐12 and S. typhimurium LT2. This inversion resembles the inversion between S. typhimurium and E. coli, but is longer at both ends.