bluuebunny/crossref_metadata_2025 huggingface dataset:
The phosphorylation site of the Kdp‐ATPase of Escherichia coli: site‐directed mutagenesis of the aspartic acid residues 300 and 307 of the KdpB subunit
Authors: Puppe Wolfram , Siebers Annette , Altendorf Karlheinz
Published: October 2006

The potassium‐translocating Kdp‐ATPase of Escherichia coli shares common functional properties with eukaryotic P‐type ATPases. The KdpB subunit has been identified as the catalytic subunit forming the phosphorylated intermediate. Substitution of Asp 307 in KdpB by Glu, Asn, Gin, Tyr, His, Ala or Ser by site‐directed mutagenesis and the subsequent transfer of the point mutations to the chromosome revealed that the mutants were not functioning with respect to cell growth at low K’ concentrations and ATPase activity as well as phosphorylation capacity of the purified Kdp complex. These findings indicate that Asp‐307 in KdpB is the phosphorylation site of the Kdp‐ATPase. In contrast, replacement of the close but non‐conserved Asp‐300 by Asn or Glu has no immediate influence on the enzyme functions tested. However, the Km for K+ of the ATPase activity has been increased 30‐fold compared with the wild‐type enzyme.
Functional analysis of the gas vesicle gene cluster of the halophilic archaeon Haloferax mediterranei defines the vac‐region boundary and suggests a regulatory role for the gvpD gene or its product
Authors: Englert Christoph , Wanner Gerhard , Pfeifer Felicitas
Published: October 2006

A series of deletions introduced into the gvp gene cluster of Haloferax mediterranel, comprising 14 genes involved in gas vesicle synthesis (mc‐vac‐region), was investigated by transformation experiments. Gas vesicle production and the expression of the gvpA gene encoding the major gas vesicle protein, GvpA, was monitored in each Haloferax volcanii transformant. Whereas transformants containing the entire mc‐vac‐region produced gas vesicies (Vac+), various deletions in the region 5′ to gvpA (encompassing gvpD‐gvpM) or 3′ to gvpA (containing gvpC, gvpN and gvpO) revealed Vac− transformants. All these transformants expressed gvpA and contained the 8 kDa GvpA protein as shown by Western analysis. However, transformants containing the gvpA gene by itself indicated a lower level of GvpA than observed with each of the other transformants. None of these transformants containing deletion constructs assembled the GvpA protein into gas vesicles. In contrast, transformants containing a construct carrying a 918 bp deletion internal to gvpD exhibited a tremendous gas vesicle overproduction, suggesting a regulatory role for the gvpD gene or Its product. This is the first assignment of a functional role for one of the 13 halobacterial gvp genes found in addition to gvpA that are involved in the synthesis of this unique structure.
A reorganized Candida albicans DNA sequence promoting homologous non‐integrative genetic transformation
Authors: Herreros E. , Garcia‐Sáez M. I. , Nombela C. , Sánchez M.
Published: October 2006

In order to develop plasmids adequate for non‐integrative genetic transformation of Candida albicans, a DNA fragment of 15.3 kb was cloned from this organism on the basis of its capacity to convert the integrative Saccharomyces cerevisiae vector Ylp5 into a non‐integrative one. Southern hybridization analysis, carried out with a labelled DNA probe of 3.6 kb derived from the cloned fragment, showed that it consisted of C. albicans DNA, the hybridization pattern indicating that the corresponding sequences were homologous to several chromosomal regions. The size of the C. albicans DNA promoting autonomous replication In S. cerevisiae was substantially reduced by subcloning. A 5.1 kb subfragment, defined by BamHI and SalI restriction sites, retained autonomous replication sequences (ARS) functional in the heterologous S. cerevisiae system and in C. albicans, when inserted in plasmid constructions that carried a S. cerevisiae trichodermin‐resistance gene (tcm1) as selection marker. C. albicans transformants were both of the integrative and the non‐integrative type and the plasmids recovered from the latter very often carried a reorganized ARS, indicating that recombination of the inserted ARS DNA had occurred in the homologous host. Successive reorganizations of the ARS insert in C. albicans eventually led to a more stable and much smaller fragment of 687 bp that was subsequently recovered unchanged from transformants. Sequence analysis of the 687 bp fragment revealed four 11‐base blocks, rich in A+T, that carried the essential consensus sequence considered relevant for yeast ARS elements in addition to other features also described as characteristic of yeast replication origins.
Broad‐host‐range Rhizobium species strain NGR234 secretes a family of carbamoylated, and fucosylated, nodulation signals that are O‐acetylated or sulphated
Authors: Price N. P. J. , RelicA B. , Talmont F. , Lewin A. , Promé D. , Pueppke S. G. , Maillet F. , Dénarié J. , Promé J.‐C. , Broughton W. J.
Published: October 2006

Rhizobium species strain NGR234 is the most promiscuous known rhizobium. In addition to the non‐legume Parasponia andersonii, it nodulates at least 70 genera of legumes. Here we show that the nodulation genes of this bacterium determine the production of a large family of Nod‐factors which are N‐acylated chitin pentamers carrying a variety of substituents. The terminal non‐reducing glucosamine is N‐acylated with vaccenic or palmitic acids, is N‐methylated, and carries varying numbers of carbamoyl groups. The reducing N‐acetyl‐glucosamine residue is substituted on position 6 with 2‐O‐methyl‐L‐fucose which may be acetylated or sulphated or non‐substituted. All three internal residues are N‐acetylated. At pico‐ to nanomolar concentrations, these signal molecules exhibit biological activities on the tropical legumes Macroptilium and Vigna (Phaseoleae), as well as on both the temperate genera Medicago (Trifoliae) and Vicia (Viciae). These data strongly suggest that the uniquely broad host range of NGR234 is mediated by the synthesis of a family of varied sulphated and non‐sulphated lipo‐oligosaccharide signals.
Molecular analysis of the aroA gene of Pasteurella multocida and vaccine potential of a constructed aroA mutant
Authors: Homchampa P. , Strugnell R. A. , Adler B.
Published: October 2006

The aroA gene from Pasteurella multocida was cloned by complementation of the Escherichia coli aroA mutant AB2829 with a DNA library constructed in pUC18. The nucleotide sequence of the P. multocida aroA gene indicated an open reading frame encoding a protein of 441 amino acids, which showed a high degree of homology with the amino acid sequences of various other bacterial AroA proteins. The cloned P. multocida aroA gene was inactivated by insertion of a kanamycin‐resistance gene and reintroduced by allelic exchange into the chromosome of P. multocida using the suicide vector pJM703.1. The P. multocida aroA mutant was highly attenuated in a mouse model. Mice immunized intraperitoneally with two doses of live P. multocida aroA mutant were completely protected against a lethal parental strain challenge.
Effect of lac repressor oligomerization on regulatory outcome
Authors: Chakerian Artemis E. , Matthews Kathleen S.
Published: October 2006

Regulatory outcome in a bacterial operon depends on the interactions of all the components which influence mRNA production. Levels of mRNA can be altered profoundly by both negative and positive regulatory elements which modulate initiation of transcription. The occupancy of regulatory sites on the DNA by repressors and activators is determined not only by the affinity of these proteins for their cognate site(s) but also by the oligomeric state of the regulatory protein. The lac operon in Escherichia coli provides an excellent prototypic example of the influence of protein assembly on the transcriptional status of the associated structural genes. DNA loop formation is essential for maximal repression of the lac operon and is contingent upon the presence of multiple operator sites in the DNA and the ability of the repressor to self‐associate to form a bidentate tetramer. The stability of this looped complex is enhanced significantly by DNA supercoiling. Tetramer assembly from dimers apparently occurs via interactions of a‘leucine zipper’motif in the C‐terminal domain of the protein, and the tetramer is essential to formation of looped complexes. Furthermore, analysis of the DNA‐binding characteristics of dimeric mutants has established that the monomer‐dimer association and dimer‐DNA binding (monomer does not bind to DNA) are coupled equilibria. Thus, dimer assembly is essential for generating a DNA‐binding unit, and tetramer assembly is required for formation of the stable looped DNA structure that maximally represses mRNA synthesis. Protein‐protein interactions therefore play a pivotal role in the regulatory activities of the lac repressor and must be considered when analysing the activities of any oligomeric DNA‐binding protein.
Escherichia coli HIyT protein, a transcriptional activator of haemolysin synthesis and secretion, is encoded by the rfaH (sfrB) locus required for expression of sex factor and lipopolysaccharide genes
Authors: Bailey Marc J. A. , Koronakis Vassilis , Schmoll Thomas , Hughes Colin
Published: October 2006

Synthesis and secretion of the 110kDa haemolysin toxin of Escherichia coli and other pathogenic Gram‐negative bacteria are governed by the four genes of the hly operon. We have identified, by transposon mutagenesis, an E. coli cellular locus, hlyT, required for the synthesis and secretion of haemolysin encoded in trans by intact hly operons carrying the hly upstream regulatory region. Mutation of the hlyT locus specifically reduced the level of hlyA structural gene transcript 20‐100‐fold and thus markedly lowered both intracellular and extracellular levels of the HlyA protein. Genetic and structural analysis of the hlyT locus mapped it at co‐ordinate 3680 kbp (minute 87) on the chromosome adjacent to the fadBA operon, and identified it specifically as the rfaH (sfrB) locus which is required for transcription of the genes encoding synthesis of the sex pilus and also the lipopolysaccharide core for attachment of the O‐antigen of E. coli and Salmonella. Expression of the hly operon in the E. coli hlyT mutant was restored in trans by both the hlyT and rfaH genes, suggesting that the rfaH gene is an important activator of regulon structures that are central to the fertility and virulence of these pathogenic bacteria. DNA sequencing of the hlyT locus identifies the HlyT/RfaH transcriptional activator as a protein of 162 amino acids (Mr 18325) which shows no identity to characterized transcription factors.
A bifunctional xylanase encoded by the xynA gene of the rumen cellulolytic bacterium Ruminococcus flavefaciens 17 comprises two dissimilar domains linked by an asparagine/glutamine‐rich sequence
Authors: Zhang Jun‐Xian , Flint Harry J.
Published: October 2006

The nucleotide sequence of the xynA gene of Ruminococcus flavefaciens 17 was determined and found to consist of a 2862 bp open reading frame beginning with a TTG start codon. The predicted product, XYLA, consisted of distinct amino‐terminal (A) and carboxy terminal (C) domains (248 amino acids, including a putative signal sequence, and 332 amino acids, respectively) linked by a repetitive sequence (B, 374 amino acids) extraordinarily rich in asparagine (45%) and glutamine (26%) residues. Domains A and C were shown to be capable of expressing xylanase activity independently of each other when suitably truncated derivatives of the xynA coding region were expressed as lacZ fusions. The activities associated with the two domains were shown to differ with respect to the average size of hydrolysis products formed from oat‐spelt xylan, with domain C releasing relatively more xylose and domain A more xylo‐oligosaccharides. The amino acid sequence of domain A of XYLA closely resembled that of the Bacillus pumilus xynA enzyme (45% identical residues). On the other hand domain C showed significant similarity (33% to 40% identical residues) to a different group of bacterial xylanases and exoglucanases exemplified by the Caldocellum saccharolyticum xynA and celB products. The xynA product is, therefore, a bifunctional enzyme having two dissimilar catalytic domains capable of acting on xylan.
Analysis of the promoters and upstream sequences of nifA1 aud nifA2 in Rhodobacter capsulatus; activation requires ntrC but not rpoN
Authors: Foster‐Hartnett Dawn , Kranz Robert G.
Published: October 2006

Transcription of Rhodobacter capsulatus genes encoding the nitrogenase polypeptides (nifHDK) is repressed by fixed nitrogen and oxygen. R capsulatus nifA1 and nifA2 encode identical NIFA proteins that activate transcription of nifHDK and other nif genes. In this study, we report that nifA1‐lacZ and nifA2‐lacZ fusions are repressed in the presence of NH3 and activated to similar levels under nitrogen‐deficient conditions. This nitrogen‐controlled activation was dependent on R. capsulatus ntrC (which encodes a transcriptional activator) but not rpoN (which encodes an RNA polymerase sigma factor). We have used primer extension analyses of nifA1, nifA2 and nifH and deletion analyses of nifA1 and nifA2 upstream regions to define likely promoters and cis upstream activation sequences required for nitrogen control of these genes. Primer extension mapping confirmed that ntrC but not rpoN is required for nifA1 and nifA2 activation, and that nifA1 and nifA2 do not possess typical RPON‐activated promoters.
Mobilization protein‐DNA binding and divergent transcription at the transfer origin of the Thiobacillus ferrooxidans pTF1 plasmid
Authors: Drolet M. , Lau P. C. K.
Published: October 2006

The possible interaction of the trans‐acting mobilization proteins, MobL and MobS, at the cognate origin of transfer (oriT) region of the Thiobacillus ferrooxidans plasmid pTF1 has been investigated. In gel retardation assays with crude protein extracts from overproducing strains, a truncated MobL (c. 28 kDa) as well as its native protein (42 kDa), but not the 11 kDa MobS protein, were found to bind specifically to a 42‐mer oligonucleotide which represents the transferred DNA strand of the minimal oriT fragment of pTF1. In vivo, the binding of MobL was studied by monitoring catechol 2,3‐dioxygenase (xylE) activities driven by promoters of the divergently transcribed mobL and mobS genes. The mob promoter sequences were found to resemble the Escherichia coliσ70‐dependent consensus promoter elements. The‘‐10’recognition sequences of mobL and one of the two mobS promoters overlap except for one base and they are positioned within the putative‘hairpin’structure in the minimal oriT sequence. In accordance with the twin supercoil‐domain model of Liu and Wang (1987) which suggests that transcription can generate local variations in DNA superhelicity, we propose a possible physiological role of DNA supercoiling in the transfer origin with reference to divergent transcription of mobL and mobS genes.
The Fis protein: it's not just for DNA inversion anymore
Authors: Finkel S. E. , Johnson R. C.
Published: October 2006

Higher‐order nucleoprotein complexes are associated with many biological processes. In bacteria the formation of these macromolecular structures for DNA recombination, replication, and transcription often requires not only the participation of specific enzymes and co‐factors, but also a class of DNA‐binding proteins collectively known as‘nucleoid‐associated’or‘histone‐like’proteins. Examples of this class of proteins are HU, Integration Host Factor, H‐NS, and Fis. Fis was originally identified as the factor for inversion stimulation of the homologous Hin and Gin site‐specific DNA recombinases of Salmonella and phage Mu, respectively. This small, basic, DNA‐bending protein has recently been shown to function in many other reactions including phage λ site‐specific recombination, transcriptional activation of rRNA and tRNA operons, repression of its own synthesis, and oriC‐directed DNA replication. Cellular concentrations of Fis vary tremendously under different growth conditions which may have important regulatory implications for the physiological role of Fis in these different reactions. The X‐ray crystal structure of Fis has been determined and insights into its mode of DNA binding and mechanisms of action in these disparate systems are being made.
Role of the RNA polymerase α subunit in transcription activation
Authors: Ishihama Akira
Published: October 2006

The N‐terminal two‐thirds of the α subunit of Escherichia coli RNA polymerase plays an essential role in the initiation of subunit assembly, by gathering two large subunits, β and β', together into a coreenzyme complex. One group of RNA polymerase mutants deficient in response to transcription activation carries mutations in the C‐terminal region of the α subunit, indicating that the C‐terminal region of the a subunit is involved in protein‐protein contact in positive control of transcription. A set of activators (class I transcription factors) which make contact with this contact site I region on RNA polymerase a subunit bind in most cases to DNA upstream of the promoter ‐35 signal. Genetic fine mapping indicates that a cluster of subsites exists in the contact site I region, each interacting with a set of the class I factors and each consisting of a structure formed by only 5‐10 amino acid residues.
Characterization of the micro‐environment of Salmonella typhimurium–containing vacuoles within MDCK epithelial cells
Authors: Portillo F. Garcia–del , Foster John W. , Maguire Michael E. , Finlay B. Brett
Published: October 2006

Salmonella typhimurium has the capacity to enter into and multiply within epithelial cells. During the entire intracellular stage, bacteria are enclosed within a vacuole. To characterize the micro–environment of the bacteria–containing vacuoles, we have used a new method to measure the expression levels of several S. typhimurium genes in intracellular bacteria within Madin–Darby canine kidney (MDCK) epithelial cells. Our study was based on the determination of ß–galactosidase activity derived from lacZ transcriptional fusions using the highly sensitive substrate fluorescein–di–ß–D–galactoside (FDG). Expression of the iroA and mgtB genes (induced by Fe2+ and Mg2+ limitation respectively), and cadA (induced by pH 6.0 in the presence of lysine, with enhanced expression under anaerobiosis) were characterized at different post–infection times. High intracellular expression levels were detected for the iroA and mgtB genes, suggesting that the concentrations of free Fe2+ and Mg2+ in the vacuole may be low. cadA actitvity was detected only at early post–infection times (4 h), suggesting that the vacuole may have a mild–acidic pH, and oxygen and lysine present at this time. Globally, the results reported indicate that the use of a highly sensitive ß–galactosidase substrate can provide information about the micro–enviroment within which an intracellular pathogen, such as S. typhimurium, resides.
Site–directed mutagenesis of an amino acid residue in the bacteriophage P2 Ogr protein implicated in interaction with Escherichia coli RNA polymerase
Authors: King Rodney A. , Anders Douglas L. , Christie Gail E.
Published: October 2006

The P2 ogr gene encodes a 72–amino–acid protein required for P2 late gene expression. This gene was defined originally by a class of compensatory mutations which overcome the block to P2 late transcription imposed by a host mutation, rpoA109, in the gene encoding the a subunit of Escherichia coli RNA polymerase. Spontaneous compensatory ogr mutations substitute a Cys for a Tyr residue at amino acid 42 in the Ogr polypeptide. Using suppression of an ogr amber mutation and site–directed oligonucleotide mutagenesis, we have studied the effect of amino acid substitutions at this position in Ogr. Substitution of charged residues at this site renders Ogr protein inactive, in rpoA+ and rpoA109 strains. While 11 different amino acids are capable of replacing the wild–type Tyr–42 to allow P2 growth to varying degrees in a wild–type E. coli strain, only three of these allow phage growth in strains carrying the rpoA109 mutation. Phages carrying Cys or Ala in place of Tyr–42 gave burst sizes at least as high as P2 ogr+ in a rpoA+ strain; a Gly substitution also allowed P2 to grow in either a rpoA+ or rpoA109 background, but markedly reduced the burst size. These results are consistent with a direct interaction between Ogr and the α subunit of E. coil RNA polymerase in positive control of P2 late transcription, and indicate that the block imposed by the rpoA109 mutation is due to steric hindrance.
Differential expression of nodS accounts for the varied abilities of Rhizobium fredii USDA257 and Rhizobium sp. strain NGR234 to nodulate Leucaena spp.
Authors: Krishnan H. B. , Lewin A. , Fellay R. , Broughton W. J. , Pueppke S. G.
Published: October 2006

Transfer of a cosmid containing nodSU from Rhizobium sp. NGR234 to Rhizobium fredii USDA257 expands the host range for nodulation to include the perennial tropical legumes, Leucaena leucocephala and Leucaena diversifolia. Complementation experiments with a series of subclones established that nodS and its associated nod‐box promoter from NGR234 are sufficient to confer this extended host‐range phenotype to L. leucocephala. Strain USDA257 contains its own copy of nodSU, including upstream nod‐box sequences. Although both nucleotide and deduced amino acid sequences of the reading frames are homologous between the two strains, there are gaps within the promoter region and the 5′‐end of nodS of USDA257. Consequently, the deduced NodS protein of USDA2S7 is shorter than its counterpart from NGR234, and the distance between the nod‐box and the initiation codon is greater. A 36 bp deletion encompasses the extreme right border of the USDA257 nod‐box and extends into the upstream leader sequence. Transcriptional fusions with both nod‐boxes confirmed that the promoter from NGR234 is flavonoid‐inducible, and that the nod‐box from USDA257 is not. These observations were corroborated by Northern analysis with a nodS‐containing Xhol fragment as hybridization probe. Flavonoid‐induced cells of NGR234 gave an intense signal, but those of USDA257 yielded only a weak trace of hybridization. EcoRl fragments with homology to nodSU of USDA257 are present in 17 of 35 tested strains, including several representatives ofBradyrhizobium japonicum, Rhizobium sp., R. loti, and R. fredii. Two wild‐type, leucaena‐nodulating strains of Rhizobium sp. lack this homology. We conclude that a genetic defect in expression of nodS accounts for the inability of USDA257 to nodulate leucaena and that diverse rhizobia may have evolved alternative mechanisms to nodulate this legume species.
The effect of ribosomal protein S1 from Escherichia coli and Micrococcus luteus on protein synthesis in vitro by E. coli and Bacillus subtilis
Authors: Farwell Mary A. , Roberts Mark W. , Rabinowitz Jesse C.
Published: October 2006

We have designed a set of nine plasmids containing the Bacillus pumilis cat gene with one of three Shine‐Dalgarno (SD) sequences (weak, strong or stronger) and one of three initiation codons (AUG, GUG or UUG). These constructions have been used to determine the effect of ribosomal protein S1, SD and initiation codon sequences and Escherichia coli ribosomal protein S1 on translation in vitro by E. coli and B. subtilis ribosomes. Translation of these nine constructions was determined with three types of ribosomes: E. coli containing ribosomal protein S1, E. coli depleted of S1, and B. subtilis which is naturally free of S1. E. coli ribosomes were able to translate all nine transcripts with variable efficiencies. B. subtilis and S1 ‐depleted E. coli ribosomes were similar to each other and differed from non‐depleted E. coli ribosomes in that they required strong or stronger SD sequences and were unable to translate any of the weak transcripts. Addition of S1 from either E. coli or Micrococcus luteus, a Gram‐positive bacterium, enabled S1‐depleted E. coli ribosomes to translate mRNAs with weak SD sequences but had no effect on B. subtilis ribosomes. AUG was the preferred initiation codon for all ribosome types; however, B. subtilis ribosomes showed greater tolerance for the non‐AUG codons than either type of E. coli ribosome. The presence of a strong or stronger SD sequence increased the efficiency by which E. coli ribosomes could utilize non‐AUG codons. The effect of non‐AUG initiation codons was dependent on the SD with which they were associated, but the effect of the SD sequence was independent of the initiation codon. We conclude that the presence of a strong SD sequence alone is able to substitute for at least part of the activities of ribosomal protein S1, and that ribosomes that exhibit S1 ‐independent translation on such messages also show less reliance on AUG as an initiation codon.
High copy number of the pUC plasmid results from a Rom/Rop‐suppressible point mutation in RNA II
Authors: Lin‐Chao Sue , Chen Wen‐Tsuan , Wong Ten‐Tsao
Published: October 2006

The plasmids pUC18 and pUC19 are pBB322 derivatives that replicate at a copy number several fold higher than the parent during growth of Escherichia coli at 37°C. We show here that the high copy number of pUC plasmids results from a single point mutation in the replication primer, RNA II, and that the phenotypic effects of this mutation can be suppressed by the Rom (RNA one modulator)/Rop protein or by lowering the growth temperature to 30°C. The mutation's effects are enhanced by cell growth at 42°C, at which copy number is further increased. During normal cell growth, the pUC mutation does not affect the length or function of RNA I, the antisense repressor of plasmid DNA replication, but may, as computer analysis suggests, alter the secondary structure of pUC RNA II. We suggest that the pUC mutation impedes interactions between the repressor and the primer by producing a temperature‐dependent alteration of the RNA II conformation. The Rom/Rop protein may either promote normal folding of the mutated RNA II or, alternatively, may enable the interaction of sub‐optimally folded RNA II with the repressor.
Bacteriophage P4 immunity controlled by small RNAs via transcription termination
Authors: Dehò Gianni , Zangrossi Sandro , Sabbattini Pierangela , Sironi Gianpiero , Ghisotti Daniela
Published: October 2006

Satellite bacteriophage P4 immunity is encoded within a short DNA region 357 bp long containing the promoter PLE and 275 bp downstream. PLE is active both in the early post‐infection phase, when genes necessary for P4 lytic cycle are transcribed from this promoter, and in the lysogenic condition, when expression of the above genes is prevented by prophage immunity.

In order to understand how P4 immunity is elicited, we have characterized the transcription pattern during the establishment and the maintenance of the satellite phage P4 lysogenic condition. We found that prophage transcription starting at PLE ends prematurely and the transcripts do not extend beyond 300‐400 nucleotides downstream of PLE. Thus P4 immunity acts by causing premature transcription termination rather than by repressing transcription initiation.

The P4 immunity region is transcribed in the prophage, but it does not seem to be translated; this region contains two elements (seqA and seqB) of a palindromic sequence. In addition to transcripts about 300 nucleotides long, P4 prophage produces a family of shorter transcripts, about 80 nucleotides long, containing seqA or seqB. Evidence is presented suggesting that SeqB RNA is the trans‐acting immunity factor, and that interaction of SeqB RNA with the complementary nascent RNA containing seqA may be involved in bringing about premature transcription termination.
The phage RNA polymerases are related to DNA polymerases and reverse transcriptases
Authors: McAllister William T. , Raskin Curtis A.
Published: October 2006

The single subunit DNA‐dependent RNA polymerase (RNAP) that is encoded by bacteriophage T7 is the prototype of a class of relatively simple RNAPs that includes the RNAPs of the related phages T3 and SP6, as well as the mitochondrial RNAPs. The T7 enzyme has been crystallized, and recent genetic and biochemical analyses have facilitated an interpretation of this structure. A growing body of evidence suggests that the phage‐like RNAPs are related to other nucleotide polymerases such as DNA polymerases, RNA‐dependent RNA polymerases, and reverse transcriptases. In this work, we review information concerning the structure and function of T7 RNAP, and evidence in support of its assignment to a broader class of nucleotide polymerases.
Structural and functional analyses of the FinP antisense RNA regulatory system of the F conjugative piasmid
Authors: van Biesen Tim , Soderbom Fredrik , Wagner E. Gerhart H. , Frost Laura S.
Published: October 2006

The efficiency of conjugation of F‐like plasmids is regulated by the FinOP fertility inhibition system. The transfer (tra) operon is under the direct control of the TraJ transcriptional activator which, in turn, is negatively regulated by FinP, an antisense RNA, and FinO, a 22 kDa protein. Recently, FinO has been shown to extend the chemical stability of FinP in vivo in the absence of traJ mRNA. The in vitro secondary structures of both the FinP and TraJ RNAs were determined by the use of single‐ and double‐strand‐specific nucleases; both RNAs were found to have double stem‐loop structures that are complementary to each other and, therefore, FinP RNA and TraJ RNA have the potential to form a duplex with each other. This was verified by in vitro binding experiments. The reaction was shown to be bimolecular with an apparent rate constant (Kapp) of 5 × 105M−1 s−1, a value that is similar to those found for other natural antisense RNA systems. Preliminary evidence for the in vivo formation of the FinP–TraJ RNA duplex was obtained by primer extension of the traJ mRNA; the presence of both FinO and FinP was required to cause a dramatic reduction in the steady‐state level of traJ mRNA, perhaps as a result of RNase III degradation of the resulting RNA duplex.
Isolation and characterization of the secE homologue gene of Bacillus subtilis
Authors: Jeong Sang Min , Yoshikawa Hirofumi , Takahashi Hideo
Published: October 2006

A 4.0 kb EcoRI fragment of Bacillus subtilis conferring thiostrepton resistance was cloned and characterized. By nucleotide sequencing of the relevant region, six open reading frames were established, which corresponded to a part of spoOH, a ribosomal protein gene (rpmG), an unidentified open reading frame (orfE), a transcription antiterminator gene nusG, and ribosomal protein genes rplK and rplA. The orfE‐encoded 59‐amino‐acid polypeptide had a low, but significant, sequence similarity with the car‐boxy‐terminal region of the Escherichia coli SecE protein. A cold‐sensitive secE mutation of E. coli was complemented by the plasmid‐borne orfE sequence. Furthermore, the normal processing of a proOmpA protein was observed when the secE cold‐sensitive strain carried an orfE plasmid, indicating that orfE is the secE homologue of B. subtilis. The B. subtilis secE has only one transmembrane sequence compared to the three in E. coli.
Analysis of the transfer region of the Streptomyces plasmid SCP2*
Authors: Brolle D.‐F. , Pape H. , Hopwood D. A. , Kieser T.
Published: October 2006

plJ903, a bifunctional derivative of the 31.4 kb low‐copy number, conjugative Streptomyces plasmid SCP2, was mutagenized in Streptomyces lividans using Tn4560. Mutant plasmids differing in their transfer frequencies, chromosome mobilization abilities, pock formation, and complementation properties were isolated. The mutations defined five transfer‐related genes, traA, traB, traC, traD and spd, clustered in a region of 9 kb. The deduced sequences of the putative TraA and TraB proteins showed no overall similarity to known protein sequences, but the phenotype of traA mutant plasmids and sequence motifs in the putative TraA protein suggested that it might be a DNA helicase.
Immunophilins: structure—function relationship and possible role in microbial pathogenicity
Authors: Hacker Jörg , Fischer Gunter
Published: October 2006

Immunophilins are housekeeping proteins present in a wide variety of organisms. Members of two protein superfamilies, cyclophilins (Cyps) and FK506‐binding proteins (FKBPs) belong to this class of immunophilins. Despite the fact that the amino acid sequences of Cyp and FKBPs do not exhibit noticeable homology to each other, proteins of both classes are able to ligate immunosuppressive peptide derivatives. Cyps form complexes with the cyclic undecapeptide cyclosporin A and FKBPs are able to bind FK506 as well as rapamycin, both of which have a pipecolyk bond within their structure. In a ligand‐bound form, immunophilins interfere with signal transduction in T cells. In addition, immunophilins have peptidyl prolyl cis‐trans isomerase (PPlase) activity and are able to accelerate the rate of conformational events in proline‐containing polypeptides. Microorganisms produce proteins that exhibit extensive sequence homologies to cyclophilins and FKBPs of higher organisms and which have considerable PPlase catalytic activity. While cyclophilins seem to be present in most if not all microbial species investigated, FKBPs are produced by yeasts as well as by a number of pathogenic bacteria, such as Legionella pneumophila, Chlamydia trachomatis and Neisseria meningitidis. The Mip protein of L. pneumophila is a virulence factor that plays an essential role in the ability of the bacteria to survive and multiply in phagocytic cells. Some results are summarized on the structure and putative functions of immunophilins and place special emphasis on the contribution of these polypeptides to the virulence of pathogenic microorganisms.
Cell‐cycle‐specific initiation of replication
Authors: Nordström Kurt , Austin Stuart J.
Published: October 2006

The following characteristics are relevant when replication of chromosomes and plasmids is discussed in relation to the cell cycle: the timing or replication, the selection of molecules for replication, and the coordination of multiple initiation events within a single cell cycle. Several fundamentally different methods have been used to study these processes: Meselson—Stahl density‐shift experiments, experiments with the so‐called‘baby machine', sorting of cells according to size, and flow cytometry. The evidence for precise timing and co‐ordination of chromosome replication in Escherichia coli is overwhelming. Similarly, the high‐copy‐number plasmid ColE1 and the low‐copy‐number plasmids R1/R100 without any doubt replicate randomly throughout the cell cycle. Data about the low‐copy‐number plasmids F and P1 are conflicting. This calls for new types of experiments and for a better understanding of how these plasmids control their replication and partitioning.
Two distinct ATP‐binding domains are needed to promote protein export by Escherichia coli SecA ATPase
Authors: Mitchell Christine , Oliver Donald
Published: October 2006

Six putative ATP‐binding motifs of SecA protein were altered by oligonucleotide‐directed mutagenesis to try to define the ATP‐binding regions of this multifunctional protein. The effects of the mutations were analysed by genetic and biochemical assays. The results show that SecA contains two essential ATP‐binding domains. One domain is responsible for high‐affinity ATP binding and contains motifs AO and BO, located at amino acid residues 102‐109 and 198‐210, respectively. A second domain is responsible for low‐affinity ATP binding and contains motifs A3 and a predicted B motif located at amino acid residues 503‐511 and 631‐653, respectively. The ATP‐binding properties of both domains were essential for SecA‐dependent translocation ATPase and in vitro protein translocation activities. The significance of these findings for the mechanism of SecA‐dependent protein translocation is discussed.
Meningococcal Opa and Opc proteins: their role in colonization and invasion of human epithelial and endothelial cells
Authors: Virji Mumtaz , Makepeace Katherine , Ferguson David J. P. , Achtman Mark , Moxon E. Richard
Published: October 2006

Neisseria meningitidis (Nm) isolates from disease or during carriage express, on their outer membranes, one or more of a family of closely related proteins designated Opa proteins. In this study, we have examined the potential rotes of Nm Opa proteins in bacterial attachment and invasion of endothelial as well as epithelial cells and compared the influence of Opa proteins with that of Ope protein, which has been previously shown to increase bacterial interactions with eukaryotic cells. Several variants expressing different Opa proteins (A, B, D) or Opc were selected from a culture of capsule‐deficient non‐piliated bacteria of strain C751. Although the Opa proteins increased bacterial attachment and invasion of endothelial cells, Opc was the most effective protein in increasing bacterial interactions with these cells. In contrast, attachment to several human epithelial cells was facilitated at least as much by OpaB as Opc protein. OpaA was largely without effect whereas OpaD conferred intermediate attachment. OpaB also increased invasion of epithelial cells; more bacteria were internalized by Chang conjunctival cells compared with Hep‐2 larynx carcinoma or A549 lung carcinoma cells. Monoclonal antibody reacting with OpaB inhibited bacterial interactions with the host cells. Opa‐mediated interactions were also eliminated or significantly reduced in variants expressing capsule or those with sialylated lipopolysaccharide. These data are consistent with the notion that environmental factors controlling capsule and lipopolysaccharide phenotype may modulate bacterial interactions mediated by these OM proteins. In permissive microenvironments, some Opa proteins may be important in bacterial colonization and translocation in addition to Opc. The data also support the notion that Nm Opa may confer tissue tropism.
Cloning and sequence analysis of an EnvCD homologue in Pseudomonas aeruginosa: regulation by iron and possible involvement in the secretion of the siderophore pyoverdine
Authors: Poole Keith , Heinrichs David E. , Neshat Shadi
Published: October 2006

Pseudomonas aeruginosa strain K437 is defective in the production of a 90 kDa ferripyoverdine receptor and is unable to grow in an iron‐deficient medium in the presence of the non‐metabolizable iron chelator 2,2′‐dípyrídyl (0.25mM). An attempt to clone the ferripyoverdine receptor gene was made by complementing this growth defect. A number of clones restoring growth of K437 on dipyridyl‐containing medium were obtained and several of these restored moderate expression of the 90 kDa receptor. A 5.5 kb Xhol‐HindIII fragment derived from one of these clones was similarly capable of complementing the dipyridyl growth defect although it failed to restore expression of the 90 kDa ferripyoverdine receptor. Nucleotide sequencing of the 5.5 kb fragment revealed two large open reading frames (ORFs), designated ORFA and ORFB, which appeared to form an operon and were capable of encoding products of 41 kDa and 112 kDa, respectively. Using a phage T7‐based expression system, products of 42 kDa and c. 108 kDa were produced from the cloned DNA, confirming that the ORFs were, indeed, expressed. The cloned ORFAB operon was inducible under conditions of iron limitation in both P. aeruginosa and Escherichia coli. In addition, mutants expressing ORFAB constitutively were constitutive for pyoverdine and ferripyoverdine receptor production suggesting that components of the pyoverdine‐mediated iron‐transport system are co‐regulated with ORFAB. The predicted products of ORFA and ORFB showed significant homology to the Escherichia coli EnvC and EnvD polypeptides which are reportedly involved in septum formation. In addition, the ORFB product showed moderate homology to the CzcA polypeptide identified as a component of a membrane‐associated plasmid‐encoded cation efflux system in Alcaligenes eutrophus. Using in vitro mutagenesis and gene replacement, ORFA‐ and ORFB‐deficient mutants of K372, the parent strain of K437, were constructed. These mutants were unable to grow on iron‐deficient minimal medium containing 0.25 mM dipyridyl although they expressed the ferripyoverdine receptor and were proficient in pyoverdine‐mediated iron uptake. Despite the homology of the ORFA and ORFB products to EnvC and EnvD, respectively, the ORFA‐ORFB‐deficient mutants were not defective in septum formation. Introduction of an ORFB mutation into the pyoverdine‐producing strain ML5087 resulted in poor growth of the mutant in an iron‐limited medium. The same mutation in a pyoverdine‐deficient derivative of ML5087 did not adversely affect growth. These data are discussed in the context of a possible role in pyoverdine secretion for the ORFAB products.
Interactions of wild‐type and mutant AmpR of Citrobacter freundii with target DNA
Authors: Bartowsky Eveline , Normark Staffan
Published: October 2006

The AmpR transcriptional activator for the chromosomal ampCβ‐lactamase gene of Citrobacter freundii was found to interact with an operator sequence located in the 5′ half of the 38 bp region protected by AmpR in DNase I footprinting experiments. AmpR binding was associated with significant DNA bending of target DNA. A glycine to glutamic acid alteration at position 102 in AmpR converts AmpR into a transcriptional activator even in the absence of β‐lactam inducer. AmpRG102E interacted with the operator binding sequence and induced DNA bending. A glycine to lysine alteration at residue 102 completely abolished the ability of AmpR to transcriptionally affect the ampC promoter, i.e. to repress in the absence of β‐lactam inducer and induce in the presence of β‐lactam. Nevertheless, AmpRG102K could repress the oppositely orientated ampR promoter. AmpRG102K could also specifically interact with the operator but the resulting complex migrated faster in gel retardation experiments and no significant DNA bending was observed.
Isolation of the periplasm of Neisseria gonorrhoeae
Authors: Judd Ralph C. , Porcella Stephen F.
Published: October 2006

The periplasm of Neisseria gonorrhoeae should be similar to other Gram‐negative bacteria, but no published reports confirm this assumption. We used a periplasmic isolation procedure developed in Escherichia coli to release the periplasmic contents of N. gonorrhoeae. The resultant periplasmic extract lacked lipopolysaccharide, protein markers of inner or outer membranes, surface‐radiolabelled protein components, or ribosomal proteins. The periplasmic extract contained a single haem protein believed to be a c‐type cytochrome known to exist in the periplasm of other Gram‐negative species, and retained significant alkaline phosphatase activity. The dominant protein species released in the periplasmic extract was the gonococcal homologue of elongation factor Tu, a major component released in similar periplasmic extracts of E. coli. These data showed that the extraction procedure selectively released periplasmic components and that the gonococcal periplasm was comparable to that of E. coli. Further analysis of the gonococcal periplasm may provide important insights into the physiology of this pathogen of humans.
Transcriptional organization of the Escherichia coli pilus adhesin K99
Authors: Inoue Olivia J. , Lee John H. , Isaacson Richard E.
Published: October 2006

The production of the Escherichia coli pilus adhesin K99 requires the expression of eight unique proteins: FanA‐H. The transcriptional organization of the K99 operon was investigated by Northern blot analysis. Four RNAs of 0.54, 1.4, 2.5 and 3.5kb were detected. When a fanC probe was used all four RNAs were detected while the use of fanD, fanF and fanG probes detected two RNAs each. Using several deletion and TnphoA insertion mutants it was concluded that there were seven unique K99‐specific transcripts, several of which were of the same approximate sizes (1.4 and 2.5 kb). It also was concluded that K99 was comprised of at least three complementation groups, two of which were regulated by catabolite repression.
Structural variation in the O‐specific polysaccharides ofKlebsiella pneumoniaeserotype O1 and O8 lipopolysaccharide: evidence for clonal diversity inrfbgenes
Authors: Kelly Robert F. , Severn Wayne B. , Richards James C. , Perry Malcolm B. , MacLean Leann L. , Tomás Juan M. , Merino Susana , Whitfield Chris
Published: October 2006

The O‐polysaccharide fraction of the lipopolysaccharide fromKlebsiella pneumoniaeserotype O8 was found to comprise two galactose‐containing homopolymers. Structural analysis, using chemical and high‐field nuclear magnetic resonance (NMR) techniques, established that theK. pneumoniaeO8 polysaccharides are composed of the linear, disaccharide repeating units

OAc

1

2/6

→3)‐β‐d‐Galf‐(1 →3)‐α‐d‐Galp‐(1→d‐Galactan I‐OAc

→3)‐α‐d‐Galp‐(1 →3)‐β‐d‐Galp‐(1→d‐Galactan II.

K. pneumoniaeO8 mutant RFK‐1 was isolated by resistance to phage KO1‐2; strain RFK‐1 expressed onlyd‐galactan I‐OAc. The1H‐ and13C‐NMR resonances from this O‐polysaccharide indicate that all of theO‐acetyl groups within theK. pneumoniaeO8 polysaccharide are carried ond‐galactan I andO‐acetylation occurs only on the β‐d‐galactofuranose residues; 60% of the available β‐d‐galactofuranose residues are non‐acetylated. TheO‐acetylation of the remaining residues is equally distributed between the O‐2 and O‐6 positions. The carbohydrate backbone structures in the O8 polysaccharide are identical tod‐galactan I and II expressed byK. pneumoniaeO1, accounting for the antigenic cross‐reaction between strains belonging to serotypes O1 and O8. However, the O1 polysaccharides are not acetylated and theO‐acetyl groups present in theK. pneumoniaeserotype O8 polysaccharides provide a structural basis for their recognition as distinct serotypes. Therfb(O‐polysaccharide biosynthesis) gene cluster ofK. pneumoniaeserotype O1 determines the synthesis ofd‐galactan I.rfbKpo1‐specific gene probes were used to examine conservation in therfbgene clusters of otherK. pneumoniaeserotypes which produced‐galactan I. Six O1 strains were examined and all showed hybridization withrfbKpO1probes under conditions of high stringency. Three serotype O2 strains produced‐galactan I and these strains also contained DNA sequences recognized byrfbKpO1probes under high stringency. The physical maps of these homologousrfbchromosomal regions showed some polymorphism. Surprisingly, therfbKpO8region fromK. pneumoniaeserotype O8 was only recognized byrfbKpO1probes under low‐stringency hybridization conditions, providing evidence for two substantially different clonal groups ofrfbgenes fromK. pneumoniaestrains with structurally related O‐antigens.
Activation and mechanism of Clostridium septicum alpha toxin
Authors: Ballard J. , Sokolov Y. , Yuan W.‐L. , Kagan B. L. , Tweten R. K.
Published: October 2006

Clostridium septicum produces a single lethal factor, alpha toxin (AT), which is a cytolytic protein with a molecular mass of approximately 48kDa. The 48kDa toxin was found to be an inactive protoxin (ATpro) which could be activated via a carboxy‐terminal cleavage with trypsin. The cleavage site was located approximately 4kDa from the carboxy‐terminus. Proteolytically activated ATpro had a specific activity of approximately 1.5 × 106 haemolytic units mg‐1. The trypsin‐activated toxin (ATact) was haemolytic, stimulated a prelytic release of potassium ions from erythrocytes which was followed by haemoglobin release, induced channel formation in planar membranes and aggregated into a complex of Mr >210000 on erythrocyte membranes. ATpro did not exhibit these properties. ATact formed pores with a diameter of at least 1.3‐1.6 nm. We suggest that pore formation on target cell membranes is responsible for the cytolytic activity of alpha toxin.