bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

SummaryThe 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

SummaryTransfer 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

SummaryWe 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

SummaryThe 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

SummarySatellite 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

SummaryThe 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

SummaryThe 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

SummaryA 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

SummaryplJ903, 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

SummaryImmunophilins 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

SummaryThe 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

SummarySix 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

Summary 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

Summary 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

SummaryThe 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.