bluuebunny/crossref_metadata_2025 huggingface dataset:
Organization of genes encoding membrane proteins of the Escherichia coli ferrienterobactin permease
Authors: Chenault S. S. , Earhart C. F.
Published: October 2006

Transposon mutagenesis and plasmid complementation studies have identified two genes, fepD and fepG, which are essential for ferrienterobactin transport in Escherichia coli. These genes mapped In the enterobactin gene cluster and genetic evidence indicated that they are transcribed as part of an operon (fepD, fepG, fepC). The nucleotide sequence of fepD was determined; it could encode a hydrophobic 33.8kDa protein with sequence homologies to other iron and vitamin B12 transport proteins. Also identified, between fepD and fepB, was an open reading frame (ORF43) with no detectable function; its 43 kDa protein product (P43) was seen on polyacrylamide gels. The fepD–C operon and ORF43 were divergently transcribed from a 110bp region containing a binding site for the repressor protein Fur.
Nucleotide sequence and genetic organization of the ferric enterobactin transport system: homology to other peripiasmic binding protein‐dependent systems in Escherichia coli
Authors: Shea C. M. , McIntosh M. A.
Published: October 2006

The nucleotide sequence of the Escherichia coli fep genomic region has been determined. Three new loci were identified. One of these, P43, encodes a membrane protein that is not essential for ferric enterobactin transport. Two others, fepD and fepG, were found to be essential for transport and their translational products showed extensive homology to other integral membrane proteins involved in TonB‐dependent transport processes. The FepC amino acid sequence suggested a peripheral membrane location and revealed conserved ATP‐binding domains. Together these data indicate that ferric enterobactin is transported through a typical periplasmic binding protein‐dependent system, in addition, the transcriptional organization of these genes was examined and primer extension analysis identified a single iron‐regulated bidirectional promoter between the P43 gene and the fepDGC operon.
Pro‐peptide as an intermolecular chaperone: renaturation of denatured subtilisin E with a synthetic pro‐peptide
Authors: Ohta Y. , Hojo H. , Aimoto S. , Kobayashi T. , Zhu X. , Jordan F. , Inouye M.
Published: October 2006

The ammo‐terminal pro‐sequence consisting of 77 amino acid residues is required to guide the folding of secreted subtilisin E, a serine protease, into active, mature enzyme (Ikemura et al., 1987). Furthermore, denatured subtilisin E can be folded to active enzyme in an intermolecular process with the aid of an exogenously added pro‐subtilisin E, the active site of which was mutated (Zhu et al., 1989). In this report, we have synthesized the pro‐peptide of 77 residues (corresponding to ‐1 to ‐77 in the sequence, where residue +1 is the N‐terminal amino acid residue of the mature protein), and have found that it could intermolecularly complement the folding of denatured subtilisin E to active enzyme. Furthermore, we have found that the synthetic pro‐peptide exhibits specific strong binding to the active mature enzyme by inhibiting it competitively at its active centre with an upper limit to a Ki of 5.4 × 10−7. In contrast, synthetic pro‐peptides corresponding to ‐44 to ‐77, ‐1 to 64 and ‐1 to ‐43 inhibited the enzyme with Ki values weaker by two orders of magnitude. The results indicate that the sequence extending from ‐1 to ‐77 is essential for specificity of interaction, perhaps generating a conformation that accounts for both roles found hitherto, i.e. specific binding to the active centre, and guiding of the refolding to active enzyme. Thus these results suggest that the pro‐peptide functions as an intermolecular chaperone.
The role of pili in the interactions of pathogenic Neisseria with cultured human endothelial cells
Authors: Virji M. , Kayhty H. , Ferguson D. J. P. , Alexandrescu C. , Heckels J. E. , Moxon E. R.
Published: October 2006

The influence of the two surface structures of Neisseria meningitidis, capsule and pili, in bacterial interactions with human endothelial cells was investigated. Increased association correlated with the presence of pili on bacteria while capsule type had no apparent effect. Strains expressing both Class I and Class II pili associated with endothelial cells in significantly larger numbers compared with the non‐piliated variants of the same strains (>10×). Variants of Neisseria gonorrhoeae strain P9 expressing antigenically distinct pili also associated with endothelial cells in larger numbers (>30×) compared with the non‐piliated variant. Electron microscopic studies confirmed these data and showed that gonococci were internalized more frequently compared with meningococci. One consequence of increased association was an increase in the cytopathic effect of bacteria on the target cells.
Site‐specific insertion of genes into integrons: role of the 59‐base element and determination of the recombination cross‐over point
Authors: Hall R. M. , Brookes D. E. , Stokes H. W.
Published: October 2006

From examination of published DNA sequences of genes found inserted at a specific site in integrons, all genes are shown to be associated, at their 3′ ends, with a short imperfect inverted repeat sequence, a 59‐base element or relative of this element. The similarity of the arrangement of gene inserts in the integron and in the Tn7 transposon family is described. A refined consensus for the 59‐base element is reported. Members of this family are highly diverged and the relationship of a group of longer elements to the 59‐base elements is demonstrated. The ability of 59‐base elements of different length and sequence to act as sites for recombination catalysed by the integron‐encoded DNA integrase is demonstrated, confirming that elements of this family have a common function. The ability of elements located between gene pairs to act as recombination sites has also been demonstrated. The recombination cross‐over point has been localized to the GTT triplet which is conserved in the core sites, GTTRRRY, found at the 3′ end of 59‐base elements. Recombination at the core site found in inverse orientation at the 5′ end of the 59‐base elements was not detected, and the sequences responsible for orientation of the recombination event appear to reside within the 59‐base element.

A model for site‐specific insertion of genes into integrons and Tn 7‐iike transposons is proposed. Circular units consisting of a gene associated with a 59‐base element are inserted into an ancestral element which contains neither a gene nor a 59‐base element. Further genes can be inserted at one or both of the composite sites generated by the first gene‐insertion
The rifampicin‐inducible genes srn6 from F and pnd from R483 are regulated by antisense RNAs and mediate plasmid maintenance by kiiling of plasmid‐free segregants
Authors: Nielsen A. K. , Thorsted P. , Thisted T. , Wagner E. G. H. , Gerdes K.
Published: October 2006

The gene systems srnB of plasmid F and pnd of plasmid R483 were discovered because of their induction by rifampicin. Induction caused membrane damage, RNase I influx, degradation of stable RNA and, consequently, cell killing. We show here that the srnB and pnd systems mediate efficient stabilization of a mini‐R1 test‐plasmid. We also show that the killer genes srnB′ and pndA are regulated by antisense RNAs, and that the srnC‐ and pndB‐encoded antisense RNAs, denoted SrnC‐ and PndB‐RNAs, are unstable molecules of approximately 60 nucleotides. The srnB and pndA mRNAs were found to be very stable. The differential decay rates of the inhibitory antisense RNAs and the killer‐gene‐encoding mRNAs explain the induction of these gene systems by rifampicin. Furthermore, the observed plasmid‐stabilization phenotype associated with the srnB and pnd systems is a consequence of this differential RNA decay: the newborn plasmid‐free cells inherit the stable mRNAs, which, after decay of the unstable antisense RNAs, are translated Into killer proteins, thus leading to selective killing of the plasmid‐free segregants. Thus our observations lead us to conclude that the F srnB and R483 pnd systems are phenotypically indistinguishable from the R1 hok/sok system, despite a 50% dissimilarity at the level of DNA sequence.
Simultaneous expression of a bacteriophage Mu transposase and repressor: a way of preventing killing due to mini‐Mu replication
Authors: Toussaint A. , Expert D. , Desmet L.
Published: October 2006

In vitro studies of bacteriophage Mu transposition have shown that the phage‐encoded transposase and repressor bind the same sequences on the phage genome. We attempted to test that prediction in vivo and found that Mu repressor directly inhibits transposition. We also found that, in the absence of repressor, constitutive expression of Mu transposition functions pA and pB is lethal in Escherichia coli strains lysogenic for a mini‐Mu and that this is the result of intensive replication of the mini‐Mu. These findings have important consequences where such mini‐Mus are used as genetic tools. We also tested whether in Erwinia chrysanthemi the effect of transposition functions on a resident mini‐Mu was the same as in E. coli. We observed that expression of pA alone was lethal in E. chrysanthemi and that a large fraction of the survivors underwent precise excision of the mini‐Mu.
The molecular basis of carbon‐starvation‐induced general resistance in Escherichia coli
Authors: Matin A.
Published: October 2006

At the onset of starvation Escherichia coli undergoes a temporally ordered program of starvation gene expression involving 40–80 genes which some four hours later yields cells possessing an enhanced general resistance. Two classes of genes are induced upon carbon starvation: the cst genes, requiring cyclic AMP, and the pex genes, not requiring this nucleotide for induction. The cst genes are not involved in the development of the resistant state and are concerned with escape from starvation, while the pex gene induction appears to be associated with resistance. Many of the latter are induced in response to a variety of starvation conditions. They include heat shock and oxidation resistance genes, and some utilize minor, stationary‐phase‐specific sigma factors for induction during starvation. The protective role of stress proteins may be due to their ability to rescue misfolded macromolecules. The starvation promoters can be potentially useful for selective expression of desired genes in metabolically sluggish populations, e.g. in high‐density industrial fermentations and in situ bioremediation.
The regulation of genetic competence in Bacillus subtilis
Authors: Dubnau D.
Published: October 2006

Genetic competence develops as a global response of Bacillus subtilis to the onset of stationary phase, in glucose‐minimal salts‐based media. The onset of competence is accompanied by the expression of several late gene products that are required for the binding, processing and uptake of transforming DNA. A number of regulatory genes have been identified that are needed for the appropriate synthesis of the late gene products. The regulatory gene products include a number of known transcription factors, as well as several members of the bacterial two‐component regulatory system. Genetic analysis has suggested a scheme for the flow of regulatory information signaling the onset of competence. Most of these regulatory products appear to be involved in the response to nutritional status, while the components responsible for growth stage and cell‐type‐specific control remain unknown. The general implications of this scheme for post‐exponential expression are discussed.
Evolution of the MIP family of integral membrane transport proteins
Authors: Pao G. M. , Wu L.‐F. , Johnson K. D. , Höfte H. , Chrispeels M. J. , Sweet G. , Sandal N. N. , Saier M. H.
Published: October 2006

Six integral membrane proteins of bacterial, animal, and plant origin, which are believed to function in solute transport, share sequence identity and are grouped together as members of the MIP family. These include the Escherichia coli glycerol facilitator, the major intrinsic protein from bovine lens fibre junction membranes, a plant tonoplast membrane protein, a soybean protein from the peribacteroid membrane, and a Drosophila neurogenic protein. These proteins, each of which appears to consist of six transmembrane helical segments per subunit, apparently arose by internal duplication of a three‐transmembrane segment. Phylogenetic‘trees’interrelating these proteins and segments are presented.
Molecular characterization of an operon (hyp) necessary for the activity of the three hydrogenase isoenzymes in Escherichia coli
Authors: Lutz S. , Jacobi A. , Schlensog V. , Böhm R. , Sawers G. , Böck A.
Published: October 2006

The 58/59 min region of the Escherichia coli chromosome contains two divergently oriented gene clusters coding for proteins with a function in hydrogenase formation. One cluster (the hyc operon), transcribed counterclockwise with respect to the E. coli chromosome, codes for gene products with a structural role in hydrogenase 3 formation (Böhm et al., 1990). The nucleotide sequence of the divergently transcribed operon (hyp) has been determined. It contains five genes, all of which are expressed in vivo in a T7 promoter/polymerase system, and the sizes of the synthesized products correspond with those predicted from the amino acid sequence. Complementation analysis of previously characterized mutants showed that the hypB, hypC and hypD genes have a function in the formation of all three hydrogenase isoenzymes, lesions in hypB being complemented by high nickel ion concentration in the medium. Prevention of hypBCDE gene expression led to an altered electrophoretic pattern of hydrogense 1 and 2 constituent subunits, indicating increased chemical or proteolytic susceptibility. Under fermentative growth conditions, operon expression was governed by an NtrA‐dependent promoter lying upstream of hypA working together with an fnr gene product‐dependent promoter which was localized within the hyp A gene. The latter (operon‐internal) promoter is responsible for hypBCDE transcription under non‐fermentative conditions when the ‐24/‐12 NtrA‐dependent promoter upstream of hyp A is silent.
Purification and characterization of two‐subunit cytochrome aa3 from Bacillus cereus
Authors: Garcia‐Horsman J. A. , Barquera B. , Gonzalez‐Halphen D. , Escamilla J. E.
Published: October 2006

Cytochrome c‐oxidase type aa3 (EC 1.9.3.1.) was purified to homogeneity from vegetative Bacillus cereus by ion‐exchange and hydroxylapatite chromatography in the presence of Triton X–100. Gel filtration analysis suggested a dimeric structure apparently 172kDa in size; however, only a monomer of 81kDa was detected when analysed by non‐denaturing gel electrophoresis. Denaturing gel electrophoresis analysis of the protein showed the presence of two subunits (51 and 30kDa), Atomic absorption and visible spectroscopy showed typical aa3 redox centres with haem a iron and copper in a ratio of 22 nmol and 35ng‐atom per mg protein, respectively. No haem c was found associated with the purified enzyme in the conditions reported here. Oxidase activity was fully reconstituted by phospholipids in the presence of N, N, N′ N′‐tetramethyl‐p‐phenylenediamine or reduced yeast cytochrome c (but not horse cytochrome c) as electron donors. This activity was abolished by cyanide and carbon monoxide.
Characterization of the yellow‐pigment genes of Erwinia herbicola
Authors: Lee L.‐Y. , Liu S.‐T.
Published: October 2006

A 6.7kb DNA fragment containing the pigment genes of Erwinia herbicola Eho13 has been cloned into Escherichia coli. These genes were chromosomally encoded in E. herbicola. The entire DNA fragment could be divided into at least three regions. Deletions in Region I resulted in a non‐pigmented phenotype, a deletion in Region II resulted in a pink/yellow phenotype, deletions in Region III resulted in either a pink or a non‐pigmented phenotype. Tn 1000 insertions in the same regions, however, gave different phenotypes. Insertions in Region U produced a pink phenotype. Insertions in Region III resulted in either a light‐yellow or a non‐pigmented phenotype. Minicell studies showed that the 6.7kb DNA fragment encoded at least five proteins (50kDa, 42kDa, 36kDa, 35kDa and 34kDa). A 2.7kb Hin dIII deletion in Region I caused the disappearance of these proteins, suggesting that this 2.7 kb fragment may play a regulatory role in pigment synthesis. Our results also showed that a 4.1kb Eco RV fragment consisted of Region I and a part of Region II complemented a pink/yellow clone of Eho10 (pHL545), suggesting that the pigments of Eho13 and Eho10 were probably similar or identical.
The use of a rare codon specifically during development?
Authors: Leskiw B. K. , Bibb M. J. , Chater K. F.
Published: October 2006

A range of circumstantial evidence suggests that in Streptomyces spp., genes required for vegetative growth do not contain the leucine codon TTA. Instead, the codon seems to be confined to a few genes necessary during differentiation, when the colonies begin to produce aerial hyphae and antibiotics. Thus, mutations in bldA, the structural gene for tRNALeuTTA, do not retard vegetative growth, but they prevent normal aerial mycelium and antibiotic production. Most of the known TTA‐containing genes specify regulatory or resistance proteins associated with antibiotic‐production clusters. Possibly the ability to translate the UUA codons in mRNA from such genes is confined to late stages of colony development. Factors that might have contributed to the evolution of this unusual situation are discussed.
Cloning, characterization and expression of the Zymononas mobilis eda gene that encodes 2‐keto‐3‐deoxy‐6‐phosphogluconate aldolase of the Entner‐Doudoroff pathway
Authors: Conway T. , Fliege R. , Jones‐Kilpatrick D. , Liu J. , Barnell W. O. , Egan S. E.
Published: October 2006

The eda gene that encodes 2‐keto‐3‐deoxy‐6‐phos‐phogluconate aldolase of the Entner‐Doudoroff pathway was cloned from Zymomonas mobilis by genetic complementation of an Escherichia coli mutant. The gene is present in a single copy on the Z mobilis genome and is not tightly linked to the edd gene. Nucleotide sequence analysis of the eda region revealed that the structural gene is 627 bp long and capable of encoding a protein of 208 amino acids with a deduced molecular weight of 21 505. The eda gene is monocistronic and is transcribed from a single promoter. The transcriptional initiation site was determined and an improved consensus promoter sequence for Z. mobilis was derived. High‐level expression of the eda gene can be attributed to very efficient translational initiation caused by the high quality of the ribosome‐binding site and stability of the mRNA, which has a decay rate of 7.6 min. A comparison of highly expressed Z mobilis genes indicated that the relative quality of the ribosome‐binding sites of these genes might play an important role in determining the level of enzyme synthesis. This possibility is discussed with regard to the role of gene expression in coordinating the enzyme levels of the Entner‐Doudoroff glycolytic pathway.
Genetic diversity among the T‐protein genes of group A streptococci
Authors: Jones K. F. , Schneewind O. , Koomey J. M. , Fischetti V. A.
Published: October 2006

T protein is a trypsin‐ and pepsin‐resistant molecule on the surface of group A streptococci used as a serological tool to differentiate streptococci of this group. The purpose of this study was to determine the relatedness among the T protein genes of the 25 known T serotypes. DNA probes were constructed which represented various regions of the structural gene for the T6 protein, tee6. The probes were assayed for their ability to hybridize Hin dlll digests of chromosomal DNA from the 25 different T serotypes. Probe pTEE6.3, coding for the entire T6 protein, and pTEE61–299, coding for the amino‐terminal half of T6, displayed the highest amount of homology, each binding to 10 of 25 T serotypes. Probes coding for sequences in the carboxy‐terminal half of T6 showed considerably less homology among T serotypes with one probe hybridizing with only three out of 25. A synthetic oligonucleotide coding for the carboxy‐terminal hydrophobic domain of T6, an area conserved to some degree among several bacterial surface proteins, showed homology with only seven out of 25 T serotypes. Hybridization with sequences outside the tee6 coding area provided additional information on the relatedness of certain sets of T serotypes according to restriction‐fragment size heterogeneity. Clearly, there is considerable diversity among T‐serotype genes. The data suggest that two or more families of structurally variant T proteins exist, which share only the property of proteolytic resistance and/or, perhaps, some biological function.
A protein kinase C‐like activity in Escherichia coli
Authors: Norris V. , Baldwin T. J. , Sweeney S. T. , Williams P. H. , Leach K. L.
Published: October 2006

The protein kinase C (PKC) family comprises calcium‐ and phospholipid‐dependent kinases whose activity is stimulated by diacylglycerol and tumour‐promoting phorbol esters such as 12‐tetradecanoyl phorbol‐13‐acetate (TPA). In the Gram‐negative bacterium Escherichia coli, functional similarity to PKC was demonstrated in crude extracts by calcium and phospholipid‐dependent, TPA‐stimulated phosphorylation of a small number of endogenous substrates. Activity was reduced by sphingosine, a known inhibitor of eukaryotic PKC. Structural similarity to PKC was demonstrated in crude and partially purified bacterial extracts by cross‐reactivity with several monoclonal antibodies. This revealed isozyme‐specific homology between a protein(s) of relative molecular mass 80–85000 in E. coli and the α‐and γ‐isozymes, but probably not the β‐isozyme, of eukaryotic PKC.
Molecular action of tricholin, a ribosome‐inactivating protein isolated from Trichoderma viride
Authors: Lin A. , Chen C.‐K. , Chen Y.‐J.
Published: October 2006

An extracellular protein was isolated from a species of soil‐borne fungi (Trichoderma viride) and its amino acid composition has been determined. The protein is acidic with a molecular mass of 14 200 daltons and is given the trivial name tricholin. Tricholin is a potent inhibitor of cell‐free protein synthesis. When rabbit reticulocyte lysate was incubated with tricholin at a concentration of 6.3 × 10−7 M, it completely abolished the capacity of the lysate to support protein synthesis. The inhibition appears to be due to its reaction to ribosomes, since it generates a specific cleavage product, an α‐sarcin RNA fragment, from reticulocyte ribosomal RNA. This reaction to ribosomes mimics that of α‐sarcin. The antibody of α‐sarcin strongly cross‐reacts with tricholin, while the antibody of tricholin shows a weak reaction with α‐sarcin.
A cytosine‐ over guanosine‐rich sequence in RNA activates rho‐dependent transcription termination
Authors: Rivellini F. , Alifano P. , Piscitelli C. , Blasi V. , Brunt C. B. , Carlomagno M. S.
Published: October 2006

We have constructed an expression vector carrying the Escherichia coli his operon control region to study the ability of defined segments of DNA to cause rho tactor‐mediated transcription termination both in vivo and in vitro. We have previously identified a consensus motif consisting of a region of high cytosine over guanosine content common to several cryptic intracistronic transcription termination elements unmasked by polar mutations. We show that a DNA fragment possessing features similar to the ones previously identified is capable of causing rho‐mediated release of transcripts in vivo and in vitro. The efficiency of termination depends on the length and relative cytosine over guanosine ratio of the element.
The tol gene products and the import of macronmolecules into Escherichia coli
Authors: Webster R. E.
Published: October 2006

Genetic studies have identified a number of genes whose products appear to be required for the transport of the group A colicins and the single‐stranded DNA of certain filamentous bacteriophages into Escherichia coli. Mutations in these genes allow normal binding of the colicins to their outer‐membrane receptors and of the bacteriophage to the tip of specific conjugative pili, but do not allow translocation of the macromolecules to their target. These mutations have been designed‘tolerant’(tol) mutations and the protein products specified by these genes appear to comprise part of a transport system known as the Tol import system. Some of these genes have been isolated, sequenced and their protein products localized to the membranes or periplasm of E. coli. Information is also available regarding the domains of the colicins or phage proteins which interact with the Tol proteins. A preliminary model of the location and possible interactions of the Tol proteins is presented.
Structure‐function analysis of the K88ab fimbrial subunit protein from porcine enterotoxigenic Escherichia coli
Authors: Pedersen P. A.
Published: October 2006

Several in‐frame linker insertions have been made in various positions in the faeG gene encoding the K88ab fimbrial subunit protein from porcine enterotoxigenic Escherichia coli. The effects of the linker insertions have been investigated with regard to the ability of the mutated fimbrial subunits to be exported to the surface of the bacterial cell and assembled into a fimbrial structure. The structure/function relationship of the subunit protein is discussed in the light of the phenotypes of the mutations constructed, secondary structure predictions, and hydrophilicity plots.
Gonococcal penicillin‐binding protein 3 and the surface‐exposed 44kDa peptidoglycan‐binding protein appear to be the same molecule
Authors: Shafer W. M. , Judd R. C.
Published: October 2006

The outer membrane of Neisseria gonorrhoeae contains a 44000 datton (44kDa) surface‐exposed protein which has the reported ability to form covalent interactions with peptidoglycan (PG). This PG‐binding outer‐membrane protein (OMP) appears to be highly conserved since it has been detected in all isolates examined. It also appears to be invariant since its primary structure among strains gives evidence of being identical (Judd et al., 1991). While studying the interaction of gonococcal penicillin‐binding proteins (PBPs) with human lysosomal cathepsin G, we noticed that the 44kDa PG‐binding OMP exhibited certain properties similar to PBP3. In this study we sought to obtain biochemical evidence to ascertain whether these proteins were the same. We found that both proteins fractionated with other sarkosyl‐insoluble OMPs and that they exhibited similar susceptibility to cleavage in situ by enzymatically active cathepsin G. Moreover, a purified preparation of the 44kDa OMP was found to covalently bind radiolabelled benzylpenicillin in vitro. Thus, the data presented herein suggest that the 44kDa PG‐binding OMP and PBP3 are the same OMP.
Molecular cloning of a gene involved in lipooligosaccharide biosynthesis and virulence expression by Haemophilus influenzae type B
Authors: Cope L D. , Yogev R. , Mertsola J. , Latimer J. L. , Hanson M. S. , McCracken G. H. , Hansen E. J.
Published: October 2006

A wild‐type Haemophilus influenzae type b (Hib) genomic DNA library was constructed in the plasmid shuttle vector pGJB103. A virulence‐deficient lipooligosaccharide (LOS) mutant of Hib was used as a recipient for genetic transformation to screen this Hib genomic DNA library for genes involved in LOS expression. A recombinant plasmid containing a 7.8 kb Pstl fragment of Hib DNA was shown to transform this LOS mutant to reactivity with a monoclonal antibody (mAb) specific for a wild‐type LOS epitope. Transformation of two different virulence‐deficient LOS mutants with a 4.4kb BglII fragment of this recombinant plasmid yielded transformants which expressed LOS that bound the wild‐type LOS‐specific mAb and yielded profiles in sodium dodecyl sulphate/polyacrylamide gradient gel electrophoresis different from those of the original LOS mutants. These transformants with structurally altered LOS molecules also exhibited increased virulence in an animal model for invasive Hib disease. The virulence‐transforming ability was further localized to a 1.8kb BglII‐AlwNI fragment of the Hib DNA insert. Nucleotide sequence analysis indicated the presence of a single large open reading frame within this fragment. This open reading frame contained 19 consecutive repeats of the tetramer CAAT near the 5’end. Linker insertion mutagenesis was used to demonstrate directly the involvement of this open reading frame in both LOS biosynthesis and virulence expression by Hib.
Characterization of legiolysin (lly), responsible for haemolytic activity, colour production and fluorescence of Legionella pneumophila
Authors: Wintermeyer E. , Rdest U. , Ludwig B. , Debes A. , Hacker J.
Published: October 2006

A genomic library of Legionella pneumophila, the causative agent of Legionnaires’disease in humans was constructed in Escherichia coli K12 and the recombinant clones were tested for haemolysis and other phenotypic properties. Seven clones were identified which were able to confer haemolysis of human, sheep, and canine erythrocytes but which were unable to mediate proteolytic activities or cyto‐toxic effects on CHO‐ or Vero cells. Clones that exhibited this haemolytic property were also able to produce a brown colour and a yellow‐green fluorescence activity detected on M9 plates containing tyrosine. The genetic determinant encoding these properties, termed legiolysin (lly) was mapped by Tn 1000 mutagenesis and by subcloning experiments. Southern hybridization with an lly‐specific gene probe showed that this determinant is part of the genome of L. pneumophila but is not identical to a protease gene of L. pneumophila which also mediates haemolysis. Minicell analysis of lly‐specific plasmids exhibited a protein of 39kDa. Polyclonal antibodies generated against a LacZ‐Lly hybrid protein also recognized a 39kDa protein produced either by the recombinant legiolysin‐positive E. coli K12 clones or by L. pneumophila wild‐type strains.
Chromosome strand segregation during sporulation in Bacillus subtilis
Authors: Errington J. , Wake R. G.
Published: October 2006

After the initiation of spore formation In Bacillus subtilis, the products of the final round of DNA replication segregate into two cells, i.e. the prespore and the mother cell. The prespore, which is known to contain a single completed chromosome, develops into a mature endospore which can be readily separated from mother cells and non‐sporulating cells on the basis of its resistance properties. We have used a procedure originally developed to label the terminus region of the B. subtilis chromosome to specifically label the newly synthesized strands of DNA during the final round of DNA replication before sporulation. We have purified prespore DNA and used strand‐specific probes to measure the radioactivity incorporated. The results show that the sister chromosomes segregate at random into the prespore. This result has implications for the segregation of chromosomes during vegetative growth and for the generation of cellular asymmetry during sporulation.
Selenocysteyl‐tRNA occurs in the diatom Thalassiosira and in the ciliate Tetrahymena
Authors: Hatfield D. L. , Lee B. J. , Price N. M. , Stadtman T. C.
Published: October 2006

Selenocysteyl‐tRNAs that decode UGA were identified previously in animal and bacterial cells and the genes for these tRNAs have been shown to be widespread in animals and eubacteria. In the present study, we identify a selenocysteyl‐tRNA that codes for UGA in Thalassiosira pseudonana, which is a diatom, and in Tetrahymena borealis, which is a ciliate. The fact that these very diverse unicellular organisms also contain a selenocysteyl‐tRNA suggests that selenocysteine‐containing proteins and the use of UGA as a codon for selenocysteine are widespread, if not ubiquitous, in nature.
A cloned ompR‐like gene of Streptomyces lividans 66 suppresses defective melC1, a putative copper‐transfer gene
Authors: Tseng H.‐C. , Chen C. W.
Published: October 2006

Expression of tyrosinase in Streptomyces requires functional MelC1 protein, which is postulated to transfer copper to apotyrosinase. We have previously isolated a mutant of Streptomyces lividans, HT32, that phenotypically suppressed mutations in cloned melC1 (H.‐C. Tseng and C. W. Chen, in preparation). Plasmid pLUS132, containing an ATG to ATA transition at the initiation codon of melC1, was used for cloning the suppressor gene from HT32. A 1687bp suppressor DNA was isolated that contained two characteristic Streptomyces coding sequences: a 217‐amino‐acid open reading frame (cutR) and a truncated open reading frame (cutR) downstream. Subcloning analysis attributed the phenotypic suppression activity to the putative cutR gene from HT32. The putative CutR exhibited similarity to the response regulator OmpR of the osmoregulatory signal‐transduction system in Escherichia coli. The truncated CutS resembled, to a lesser degree, the N‐terminus of EnvZ, the histidine protein kinase counterpart of OmpR. DNA hybridizing to the cloned cutR‐cutS sequence was detected in 16 other Streptomyces species. We postulate that the putative cutR‐cutS operon regulates copper metabolism in Streptomyces.
The glucose permease of the phosphotransferase system of Bacillus subtilis: evidence for IIGlc and IIIGlc domains
Authors: Gonzy‐Tréboul G. , de Waard J. H. , Zagorec M. , Postma P. W.
Published: October 2006

Glucose is taken up in Bacillus subtilis via the phosphoenolpyruvate:glucose phosphotransferase system (glucose PTS). Two genes, orfG and ptsX, have been implied in the glucose‐specific part of this PTS, encoding an Enzyme IIGlc and an Enzyme IIIGlc, respectively. We now show that the glucose permease consists of a single, membrane‐bound, polypeptide with an apparent molecular weight of 80000, encoded by a single gene which will be designated ptsG. The glucose permease contains domains that are 40‐50% identical to the IIGlc and IIIGlc proteins of Escherichia coli. The B. subtilis IIIGlc domain can replace IIIGlc in E. coli crr mutants in supporting growth on glucose and transport of methyl α‐glucoside.

Mutations in the IIGlc and IIIGlc domains of the B. subtilis ptsG gene impaired growth on glucose and in some cases on sucrose. ptsG mutants lost all methyl α‐glucoside transport but retained part of the glucose‐transport capacity. Residual growth on glucose and transport of glucose in these ptsG mutants suggested that yet another uptake system for glucose existed, which is either another PT system or regulated by the PTS.

The glucose PTS did not seem to be involved in the regulation of the uptake or metabolism of non‐PTS compounds like glycerol. In contrast to ptsl mutants in members of the Enterobacteriaceae, the defective growth of B. subtilis ptsl mutants on glycerol was not restored by an insertion in the ptsG gene which eliminated IIGlc. Growth of B. subtilis ptsG mutants, lacking IIGlc, was not impaired on glycerol. From this we concluded that neither non‐phosphorylated nor phosphorylated IIGlc was acting as an inhibitor or an activator, respectively, of glycerol uptake and metabolism.
Regulation of methionine synthesis in Escherichia coli
Authors: Weissbach H. , Brot N.
Published: October 2006

The biosynthesis of methionine in Escherichia coli is under complex regulation. The repression of the biosynthetic pathway by methionine is mediated by a repressor protein (MetJ protein) and S‐adenosyl‐methionine which functions as a corepressor for the MetJ protein. Recently, a new regulatory locus, metR has been identified. The MetR protein is required for both metE and metH gene expression, and functions as a transactivator of transcription of these genes. MetR is a unique prokaryotic transcription activator in that it possesses a leucine zipper motif, first described for eukaryotic DNA‐binding proteins. The transcriptional activity of MetR is modulated by homocysteine, the metabolic precursor of methionine. Finally, it is known that vitamin B12 can repress expression of the metE gene. This effect is mediated by the MetH holoenzyme, which contains a cobamide prosthetic group.
Regulation of capsular polysaccharide synthesis in Escherichia coli K12
Authors: Gottesman S. , Stout V.
Published: October 2006

Synthesis of the capsular polysaccharide colanic acid in Escherichia coli K12 Is regulated by a complex network of regulatory proteins. This regulation is expressed at the level of transcription of the cps (capsular polysaccharide synthesis) genes. Two positive regulators, RcsA and RcsB, are necessary for maximal capsule expression. The availability of RcsA is normally limited because the RcsA protein is rapidly degraded by the Lon ATP‐dependent protease. Therefore Lon acts, indirectly, as a negative regulator of capsule synthesis. The sequence predicted for RcsB suggests that it is the effector component of a two‐component system; a protein with homology to sensors, RcsC, also plays a role in capsule regulation. We propose a model for capsule synthesis in which RcsA interacts with RcsB to stimulate transcription of the cps genes. The mechanism of regulation of colanic acid synthesis in E. coli may apply to other capsules in a variety of Gram‐negative bacteria.
Thirty‐three nucleotides of 5′ flanking sequence including the ‘TATA’ box are necessary and sufficient for efficient U2 snRNA transcription in Schizosaccharomyces pombe
Authors: Dandekar T. , Tollervey D.
Published: October 2006

We have sequenced the 5′ flanking region of the U2 gene and compared this with the 5′ flanking sequences of other snRNA genes from Schizosac‐charomyces pombe. This revealed no regions of clear homology 5′ to a region surrounding the ‘TATA’ box at −32 to −29. Deletion analysis shows that a 5′ flanking region extending to only −33 is sufficient for accurate and efficient transcription of U2 in Schizosaccharomyces pombe.
A gene in the archaebacterium Sulfolobus solfataricus that codes for a protein equivalent to the alpha subunits of the signal recognition particle receptor in eukaryotes
Authors: Ramirez C. , Matheson A. T.
Published: October 2006

We have sequenced a gene in the archaebacterium Sulfolobus solfataricus that codes for a protein that shows sequence similarity to the alpha subunit of the signal recognition particle receptor or docking protein In eukaryotes and the product of the ftsY gene in Escherichia coli. Comparison of the Sulfolobus‘docking protein’ with its eukaryotic and eubacterial counterparts showed that the region of highest sequence similarity corresponds to a GTP‐binding site. The presence of this gene in archaebacteria suggests that some of the components involved in protein transport have been conserved in the three kingdoms.
Both genes for EF‐1α in Candida albicans are translated
Authors: Sundstrom P. , Irwin M. , Smith D. , Sypherd P. S.
Published: October 2006

In previous work, we showed that Candida albicans has two genes, TEF‐1 and TEF‐2, which encode identical polypeptides for the highly conserved, essential, protein synthesis factor EF‐1α (Breviario et al., 1988). This result prompted questions as to whether C. albicans preferentially uses one of the genes over the other and whether both genes are actually translated into protein. Gene‐specific sequence differences in the untranslated portion of each gene made it possible to prepare gene‐specific oligonucleotide hybridization probes. Results with the probes showed that the relative steady‐state mRNA levels of the two genes were equivalent and that the mRNA for each gene was present in active translation complexes.
Absence of acetohydroxy acid synthase III in Salmonella typhimurium is due to early termination of translation within the ilvl gene
Authors: Ricca E. , Lago C. T. , Sacco M. , De Fellce M.
Published: October 2006

The cryptic ilvlH locus of Salmonella typhimurium has genetic information for two distinct subunits of acetohydroxy acid synthase III. We show that the ilvH‐encoded subunit is normally translated and the lack of activity is due to early termination of translation within the promoter‐proximal ilvl gene. Analysis of the 5’region of the operon led to identification of the promoter and the amino‐terminal part of ilvl. Expression of this gene in a mutant producing acetohydroxy acid synthase is due to a transversion which creates a UUA (leucine) codon in the place of a UGA (stop) codon present in position 12 of the wild‐type coding region.
The mdoA locus of Escherichia coli consists of an operon under osmotic control
Authors: Lacroix J.‐M. , Loubens I. , Tempête M. , Menichi B. , Bohin J.‐P.
Published: October 2006

In Escherichia coli, the 5 kb mdoA locus is involved in the osmotically controlled biosynthesis of periplasmic membrane‐derived oligosaccharides (MDOs). The structure of this locus was analysed by in vitro cassette insertion, transposon mutagenesis, and gene‐fusion analysis.

A ‘neo’ cassette, derived from the neomycin phosphotransferase II region of transposon Tn5, was inserted into mdoA, borne by a multicopy plasmid. This plasmid was shown to complement two previously described mdoA mutations, depending on the orientation of the exogenous gene. Thus, the gene altered by these mutations could be expressed under the control of the exogenous promoter. Moreover, the ‘neo’ cassette inactivated another, uncharacterlzed, mdo gene, because when this insertion was transferred into the chromosome MDO synthesis was abolished.

The existence of a second gene was confirmed by complementation analysis with a collection of Tn1000 insertions into mdoA. Two groups were defined, and the two genes are organized into an operon (mdoGH). This conclusion was reached because Tn1000 insertions in the first gene displayed a polar effect on the expression of the second gene.

An active gene fusion was obtained on a multicopy plasmid between the beginning of mdoH and lacZ. The hybrid β‐galactosidase activity followed the same osmotically controlled response as that described for of MDO synthesis. This regulation was unaffected by the presence, or absence, of MDOs In the periplasm. Finally, the amount of mdoA‐specific mRNAs, determined by dot blot hybridization, decreased when the osmolarity of the growth medium increased.
Location of the RNA‐processing enzymes RNase III, RNase E and RNase P in the Eschenchia coli cell
Authors: Miczak A. , Srivastava R. A. K. , Apirion O.
Published: October 2006

Cells overexpressing the RNA‐processing enzymes RNase III, RNase E and RNase P were fractionated into membrane and cytoplasm. The RNA‐processing enzymes were associated with the membrane fraction. The membrane was further separated to inner and outer membrane and the three RNA‐processing enzymes were found in the inner membrane fraction. By assaying for these enzymatic activities we showed that even in a normal wild‐type strain of Escherichia coli these enzymes fractionate primarily with the membrane. The RNA part of RNase P is found in the cytosolic fraction of cells overexpressing this RNA, while the overexpressed RNase P protein sediments with the membrane fraction; this suggests that the RNase P protein anchors the RNA catalytic moiety of the enzyme to a larger entity. The implications of these findings for the cellular organization of the RNA‐processing enzymes in the cell are discussed.
Regulation of sod genes in Escherichia coli: relevance to superoxide dismutase function
Authors: Fee J. A.
Published: October 2006

This review is concerned with the effects of environmental perturbations on the expression of the two superoxide dismutase (SOD) genes in Escherichia coli (sodA, MnSOD; sodB, FeSOD). Early studies using SOD activity, showed that MnSOD levels respond to changes in oxygen tension, type of substrate, redox active compounds, iron concentration, the nature of the terminal oxidant, and the redox potential of the medium. FeSOD levels appeared nominally insensitive to these perturbations. More recent molecular genetic studies revealed that sodA expression is subject to regulation by three major regulatory systems: fur (ferric uptake regulation) and arcA arcB (aerobic respiratory control) mediate repression of sodA, while a relatively new system, soxR soxS (superoxide response), mediates activation of sodA expression. By contrast, sodB expression, which is much less studied at this time, appears to be positively activated in trans by fur. A rudimentary gene regulation model is presented which rationalizes past observations, is experimentally testable, and should serve as a guide to future research in this area.
Activation of cholera toxin and Escherichia coli heat‐labile enterotoxins by ADP‐ribosylation factors, a family of 20 kDa guanine nucleotide‐binding proteins
Authors: Moss J. , Vaughan M.
Published: October 2006

Cholera toxin and Escherichia coli heat‐labile enterotoxins are responsible, in part, for the symptomatology of cholera and traveller's diarrhoea, respectively. Effects of the toxins result from ADP‐ribosylation of regulatory guanine nucleotide‐binding (G) proteins; the ADP‐ribosylated G protein is stabilized in an activated state, resulting in prolonged effects on its target. Toxin‐catalysed ADP‐ribosylation is stimulated in vitro by a family of guanine nucleotide‐binding proteins, c. 20 kDa, termed ADP‐ribosylation factors or ARFs. In the presence of GTP, but not GDP or adenine analogues, ARFs serve as allosteric activators of the toxin. The effects are amplified by certain phospholipids and detergents which promote guanine nucleotide binding. Six different mammalian ARF genes have been identified. They encode highly conserved, ubiquitous proteins of 175 to 181 amino acids, containing consensus domains responsible for guanine nucleotide binding. Differences in amino acid sequences are localized near the amino terminus and in the carboxy half of the protein. Although the physiological functions of ARFs have not been precisely defined, their immunological localization to the Golgi is consistent with a role in the regulated orderly movement of newly synthesized proteins from the endoplasmic reticulum, through the Golgi system to their ultimate destination.
Escherichia coli molecular genetic map (1500 kbp): update II
Authors: Médigue C. , Viari A. , Hénaut A. , Danchin A.
Published: October 2006

The DNA sequence data for Escherichia coli deposited in the EMBL library (release 27), together with miscellaneous data obtained from several laboratories, have been localized on an updated and corrected version of the restriction map of the chromosome generated by Kohara et al. (1987) and modified by others. This second update adds a further 500 kbp, increasing the amount of the E. coli chromosome sequenced to about one third of the total: 1510 kbp of sequenced DNA is included in the present data base. The accuracy of the map is assessed, and allows us to propose a precise genetic map position for every sequenced gene. The location of rare‐cutting sites such as AvII, NotI and SfiI have also been included in the update in order to combine the data obtained from different sources into one single file. The distribution of palindromic sequences (to which most restriction sites belong) has been studied in coding sequences. There appears to be a significant counter‐selection against several such sequences in E. coli coding sequences (but not in other organisms such as Saccharomyces cerevisiae), suggesting the existence of constraints on DNA structure in E. coli, perhaps indicative of a functional role for horizontal gene transfer, preserving coding sequences, in this type of bacteria.