bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression patterns of genes encoding elastase and controlling mucoidy: co‐ordinate regulation of two virulence factors in Pseudomonas aeruginosa isolates from cystic fibrosis
Authors: Mohr C. D. , Rust L. , Albus A. M. , Iglewski B. H. , Deretic1 V.
Published: October 2006

Transcriptional patterns of lasB and algD were compared in isogenic mucoid and non‐mucoid Pseudomonas aeruginosa isolates from cystic fibrosis patients. The lasB gene encodes elastase, a major proteolytic enzyme secreted by P. aeruginosa, while algD is required for the synthesis of alginate, an exopolysaccharide frequently overproduced by strains infecting cystic fibrosis patients. A possible coregulation at the transcriptional level of these major virulence determinants was analysed. The lasB and algD genes showed inverse levels of promoter activity. The lasB promoter was active in non‐mucoid cells and inactive in mucoid cells (in four out of five tested pairs), while the algD promoter was active in mucoid cells and silent in non‐mucoid cells in all cases. When PAO568, a model strain for the analysis of control of the alginate system, was grown under conditions promoting mucoidy, the algD promoter was activated, whereas lasB mRNA could not be detected. This effect was reversed when the cells were grown in a medium suppressing mucoidy. Insertional inactivation of algR, a member of the signal‐transduction systems regulating algD transcription, although abolishing algD expression and rendering cells non‐mucoid, did not alter the nature of the induction and repression patterns of lasB seen in the parental strain PAO568. These results suggest that the lasB gene and the alginate system are co‐ordinately regulated at a level parallel to or above the algR gene.
Genetic analysis of the cytochrome c‐aa3 branch of the Bradyrhizobium japonicum respiratory chain
Authors: Bott M. , Bolliger M. , Hennecke H.
Published: October 2006

Further genetic evidence is provided here that Bradyrhizobium japonieum possesses a mitochondria‐like electron‐transport pathway: 2[H]→UQ→bc1→c→aa3→O2. Two Tn5‐induced mutants, COX122 and COX132, having cytochrome c oxidase‐negative phenotypes, were obtained and characterized. Mutant COX122 was defective in a novel gene, named cycM, which was responsible for the synthesis of a c‐type cytochrome with an Mr, of 20000 (20K). This 20K cytochrome c appeared to catalyse electron transport from the cytochrome bc1, complex to the aa3‐type terminal oxidase and, unlike mitochondrial cytochrome c, was membrane‐bound in B. japonicum. The Tn5 insertion of mutant COX132 was localized in coxA, the structural gene for subunit I of cytochrome aa3. This finding also led to the cloning and sequencing of the corresponding wild‐type coxA gene that encoded a 541 ‐amino‐acid protein with a predicted Mr of 59247. The CoxA protein shared about 60% sequence identity with the cytochrome aa3 subunit I of mitochondria. The B. japonicum cycM and coxA mutants were able to fix nitrogen in symbiosis with soybean (Fix+). In contrast, mutants described previously which lacked the bc1 complex did not develop into endosymbiotic bacteroids and were thus Fix−. The data suggest that a symbiosis‐specific respiratory chain exists in B. japonicum in which the electrons branch off at the bc1 complex.
Attenuated mutants of the intracellular bacterium Listeria monocytogenes obtained by single amino acid substitutions in listeriolysin O
Authors: Michel E. , Reich K. A. , Favier R. , Berche P. , Cossart P.
Published: October 2006

Listeriotysjn O (LLO), a major virulence factor of the intracellular bacterium Listens monocytogenes, shares with other known ‘thiol‐activated toxins’ a conserved undecapeptide, ECTGLAWEWWR, located in the C‐terminal region of the protein and containing the unique cysteine of the molecule. Single amino acid substitutions were created in this region to study the role of cysteine and tryptophan residues in the lytic activity of LLO as well as in the virulence of the bacterium. Transformation of a transposon‐induced non‐haemolytic mutant with plasmids carrying the mutated genes allowed allele exchange and transfer of mutations on to the chromosome by in vivo recombination. The mutant strains secreted a full‐length 59 kilodalton LLO. A decrease of 25% in the haemolytic activity in culture supernatants was observed in the case of mutation Cys‐484 to Ala and of 80% for mutation Cys‐484 to Ser. Mutations Trp‐491 and Trp‐492 to Ala decreased activity by, respectively, 95% and 99.9%. LLOs produced by the mutants, as the wild type, were active at low pH, inhibited by cholesterol, and able to bind to cell membranes. A close relationship was found between virulence of mutants in the mouse model and haemolytic activity in their culture supematants. These results demonstrate that the thiol group of Cys‐484 is not essential for either haemolytic activity in vitro or virulence in vivo. In contrast, Trp‐492 appears to be required for both haemolytic activity and virulence. The finding that the nearly non‐haemolytic mutant Trp‐492‐Ala persisted in the spleen for several days after inoculation indicates that mutagenesis of a virulence determinant can attenuate virulence and provides a novel approach to the development of live vaccine strains.
Functions of the flagellar modes of rotation in bacterial motility and chemotaxis
Authors: Eisenbach M.
Published: October 2006

Bacteria swim by rotating their flagella, the rotation being due to a motor located at the base of each flagellum. In this paper the correlation between motor function and mode of swimming is reviewed, with special emphasis on recent data that indicate that the motor is a three‐state device. Novel findings with regard to the motor function and bioenergetics are surveyed, and mechanisms are proposed to account for these findings.
Mapping of sequenced genes (700 kbp) in the restriction map of the Escherichia coli chromosome
Authors: Médigue C. , Bouché J. P. , Hénaut A. , Danchin A.
Published: October 2006

This paper describes software (written in Pascal and running on Macintosh computers) allowing localization of unknown DNA fragments from the Escherichia coli chromosome on the restriction map established by Kohara et al. (1987). The program identifies the segment's map position using a restriction pattern analysis obtained with all, or some, of the eight enzymes used by Kohara et al. (1987). Therefore, the sequenced genes available in the EMBL library may be localized on the E. coli chromosome restriction map. This allowed correction of the map (mainly by introducing missing sites in the published maps) at the corresponding positions. Analysis of the data indicates that there is only a very low level of polymorphism, at the nucleotide level, between the E. coli K12 strains used by the various laboratories involved in DNA sequencing. The program is versatile enough to be used with other genomes.
Mucoid Pseudomonas aeruginosa in cystic fibrosis: mutations in the muc loci affect transcription of the algR and algD genes in response to environmental stimuli
Authors: Deretic V. , Govan J. R. W. , Konyecsni W. M. , Martin D. W.
Published: October 2006

Increased levels of alginate biosynthesis cause mucoidy in Pseudomonas aeruginosa, a virulence factor of particular importance in cystic fibrosis. The algR gene product, which controls transcription of a key alginate biosynthetic gene, algD, is homologous to the activator members of the two‐component, environmentally responsive systems (NtrC, OmpR, PhoB, ArcA, etc). In this report, we show that mutations in the muc loci, (muc‐2, muc‐22, and muc‐23, in the standard genetic P. aeruginosa strain PAO, as well as a mapped muc allele in an isolate from a cystic fibrosis patient) affect transcription of algD and algR. This influence was strongly dependent on environmental factors. Regulation by nitrogen was observed in all strains examined, but the absolute transcriptional levels, determining the mucoid or non‐mucoid status, were strain (muc allele)‐dependent. Increased concentrations of NaCl in the medium, an osmolyte which is elevated in cystic fibrosis lung secretions, resulted in an increased algD transcription and mucoid phenotype in a muc‐2 strain; the same conditions, however, produced a nonmucoid phenotype in the muc‐23 background and abolished algD transcription. Mutations in the muc loci may cause mucoidy by deregulating the normal response of the alginate system to environmental stimuli.
Molecular characterization of the nodulation gene, nodT, from two biovars of Rhizobium leguminosarum
Authors: Surin B. P. , Watson J. M. , Hamilton W. D. O. , Economou A. , Downie J. A.
Published: October 2006

DNA sequencing of the nodlJ region from Rhizobium leguminosarum biovar trifolii revealed the nodT gene immediately downstream of nodJ. DNA hybridizations using a nodT‐specific probe showed that nodT is present in several R. leguminosarum strains. Interestingly, a flavonoid‐inducible nodT gene homologue in R. leguminosarum bv. viciae is not in the nodABCIJ operon but is located downstream of nodMN. The sequence of the nodT gene from bv. viciae was determined and a comparison of the predicted aminoacid sequences of the two nodT genes shows them to be conserved; the predicted protein sequences appear to have a potential transit sequence typical of outer‐membrane proteins. Mutations affecting nodT in either biovar had no observed effect on nodulation of the legumes tested.
Export and processing analysis of a fusion between the extracellular heat‐stable enterotoxin and the periplasmic B subunit of the heat‐labile enterotoxin in Escherichia coli
Authors: Guzmán‐Verduzco L.‐M. , Kupersztoch Y. M.
Published: October 2006

As an initial approach in the study of the mechanism of secretion of the extracellular heat‐stable enterotoxin of Escherichia coli (STA), and in order to use this polypeptide as an extracellular carrier we previously constructed a fusion between the complete STA toxin (pre‐pro‐STA) and the mature B subunit of the periplasmic heat‐labile enterotoxin (LTB); the resulting STA‐LTB hybrid was not secreted to the extracellular environment, and cells expressing the hybrid lysed at temperatures above 35°C. In this work we have established that the hybrid is initially detected as pre‐pro‐STA‐LTB and converted to pro‐STA‐LTB, which lacks the 19amino acids that share the properties of a signal peptide; the sequenced 17 amino‐terminal residues of pro‐STA‐LTB defined the processing site of pre‐pro‐STA‐LTB at pro_3phe_2ala_1 ↓ gln+1. This process was sensitive to an energy uncoupler (CCCP) and was correlated with translocation of pro‐STA‐LTB across the inner membrane. Additionally, we are able to show that although pre‐pro‐STA‐LTB is processed at 37°C and 29°C, it is more efficiently processed at the latter temperature. At 37°C, pro‐STA‐LTB was poorly released into the periplasm, resulting in accumulation of this protein, pre‐pro‐STA‐LTB, and pre‐β‐lactamase in the inner membrane, and in cell lysis. In contrast, at 29°C pro‐STA‐LTB was localized in the periplasm and in the inner membrane, and pre‐pro‐STA‐LTB and pre‐β‐lactamase did not accumulate; however, translocation of periplasmic pro‐STA‐LTB across the outer membrane still did not occur, and a second processing step that would eliminate the pro segment from pro‐STA‐LTB was never observed. Thus, the fusion of pre‐pro‐STA and LTB resulted in a polypeptide that, while incompatible with secretion to the extracellular medium, is exported to the periplasm in a temperature‐conditional fashion. This latter observation is consistent with an STA secretion pathway whereby pre‐pro‐STA is first processed to periplasmic pro‐STA by the removal of 19‐amino‐acid signal peptide.
A soluble 60 kilo Dalton antigen of Chlamydia spp. is a homologue of Escherichia coli GroEL
Authors: Bavoil P. , Stephens R. S. , Falkow S.
Published: October 2006

Two major 60kD protein species can be separated by differential detergent extraction in Chlamydia spp. A Sarkosyl‐soluble 60kD protein is (i) structurally and antigenically distinct from the previously characterized 60kD Omp2 outer membrane protein; and (ii) antigenically related to a bacterial common antigen of similar molecular weight which includes a 65 kD myco‐bacterial antigen and the GroEL heat‐shook protein of Escherichia coli. Among GroEL homologues, the chlamydial protein (chl‐GroEL) uniquely displays affinity towards immobilized thiol groups. The significance of this property is discussed with respect to the synthesis and assembly of the chlamydial disulphide‐rich cell wall late in the growth cycle. Chl‐GroEL is identical to the Triton X‐100‐soluble, ocular delayed‐type hypersensitivity agent (Morrison et al., 1989), an essential component in the development of blinding trachoma. An autoimmune mechanism for chronic chlamydial diseases based on chl‐GroEL homology to host proteins is hypothesized.
Transcriptional regulation of the cytR repressor gene of Escherichia coli: autoregulation and positive control by the cAMP/CAP complex
Authors: Gerlach P. , Valentin‐Hansen P. , Bremer E.
Published: October 2006

The Escherichia coli cytR‐encoded repressor protein (CytR) controls the expression of several genes involved in nucleoside and deoxynucleoside uptake and metabolism. The cytR promoter was identified by determining the transcriptional initiation site of the cytR gene. A chromosomal cytR‐lacZ+ operon fusion was isolated and used to study the regulation of cytR. We show that cytR expression is negatively controlled by the CytR protein and positively affected by the cAMP/CAP complex. Foot printing studies with purified CAP protein revealed two CAP binding sites upstream of the cytR promoter. A previousty described mutation (cytR*) in the cloned cytR gene, which results in the phenotypic suppression of a CytR operator mutation in the tsx P2 promoter, was analysed. DN A sequence analysis of the cytR* mutation revealed a G‐C to an A‐T base pair transition at position ‐34 bp relative to the translational initiation site of cytR. This point mutation activates a cryptic promoter that is stronger than the wild‐type cytR promoter and leads to overproduction of the CytR repressor.
Identification of a 180kD protein in Escherichia coli related to a yeast heavy‐chain myosin
Authors: Casaregola S. , Norris V. , Goldberg M. , Holland I. B.
Published: October 2006

A high molecular‐weight protein from Escherichia coli sharing structural ho mo logy at the protein level with a yeast heavy‐chain myosin encoded by the MYO1 gene is described. This 180kD protein (180‐HMP) can be enriched in cell fractions following the procedure normally utilized for the purification of non‐muscle myosins. In Western blots this protein cross‐reacts with a monoclonal antibody against yeast heavy‐chain myosin. Moreover, antibodies raised against the 180kD protein cross‐react with the yeast myosin and with a myosin heavy chain from chicken. Recognition by anti‐180‐HMP antibodies of an overexpressed fragment of yeast myosin encoded by MYO1 allows the localization of one of the shared epitopes to a specific region around the ATP binding site of the yeast myosin heavy chain. The existence of a high molecular‐weight protein with structural similarity to myosin in E. coli raises the possibility that such a protein might generate the force required for movement in processes such as nucleoid segregation and cell division.
Mechanism for induction of adaptive mutations in Escherichia coli
Authors: Boe L.
Published: October 2006

When bacterial cells are subjected to a strong selective pressure it often induces specific mutations. Here a model is considered in which errors are introduced at random in one of the strands of the DNA molecule: a nick in one of the strands can initiate strand displacement rendering a region of the chromosome single‐stranded. Upon conversion back to double‐stranded DNA there is a certain probability of introducing errors creating a heteroduplex. If an error results in the production of an mRNA molecule encoding a product which provides a selective advantage, growth will be stimulated and the mutation can be immortalized by chromosomal replication. Otherwise, the error can be corrected by the DNA ‘proofreading’ enzymes.
A physical map of the Mycoplasma genitalium genome
Authors: Colman S. D. , Hu P.‐C. , Litaker W. , Bott K. F.
Published: October 2006

We report the construction of a physical map of the genome of the human pathogen Mycoplasma genitalium through the use of pulse‐field gel electrophoresis. The small size and relative simplicity of this genome permit the arrangement of restriction fragments without having to construct linking clones. The size of the genome has been calculated to be approximately 600 kb and several important genetic determinants have been assigned specific loci on the map.
Cloning and sequence analysis of linear plasmid telomeres of the bacterium Borrelia burgdorferi
Authors: Hinnebusch J. , Bergstrom S. , Barbour A. G.
Published: October 2006

Summary

Borrelia burgdorferi, the Lyme disease agent, has double‐stranded linear plasmids with covalently closed ends. DNA at the ends, or telomeres, of two linear plasmids of B. burgdorferi strain B31 was examined. Telomeric sequences from both ends of a 16kb linear plasmid and from one end of a 49kb linear plasmid were cloned and sequenced. An 18bp AT‐rich inverted repeat was found at each end of the 16kb linear plasmid. The sequences of the two ends of this plasmid were different beyond these short inverted terminal repeats. The cloned end of the 49kb linear plasmid had sequence identity with one end of the 16kb linear plasmid. The end sequence common to both plasmids contained a series of phased, short direct repeats and a 52bp palindrome adjacent to a highly AT‐rich region. These findings indicate that Borrelialinear plasmid telomeres have structural features different from those of other known replicons.
Analysis of three nodD genes in Rhizobium leguminosarum biovar phaseoli; nodD 1 is preceded by nolE, a gene whose product is secreted from the cytoplasm
Authors: Davis E. O. , Johnston A. W. B.
Published: October 2006

In a strain of Rhizobium leguminosarum biovar phaseoli, three copies of the regulatory nodulation gene nodD were identified on the Sym plasmid and sequenced. Two were closely linked to each other and the third was near, but not adjacent, to the nodABC genes. Each of these nodD genes could correct the Nod defect of a nodD mutant strain of R. leguminosarum biovar viciae on peas. A truncated form of nodD2 could also correct this mutant, indicating that the C‐terminus of NodD2 is not needed for inducing activity. Upstream of nodD1 and in the same operon is a newly described gene, nolE, whose product appears to be exported into the periplasm. Close to nodD2 is another gene, nolP, with no known counterpart in other rhizobia. Both nolP and nolE‐nodD1 are preceded by ‘nod‐box’ sequences and, in the former case, there appear to be two tandemly repeated nod‐box sequences. Mutations in each of the nodD genes and in the nolE and nolP genes did not abolish nodulation or nitrogen fixation on beans.
Localized transposon Tn5 mutagenesis of the photosynthetic gene cluster of Rhodobacter sphaeroides
Authors: Coomber S. A. , Chaudhri M. , Connor A. , Britton G. , Hunter C. N.
Published: October 2006

Four genes essential for bacteriochlorophyll biosynthesis were known to be encoded within a 45 kb region of the Rhodobacter sphaeroides genome, the boundaries of which are defined by puh and puf genes for reaction‐centre and light‐harvesting LH1 complexes. The cluster is represented by eight overlapping inserts cloned in the mobilizable vector pSUP202. We have used localized transposon Tn5 mutagenesis to characterize this cluster further; a total of 87 independent insertions were generated which identify nine genes for bacteriochlorophyll biosynthesis, six for carotenoid biosynthesis, and puhA encoding the reaction‐centre H subunit. This work provides an essential framework for a detailed study of the structure and expression of genes for photosynthesis in this bacterium.
Sequence of the peh gene of Erwinia carotovora: homology between Erwinia and plant enzymes
Authors: Hinton J. C. D. , Grll D. R. , Lalo D. , Plastow G. S. , Salmond G. P. C.
Published: October 2006

Polygalacturonase (Peh) and other pectolytic enzymes play a crucial role in the maceration of vegetables by soft rot Erwinia spp. We have sequenced the peh gene of Erwinia carotovora subsp. carotovora, and identified its product as a precursor of molecular weight 42639, and a mature protein of molecular weight 42200. A putative KdgR‐binding site was identified in the region 5’to the peh gene. The Peh protein showed significant homology with Peh from tomato. In addition, we have found homologies between pectin methylesterase and pectate lyase from Erwinia and their counterparts in tomato. These homologies are described, and their significance discussed.
Mycoplasma pneumoniae DNA gyrase genes
Authors: Colman S. D. , Hu P.‐C. , Bott K. F.
Published: October 2006

We have identified a clone from a λEMBL3 library containing a 19kb insert of Mycoplasma pneumoniae DNA which includes the genes that encode both subunits of DNA gyrase. The gyrB gene and the 5’end of the gyrA gene have been subcloned into M13. The gryB gene is 1953bp in length and overlaps the gryA gene by a single base. The nucleotide sequence of these subclones has significant homology to previously reported gyrase genes. In terms of the size of gyrB gene and its proximity to the gyrA gene, M. pneumoniae is more similar to Bacillus subtilis than to Escherichia coli.
Genetic variation at the omp2 porin locus of the brucellae: species‐specific markers
Authors: Ficht T. A. , Bearden S. W. , Sowa B. A. , Marquis H.
Published: October 2006

The omp2 locus of Brucella abortus is composed of two closely related genes (omp2a and omp2b) that encode, and potentially both express, homologous porin proteins. Genetic variation at this locus is revealed in the form of restriction‐fragment‐length polymorphisms which can be used to distinguish the type strains of all six Brucella species. Five of the six species contain single copies of omp2a and omp2b, whereas Brucella ovis appears to have two copies of the omp2a gene. The implications of these results with regard to the physiological functions of the omp2a and the omp2b gene products, phylogeny of the genus, and species‐specific adaptation are discussed.
Characterization of the virB operon of an Agrobacterium tumefaciens Ti plasmid: nucleotide sequence and protein analysis
Authors: Shirasu K. , Morel P. , Kado C. I.
Published: October 2006

The virulence regulon of the Agrobacterium tumefaciens TiC58 plasmid is composed of six operons, virA, virB, virG, virC, virD and virE, which direct the transfer of T‐DNA into plant cells. The 9.5kbp virB operon is the largest of these operons and its entire nucleotide sequence was determined and found to contain eleven open reading frames (ORFs). Gene fusions of each VirB ORF to T7φ10 were made and over‐expressed in Escherichia coli to confirm that they encode proteins of predicted size. Hydrophobic analysis of these peptide sequences revealed nine proteins that contain hydrophobic spanning regions including signal‐peptide‐like sequences. These data suggest that the majority of VirB proteins may associate with bacterial cell membranes, while the two additional proteins possess a potential ATP‐binding site. Strong homologies in amino acid sequences were observed between nopaline‐ and octopine‐type plasmids. Specific differences in amino acid sequence encoded by VirB ORFs of nopaline and octopine Ti plasmid and a functional role of the gene products are discussed.
Apo forms of cytochrome C550 and cytochrome cd1 are translocated to the periplasm of Paracoccus denitrificans in the absence of haem incorporation caused by either mutation or inhibition of haem synthesis
Authors: Page M. D. , Ferguson S. J.
Published: October 2006

An apo form of cytochrome C550 can be detected by immunoblotting cell‐free extracts of a mutant of Paracoccus denitrificans that is deficient in c‐type cytochromes. This apoprotein is found predominantly in the periplasm, the location of the holocytochrome in the wild‐type organism, indicating that translocation of the polypeptide occurs in the absence of haem attachment. The polypeptide molecular weight, as judged by sodium dodecyl sulphate/polyacrylamide gel electrophoresis, is indistinguishable from that of the holoprotein and the chemically prepared apoprotein; this suggests that the N‐terminal signal sequence is removed in the mutant as in the wild‐type organism. In the presence of levulinic acid, an inhibitor of haem biosynthesis, apocytochrome c550 and aponitrite reductase (cytochrome cd1) accumulated in the periplasm of wild‐type cells. Synthesis of these apoproteins was blocked by chloramphenicol. Thus in P. denitrificans the synthesis of these polypeptides is neither autoregulated nor regulated by the availability of haem. That the apoproteins appear in the periplasm argues against the possibility of polypeptide/haem co‐transport from cytoplasm to periplasm. These observations are related to, and contrasted with, the biosynthesis of c‐type cytochromes in eukaryotic cells.
Immunocytochemical analysis of P‐fimbrial structure: localization of minor subunits and the influence of the minor subunit FsoE on the biogenesis of the adhesin
Authors: Riegman N. , Hoschützky H. , van Die I. , Hoekstra W. , Jann K. , Bergmans H.
Published: October 2006

Antibodies recognizing the non‐adhesive minor P‐fimbrial subunrt protein E and the P‐fimbrial adhesin were used in an immunocytochemical analysis of P‐fimbrial structure. It was demonstrated that P‐fimbriae of the serotypes F71, F72 and F11 carry their adhesin in a complex with protein E. These complexes are commonly found at the tip of the fimbrial structure. In P‐fimbriae of serotype F9, expressed by the uropathogenic Escherichia coli strain 21086, adhesin‐protein E complexes are localized at the tips as well as along the shafts of the fimbriae. Protein E of F71 fimbriae (FsoE) plays a catalysing role in the biogenesis of the adhesin, but has no effect on the eventual localization of the adhesin.
COBIOTECH's Role in Biotechnology Worldwide
Authors: Gerhardt Philipp
Published: October 2006

Curious or sceptical friends often ask, ‘What and why is COBIOTECH? What is it doing?’. After serving as President during its formative first years, I attempt here to provide information for the curious and allay concerns of the sceptics about COBIOTECH's emerging role in biotechnology internationally.
Post‐transcriptional control in the polycistronic operon environment: studies of the atp operon of Escherichia coli
Authors: McCarthy J. E. G.
Published: October 2006

Post‐transcriptional control mechanisms assume special significance in polycistronic operons. Differential gene expression in the atp operon of Escherichia coli is primarily attributable to translational control and, to a lesser extent, to control of mRNA stability. At the same time, the polycistronic environment influences, to varying degrees, the relative importance of the different types of post‐transcriptional control. The present article briefly reviews more recent results obtained through studies of the atp operon. Investigations of the pathway and kinetics of mRNA decay have yielded new information about the role of degradative mechanisms in the overall scheme of control. Moreover, translational coupling has been shown to feature as a major form of interaction between the atp genes. The relevance of these and other data is discussed in the wider context of the post‐transcriptional control mechanisms generally available to E. coli.
Overproduction and purification of the Tn10‐specified inner membrane tetracycline resistance protein Tet using fusions to β‐galactosidase
Authors: Hickman R. K. , McMurry L. M. , Levy S. B.
Published: October 2006

Tetracycline resistance in the Enterobacteriaceae is mediated by a number of genetically related, usually plasmid‐borne, determinants which specify an efflux system involving an inner membrane protein, Tet. Attempts to overproduce the Tn 10 (Class B)‐encoded Tet in Escherichia coli by cloning the structural gene tet downstream of the λPL promoter under regulation by temperature‐sensitive λ repressor c 1857 were unsuccessful; induction at 42°C resulted in filamentous, non‐viable cells containing little detectable overproduction of the protein. However, cells containing tet fused to lacZ were resistant to tetracycline at 30°C and synthesized modest amounts of a large fusion protein when induced at 42°C. Fusion of the N‐terminal half or the first 38 amino adds of tet to lacZ did lead to increased production of fusion proteins. Fusions could be purified by size or by LacZ immunoaffinity or substrate‐affinity chromatography. In the latter method, selected detergents were required to counteract nonspecific binding of Tet to the adsorbant. Amino acid sequencing of the N‐terminus of Tet–LacZ fusion proteins indicated that most molecules were blocked at this terminus. The sequence of an unblocked subpopulation was consistent with that expected from the nucleotide sequence. A collagen peptide linker, genetically placed between tet and lacZ, allowed recovery of purified Tet protein after collagenase treatment of the purified fusion protein.
Cell‐type‐specific genes expressed late in Dictyostelium development show markedly different responses to 3′ 5′ cyclic AMP
Authors: Richards A. J. , Corney A. J. , Hames B. D.
Published: October 2006

The maintenance of late gene expression by 3′ 5′ cyclic AMP was re‐examined using several newly isolated cell‐type‐specific genes. Expression of all the prespore‐enriched genes ceased immediately upon disaggregation of developing cells and pre‐existing mRNA was rapidly degraded. For most genes, cAMP had little or no effect either alone or in combination with conditioned medium factors. The expression of the non‐cell‐type‐specific genes 7E and 2C also ceased upon cell disaggregation but cAMP triggered a full re‐induction of expression although the timing of the response differed markedly between these two genes. In contrast to earlier interpretations, these data argue that for none of these late prespore genes is cAMP alone sufficient for the maintenance of expression. The responses of the two prestalk mRNAs examined were gene‐specific. Prestalk 5D mRNA decayed slowly upon disaggregation and was partially stabilized by cAMP whereas prestalk 5G mRNA increased upon disaggregation and was inhibited by cAMP.
Identification and sequence analysis of the gene encoding the transcriptional activator of the formate hydrogenlyase system of Escherichia coli
Authors: Schlensog V. , Böck A.
Published: October 2006

Through complementation of a trans‐acting regulatory mutation a gene has been cloned whose product is required for the formate induction of the anaerobic expression of the formate hydrogenlyase structural genes. By restriction analysis, and from the size of the encoded protein, the gene could be identified as being equivalent to fhlA described by Sankar et al. (1988). The nucleotide sequence of the fhlA gene was determined and it was shown to code for a protein with a calculated Mr of 78467. Analysis of the derived amino acid sequence showed that the carboxy‐terminal domain of FHLA shares considerable sequence similarity with NIFA and NTRC, which are the ‘regulators’ of two‐component regulatory systems. Carboxy‐terminal truncation of, and introduction of amino‐terminal deletions in, the fhlA gene delivered inactive gene products. When overexpressed, FHLA mediates activation of expression of the formate dehydrogenase and hydrogenase structural genes in the presene of formate also under aerobic growth conditions. FHLA appears to bind to the upstream regulatory sequence (URS) in the 5′ flanking region of the fdhF gene since activation of fdhF expression was dependent on the presence of the URS.
The role of direct oligonucleotide repeats in gonococcal pilin gene variation
Authors: Hill S. A. , Morrison S. G. , Swanson J.
Published: October 2006

Previous studies indicate that gonococcal pilin phase and antigenic variation occur by intragenomic pilin gene recombination, the outcome of which resembles that of gene conversion. During such transitions, the expressed complete pilin gene (pilE) acquires a novel sequence corresponding to that of a silent pilin gene (pilS). In the present study, we find that internal deletions of pilE can produce pilus−/pilus+ phase transitions: direct oligonucleotide repeats in the pilin‐encoding portion of pilE bracket the deleted segments. A novel, orthodox pilE is formed upon repair of the internal deletions, with pilS sequence probably acting as a template for repair. Such deletion/repair of pilE is suggested as a principal mechanism underlying gonococcal pilus variation.
Lipid involvement in protein translocation in Escherichia coli
Authors: de Vrije G. J. , Batenburg A. M. , Killian J. A. , de Kruijff B.
Published: October 2006

Signal peptides play an essential role in protein trans‐location. This review summarizes the current knowledge of the structure of signal peptides and signal peptide‐lipid interactions and addresses the possibility that signal peptide‐lipid interactions initiate membrane translocation of precursor proteins. A new model for protein translocation in Escherichia coli is proposed, which includes as central features con‐formational changes of the signal peptide and signal‐peptide‐induced local changes in membrane organization (non‐bilayer lipids).
Identification and cloning of nodulation genes and host specificity determinants of the broad host‐range Rhizobium leguminosarum biovar phaseoli strain CIAT899
Authors: Vargas C. , Martinez L. J. , Megias M. , Quinto C.
Published: October 2006

Rhizobium Ieguminosarum biovar phaseoli type II strain CIAT899 nodulates a wide range of hosts: Phaseolus vulgaris (beans), Leucaena esculenta (leucaena) and Macroptilium atropurpureum (siratro). A nodulation region from the symbiotic plasmid has been isolated and characterized. This region, which is contained in the overlapping cosmid clones pCV38 and pCV117, is able to induce nodutes in beans, leucaena and siratro roots when introduced in strains cured for the symbiotic plasmid, pSym. In addition, this cloned region extends the host range of Rhizobium meliloti and R. leguminosarum biovar (bv.) trifolii wild‐type strains to nodulate beans. Analysis of constructed subclones indicates that a 6.4 kb Hin dlll fragment contains the essential genes required for nodule induction on all three hosts. Rhizobium leguminosarum bv. phaseoli type I strain CE3 nodulates only beans. However, CE3 transconjugants harbouring plasmid pCV3802 (which hybridized to a nodD heterologous probe), were capable of eliciting nodules on leucaena and siratro roots. Our results suggest that the CIAT899 DNA region hybridizing with the R. meliloti nodD detector is involved in the extension of host specificity to promote nodule formation in P. vulgaris, L. esculenta and M. atropurpureum.
Isolation and characterization of efficient plasmid transformation mutants of Mycobacterium smegmatis
Authors: Snapper S. B. , Melton R. E. , Mustafa S. , Kieser T. , Jr W. R.Jacobs
Published: October 2006

Recent development of vectors and methodologies to introduce recombinant DNA into members of the genus Mycobacterium has provided new approaches for investigating these important bacteria. While most pathogenic mycobacteria are slow‐growing, Mycobacterium smegmatis is a fast‐growing, non‐pathogenic species that has been used for many years as a host for mycobacteriophage propagation and, recently, as a host for the introduction of recombinant DNA. Its use as a cloning host for the analysis of mycobacterial genes has been limited by its inability to be efficiently transformed with plasmid vectors. This work describes the isolation and characterization of mutants of M. smegmatis that can be transformed, using electroporation, at efficiencies 104 to 105 times greater than those of the parent strain, yielding more than 105 transformants per μg of plasmid DNA. The mutations conferring this efficient plasmid transformation (Ept) phenotype do not affect phage transfection or the integration of DNA into the M. smegmatis chromosome, but seem to be specific for plasmid transformation. Such Ept mutants have been used to characterize plasmid DNA sequences essential for replication of the Mycobacterium fortuitum plasmid pAL5000 in mycobacteria by permitting the transformation of a library of hybrid plasmid constructs. Efficient plasmid transformation of M. smegmatis will facilitate the analysis of mycobacterial gene function, expression and replication and thus aid in the development of BCG as a multivalent recombinant vaccine vector and in the genetic analysis of the virulence determinants of pathogenic mycobacteria.
Molecular characterization of pmbA, an Escherichia coli chromosomal gene required for the production of the antibiotic peptide MccB17
Authors: Rodríguez‐Sáinz M. C. , Hernández‐Chico C. , Moreno F.
Published: October 2006

Microcin B17 (MccB17) is a peptide antibiotic produced by Escherichia coli strains harbouring plasmid pMccB17. We have isolated two mutations that strongly reduce the production of MccB17. These mutations, which map at 96 min on the E. coli chromosome, define a new gene that we have caiied pmbA. A chromosomal DNA fragment of about 13kb, including the wild‐type pmbA allele, was cloned into a mini‐Mu piasmid vector. pmbA was located within the cloned DNA fragment by insertional mutagenesis and deletion analysis. The nucleotide sequence of a 1.7 kb DNA region containing the gene was determined. pmbA encodes a hydrophilic protein of 450‐amino‐acid residues with a predicted molecular size of 48375 D, which was visualized in polyacrylamide gels. Protein profiles of cellular envelope and soluble fractions from cells with plasmids overproducing PmbA indicated that it is cytoplasmic. Physiological experiments suggested that pmbA mutants synthesize a molecule (pro‐MccB17) able to inhibit DNA replication but unable to the released from cells. We propose that PmbA facilitates the secretion of the antibiotic by completing its maturation.
The Escherichia coli unc transcription terminator enhances expression of uncC, encoding the e subunit of F1‐ATPase, from plasmids by stabilizing the transcript
Authors: Patel A.M. , Dallmann H.G. , Skakoon E.N. , Kapala T.D. , Dunn S.D.
Published: October 2006

The effect of the unc transcription terminator on expression of uncC, encoding the e subunit of Escherichia coli F1‐ATPase, from plasmids was studied. The cloned sequence in pTK1 included the uncC ribosome binding site, the uncC structural gene, and unc transcription terminator. The cloned region in pSD37 was similar, but lacked the unc transcription terminator. Transformants carrying pTK1 produced the e subunit of F1‐ATPase, encoded by uncC, in 10‐fold greater abundance than transformants carrying pSD37. Northern blots revealed similar differences in the steady‐state uncC mRNA levels. The half‐life of the message transcribed from pTK1 was 90–100 seconds, while that from pSD37 was 25–30 seconds. These studies indicate the importance of message stabilization through features at the 3 end of the transcript in ensuring adequate production of. We have exploited this stabilization to develop a simple, efficient, and gentle method of purifying the overproduced subunit.
Molecular evolution of class A β‐lactamases: phylogeny and patterns of sequence conservation
Authors: Pastor N. , Piñero D. , Valdés A. M. , Soberón X.
Published: October 2006

We present a multiple alignment of the amino acid sequences of eight class A β‐lactamases and utilized it to propose a phylogeny, based on the nucleotide sequences of their corresponding genes. We have also used the alignment, together with the α‐carbon co‐ordinates of the Staphylococcus aureus protein, to search systematically for neighbouring residues that share the same pattern of conservation among the different members of the protein family. The distribution of invariant residues and of groups of residues with co‐ordinate changes map, predominantly, at the region of the active site and at interfaces between structural elements, respectively. We have also contrasted the distribution of conserved residues with the positions which are known to differ in mutants and variants of class A β‐lactamases.
Larvicidal activity of chimeric Bacillus thuringiensis protoxins
Authors: Raymond K. C. , John T. R. , Bulla L. A.
Published: October 2006

Bacillus thuringiensis subspecies kurstaki (Btk) and subspecies berliner (Btb) both produce lepidopteran‐specific larvicidal protoxins with different activities against the same insect species. Toxic activity resides in the amino‐terminal half of both protoxins, whereas the carboxy‐terminal half of the molecules is not required for toxicity. The protoxins are 90% homologous, with a major cluster of differences in the amino‐terminal half, and a 26 consecutive amino‐acid insertion within the carboxy‐terminal half of the Btk protoxin. Protoxin chimeras composed of the amino‐terminal half of one subspecies and the carboxy‐terminal half of the other were generated. Wild‐type and chimeric protoxins were compared in bioassays against tobacco horn worm larvae. The amino‐terminal half, the toxin itself, dictates specific larvicidal activity.
Identification and characterization of a gene and protein required for glycosylation in the yeast Golgi
Authors: Devlin C. , Ballou C. E.
Published: October 2006

The MNN2 gene of Saccharomyces cerevisiae has been cloned by complementation of the mnn2 mutant phenotype scored by a change in cell surface carbohydrate structure resulting from a lack of α1→2‐man‐nose branching in the outer chain. The gene was subcloned as a 3 kb DNA fragment that integrated at the MNN2 locus, and a gene disruption yielded the mnn2 phenotype. A lacZ–MNN2 gene fusion protein, produced in Escherichia coli, was used to raise a specific antiserum that recognized a 65kD wild‐type yeast protein. This MMN2 gene product lacks N‐linked carbohydrate but appears to be an integral membrane protein. Overproduction of MNN2p does not enhance the α1→2‐mannosyltransferase activity of yeast cells. The results suggest that MNN2p is a Golgi‐associated protein that is involved in mannoprotein sorting rather than glycosylation.
Corrected sequence of the mannitol (mtl) operon in Escherichia coli
Authors: Jiang W. , Wu L.‐F. , Tomich J. , Saier M.H. , Niehaus W.G.
Published: October 2006

The previously published sequences of the operator‐promoter region of the mannitol operon of Escherichla coli and of the mtlD gene have been found to contain a number of errors. The major conclusions reported previously were correct, but additionally it is now clear that a C‐terminal portion of mannitol‐1‐phosphate dehydrogenase (the mtlD gene product) exhibits significant sequence identity with an amino‐terminal region of human liver fructose‐6‐phoshate‐2‐kinase; fructose‐2,6‐bishosphatase.
Triple post‐transcriptional control
Authors: Bechhofer D. H.
Published: October 2006

The ermC gene confers resistance to MLS antibiotics in a Bacillus subtilis host. Synthesis of the ermC gene product, a ribosomal RNA methylase, is inducible by the addition of subinhibitory concentrations of erythromycin. Regulation of ermC gene expression occurs at the post‐transcriptional level in three ways: translational attenuation, translational autoregulation, and messenger RNA stabilization.
Escherichia coli molecular genetic map (1000Kbp):update
Authors: Medigue C. , Hénaut A. , Danchin A.
Published: October 2006

Summery

The sequenced genes from Escherichia coli that are available in the EMBL library (release 21) have been localized on an updated and corrected version of the restriction map of the chromosome generated by kohara et al. (1987). One thousand kbp of sequenced DNA are incorporated in this update; this is equivalent to 23% of the total genome. The accuracy of the map is assessed, and it is corrected and updated where appropriate, A significant number of sites were missing from the original map, mainly involving two of the eight enzymes used by Kohara et al. (1987), ie. Pvull and EcoRV, The nucleotide environment of such missing sites was examined and, using an Artificial intelligence approach, it appears that the site for these enzymes is sensitive to context effects. Several genes of known position on the E. coli chromosome could not be placed on the restriction map; this suggests that additional gaps are likely to exists on the restriction map, in addition to the original seven identified by Kohara et al. We have also obtained information about the probable direction of transcription of chromosomal genes with respect to the map. Most genes are transcribed in the same direction as the replication forks, particularly around oriC at 84 min
Evidence that conserved residues Cys‐62 and Cys‐154 within the Azotobacter vinelandii nitrogenase MoFe protein α‐subunit are essential for nitrogenase activity but conserved residues His‐83 and Cys‐88 are not
Authors: Dean D. R. , Setterquist R. A. , Brigle K. E. , Scott D. J. , Laird N. F. , Newton W. E.
Published: October 2006

Metallocluster extrusion requirements, interspecies MoFe‐protein primary sequence comparisons and comparison of the primary sequences of the MoFe‐protein subunits with each other have been used to assign potential P‐cluster (Fe‐S cluster) domains within the MoFe protein. In each β unit of the MoFe protein, subunit domains, which include potential Fe‐S cluster ligands Cys‐62, His‐83, Cys‐88 and Cys‐154, and β‐subunit domains, which include potential Fe‐S cluster ligands Cys‐70, His‐90, Cys‐95 and Cys‐153, are proposed to comprise nearly equivalent P‐cluster environments located adjacent to each other in the native protein. As an approach to test this model and to probe the functional properties of the P clusters, amino acid residue substitutions were placed at the α‐ subunit Cys‐62, His‐83, Cys‐88 and Cys‐154 positions by site‐directed mutagenesis of the Azotobacter vinelandii nifD gene. The diazotrophic growth rates. MoFe—protein acetylene‐reduction activities, and whole‐cell S 3/2 electron paramagnetic resonance spectra of these mutants were examined. Results of these experiments show that MoFe‐protein α‐submit residues, Cys‐62 and Cys‐154, are probably essential for MoFe‐protein activity but that His‐83 and Cys‐88 residues are not. These results indicate either that His‐83 and Cys‐88 do not provide essential P‐cluster ligand or that a new cluster‐ligand arrangement is formed in their absence.
Cysteine‐rich outer membrane proteins of Chlamydia trachomatis display compensatory sequence changes between biovariants
Authors: Allen J. E. , Cerrone M. C. , Beatty P. R. , Stephens R. S.
Published: October 2006

Two cysteine‐rich proteins of Chlamydia trachomatis are essential structural components of the unique outer membrane of the infectious elementary body. These 58000 (outer membrane protein 2; OMP2) and 15000 (OMP3) proteins also differ structurally and chemically between biovariants that differ in invasive capability. We have identified the gene for OMP3 and sequenced both trachoma and lymphogranuloma venereum (LGV) omp3 genes. We have previously sequenced omp2 from the LGV biovar and now describe the omp2 sequence for a trachoma biovariant. Amino acid sequence differences between biovariants were few but, significantly, these changes have altered the charge of both OMP2 and OMP3 such that the net charge of each protein differs between biovariants. These compensatory charge alterations have implications for the outer membrane organization of these proteins. In addition, examination of the OMP3 sequence suggests that OMP3 may be a lipoprotein.