bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of a Mycobacterium tuberculosis insertion sequence belonging to the IS3 family
Authors: McAdam R. A. , Hermans P. W. M. , van Soolingen D. , Zainuddin Z. F. , Catty D. , van Embden J. D. A. , Dale J. W.
Published: October 2006

A repetitive element (IS986), previously isolated from Mycobacterium tuberculosis and shown to detect multiple restriction fragment‐length polymorphisms (RFLPs), has been sequenced. It consists of a potential insertion sequence of 1358bp, with 30‐bp inverted repeat ends. IS986 has four potentially significant open reading frames (ORFs): ORFa1, ORFa2 and ORFb on one strand and ORFc on the complementary strand. The sequences of the potential translated products identify IS986 as a member of the IS3 family, with an apparent frameshift between ORFa1 and ORFa2. IS986 has potential as a highly specific probe for detection and typing of M. tuberculosis, as well as for transposon mutagenesis of mycobacteria. The sequence of IS986 is virtually identical to that of another recently described element, IS6110 (Thierry et al., 1990).
Elimination of the disulphide bridge in fragment B of diphtheria toxin: effect on membrane insertion, channel formation, and ATP binding
Authors: Stenmark H. , Olsnes S. , Madshus I. H.
Published: October 2006

Active diphtheria toxin consists of two disulphide‐linked fragments, termed A and B. Fragment B, which contains an internal disulphide bridge, facilitates translocation of the enzymatically active fragment A to the cytosol of eukaryotic cells. In this process cation‐selective channels are formed. An in vitro translated full‐length mutant lacking the internal disulphide bridge (A‐58**) was functionally indistinguishable from its disulphide‐containing counterpart (A‐58) with respect to trypsin sensitivity, receptor binding, A‐fragment translocation, and channel formation. In contrast, the B fragment of A‐58** (B‐36**) was slightly less trypsin resistant than the S‐S‐Containing B fragment, B‐36, and was 300‐fold less efficient than B‐36 in permeabilizing cells. When first dialysed and then reconstituted with A fragment, B fragment without disulphide bridge yielded a less‐active toxin than did wild‐type B fragment. We conclude that the disulphide bridge in fragment B is not necessary for toxicity, as earlier believed, and that channel formation may play a role in membrane translocation.
IS801, an insertion sequence element isolated from Pseudomonas syringae pathovar phaseolicola
Authors: Romantschuk M. , Richter G. Y. , Mukhopadhyay P. , Mills D.
Published: October 2006

A transposable element, designated IS801, was isolated from strain LR781 of Pseudomonas syringae pathovar phaseolicola in two independent events using the entrapment plasmid, pUCD800. IS801 is 1517 base pairs in length and contains open reading frames that potentially encode proteins of 311 and 172 amino acids, as well as smaller proteins. Unlike most other prokaryotic transposable elements, IS801 lacks terminal repeats. Sequence analysis revealed two target pentamers for IS801 insertion that differ by one base pair. One copy of IS801 generated a perfect duplication of its target, TGAAC. The second copy of IS801 was flanked by the target, TGGAC, at one end, and TGAAC at the other end. A third copy of IS801 was cloned from pMMC7105, an indigenous plasmid of strain LR781, and it was flanked by copies of the pentamer TGAAC.
nolC, a Rhizobium fredii gene involved in cultivar‐specific nodulation of soybean, shares homology with a heat‐shock gene
Authors: Krishnan H. B. , Pueppke S. G.
Published: October 2006

Rhizobium fredii strain USDA257 does not nodulate soybean (Glycine max (L.) Merr.) cultivar McCall. Mutant 257DH5, which contains a Tn5 insert in the bacterial chromosome, forms nodules on this cultivar, but acetylene‐reduction activity is absent. We have sequenced the region corresponding to the site of Tn5 insertion in this mutant and find that it lies within a 1176 bp open reading frame that we designate nolC. nolC encodes a protein of deduced molecular weight 43564. Nucleotide sequences homologous to nolC are present in several other Rhizobium strains, as well as Agrobacterium tumefaciens, but not in Pseudomonas syringae pathovar glycinea. nolC lacks significant sequence homology with known genes that function in nodulation, but is 61% homologous to dnaJ, an Escherichia coli gene that encodes a 41 kDa heat‐shock protein. Both R. fredii USDA257 and mutant 257DH5 produce heat‐shock proteins of 78, 70, 22, and 16kDa. A 4.3kb EcoRI–HindIII subclone containing nolC expresses a single 43 kDa polypeptide in mini‐cells. A longer, 9.4kb Eco RI fragment expresses both the 43kDa polypeptide and a 78kDa polypeptide that corresponds in size to that of the largest heat‐shock protein. Thus, although nolC has strong sequence homology to dnaJ and appears to be linked to another heat‐shock gene, it does not directly function in the heat‐shock response.
A family of clostridial and streptococcal ligand‐binding proteins with conserved C‐terminal repeat sequences
Authors: Wren B. W.
Published: October 2006

Analysis of the derived amino acid sequences of toxins A and B from Clostridium difficile has identified an extraordinarily large number of repeat amino acid units in the C‐terminal regions of the proteins. Nearly one third of each of the proteins consist of repeating units which appear, at least in the case of toxin A, to be responsible for carbohydrate binding. Similar repeat units are also found in the C‐terminal region of four glucosyltransferases from Streptococcus mutans and Streptococcus downel, and in four lytic enzymes from Streptococcus pneumoniae and its bacteriophages (HB‐3, Cp‐1 and Cp‐9). In each case the repeats constitute the ligand‐binding portion of the respective enzymes. A glucan‐binding protein from S. mutans, which lacks enzymatic activity, has similar repeats spanning almost the entire molecule. This family of ligand‐binding proteins appears to be of modular design, with one module consisting of a repetitive ligand‐binding domain located in the C‐terminal region and the other module(s) providing enzymatic functions.
Translational coupling varying in efficiency between different pairs of genes in the central region of the atp operon of Escherichia coli
Authors: Hellmuth K. , Rex G. , Surin B. , Zinck R. , McCarthy J. E. G.
Published: October 2006

A series of afp::lacZ fusions has been constructed for use in a study of translational coupling in the central region of the Escherichia coli atp operon. Five genes, atpE, atpF, atpH, atp A and atpG, were shown to be translationally coupled to various degrees of tightness. A new lac promoter vector, compatible with the afp::lacZ fusion vectors, was used to express individual atp genes in the same hosts as the fusion genes. The H+‐ATPase subunits thus synthesized exercised no significant trans‐regulation on the expression of the atp::lacZ fusions, indicating that the coupling is primarily cis. The mechanism of this coupling was investigated using in vitro mutagenesis. At least in the case of the pair atpHA, coupling seems to involve facilitated binding of fresh ribosomes to the atpA translational initiation regions.
Isolation and properties of the RepA1 protein of the IncFII replicon, RepFIC
Authors: Maas R. , Oppenheim J. , Saadi S. , Fuchs T. , Maas W. K.
Published: October 2006

The initiator protein RepA1 of the IncFII replicon RepFIC derived from the enterotoxin plasmid EntP307 has been cloned under the control of the λ Pl promoter. This has enabled us to overproduce this protein and study its properties. Here we show that RepA1 is a soluble basic protein with an experimentally determined molecular weight of 40000. Deletion analysis indicates that the overproduced protein originates from the open reading frame which we previously designated as coding for RepA1. We have also shown that the replication function of the replicon RepFIC depends on the intact RepA1 coding frame.
A sugar‐specific porin, ScrY, is involved in sucrose uptake in enteric bacteria
Authors: Schmid K. , Ebner R. , Jahreis K. , Lengeler J. W. , Titgemeyer F.
Published: October 2006

During the molecular analysis of a plasmid‐coded sucrose metabolic pathway of enteric bacteria, a gene, scrY, was found whose product, ScrY, had all the properties of a bacterial porin (Schmid et al, 1988). Loss of this protein (Mr 58kDa), localized in the outer membrane, led, as shown here, to an increase in the apparent Km for sucrose transport in whole cells from 10 μM in wild‐type cells to 300 μM in mutant cells. This contrasts with the Km for sucrose phosphorylation as measured in membrane vesicles from mutant and wild‐type cells, which remained unchanged at about 10 μM, and reflects the activity of the sucrose‐specific Enzymell of the phosphoenolpyruvate‐dependent carbohydrate:phosphotransferase system (PTS) responsible for uptake through the inner membrane. Furthermore, the presence of ScrY restored growth on maltodextrins in cells devoid of LamB, thus complementing the lack of this maltoporin. The amino acid sequence deduced from the DNA sequence was determined for the plasmid‐coded and the ScrY porin coded in the chromosome of Klebsiella pneumoniae. Both show high identity (86%) to each other, and to the channel domain of LamB, further corroborating the conclusion that they constitute porins.
Pleiotropic changes resulting from depletion of Era, an essential GTP‐binding protein in Escherichia coli
Authors: Lerner C. G. , Inouye M.
Published: October 2006

Phenotypic analysis of a temperature‐sensitive era mutant strain indicates that Escherichia coli cells depleted of Era undergo many physiological changes. At 43° C., a completely non‐permissive temperature, growth is arrested because of loss of the gene and depletion of the Era protein. Depletion of Era at 43° C results in depressed synthesis of heat‐shock proteins DnaK, GroEL/ES, D33.4 and C62.5, lack of thermal induction of ppGpp pool levels, and increased capacity for carbon source metabolism through the citric acid cycle. Thus, in addition to inhibition of cell growth and viability, ioss of Era function results in pleiotropic changes including abnormal adaptation to thermal stress.
The surface‐located YopN protein is involved in calcium signal transduction in Yersinia pseudotuberculosis
Authors: Forsberg Å. , Viitanen A.‐M. , Skurnik M. , Wolf‐Watz H.
Published: October 2006

The low‐calcium response (Icr) is strongly conserved among the pathogenic Yersinia species and is observed when the pathogen is grown at 37°C in Ca2+‐depleted medium. This response is characterized by a general metabolic downshift and by a specific induction of virulence‐plasmid‐encoded yop genes. Regulation of yop expression is exerted at transcriptional level by a temperature‐regulated activator and by Ca2+‐regulated negative elements. The yopN gene was shown to encode a protein (formerly also designated Yop4b) which is surface‐located when Yersmia is grown at 37°C. yopN was found to be part of an operon that is induced during the low‐calcium response. Insertional inactivation of the yopN gene resulted in derepressed transcription of yop genes. A hybrid plasmid containing the yopN gene under the control of the tac promoter fully restored the wild‐type phenotype of the yopN mutant. Thus the surface‐located YopN somehow senses the calcium concentration and transmits a signal to shut off yop transcription when the calcium concentration is high.
The RecA protein as a recombinational repair system
Authors: Cox M. M.
Published: October 2006

The Escherichia coli RecA protein plays a central role in homologous genetic recombination, recombinational repair, and several other processes in bacteria. In vitro, an extended filament involving thousands of RecA monomers promotes a reaction in which individual ONA strands switch pairing partners (DNA strand exchange). This reaction has been extensively studied as a paradigm for the central steps in recombination. Because the strand‐exchange reaction is relatively simple and Isoenergetic, the complexity of the RecA system that carries it out has led to controversy about the functional significance of many fundamental properties of RecA. Filamentous protein structures involving thousands of RecA monomers, which hydrolyse 100 ATPs per base pair of heteroduplex DNA formed, are hard to rationalize in the context of recombination between two homologous DNAs. The thermodynamic barriers to strand exchange are much too small. These molecular features of the system can be easily rationalized, however, by shifting the focus to DNA repair.
Cassette mutagenesis implicates a helix‐turn‐helix motif in promoter recognition by the novel RNA polymerase Sigma factor σ54
Authors: Coppard J. R. , Merrick M. J.
Published: October 2006

Cassette mutagenesis has been used to study the role of a helix‐turn‐helix (HTH) motif in the novel RNA polymerase sigma factor σ54 of Klebsiella pneumoniae. Of the four residues which are predicted to be solvent‐exposed in the second helix, the first (Glu‐378) tolerated all substitutions, and some mutations of this residue increased expression from σ54‐dependent promoters. Certain substitutions in the third exposed residue (Ser‐382) produced a promoter‐specific phenotype and all substitutions in the fourth residue (Arg‐383) inactivated the protein, identifying this residue as being likely to be involved in base‐specific interactions with the promoter. In vivo foot‐printing indicated that the inactive HTH mutants of a54 were defective in interaction with both the ‐24 and ‐12 regions of the gln Ap2 promoter.
Analysis of an anaerobically induced promoter for the cobalamin biosynthetic genes in Salmonella typhimurium
Authors: Richter‐Dahlfors A. A. , Andersson D. I.
Published: October 2006

We have identified an anaerobicaily induced promoter for the cobalamin biosynthetic (cob) genes. In a plasmid the Cob promoter showed two of the three types of control of the intact chromosomal Cob operon: anaerobic induction and cAMP stimulation. Cobalamin repression was observed only in promoter fragments which included sequences far downstream of the transcription start site, suggesting that this control is post‐transcriptional. One anaerobically induced transcript was identified and its 5′ end was determined. Deletion mapping showed that 60 nucleotides upstream of the start site were sufficient for anaerobic synthesis of this transcript. Upstream of the transcription start site a putative σ70‐dependent ‐10 recognition sequence was identified; however, no consensus ‐35 region was observed.
Sequential activation of dual promoters by different Sigma factors maintains spoVJ expression during successive developmental stages of Bacillus subtilis
Authors: Foulger D. , Errington J.
Published: October 2006

The spoVJ gene of Bacillus subtilis encodes a 36 k Da protein and is expressed only in the mother cell. spoVJ has an interesting pattern of regulation during sporulation because it is expressed from sequentially activated promoters. These promoters, designated P1 and P2, are under the control of different sigma factors, σEand σK, which become active at separate times during sporulation. Removal of promoter P1, leaving promoter P2 active, resulted in about a 30‐minute delay in the formation of heat‐resistant spores and demonstrated that the expression of spoVJ from both promoters is essential for normal sporulation. A comparison is made between the sequences of the spoVJ promoters and the promoters of other genes dependent upon σE and σk.
Genetic analysis of the rfb region of Shigella flexneri encoding the Y serotype O‐antigen specificity
Authors: Macpherson D. F. , Morona R. , Beger D. W. , Cheah K.‐C. , Manning P. A.
Published: October 2006

The gene cluster (rfb region) which determines the biosynthesis of the Shigella flexneri O‐antigen of the Y serotype specificity was cloned from a S. flexneri serotype 2a strain. Two plasmids, pPM2212 and pPM2213, which conferred O‐antigen biosynthesis were generated from separate cosmid clones by deletion with Cla l. These plasmids expressed O‐antigen in Escherichia coli K12 like that of the parental strain, as assessed by reactions to antisera in colony and Western immunoblots, sensitivity to bacteriophage Sf6, and by silver staining of lipopolysaccharides separated by sodium dodecyl sulphate/polyacryl‐amide gel electrophoresis. These plasmids also mediated O‐antigen expression in an E. coli K12 rfb‐delete background, indicating that all the necessary genes have been cloned. A detailed restriction map of the region has been constructed and analysis of various subclones has allowed the limits of the coding region for O‐antigen biosynthesis to be defined to a maximum of 11kb. Expression of these plasmids demonstrates a novel phenotype associated with control of lipopolysaccharide chain length. The gene(s) responsible maps adjacent to, but separate from, those associated with the biosynthesis of the O‐antigen unit. Analysis of plasmid‐encoded proteins in minicells and maxicells has facilitated the construction of a physical map. Finally, plasmid pPM‐2212 was used to probe a collection of S. flexneri serotypes by Southern hybridization. With the exception of serotype 6, which appears to be unrelated, a similar pattern was found in all serotypes.
Anthrax toxin protective antigen: low‐pH‐induced hydrophobicity and channel formation in liposomes
Authors: Koehler T. M. , Collier R. J.
Published: October 2006

To probe the role of the protective antigen (PA) component of anthrax toxin in toxin entry into animals cells, we examined the membrane channel‐forming properties and hydrophobicity of intact and trypsin‐cleaved forms of the protein at various pH values. At neutral pH neither form caused release of entrapped K+ from unilamellar lipid vesicles. At pH values below 6.0, however, K+ was rapidly released upon addition of either the nicked PA (PAN) or the 63kDa tryptic fragment of PA (PA63), which has been implicated in the toxin entry process. Under the same conditions intact PA exhibited only weak channel‐forming activity, and PA20, the complementary tryptic fragment, showed no such activity. Both PA and PA63 exhibited enhanced hydrophobicity at acidic pH values, but the enhancement was greater and the pH threshold higher with PA63. Our findings indicate that proteolytic removal of PA20 from intact PA enables the residual protein, Pasb63, to adopt a conformation at mildly acidic pH values that permits it to insert readily and form channels in membranes. Thus acidic conditions within endocytic vesicles may trigger membrane insertion of PA63, which in turn promotes translocation of ligated effector moieties, edema factor or lethal factor, across the vesicle membrane into the cytosol.
The pathogenic mechanisms of Shiga toxin and the Shiga‐like toxins
Authors: Tesh V. L. , O'Brien A. D.
Published: October 2006

It is now well documented that some enteric bacteria which cause diarrhoeal and/or dysenteric disease produce, at high levels, one or more of a family of protein toxins referred to as Shiga toxin and Shiga‐like toxins (SLTs; alternatively called verocytotoxins or VTs). Within the past few years, there have been considerable advancements made in our understanding of the biochemistry and molecular biology of Shiga toxin and SLTs. However, the precise role of the toxins in mediating colonic disease, as well as their contribution to the development of extra‐intestinal sequelae (e.g. the haemolytic uraemic syndrome and neurological disorders), remain less clear. In thisMicroReview, we will briefly summarize recent progress in Shiga toxin‐ and SLT‐related research and present evidence supporting the concept that these toxins contribute to pathogenesis by directly damaging vascular endothelial cells, thereby disrupting the homeostatic properties of these cells. We will also discuss data which suggest that toxin‐mediated damage in the kidney may not be limited to glomerular endothelial cells but may include tubular epithelial cells. Thus, the role of the toxins in renal disease may not be limited to the glomeruli, as was initially hypothesized when the association of infection with toxin‐producing strains and the development of acute renal failure was established.
The opacity proteins of Neisseria gonorrhoeae strain MS11 are encoded by a family of 11 complete genes
Authors: Bhat K. S. , Gibbs C. P. , Barrera O. , Morrison S. G. , Jähnig F. , Stern A. , Kupsch E.‐M. , Meyer T. F. , Swanson J.
Published: October 2006

Variants of Neisseria gonorrhoeae MS11 show distinct colony morphologies because of the expression of a class of surface components called opacity (Opa, PII) proteins. Southern analyses combined with molecular cloning of genomic DNA from a single variant of MS11 has identified 11 opa genes contained in separate loci. These opa genes code for distinct opacity proteins which are distinguishable at their variable domains. The opa gene analyses were also extended to divergent variants of MS11. These studies have shown that, during in vitro and in vivo culture, 10 of the 11 opa genes did not undergo significant change in their primary sequence. However, in these variants, one gene (opaE) underwent non‐reciprocat inter‐opa recombinations to generate newer Opa variants. Phylogenic analysis of the opa gene sequences suggests that the opa gene family have evolved by a combination of gene duplication, gene replacement and partial inter‐opa recombination events.
Identification of glyA as a symbiotically essential gene in Bradyrhizobium japonicum
Authors: Rossbach S. , Hennecke H.
Published: October 2006

A Bradyrhizobium japonicum Tn5 mutant (strain 3160) induced numerous, tiny, white nodules which were dispersed over the whole root system of its natural host plant, soybean (Glycine max). These ineffective, nitrogen non‐fixing pseudonodules were disturbed at a very early step of bacteroid and nodule development. Subsequent cloning and sequencing of the DNA region mutated in strain 3160 revealed that the Tn5 insertion mapped in a gene that had 60% homology to the Escherichia coli glyA gene coding for serine hydroxymethyitransferase (SHMT; E.C. 2.1.2.1.). SHMT catalyses the biosynthesis of glycine from serine and the transfer of a one‐carbon unit to tetrahydrofolate. The B. japonicum glyA region was able to fully complement the glycine auxotrophy of an E. coli glyA deletion strain. Although the Tn5 insertion in B. japonicum mutant 3160 disrupted the glyA coding sequence, this strain was only a bradytroph (i.e. a leaky auxotroph). Thus, B. japonicum may have an additional pathway for glycine biosynthesis. Nevertheless, the glyA mutation was responsible for the drastic symbiotic phenotype visible on plants. It may be possible, therefore, that a sufficient supply with glycine and/or a functioning C1 ‐metabolism are indispensable for the establishment of a fully effective, nitrogen‐fixing root nodule symbiosis.
Molecular characterization of the O‐acetyl transferase gene of converting bacteriophage SF6 that adds group antigen 6 to Shigella flexneri
Authors: Verma N. K. , Brandt J. M. , Verma D. J. , Lindberg A. A.
Published: October 2006

Bacteriophage SF6 antigenically converts Shigella flexneri serotype Y strains (‐;3,4) to type 3b carrying group antigen 6,3,4 by means of an O‐acetylation of the O‐antigenic polysaccharide chain. The gene for O‐acetyl transferase of bacteriophage SF6 has been cloned, identified and sequenced. The predicted O‐acetyl transferase protein encoded by this gene was found to consist of 333 amino acids, (37 185 daltons) and to have some similarity with the galactose‐1‐phosphate uridylyltransferase protein of Escherichia coli. The gene has been shown to function in a live vaccine strain of S. flexneri Y type (ΔaroD), making it a 3b type. The converted type 3b strain, SFL1104, was found to elicit significant protection against challenge by both wild‐type serotypes 3b and Y in a guinea‐pig keratoconjunctivitis model.
Control of replication of plasmid R1: the intergenic region between copA and repA modulates the level of expression of repA
Authors: Berzal‐Herranz A. , Wagner E. G. H. , Diaz‐Orejas R.
Published: October 2006

The RepA protein of plasmid R1 is rate‐limiting for initiation of R1 replication. Its synthesis is mainly regulated by interactions of the antisense RNA, CopA, with the leader region of the RepA mRNA, CopT. This work describes the characterization of several mutants with sequence alterations in the intergenic region between the copA gene and the repA reading frame. The analysis showed that most of the mutations led both to a decrease in stability of maintenance of mini‐R1 derivatives and to lowered repA expression assayed in translational repA‐lacZ fusion constructs. Destruction of the copA gene and replacement of the upstream region by the tac promoter in the latter constructs indicated that these mutations per se alter the expression of repA. In addition, we show that particular mutations in this region can directly affect CopA‐mediated control, either by changing the kinetics of interaction of CopA RNA with the RepA mRNA and/or by modifying the activity of the copA promoter. These data indicate the importance of the region analysed in the process that controls R1 replication.
Molecular characterization of the gor gene encoding glutathione reductase from Pseudomonas aeruginosa: determinants of substrate specificity among pyridine nucleotide‐disulphide oxidoreductases
Authors: Perr A. C. F. , Bhriain N. Ni , Brown N. L. , Rouch D. A.
Published: October 2006

In order to investigate the basis of functional diversity among the pyridine nucleotide‐oxidoreductases the gor gene from Pseudomonas aeruginosa PAO, which encodes glutathione reductase, was analysed. The P. aeruginosa gor gene was identified by hybridization with a short DNA sequence from the gene encoding mercuric reductase in transposon Tn501. The gene was cloned, sequenced and overexpressed in Escherichia coli. Expression of the gene enabled rescue of an E. coli gor‐ mutant, confirming the identity of the cloned gene. The predicted sequence of the gene product showed homology with other members of the pyridine nucleotide‐disulphide oxidoreductase family, and allowed determination of positions that may be involved in substrate specificity. These predictions provided information on the relationship of sequence to function, independently of structural data used in previous studies.
Response of Escherichia coli cell membranes to induction of λc1857 prophage by heat shock
Authors: Kucharczyk K. , Laskowska E. , Taylor A.
Published: October 2006

Heat shock induces protein aggregation in Escherichia coli and E. coli (λc1857). The aggregates (S fraction) appear 15 min post‐induction and are separable from membranes by sucrose density‐gradient centrifugation. The S fraction quickly disappears in wild type strains but persists in rpoH mutant with concomitant quick inner membrane destruction. We propose that: (1) the disappearance of the S fraction reflects a rpoH‐dependent processing, (2) the membrane destruction explains the lethality of the rpoH mutation at elevated temperatures; and (3) the protection of the inner membrane integrity is an important physiological function of the heat‐shock response. We assume that the S fraction of aggregated proteins represents the signal inducing the heat‐shock response.

The prophage thermo‐induction results in an increase (35 min post‐induction) in the A fraction resembling that of the adhesion zones of the membranes. This fraction is greater than the corresponding fraction from uninduced cells. The increase is mediated by the λ late genes, since it is absent in the induced E.coli (λc1857 Oam21).

Since heat shock is widely used for induction of the λ promoters in expression vectors it is possible that the formation of the protein aggregates (though transient in WT strains) and/or the fragility of membranes in rpoH mutants may be the cause of poor expression of cloned genes or may lead to mistaken localization of their expression products.
Multifunctional nature of P fimbriae of uropathogenic Escherichia coli: mutations in fsoE and feoF influence fimbrial binding to renal tubuli and immobilized fibronectin
Authors: Westerlund B. , van Die I. , Kramer C. , Kuusela P. , Holthöfer H. , Tarkkanen A.‐M. , Virkola R. , Riegman N. , Bergmans H. , Hoekstra W. , Korhonen T. K.
Published: October 2006

P fimbriae of the F71 serotype of Escherichia coli are composed of a major subunit, FsoA, and of three minor proteins named FsoG, FsoE, and FsoF. FsoG is the Galα(1–4)Gal‐specific lectin. We assessed mutated recombinant strains each deficient in one fimbrial component for adhesion to frozen sections of rat cortical kidney and to fibronectin immobilized on glass. Rat kidney lacks the Galα(1–4)Gal‐containing glycolipids. The fsoG mutant strain was as adhesive to sections of rat kidney and to fibronectin‐coated glass as was the recombinant strain expressing the complete fso gene cluster. The fsoA mutant strain was highly adhesive to fibronectin and to kidney sections. In the rat kidney, the adhesion of these strains was predominantly localized to sites of basolateral membranes of tubuli. The fsoE and the fsoF mutant strains were slightly less adhesive to kidney structures and failed to adhere to fibronectin. The fsoE, fsoF double mutant strain adhered neither to fibronectin nor to kidney sections. None of the fso recombinant strains reacted with soluble fibronectin, suggesting that the interaction is dependent on the conformation of the fibronectin molecules. Recombinant strains expressing the F72, F8, F11, F13, and F14 serovariants of the P fimbria also showed adherence to immobilized fibronectin. The results show that in addition to binding to globoseries of glycolipids via the G protein, the P fimbriae of uropathogenic E. coli exhibit a tissue‐binding property influenced by fsoE and fsoF gene products and with affinity for basolateral membranes and fibronectin.
Involvement of the syrM and nodD3 genes of Rhizobium meliloti in nod gene activation and in optimal nodulation of the plant host
Authors: Kondorosi E. , Buiré M. , Cren M. , Iyer N. , Hoffmann B. , Kondorosi A.
Published: October 2006

We identified and sequenced the regulatory syrM and nodD3 genes of Rhizobium meliloti 41. Both genes were shown to contribute to optimal nodulation of alfalfa. In R. meliloti strains carrying syrM and nodD3 on plasmid, the nod genes are expressed constitutively, resulting in host‐range extension to siratro. This is due to the presence of multiple syrM copies, suggesting that SyrM participates directly in nod gene activation. NodD3 activates nod genes in conjunction with flavonoids and enhances syrM expression, which is controlled also by its own product, NodD2, and two putative trans‐acting factors. nodD3 is regulated by SyrM, NodD1, NodD3, the repressor NoIR, and two putative factors.
Genetic and biochemical analysis of the aspartokinase from Corynebacterium glutamicum
Authors: Kalinowski J. , Cremer. J. , Bachmann B. , Eggeling L. , Sahm H. , Pühler A.
Published: October 2006

The lysC/asd gene cluster of Corynebacterium glutamicum ATCC 13032 was cloned and sequenced. The lysC locus coding for aspartokinase consists of two in‐frame overlapping genes, lysCα encoding a protein of 421 amino acids (Mr 44300) and lysCβ encoding a protein of 172 amino acids (Mr 18600). The C. glutamicum aspartokinase was purified and found to contain two proteins of Mr 47000 and Mr 18000.

A C. glutamicum mutant expressing a feedback‐resistant aspartokinase was shown to be changed in a single base pair of the lysCβ gene, leading to an amino acid exchange in the β‐subunit of the aspartokinase. In addition, the identified mutation was found to be responsible for the enhanced expression of the asd gene located downstream of lysC.
Expression of Salmonella typhi and Escherichia coli OmpC is influenced differently by medium osmolarity; dependence on Escherichia coli OmpR
Authors: Puente J. L. , Verdugo‐Rodríguez A. , Calva E.
Published: October 2006

OmpC, a major outer‐membrane protein, is highly expressed when Salmonella typhi is grown in nutrient broth (NB) of either low (NB + 0% sucrose) or high (NB + 20% sucrose) osmolarity. This contrasts with the expression of Escherichia coli OmpC, which is inhibited in low osmolarity and enhanced in high osmolarity, as has been described previously (van Alphen and Lugtenberg, 1977; Verhoef et al., 1979; Kawaji et al., 1979). Nevertheless, expression of S. typhi OmpC is dependent on the E. coli OmpR transcriptional activator. These findings suggest differences between the mechanisms of osmoregulation of gene expression in both bacteria, although common effectors appear to be shared.
Filnmentous phage assembly
Authors: Russel M.
Published: October 2006

Filamentous phages present a genetically well‐defined system for studying the ordered membrane assembly of five different phage‐encoded proteins around the circular single‐stranded DNA phage genome. Assembly occurs at high efficiency in vivo, catalysed by two phage‐encoded membrane proteins and al least one host protein, thioredoxin. This review presents a description of the virion and its cytoplasmic precursor and summarizes the results of genetic and biochemical experiments that are beginning to elucidate the rote of the three morphogenetic proteins. The recent discovery of bacterial transport proteins with homology to a phage morphogenetic protein located in the outer membrane suggests the existence of a common mechanism for moving complex macromolecules across bacterial membranes.
Analysis of structure and function of the B subunit of cholera toxin by the use of site‐directed mutagenesis
Authors: Jobling M. G. , Holmes R. K.
Published: October 2006

Oligonucleotide‐directed mutagenesis of ctxB was used to produce mutants of cholera toxin B subunit (CT‐B) altered at residues Cys‐9, Gly‐33, Lys‐34, Arg‐35, Cys‐86 and Trp‐88. Mutants were identified phenotypically by radial passive immune haemolysis assays and genotypically by colony hybridization with specific oligonucleotide probes. Mutant CT‐B poly‐peptides were characterized for immunoreactivity, binding to ganglioside GM1, ability to associate with the A subunit, ability to form holotoxin, and biological activity. Amino acid substitutions that caused decreased binding of mutant CT‐B to ganglioside GM1 and abolished toxicity included negatively charged or large hydrophobic residues for Gly‐33 and negatively or positively charged residues for Trp‐88. Substitution of lysine or arginine for Gly‐33 did not affect immunoreactivity or GM1 ‐binding activity of CT‐B but abolished or reduced toxicity of the mutant holotoxins, respectively. Substitutions of Glu or Asp for Arg‐35 interfered with formation of holotoxin, but none of the observed substitutions for Lys‐34 or Arg‐35 affected binding of CT‐B to GM1. The Cys‐9, Cys‐86 and Trp‐88 residues were important for establishing or maintaining the native conformation of CT‐B or protecting the CT‐B polypeptide from rapid degradation in vivo.
Targeting and assembly of an oligomeric bacterial enterotoxoid in the endoplasmic reticulum of Saccharomyces cerevisiae
Authors: Schonberger O. , Hirst T. R. , Pines O.
Published: October 2006

A hybrid protein consisting of the Escherichia coli lipoprotein signal sequence attached to the mature sequence of the B subunit of heat‐labile enterotoxrn (Lipo‐EtxB) was expressed in yeast and E. coli Analyses of cell lysates from Saccharomyces cerevisiae and E. coli expressing the protein revealed that both organisms were able to assemble Lipo‐EtxB into oligomers that were (i) stable in the presence of sodium dodecyl sulphate, (ii) resistant to proteinase K degradation, and (iii) able to bind to GM1‐ganglio‐side receptors. Each of these properties are characteristic of the wild‐type B subunit pentamer produced in E. coli. Assembly of Lipo‐EtxB was found to be unaffected in a sec18 mutant of S. cerevisiae, which possesses a temperature‐sensitive defect in protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus, but was found not to assemble in a sec53 mutant, which causes the misfolding of proteins targeted to the ER. A kar2‐1 mutation with a defect in the yeast homologue of BiP caused an 18‐fold reduction in Lipo‐EtxB assembly at the non‐permissive temperature in S. cerevisiae. However, introduction of the wild‐type KAR2gene on a plasmid into the kar2‐1 mutant completely suppressed the inhibition of Lipo‐EtxB assembly. This provides the first evidence that KAR2 facilitates the assembly of an oligomeric protein in yeast and thus implicates KAR2 as a‘molecular chaperone'. The possible mechanisms of enterotoxoid assembly In E. coli and S. cerevisiae are discussed.
Identification of a nifA‐like regulatory gene of Azospirillum brasilense Sp7 expressed under conditions of nitrogen fixation and in the presence of air and ammonia
Authors: Liang Y. Y. , Kaminski P. A. , Elmerich C.
Published: October 2006

A gene bank of Azospirillum lipoferum Br17 constructed in the vector λGEM11 was screened with a Bradyrhizobium japonicum nifA gene probe. A 7.3 kb EcoRI fragment carrying a nifA‐like gene was thereby isolated and subsequently used to screen a gene bank of Azospirillum brasilense Sp7 constructed in pUC18. Two EcoRI fragments of 5.6 kb and 3.6 kb covering the nifA‐homology region were found. Mutants with Nif‐phenotype were obtained by site‐directed Tn5 mutagenesis of the 5.6 kb fragment and subsequent recombination into the A. brasilense Sp7 genome. The mutations were clustered into two loci located at each extremity of the fragment. One of these loci corresponded to nifA and the other to nifB. The nucleotide sequence of nifA of A. brasilense Sp7 was determined. Comparison of the deduced amino acid sequences of NifA of A. brasilense Sp7 and NifA of B. Japonicum, Rhizobium leguminosarum biovar trifolii and Klebsiella pneumoniae confirmed that it was a nifA‐like gene. Construction of a nifA‐lacZ fusion and mapping of the RNA transcriptional start site showed that the nifA‐like gene was expressed from an unidentified promoter, under conditions of nitrogen fixation and in the presence of oxygen and ammonia.
New approaches in genome analysis by pulsed‐field gel electrophoresis: application to the analysis of Pseudomonas species
Authors: Grothuesi D. , Tümmler B.
Published: October 2006

Summary A general method for the evaluation of macrorestriction fragment patterns is presented and its applicability to the taxonomy of bacteria is demonstrated for 32 Pseudomonas species. Strains were differentiated at the species and subspecies level by genome size and macrorestriction fragment fingerprints of the chromosome that had been separated on pulsed‐field gels. The relatedness of bacteria was ascertained from the similarity of Asnl, Dral, Spel, Sspl or Xbal fragment patterns. In general, the dendrograms calculated from the genome fingerprints corresponded with the phylogenetic classification obtained from phenotypic marker or nucleic acid hybridization analysis, but several exceptions were noted. The techniques and algorithms presented herein are generally applicable to the genome analysis of bacteria, lower eukaryotes, and DNA fragments cloned in yeast artificial chromosomes.
The tonB gene of Serratia marcescens: sequence, activity and partial complementation of Escherichia coli tonB mutants
Authors: Gaisser S. , Braun V.
Published: October 2006

The TonB protein plays a key role in the energy‐coupled transport of iron siderophores, of vitamin B12, and of colicins of the B‐group across the outer membrane of Escherichia coli. In order to obtain more data about which of its particular amino acid sequences are necessary for TonB function, we have cloned and sequenced the tonB gene of Serratia marcescens. The nucleotide sequence predicts an amino acid sequence of 247 residues (Mr 27389), which is unusually proline‐rich and contains the tandem sequences (Glu‐Pro)5 and (Lys‐Pro)5. In contrast to the TonB proteins of E. coli and Salmonella typhimurium, translation of the S. marcescens TonB protein starts at the first methionine residue of the open reading frame, which is the only amino acid removed during TonB maturation and export. Only the N‐terminal sequence is hydrophobic, suggesting its involvement in anchoring the TonB protein to the cytoplasmic membrane. The S. marcescens tonB gene complemented an E. coli tonB mutant with regard to uptake of iron siderophores, and sensitivity to phages T1 and φ 80, and to colicins B and M. However, an E. coli tonB mutant transformed with the S. marcescens tonB gene remained resistant to colicins la and Ib, to colicin B derivatives carrying the amino acid replacements Val/Ala and Val/Gly at position 20 in the TonB box, and they exhibited a tenfold lower activity with colicin D. In addition, the S. marcescens TonB protein did not restore T1 sensitivity of an E. coli exbB tolQ double mutant, as has been found for the overexpressed E. coli TonB protein, indicating a lower activity of the S. marcescens TonB protein. Although the S. marcescens TonB protein was less prone to proteolytic degradation, it was stabilized in E. coli by the ExbBD proteins. In E. coli, TonB activity of S. marcescens depended either on the ExbBD or the TolQR activities.
The C‐terminal domain of the toxic fragment of a Bacillus thuringiensis crystal protein determines receptor binding
Authors: Honée G. , Convents D. , Van Rie J. , Jansens S. , Peferoen M. , Visser B.
Published: October 2006

The insecticidal crystal proteins of Bacillus thuringiensis show a high degree of specificity. In vitro binding studies with several crystal proteins demonstrated a correlation between toxicity and binding to receptors of larval midgut epithelial cells. In order to study the domain‐function relationships of the toxic fragment, hybrid crystal proteins based on CrylA(b) and CrylC were constructed. Two out of 11 hybrid proteins constructed exhibited insecticidal activity. Both displayed an insectidial spectrum similar to that of the parental crystal protein from which the C‐terminal part of the toxic fragment originated. In addition, in vitro binding studies directly demonstrated the involvement of the C‐terminal part of the toxic fragment in receptor binding. These results demonstrate that the C‐terminal part of the toxic fragment determines specific receptor binding, which in turn determines, to a large extent, the insect specificity.
The 120 kilodalton outer membrane protein (rOmp B) of Rickettsia rickettsii is encoded by an unusually long open reading frame: evidence for protein processing from a large precursor
Authors: Gilmore R. D. , Cieplak W. , Policastro P. F. , Hackstadt T.
Published: October 2006

A Rickettsia rickettsii outer surface membrane protein (rOmp B), of an apparent molecular mass of 120 kilodaltons, is a major surface antigen of the Rickettsiae that displays genus, species, and sub‐species specific antigenic determinants. The 5′ portion of this gene was found to be unstable in plasmids, but was stably cloned in a lambda vector. The nucleotide sequence of the 5′ terminus has been determined, thus completing the DNA sequence of the entire gene. Genetic analysis revealed an unusually large open reading frame with the capacity to encode a product much larger than the mature protein. A 32 kilodalton peptide from purified rickettsiae was isolated and the amino terminus was sequenced, which revealed that the peptide is encoded by the 3′ portion of this large open reading frame. This suggests a role for post‐translational processing of rOmp B from a large precursor molecule.
Differential gene expression from the Escherichia coli atp operon mediated by segmental defferences in mRNA stability
Authors: McCarthy J. E. G. , Gerstel B. , Surin B. , Wiedemann U. , Ziemke P.
Published: October 2006

The atp operon of Escherichia coli directs synthesis rates of protein subunits that are well matched to the requirements of assembly of the membrane‐bound H+‐ATPase (α3β3γ1δ1σ1a1b2c10–15). Segmental differences in mRNA stability are shown to contribute to the differential control of atp gene expression. The first two genes of the operon, atpl and atpB, are rapidly inactivated at the mRNA level. The remaining seven genes are more stable. It has previously been established that the translational efficiencies of the atp genes vary greatly. Thus differential expression from this operon is achieved via post‐transcriptional control exerted at two levels. Neither enhancement of translational efficiency nor insertion of repetitive extragenic palindromic (REP) sequences into the atplB intercistronic region stabilized atpl. We discuss the implications of these results in terms of the pathway of mRNA degradation and of the role of mRNA stability in the control of gene expression.
Analysis of Haemophilus influenzae type b lipooligosaccharide‐synthesis genes that assemble or expose a 2‐keto‐3‐deoxyoctulosonic acid epitope
Authors: Abu Kwaik Y. , McLaughlin B. E. , Apicella M. A. , Spinola S. M.
Published: October 2006

We recently isolated a recombinant phage from a Haemophilus influenzae type b (Hib) library that assembles an oligosaccharide with an apparent molecular weight of 1400 (1.4 K) on a 4.1 K Escherichia coli lipopolysaccharide (LPS) structure, producing a 5.5 K LPS species that contains a KDO (2‐keto‐deoxyoctulosonic acid) epitope. Subcloning and deletional analysis of the 14 kb Haemophilus insert showed that three overlapping restriction fragments contained within a 7.2 kb pstl–Bam HI fragment sequentially modified an E. coli 4.1 K LPS structure, generating novel species of 4.5K, 5.1K and 5.5K. Only the 5.5K species contained the KDO epitope. We confirmed the relationship between the cloned genes and Hib lipooligosaccharide (LOS) biosynthesis by constructing a mutant that expressed an altered LOS. Thus, the Hib 7.2 kb Pstl‐Bam Hl restriction fragment contained a cluster of at least three genetic loci whose products acted sequentially in LOS biosynthesis.
The bvg‐dependent promoters show similar behaviour in different Bordetella species and share sequence homologies
Authors: Scarlato V. , Prugnola A. , Aricò B. , Rappuoli R.
Published: October 2006

The expression of the virulence‐associated genes in Bordetella species is co‐ordinately regulated by the gene products encoded by the bvg locus. In Bordetella pertussis the expression of this locus is regulated by the P1, P2, P3 and P4 promoters which are located in a 350bp DNA fragment also containing the PFHA promoter. Here we report the transcriptional regulation of the bvg locus and the fha gene in Bordetella paraper‐tussis and a sequence analysis of the byg‐regulated promoters. The Pp1, Pp2, Pp4 and PpFHA promoters are indistinguishable, both in transcription initiation sites and environmental regulation, from the corresponding promoters of B. pertussis, while the Pp3 promoter is not active. Sequence homologies from nine bvg‐regulated promoters show a conserved dinucleotide, 5′‐TG‐3′, at approximately one turn of helix upstream of the ‐10 5′‐A.AaTat‐3’region, and a 5′‐TTTCC‐3’sequence in the ‐90 region. Since the nucleotide sequence of the inactive Pp3 promoter shows several base substitutions with respect to the found sequence homologies, it is likely that some of these bases play an essential role in promoter activity.
Role of plasmid multimers in mutation to tetracyline resistance
Authors: Boe L. , Marinus M. G.
Published: October 2006

As an additional system for analysing mutations that appear to be specifically induced or directed, we have used a plasmid that contains the mnt repressor gene inserted as an operon fusion with the tet gene of the plasmid pBR322. Thus, the mnt gene product acts as a negative transcriptional regulator of tet gene expression. Mutations inactivating the Mnt repressor are recessive while those destroying operator recognition (Oc) are dominant in conferring tetracycline resistance on the host. When resistance mutations were isolated on plates with high levels of tetracycline they were preferentially mnt‐ and the plasmids were monomers. Pre‐exposure to low concentrations increased the frequency of resistant mutants by 100‐ to 1000‐fold, and the mutations were now mostly Oc, located on one unit of a plasmid multimer. Recessive repressor mutations on one unit would not have been selected. We suggest that the high frequency of mutation in tandem multimeric plasmids may be caused by the formation of single‐stranded and hence highly mutable regions by homologous pairing out of register. The role of tetracycline in promoting mutations is discussed.