bluuebunny/crossref_metadata_2025 huggingface dataset:
Specificity of Bacillus thuringiensis for lepidopteran larvae: factors involved in vivo and in the structure of a purified protoxin
Authors: Arvidson H. , Dunn P. E. , Strnad S. , Aronson A. I.
Published: October 2006

The relative LD50 values in two test Lepidoptera of Bacillus thuringiensis subspecies kurstaki HD1, which contains three crylA protoxin genes, was the same as a plasmid‐cured derivative or a Bacillus cereus transcipient containing only one of the three genes. Differential rates of transcription of these genes in the original strain could account, at least partly, for this result. Strains containing only the single protoxin gene (crylA(b)) produced inclusions when grown at 25°C but not 32°C, despite transcription of this gene at both temperatures. The instability of the crylA(b) protoxin was not found in the parental B. thuringiensis subsp. kurstaki HD1 strain grown at either temperature, however, so kurstaki HD1 strains with multiple protoxin genes must produce some stabilizing factor, perhaps another protoxin.

The cryl protoxins contain a highly conserved carboxyl half which is proteolytically removed upon conversion to toxin. All of the protoxin cysteines are present in protease‐sensitive regions and they are oxidized in inclusions. Most of the disulphides appear to be essential for specificity since their reduction in the crylA(b) protoxin resulted in loss of selectivity for one of the test insects. This lack of specificity was also found for this protoxin produced by an Escherichia coli clone, probably because of the reducing conditions in these cells. Specificity was restored by reoxidation of the pure protoxin, by removal of the carboxyl half of oxidized protoxin with trypsin, or by subcloning of the toxin portion. The oxidized form of protoxins must be important for specificity, for the formation of crystalline inclusions, and probably for interactions required for the stabilization of some protoxins.
Cloning, expression and sequence analysis of the gene encoding the 120kD surface‐exposed protein of Rickettsia rickettsii
Authors: Gilmore R. D. , Joste N. , McDonald G. A.
Published: October 2006

Rickettsia rickettsii (R strain) genomic DNA was partially digested and cloned into the lambda expression vector gt11 generating a genomic clone bank. Trans‐formant plaques were screened with antisera generated against the 120 kiloDalton protein of R. rickettsii to detect those phage expressing the recombinant protein. The gene encoding the 120kD protein was localized to a 4.3 kilobase Sphl‐BamHl fragment from the recombinant phage and subcioned into pUC18 and pUC19. Full‐length expression of the recombinant protein was achieved with both orientations. The gene and flanking regions were sequenced. The p120 gene consists of 3900 base pairs coding for 1300 amino acids. A distinguishable promoter region was not identified, although there are several 5′ sequences that resemble classical prokaryotic promoters. Downstream of the termination codon for this gene lies a 726 base pair open reading frame on the opposite strand with the potential to encode a protein of approximately 27 kD. The identity of this putative gene product is unknown. The two open reading frames are separated by a 106 base pair intergenic region that consists of a stretch of dyad symmetry resembling rho‐independent transcriptional terminators.
Molecular cloning, nucleotide sequence and fine‐structural analysis of the Corynebacterium glutamicum fda gene: structural comparison of C. glutamicum fructose‐1,6‐biphosphate aldolase to class I and class II aldolases
Authors: von der Osten C. H. , Barbas C. F. , Wong C.‐H. , Sinskey A. J.
Published: October 2006

The Corynebacterium glutamicum fda gene encoding fructose‐ 1,6‐btphosphate (FBP) aldolase has been isolated by complementation of an Escherichia coli mutant. The nucleotide sequence of a 3371 bp chromosomal fragment containing the C. glutamicum fda gene was determined. The N‐terminal amino acid sequence of C. glutamicum FBP aldolase identified the correct initiation site for the fda gene, and a molecular weight of 37092 was predicted for the fda polypeptide. S1 nuclease mapping identified the transcriptional start site, and Northern hybridization analysis indicated that the fda gene encodes a single 1.3 kb transcript. The primary structure of C. glutamicum FBP aldolase shows strong homology to class 11 FBP aldolases. Conservation of primary structure was observed between class I and class II aldolases, but several residues essential for catalytic activity in class I aldolases were absent from class II aldolases.
Analysis of genes coding for the sialic acid‐binding adhesin and two other minor fimbrial subunits of the S‐fimbrial adhesin determinant of Escherichia coli
Authors: Schmoll T. , Hoschützky H. , Morschhäuser J. , Lottspeich F. , Jann K. , Hacker J.
Published: October 2006

The S fimbrial adhesin (Sfa) enables Escherichia coli to attach to sialic acid‐containing receptor molecules of eukaryotic cells. As previously reported, the genetic determinant coding for the Sfa of an E. coli O6 strain was cloned, the gene coding for the major fimbrial subunit was identified and sequenced and the S specific adhesin was detected. Here we present evidence that in addition to the major subunit protein SfaA three other minor subunit proteins, SfaG (17kD), SfaS (14 kD) and SfaH (31 kD) can be isolated from the S‐specific fimbrial adhesin complex. The genes coding for these minor subunits were identified, mutagenized separately and sequenced. Using haemagglutination tests, electron‐microscopy and quantitative ELISA assays with monoclonal anti‐SfaA and anti‐SfaS antibodies the functions of the minor subunits were determined. It was determined that SfaS is identical to the S‐specific adhesin, which also plays a role in determination of the degree of fimbriation of the cell. The minor subunit SfaH also had some influence on the level of fimbriation of the cell, while SfaG is necessary for full expression of S‐specific binding. It was further shown that the amino‐terminal protein sequence of the isolated SfaS protein was identical to the protein sequence calculated from the DNA sequence of the sfaS gene locus.
Molecular linkage of the nifl fix and nod gene regions in Rhixobium leguminosarum biovear trifolii
Authors: Iismaa S. E. , Ealing P. M. , Scott K. F. , Watson J. M.
Published: October 2006

Nucleotide sequence analysis of a 2.5kb region downstream of the nifA gene from Rhizobium leguminosarum biovar trifolii has resulted in linkage, at the DNA sequence level, of the nifEN, nifHDK, fixABCX, nifA gene cluster with the nodEF, nodD, nodABCIJ genes. Four genes have been identified within this intervening region. Immediately 3’to the nifA gene is the nifB gene and the nifB‐linked ferredoxin‐encoding fdxN gene. Downstream of fdxN in R. leguminosarum bv. trifolii and in Rhizobium meliloti, we have identified an open reading frame which has not been described previously and which we propose to designate fixU. Downstream of fixU in R. leguminosarum by. trifolii is a nod gene, nodT, which is contiguous with nodJ (B. Surin et al., manuscript in preparation). As a result of this study, the linkage relationships of 22 symbiotic genes spanning a 24 kb region of the symbiotic plasmid from R. leguminosarum bv. trifoiii are now known.
Bacterial polysaccharide capsule synthesis, export and evolution of structural diversity
Authors: Boulnoisl G. J. , Jann K.
Published: October 2006

Elaboration of a capsule composed of one of a range of acidic polysaccharides is a common feature of many bacteria, particularly those capable of causing serious infections in humans. Biochemical and genet‐cal analyses of capsule biogenesis in Escherichia coli are beginning to reveal new aspects of polysaccharide biosynthesis. Genes have been identified which are thought to encode products responsible for the trans‐location of these high molecular‐weight polysaccharides across the cytoplasmic and outer membranes, and the organization of exported polysaccharide into a capsule. Their further analysis should provide new insights into membrane biology, particularly since the genes in question are absent from the often used laboratory strains of E. coli. Genetic analysis of capsule diversity is beginning to suggest possible mechanisms for the generation of the structural diversity of polysaccharides.
The gene for the precursor of plastocyanin from the cyanobacterium Anabaena sp. PCC 7937: isolation, sequence and regulation
Authors: Van der Plas J. , Bovy A. , Kruyt F. , de Vrieze G. , Dassen E. , Klein B. , Weisbeek P.
Published: October 2006

The gene encoding plastocyanin (petE1) from Anabaena sp. PCC 7937 was isolated using two sets of mixed oligonucleotide hybridization probes derived from conserved regions in the protein. Plastocyanin is encoded as a preprotein of 139 amino acids. The amino‐terminal extension of 34 residues has all the characteristics of a signal peptide and is probably involved in translocation of preplastocyanin over the thylakoid membrane. The level of the petE1 mRNA, a single transcript of about 740 bases, was found to be severely reduced under conditions of Cu2+ deficiency. The petE1 gene was transferred to the genome of Synechococcus sp. PCC 7942, which did not appear to contain a structural gene for plastocyanin itself. The integrated gene becomes expressed at the transcriptional level, regardless of the amount of Cu2+ available.
The major outer membrane protein of Chlamydia trachomatis: critical binding site and conformation determine the specificity of antibody binding to viable chlamydiae
Authors: Conlan J. W. , Kajbaf M. , Clarke I. N. , Chantler S. , Ward M. E.
Published: October 2006

The major outer membrane protein (MOMP) is the prime candidate for the development of a chlamydial vaccine. Antibodies to the subspecies‐specific epitope neutralize chlamydial infection. Monoclonal antibodies (MAbs) to this epitope were prepared either by immunization with whole chlamydiae or with a 16 amino acid synthetic peptide. The critical binding site on the subspecies epitope for these MAbs was determined to single amino acid resolution using several hundred solid‐phase peptides. A frame shift of just one amino acid in critical binding site completely prevented antibody binding to viable chlamydiae. A single MAb to whole organisms was capable of spanning both the surface‐exposed, conformation‐dependent, subspecies epitope and a buried, conformation‐independent species epitope some 10 Å distant. Immunization with peptide generated an MAb with reduced binding constraints which permitted the antibody to bind with broadened species‐specificity at the subspecies binding site. The results show for the first time the importance of both critical binding site and conformation at the subspecies epitope. We suggest that the conformational flexibility of short, epitopic peptide vaccines may in some cases be advantageous, giving rise to extended specificity not attained with the natural protein.
Genetic analysis of phycobilisome mutants in the cyanobacterium Synechococcus species PCC 6301
Authors: Kalia R. , Lind L. K. , Gustafsson P.
Published: October 2006

The chromophoric protein phycocyanin is the major protein in the phycobilisome rod of the cyanobacterium Synechococcus 6301 (formerly designated Ana‐cystis nidulans sp. UTEX 625). The gene clusters coding for the β‐ and α‐subunits of phycocyanin are duplicated on the chromosome of Synechococcus 6301 and separated by an intergenic region 2.5 kb long. The structure of the phycocyanin operons of the phycobilisome mutant strains AN112 and AN135 was compared to that of wild‐type Synechococcus 6301. Both mutants have an altered phycobilisome structure resulting in the appearance of rods of a shorter overall length. Genetic mapping indicated that the mutant strain AN112 completely lacked the intergenic region and carried only one set of phycocyanin subunit genes. No gross structural difference in the genetic organization of the corresponding region of mutant strain AN135 was detected. Northern blot analysis and primer extension experiments were used to monitor the transcriptional pattern of the phycocyanin rod operon. It was found that AN112 had lost the 3.7 kb minor mRNA, which covers the intergenic region, and only produced two major 1.3 and 1.4 kb mRNA species. These transcripts were identified as fusion products of the 5′ end of the transcriptional unit originating from the promoter region of the left‐hand phycocyanin gene cluster and the 3′ end of the transcriptional unit covering the right‐hand phycocyanin gene cluster. The mutant strain AN 135 had a transcriptional pattern very similar to that of the wild type. The level of transcription of the major transcripts covering the phycocyanin gene clusters was similar in the wild‐type and mutant strains. The results indicate that genes coding for two of the linker proteins, namely the 30 and 33 kD poly‐peptides, are located in the intergenic region between the duplicated phycocyanin gene clusters in Synecho‐coccus 6301. The results also show that loss of the right‐hand phycocyanin promoter does not drastically impair phycocyanin synthesis.
Phospholipase C and haemolytic activities of Clostridium perfringens alpha‐toxin cloned in Escherichia coli: sequence and homology with a Baciilus cereus phospholipase C
Authors: Leslie D. , Fairweather N. , Pickard D. , Dougan G. , Kehoe M.
Published: October 2006

The Clostridium perfringens alpha‐toxin (phospholipase C) gene (cpa) has been cloned and expressed in Escherichia coli. The biological activities of the cloned gene product have been analysed and the complete nucleotide sequence of the cpa gene has been determined. The cloned cpa gene product, which is exported to the periplasm in E. coli, possesses both phospholipase C and haemolytic activities. Haemolysis is not apparent when cell extracts are incubated with isotonic suspensions of sheep erythrocytes, but can be detected and quantified readily when dilutions of the same extracts are placed in wells in sheep‐blood agar plates. Like other sequenced clostridial genes, the cpa gene has a high AT content (66.4%), exhibits a strong bias for using codons with A or T in the wobble position, and the 350 base pairs upstream from the gene have a significantly higher AT content (79.5%) than the coding region. The cpa gene encodes a 398 amino acid polypeptide with a deduced molecular weight of 45481D. This is very similar to the estimated molecular weight (Mr) of the cpa primary gene product expressed in an in vitro transcription‐translation system (Mr 46000), but larger than the cpa gene product detected in E. coli minicells, E. coli whole cells or in C. perfringens cells (Mr 43000), suggesting post‐translational processing. The 28 N‐terminal residues of the deduced alpha‐toxin sequence possess the consensus features of a signal peptide and may be removed during secretion. The deduced alpha‐toxin sequence shares significant structural homology with the phosphatidylcholine‐preferring phospholipase C of Bacillus cereus.
Molecular and mutational analysis of three genes preceding pyrE on the Escherichia coli chromosome
Authors: Poulsen P. , Andersen J. T. , Jensen K. F.
Published: October 2006

The nucleotide sequence of two kilobase pairs (kb) 5′ to the orfE‐pyrE operon has been determined. The sequence revealed two open reading frames, orfX and orfY, consisting of 286 and 274 codons, respectively, and having a transcriptional orientation opposite that of the orfE‐pyrE operon. Analysis of transcription initiations showed that the promoters of orfE and orfX constitute a pair of divergent promoters with overlapping ‐35 regions and that orfY is transcribed from an independent promoter. Translational analysis indicated that the orfs are expressed in Escherichia coli.

The orfE, orfX, and orfY genes were inactivated on the bacterial chromosome by deletion‐insertion mutagenesis using a kanamycin resistance cassette. The mutants were all viable. However, the orfE deletion caused a dramatic reduction in the level of pyrE expression and a partial pyrimidine requirement, because this mutation prevented transcription of pyrE. The orfE protein seemed without significance for pyr‐gene expression in E. coli, and the mutations in orfX and orfY were without detectable phenotypes.
Gene structure and precursor processing of a novel Bacillus subtilis spore coat protein
Authors: Aronson A. I. , Song H.‐Y. , Bourne N.
Published: October 2006

The gene for an unusual 8kD Bacillus subtilis spore coat polypeptide has been cloned and sequenced. It contains high percentages of proline, glycine and tyrosine, lacks thirteen amino acids, and is present as the carboxyl two‐thirds of an open reading frame encoding a 12 kD polypeptide. Two presumptive precursors which could be converted to the 8kD antigen by incubation with trypsin were found in extracts of cells or spores of a strain containing multiple copies of this gene. Large amounts of these coat antigens were also present in extracts of a germination‐defective mutant which is altered in spore coat structure. There was little 8kD coat protein in the mutant, however, implying that processing is dependent on proper coat assembly. This gene was mapped to the metA region of the B. subtilis chromosome, a unique location as is true for other spore coat genes. Transcription and translation occurred late in sporulation (stage V) and the upstream region contained sequences similar to those found in other spore coat genes.
Cloning and expression of plasmid DNA sequences involved in Salmonella serotype Typhimurium virulence
Authors: Norel F. , Coynault C. , Miras I. , Hermant D. , Popoff M. Y.
Published: October 2006

A 22 kb region of the 90kb virulence‐associated plasmid, plP1350, of Typhimurium strain C52 was cloned into the mobilizable vector pSUP202, yielding plasmid plP1352. This recombinant plasmid restored full virulence to plasmidless strain C53 in a mouse model. Transposon Tn5 insertion mutagenesis demonstrated the existence of two DNA sequences in plP1352 designated VirA and VirB, both of which are essential for the expression of virulence. A recombinant plasmid containing only the VirA and VirB regions markedly increased the virulence of the plasmidless strain C53, but did not confer full virulence. These results suggested that a third virulence‐associated region might be present on plP1352. Eleven proteins encoded by the 22 kb insert sequence of pl P1352 were identified in Escherichia coli SE5000 maxicells. The VirA region encoded at least two proteins with apparent molecular weights of 71000 and 28000 and the VirB region encoded two proteins of 43000 and 38000.
The Escherichia coli enterobactin biosynthesis gene, entD: nucleotide sequence and membrane localization of its protein product
Authors: Armstrong S. K. , Pettis G. S. , Forrester L. J. , Mclntosh M. A.
Published: October 2006

The nucleotide sequence of the Escherichia coli enterobactin biosynthesis gene entD has been determined. entD specifies a predicted 23579 Dalton protein containing several helical regions, a trans‐membrane segment and one positively charged domain. The EntD polypeptide was overexpressed and identified in electrophoretic gels as a membrane protein. Although results of conventional membrane fractionation techniques were inconclusive, protease accessibility studies provided evidence that EntD domains are exposed on the inner leaflet of the cytoplasmic membrane. The presence of repetitive extragenic palindromic (REP) sequences within the fepA‐entD intercistronic region was confirmed. Lack of a canonical promoter and an iron control region 5’to entD, along with RNA hybridization data, suggest that an iron‐regulated transcript contains both fepA and entD.
RFLP‐based genetic map of Phanerochaete chrysosporium ME446: lignin peroxidase genes occur in clusters
Authors: Raeder U. , Thompson W. , Broda P.
Published: October 2006

We describe the construction of an RFLP‐based genetic map of the white rot fungus Phanerochaete chrysosporium ME446. The map is deduced from the allele distributions of 38 RFLP markers in a test set of 53 meiotically derived haploid recombinants of this strain. The map includes a cellulase gene, a mating‐type locus and a family of lignin peroxidase (and related) genes that are arranged in two unlinked clusters.
The nifA gene product from Rhizobium leguminosarum biovar trifolii lacks the N‐terminal domain found in other NifA proteins
Authors: Iismaa S. E. , Watson J. M.
Published: October 2006

The nifA gene has been identified between the fixX and nifB genes in the clover microsymbiont Rhizobium leguminosarum biovar trifolii (R.I. bv. trifolii) strain ANU843. Expression of the nifA gene is induced in the symbiotic state and site‐directed mutagenesis experiments indicate that nifA expression is essential for symbiotic nitrogen fixation. Interestingly, the predicted R.I. bv. trifolii NifA protein lacks an N‐terminal domain that is present in the homologous proteins from R.I. bv. viciae, Rhizobium meliloti, Brady rhizobium japonicum, Klebsiella pneumoniae and all other documented NifA proteins. This indicates that this N‐terminal domain is not essential for NifA function in R.I. bv. trifolii.
Specificity of insertion of IS91, an insertion sequence present in α‐haemolysin plasmids of Escherichia coli
Authors: Mendiola M. V. , de la Cruz F.
Published: October 2006

We have determined the DNA sequences of eight different insertions of IS91 in a specifically engineered recipient plasmid of known DNA sequence (pSU300). The sequences at the termini of IS91 are 5′ ‐CGAG‐TAGG… CCTATCGAT. IS91 inserts specifically 5′ to either one of the tetranucleotides 5′‐GAAC or 5′‐CAAG, and always in the same relative orientation with respect to the sequence of the target. Except in one special case, no duplications of the recipient DNA were produced at the site of insertion.
Genetic evidence that DicF, a second division inhibitor encoded by the Escherichia coli dicB operon, is probably RNA
Authors: Bouché F. , Bouché J.‐P.
Published: October 2006

The dicB operon of Escherichia coli, which was previously shown to code for a small protein inhibiting cell division, expresses a second inhibitor, DicF. Inhibition by DicF requires the transcription of a short (at the most 65 nucleotides long) stretch of DNA, acts in trans, and does not require the expression of other components of the dicABCF locus. The characteristics of the DNA sequence strongly suggest that division inhibition does not involve the translation of dicF mRNA into protein.
The terminus of the Escherichia coli chromosome is flanked by several polar polar replication pause sites
Authors: François V. , Louam J. , Louarn J.‐M.
Published: October 2006

Replication of two small ‘constrained’ regions of the Escherichia coli chromosome, one bordered by replication terminator T1 and the other by T2, displays normal velocity in the normal direction whereas it is much slower in the opposite direction (de Massy et al., 1987). The presence of multiple polar terminators has been investigated, using a bacteriophage λ derivative which provides a replication origin movable to predetermined loci and inducible on demand. The amount of DNA made from this induced origin was determined by in vivo labelling and hybridization to probes of the surrounding region. A redundancy of terminator‐like sequences, or pause sites, has been disclosed. So far, two polar pause sites, in the same orientation and separated by 50 or 80 kb, have been localized on each side of the terminus region. The results are discussed in relation to previously observations indicating that these regions are refractory to genomic inversions.
Yeast 2μm vectors replicate and undergo recombination in Torulaspora delbrueckii
Authors: Compagno C. , Ranzi B. M. , Martegani E.
Published: October 2006

In order to develop a procedure for transformation of the industrial yeast Torulaspora delbrueckii, we have constructed a set of recombinant plasmids carrying Saccharomyces cerevisiae ARS and 2μm origin of replication and kanamycin‐G418 resistance gene of Tn903(601) as a selective marker. In this paper we show that S. cerevisiae ARS vectors can replicate autonomously and that vectors bearing the whole S. cerevisiae 2 μ sequence yield stable transform ants. We also present evidence to show that 2μm vectors undergo an FLP‐mediated inter‐ and intramolecular recombination, which suggests that T. delbrueckii can support the amplification and partition mechanisms of these plasmids.
Identification of Bacillus subtilis genes expressed early during sporulation
Authors: Mathiopoulos C. , Sonenshein A. L.
Published: October 2006

Labelled cDNA transcribed in vitro from early‐sporulation RNA was enriched for sporulation‐specific sequences by subtractive hybridization to an excess of vegetative RNA and used to probe libraries of Bacillus subtilis chromosomal DNA. From the initial collection of clones that coded for RNAs transcribed preferentially during sporutation, several were subcloned and studied in more detail. It was found that two clones contained sequences (dciA and dciB) that had an undetectable level of transcription during vegetative growth but had transcripts that started to appear no later than eight minutes after induction of sporulation. A third DNA segment (dciC) was expressed at a low level in vegetative cells and increased within four minutes after induction of sporulation.

The effects of spo0 mutations, i.e. mutations that prevent cells from reaching stage I of the sporulation process, were tested. Induction of the dciA and dciB transcripts was significantly reduced in strains carrying mutations in the spo0A and spo0H genes but not in a spo0B mutant strain. In addition, a product of the abrB locus, a locus in which mutations are known to partially overcome the pleiotropic effect of spo0A and spo0B mutations, seemed to be required for dciA and dciB expression.
The nucleotide sequence of the rodC operon of Bacillus subtilis
Authors: Honeyman A. L. , Stewart G. C.
Published: October 2006

The rodC1 mutation of Bacillus subtilis is a temperature‐sensitive marker which affects the levels of teichoic acid synthesis and the cellular morphology. We have determined the nucleotide sequence of the bicistronic operon which contains the rodC gene and the nucleotide sequence of the rodC1 mutant allele. The temperature‐sensitive phenotype of the rodC mutant is the result of a single base‐pair change. A cytosine to thymine transition in the non‐coding strand results in the replacement of a serine residue in the wild‐type protein with a phenylalanine residue in the mutant protein. The other gene in the operon, the rodD gene, appears to be equivalent to the gtaA gene which encodes uridine diphosphate‐glucose poly‐(glycerol phosphate) α‐glucosyl transferase, an enzyme involved in techoic acid synthesis. This is the first nucleotide sequence analysis of both the wild‐type and mutant alleles of a morphogene in B. subtilis.
Iron and virulence in Shigella
Authors: Payne S. M.
Published: October 2006

Iron limitation, a condition encountered within mammalian hosts, induces the synthesis of a number of proteins in pathogenic Shigella species. These include several outer membrane proteins, Shiga toxin, and proteins involved in the biosynthesis and transport of high‐affinity iron‐binding compounds or siderophores. Although siderophores have been shown to play a major role in the virulence of some bacterial pathogens, these compounds do not appear to be essential for the virulence of Shigella species. Unlike those pathogens which are restricted to the extracellular compartments of the host, the Shigella species invade and multiply within host cells. Alternative iron‐acquisition systems, such as the ability to utilize haem‐iron, permit growth of the intracellular bacteria. Virulent shigellae also possess a cell‐surface haem‐binding protein, and synthesis of this protein correlates with infectivity and virulence. This protein does not appear to be involved in iron acquisition. Rather, it may allow the bacteria to coat themselves with haem compounds, thus enhancing their ability to interact with target host cells.
Isolation, molecular characterization and expression of the ushB gene of Salmonella typhimurium which encodes a membrane‐bound UDP‐sugar hydrolase
Authors: Garrett A. R. , Johnson L. A. , Beacham I. R.
Published: October 2006

The UDP‐sugar hydrolase of Salmonella typhimurium has previously been reported to be located in both the inner and the outer membrane. We have cloned the gene, designated ushB, encoding this enzyme and determined its nucleotide sequence. No significant sequence homology with the periplasmic UDP‐sugar hydrolase of Escherichia coli was found at either the DNA or protein level. However, a sequence is detectable, in the E. coli genome, which weakly hybridizes with a specific ushB probe. Polypeptide analysis has allowed the identification of the Salmonella hydrolase which has an Mr of 28349 as compared to an Mr of 60767 for the E. coli hydrolase. Most of the protein (∼90%) is located in the inner membrane. Two independent membrane fractionation procedures indicate that the remainder may be associated with the outer membrane. The deduced primary structure indicates the presence of an N‐terminal signal peptide, although certain features of the region surrounding the putative processing site indicate that processing may be inefficient, or may not occur. Experiments with several inhibitors of signal peptidase function fail to demonstrate the appearance of a precursor form.
Receptor for IgA in group A streptococci: cloning of the gene and characterization of the protein expressed in Escherichia coli
Authors: Lindah G. , Åkerström B.
Published: October 2006

The gene for an IgA‐binding protein from a group A streptococcal strain was cloned and expressed in Escherichia coli. The IgA‐binding protein, called protein Arp, was purified on IgA‐Sepharose, allowing complete purification in a single step. Analysis of protein Arp by Western immunoblotting demonstrated a major IgA‐binding band, with an apparent molecular weight of 42kD. The purified protein was shown to bind serum IgA and secretory IgA, as well as monoclonal IgA of both subclasses. There was no binding to IgM, IgD or IgE, but a weak binding to IgG. Inhibition experiments with whole bacteria indicated that IgA and IgG bind at separate sites. Experiments with immunoglobulin fragments showed that protein Arp binds to the Fc region of both IgA and IgG. The equilibrium constant of the reaction between protein Arp and polyclonal human IgA was determined to be 5.6 × 108 M‐1. Amino acid sequencing of protein Arp demonstrated a direct repeat of 7 amino acids in the NH2‐terminal region, a feature previously found in several streptococcal M proteins. This suggests that protein Arp, like M proteins, may be a streptococcal virulence factor.
Molecular cloning and characterization of a genetic region from Serratia marcescens involved in DNA repair
Authors: Murphy K. E. , Braymer H. D.
Published: October 2006

We report here the molecular isolation of a DNA fragment which encodes Tag‐like activity from the Gram‐negative bacterium Serratia marcescens. A recombinant plasmid encoding Tag‐like activity was isolated from a S. marcescens plasmid gene library by complementation of an Escherichia coli tag mutant, which is deficient in 3‐methyladenine DNA glycosylase I. The clone complements E. coli tag, recA, alkA, but not alkB, mutants for resistance to the DNA‐damaging agent methyl methanesulphonate (MMS). The coding region of the Tag activity, initially isolated on a 6.5kb BamHI fragment, was defined to a 1.8kb BglII‐Smal fragment. Labelling of plasmid‐encoded proteins using maxicells revealed that the 1.8kb fragment encodes two proteins of molecular weights 42000 and 16000. Data presented here suggest that the cloned fragment encodes a DNA repair protein(s) that has similar activity to the 3‐methyladenine DNA glycosylase I of E. coli.
Mutations that alter the helix‐turn‐helix region of the spollAC protein: a Bacillus subtilis sporulation‐specific sigma factor
Authors: Yudkin M. D. , Millonig J. H. , Appleby L.
Published: October 2006

It has previously been shown that spollAC561, a mutation that diminishes the incidence of sporulation by more than six orders of magnitude, alters the residue at position 13 of the helix‐turn‐helix region of the sporulation‐specific sigma factor encoded by spoll AC from valine to methionine (Yudkin, 1987b). We have now found that four spontaneous revertants, which sporulate at an incidence of 30‐60%, all contain transitions within the codon that was altered by spo‐561. The mutant methionine is replaced by isoleucine in two revertants, and by threonine in the other two.
Cloning of xcp genes located at the 55 min region of the chromosome and involved in protein secretion in Pseudomonas aeruginosa
Authors: Filloux A. , Bally M. , Murgier M. , Wretlind B. , Lazdunski A.
Published: October 2006

Pleiotropic mutations (xcp) affecting secretion of proteins in Pseudomonas aeruginosa have been previously characterized and mapped at 0 min, 55 min and 65 min. Genomic libraries of this organism have been constructed and the genes xcp‐5 and xcp‐54, located at the 55 min region, were cloned using the adjacent met allele as a marker, and complementation of xcp strains. From our linkage and cloning analysis, the most probable gene order in this region appears to be pyrD., xcp‐5/xcp‐54/met‐9011/oru‐314/trpF/leu‐10. Restriction mapping and transposon (Tn 1725) insertion mutagenesis demonstrated that: (i) the overall size of DNA necessary for xcp expression was 9 kb, (ii) the two loci are not adjacent on the chromosome, and (iii) the two loci are expressed independently. The xcp‐5 gene has been subcloned on a 4 kb Eco RI fragment.
Regulation of bacterial gene expression by metal–protein complexes
Authors: Hennecke H.
Published: October 2006

Metal ions are essential cofactors in several transacting bacterial gene regulators. Upon binding of the metal, the receptor proteins act either as repressors of gene expression or, in other systems, as transcriptional activators. Other metal‐dependent regulatory proteins may function, directly or indirectly, as sensors of the cellular oxygen status, and may even be mediators in light‐responsive gene regulation.
Precursor/product antiport in bacteria
Authors: Poolman B.
Published: October 2006

Many microorganisms metabolize their substrates (precursors) only partially and excrete the products of the metabolism into the medium. Although uptake of precursor and exit of product can proceed as two independent steps, there is increasing evidence that these processes are often linked and that transport is facilitated by a single antiport mechanism. Features of antiport mechanisms and advantages for the organism of catalysing precursor/product antiport will be illustrated by discussing a number of well‐characterized systems. Based on precursor‐product conversion stoichiometries, structural relatedness between precursors and products, and energetic and kinetic considerations, new examples of antiport systems will be proposed.
Molecular genetic analysis of an FNR‐dependent anaerobically inducible Escherichia coli promoter
Authors: Bell A. I. , Cole J. A. , Busby S. J. W.
Published: October 2006

From the effects of 13 deletions and three linker‐scanner mutations at the Escherichia coli nirB promoter we have located sequences necessary for FNR‐dependent induction of activity by anaerobiosis and further nitrite‐dependent stimulation of expression. We describe a nirB promoter derivative that allows the cloning of ‘cassettes’ carrying different FNR‐binding sequences and experiments in which a number of point mutations were introduced into these sequences. FNR‐dependent stimulation of expression from the nirB promoter is critically dependent on the location of the FNR‐binding site, and deletion or insertion of one base pair is sufficient to disrupt promoter function. We have transferred a number of cassette FNR‐binding sequences from the nirB promoter to the unrelated melR promoter. The insertion of FNR‐binding sequences at the melR promoter is sufficient to confer fnr‐dependency on expression. However expression from these hybrid promoters is not as efficiently repressed during aerobic growth, suggesting that the function of bound FNR is dependent on the sequence context of the FNR‐binding sequence.
Generation of a cytotoxic T‐lymphocyte response using a Salmonella antigen‐delivery system
Authors: Flynn J. L. , Weiss W. R. , Norris K. A. , Seifert H. S. , Kumar S. , So M.
Published: October 2006

We have constructed a general‐use vector for the cloning and stable expression of foreign genes in the chromosome of attenuated Salmonella typhimurium. Using this chromosomal expression vector (CEV), we expressed the circumsporozoite (CS) gene of the mouse malaria Plasmodium yoelii in an aroA S. typhimurium strain. Mice immunized with CS‐expressing Salmonella recombinants mount a CS‐specific cytotoxic T‐lymphocyte (CTL) response. This is the first demonstration that attenuated Salmonella can elicit a specific CTL response to a foreign protein in mice. The ability to easily and stably express foreign genes from the Salmonella chromosome and the generation of specific CTL greatly expands the potential of Salmonella as an antigen‐delivery system.
Specific β‐lactam antibiotics inhibit secretion of lipo‐β‐lactamase in yeast
Authors: London A. , Pines O.
Published: October 2006

The β‐lactam antibiotic cloxacillin can inhibit secretion of prokaryotic lipo‐β‐lactamase into the periplasm of yeast. The results indicate that this phenomenon is specific with respect to both the antibiotic and the lipo‐β‐lactamase whose secretion is affected, strongly suggesting that this involves an interaction between the enzyme and its substrates. The effect of the antibiotic on secretion is reversible. With different β‐lactam antibiotics, the clearest difference is observed between type A and type S penicillins; the former exert a strong inhibition of secretion whereas the latter exhibit a weak effect or no effect at all. Type A penicillins have been previously shown to cause a conformational change in various β‐lactamases. Mature lipo‐β‐lactamase species in yeast were localized either to the periplasmic space or bound to the outer surface of the cytoplasmic membrane and thus exposed to periplasm. The results are consistent with the hypothesis that binding of cloxacillin to lipo‐β‐lactamase induces a conformation on the protein that is unfavourable for its release from the membrane.
CiS‐ and trans‐acting factors involved in centromere function in Saccharomyces cerevisiae
Authors: Murphy M. , Fitzgerald‐Hayes M.
Published: October 2006

The function of centromeric DNA in the yeast Saccharomyces cerevisiae has been studied in detail. Twelve of the sixteen S. cerevisiae centromeres have been sequenced to date, and a consensus sequence has been identified. This sequence consists of a central region 78 to 86 bp in length which is greater than 90% A+T, usually in runs of As and runs of Ts. The central region is flanked on one side by a highly conserved 8bp sequence and on the other side by a highly conserved 25 bp sequence which contains partial dyad symmetry around a central C/G base pair. Mutational analyses have been used to determine the importance of each subset of the consensus sequence to centromere function. A protein which binds to the 8 bp sequence and at least one that binds to the 25bp sequence have been identified. The roles of these proteins in centromere function in mitosis and meiosis are currently under investigation.
Escherichia coli cells resistant to the DNA gyrase inhibitor, ciprofloxacin, overproduce a 60 kD protein homologous to GroEL
Authors: Hallett P. , Mehlert A. , Maxwell A.
Published: October 2006

Using a variety of mutagenic methods, we have generated a series of ciprofloxacin‐resistant mutants derived from Escherichia coli strains which overproduce the DNA gyrase A protein. Many of these mutants are found to overexpress a 60 kD protein which is shown to be highly homologous in terms of N‐terminal a mi no acid sequence to the E. coli heat‐shock protein, GroEL. Other evidence confirms that the 60 kD protein is unrelated to DNA gyrase and is similar, but not identical, to GroEL.
Five genes at the 3′ end of the Klebsiella pneumoniae pulC operon are required for pullulanase secretion
Authors: Pugsley A. P. , Reyss I.
Published: October 2006

The nucleotide sequence of a 5082bp fragment of chromosomal DNA from Klebsiella pneumoniae strain UNF5023 is reported. The sequence includes the last four genes of an operon of genes specifically required for the secretion of the enzyme pullulanase. All four genes (puIL, puIM, puIN and puIO) are shown to be required for pullulanase secretion, as is a fifth gene (puIK) which extends beyond the 5′ end of the sequenced DNA. The products of the puIL, puIM, puIN and puIO genes (44kD, 18kD, 27kD and 24kD, respectively) are all predicted to have one or more hydrophobic domains typical of signal sequences and/or membrane anchors, and were all found mainly associated with the inner membranes of subfractionated cells in which the corresponding genes had been expressed from the bacteriophage T7 gene 10 promoter. The results of this study increase the number of genes which have been identified as required for pullulanase secretion to eight, in addition to genes coding for components of the general export pathway.
Multiple promoters control the regulation of the Pseudomonas aeruginosa regA gene
Authors: Storey D. G. , Frank D. W. , Farinha M. A. , Kropinski A. M. , Iglewski B. H.
Published: October 2006

Expression studies utilizing the regA promoters, fused in tandem or separately to promoterless reporter genes, indicated that regA is transcribed from two promoters (P1 and P2). Both promoters can act independently. Expression from the P1 promoter is not affected by the iron content of the medium. Expression from the P2 promoter is tightly regulated by iron.
Mini‐Mu mediates deletion‐inversions in vivo by intra‐transposon transposition
Authors: Leach D. R. F. , Okely E. A. , Percy‐Robb M. I.
Published: October 2006

We have shown that a mini‐Mu can transpose into itself in vivo to generate a circle containing only transposon sequences. This deletion‐inversion product, which has previously been observed in vitro, is formed by non‐replicative transposition and has directly repeated Mu ends. It therefore cannot undergo further rounds of transposition and retains the two copies of the target sequence duplicated in the event. Thus we have been able to confirm that a mini‐Mu can undergo non‐replicative reactions in vivo and that these generate a 5bp target site duplication, as has been shown to occur following replicative transposition and lysogenization with Mu.
A lipopeptide‐encoding sequence upstream from the IysA gene of Pseudomonas aeruginosa
Authors: Jann A. , Cavard D. , Martin C. , Cami B. , Patte J.C.
Published: October 2006

An open reading frame (ORF) of 141 bp was observed upstream from the Pseudomonas aeruginosa lysA gene. The translation product of this ORF contains a signal peptide with a lipoprotein box, fle‐Ala‐Ala‐Cys, at the predicted signal peptidase cleavage site. The Escherichia coli phoA gene without its signal sequence was fused in frame to this ORF in a broad host‐range plasmid. The resulting construct expressed a hybrid protein exhibiting alkaline phosphatase activity in phoA mutants of both E. coli and P. aeruginosa. This indicates that the ORF encodes a peptide, part of which acts as an export signal. The hybrid peptide was identified by immunoblotting with alkaline phosphatase antiserum. The accumulation of a precursor form was observed when P. aeruginosa cells carrying this gene fusion on a plasmid were treated with globomycin. Moreover, the mature form could be labelled with 2‐[3H]‐glycerol, indicating that lipidic residues may be linked to the hybrid protein. Taken together, these results strongly suggest that the ORF encodes a lipopeptide. We propose that the gene is called IppL.
The arcB gene of Escherichia coli encodes a sensor‐regulator protein for anaerobic repression of the arc modulon
Authors: Iuchi S. , Matsuda Z. , Fujiwara T. , Lin E. C. C.
Published: October 2006

The arcA (dye) and arcB genes of Escherichia coli are responsible for anaerobic repression of target operons and regulons of aerobic function (the arc modulon). The amino acid sequence of ArcA (Dye) indicated that it is the regulator protein of a two‐component control system. Here we show that ArcB is a membrane sensor protein on the basis of its deduced amino acid sequence (778 residues), hydropathicity profile, and cellular distribution. On the carboxyl end of the ArcB sequence there is an additional domain showing homology with conserved regions of regulator proteins. Deletion into this domain destroyed ArcB function. ArcB conserved a histidine residue for autophosphorylation of the sensor proteins, and aspartic residues important for the regulator proteins.
Stable expression of meningococcal class 1 protein in an antigenically reactive form in outer membranes of Escherichia coli
Authors: White D. A. , Barlow A. K. , Clarke I. N. , Heckels J. E.
Published: October 2006

The entire gene encoding the class 1 outer membrane protein of Neisseria meningitidis is located on a 2.2 kb fragment, obtained on digestion of chromosomal DNA with Xbal. This Xbal fragment from strain MC50 (subtype P1‐16), which had previously been cloned in bacteriophage M13, has been transferred to the plasmid vector pMTL20. The resulting plasmid (pPORA100) was propagated in Escherichia coli (JM109) and cell lysates were subjected to SDS‐PAGE. Western blotting with anti‐class 1 protein antibodies revealed constitutive expression of a protein of 41kD, corresponding to the class 1 protein of the parent meningococcal strain, which was absent in the E. coli control Fractionation of E. coli cells carrying the recombinant plasmid revealed that the protein was exclusively located in the outer membrane, and N‐terminal amino acid analysis of the expressed protein revealed that normal processing of the signal peptide had occurred. Immuno‐gold electron microscopy showed that the protective epitope recognized by a P1‐16 subtype‐specific monoclonal antibody was exposed in an antigenically reactive form on the surface of E. coli cells carrying plasmid pPORA100. In contrast, expression in E. coli of a second plasmid (pPORA104) lacking the coding sequence for the first 15 amino acids of the signal peptide resulted in accumulation of recombinant class 1 protein only in the cytoplasm of the cells.

Thus the presence of the meningococcal signal sequence ensures expression of this meningococcal porin protein in an antigenically native conformation in outer membranes of E. coli, while its absence results in expression of a soluble protein. Such constructs illustrate the potential use of recombinant DNA technology for the development of effective human vaccines against meningococcal infection.
Notes An inversion truncating the creA gene of Aspergillus niduians results in carbon catabolite derepression
Authors: Arst H. N. , Tollervey D. , Dowzer C. E. A. , Kelly J. M.
Published: October 2006

The creAd‐30 mutation leading to carbon catabolite derepression in Aspergillus nidulans is a pericentric inversion, having one breakpoint within the creA gene on the left arm of chromosome I and the other breakpoint between binG and yA on the right arm. The left‐arm breakpoint alters the creA transcript. The likelihood that the inversion truncates creA centrally strengthens a previous proposal that derepression is the phenotype of loss‐of‐function mutations in creA.