bluuebunny/crossref_metadata_2025 huggingface dataset:
Systemin in Solanum nigrum. The Tomato-Homologous Polypeptide Does Not Mediate Direct Defense Responses
Authors: Schmidt Silvia , Baldwin Ian T.
Published: October 2006

We extend Ryan's seminal work on the 18-amino acid polypeptide systemin in tomato's (Solanum lycopersicum) systemic wound response to the closely related solanaceous species Solanum nigrum. We compared wild-type plants to plants transformed with an inverted repeat prosystemin construct (IRSys) to silence the expression of the endogenous S. nigrum prosystemin gene. In wild-type plants elicited with wounding + oral secretions from Manduca sexta larvae, trypsin-proteinase inhibitors (TPIs) accumulated even though prosystemin transcripts were down-regulated. Neither reducing the endogenous systemin levels by RNAi nor complementing the plants with systemin by exogenously supplying the polypeptide through excised stems significantly increased TPI activity, indicating that systemin and TPIs are not correlated in S. nigrum. The performance of two herbivore species from two feeding guilds, M. sexta larvae and Myzus persicae nicotianae, did not differ between wild-type and IRSys plants, demonstrating that varying endogenous systemin levels do not alter the direct defenses of S. nigrum. Field experiments with wild-type and IRSys plants and the flea beetle Epitrix pubescens supported these glasshouse data. That levels of oral secretion-elicited jasmonic acid did not differ between wild-type and IRSys plants suggests that systemin is unlikely to mediate jasmonate signaling in S. nigrum as it does in tomato. We conclude that the tomato-homologous polypeptide does not mediate direct defense responses in S. nigrum.
Molecular analysis of the Corynebacterium glutamicum gdh gene encoding glutamate dehydrogenase
Authors: Börmann E. R. , Eikmanns B. J. , Sahm H.
Published: October 2006

The Corynebacterium glutamicum gdh gene encoding NADP‐dependent glutamate dehydrogenase (GDH) has been isolated by complementation of the Escherichia coli gdh mutant PA340. The gdh gene was subcloned into the E. coli/C. glutamicum shuttle vector pEK0 and introduced into C. glutamicum. Recombinant strains showed approximately eightfold higher specific GDH activity (15U mg protein‐1) relative to the wild type (1.8U mg protein ‐1). Physiological studies with wild‐type and recombinant C. glutamicum strains revealed no indication of significant regulation of gdh expression. The DNA sequence of 2082 bp, including the gdh gene, 5′‐, and 3′‐flanking regions, was determined. The structural gene consists of 1344 bp and codes for a polypeptide of 448 amino acid residues (Mr 49152) showing up to 53.6% identity with reported amino acid sequences of glutamate dehydrogenases from other organisms. Northern blot hybridization revealed a 1.65 kb mRNA transcript, indicating that the gdh gene of C. glutamicum is monocistronic. Transcription occurred from a G residue located 284bp upstream of the AUG considered to be the translational initiation codon.
The gene for the S7 ribosomal protein of Chlamydia trachomatis: characterization within the chlamydial sfr operon
Authors: Wagar E. A. , Pang M.
Published: October 2006

The prokaryotic ribosomal operon, str, contains open reading frames for the two elongation factors, elongation factor G (EF‐G) and elongation factor Tu (EF‐Tu), and ribosomal proteins 57 and S12. The DNA sequence and predicted amino acid sequence for S7 from Chlamydia trachomatis are presented and compared with homologues from other prokaryotes. Also, the relationship of the S7 gene to the open reading frames for ribosomal protein S12 and EF‐G is described. Significant amino acid homology is also noted when the amino‐terminal sequence of chlamydial EF‐G is compared with the cytoplasmic tetra‐cycline resistance factors, tetM and tetO, from streptococci and Campylobacter jejuni. Related findings and possible resistance mechanisms for the newly recognized tetracycline‐resistant clinical isolates of C. trachomatis are discussed.
Sequence diversity of the 1.3 kb retron (retron‐Ec107) among three distinct phylogenetic groups of Escherichia coli
Authors: Kawaguchi T. , Herzer P. J. , Inouye M. , Inouye S.
Published: October 2006

In the preceding paper, we showed that a new 1.3kb retron (retron‐Ec 107) in Escherichia coli is responsible for the biosynthesis of a branched‐RNA‐linked multicopy single‐stranded DNA (msDNA‐Ec107). Here, we show that this retron occurs in strains from different branches. A, B1, and D of a well‐defined phylogenetic tree of a collection of wild E. coli. Sequence comparisons of the retrons from these three branches were carried out. Sequence homology was well conserved among the strains within the same branch and the retron sequence from branch A was exactly the same with that from branch D, while there were 18 base substitutions between the retrons from branch B1 and A or D, resulting in seven amino acid substitutions in reverse transcriptase. No substitutions were found in the msDNA‐ and msdRNA‐coding regions, and there was no difference in the ability of msDNA production between them. These results suggest that the retron has probably been integrated into at least one of the three branches at an early stage of evolution and subsequently transferred to the other two branches, and also that the msDNA‐producing system has been conserved during evolution with some mutations in the retron.
Functional expression of the rat brown adipose tissue uncoupling protein in Saccharomyces cerevisiae
Authors: Bathgate B. , Freebairn E. M. , Greenland A. J. , Reid G. A.
Published: October 2006

The uncoupling protein (UCP) from mammalian brown adipose tissue is an integral component of the mitochondrial inner membrane where it dissipates the proton electrochemical gradient. UCP is transported into mitochondria from the cytosol but lacks a cleavable targeting peptide. We have expressed the rat UCP in Saccharomyces cerevisiae and shown that this protein, which is not normally found in yeast, is targeted to the mitochondria where it disrupts mitochondrial function, probably by uncoupling oxidative phosphorylation.

The observed growth defect is dependent upon the level of expression of UCP. When the unmodified UCP cDNA is expressed in yeast under the control of the GAL10 promoter no defect in growth is observed. We have inserted the UCP coding sequence behind the strong phosphoglycerate kinase promoter under the control of the GAL1‐10 upstream activation site and introduced a yeast consensus sequence (ATAATG) at the translation start site. We have found that UCP expressed in S. cerevisiae is targeted to mitochondria and that its expression induces a marked growth defect on non‐fermentable carbon sources in a manner dependent on induction with galactose.
Molecular cloning, iron‐regulation and mutagenesis of the irp2 gene encoding HMWP2, a protein specific for the highly pathogenic Yersinia
Authors: Carniel E. , Guiyoule A. , Guilvout I. , Mercereau‐Puijalon O.
Published: October 2006

Under iron‐starvation, the highly pathogenic Yersinia synthesize several iron‐regulated proteins including two high‐molecular‐weight polypeptides (HMWP1 and HMWP2). From the chromosome of Yersinia enterocolitica serovar O:8 (strain Ye 8081), the genes coding for the HMWP2 (irp2) and its promoter were cloned into plasmid pUC18 (plR2) and used as a probe. We show here that the irp2 gene is present only in the highly pathogenic strains and that its promoter is iron‐regulated in Escherichia coli. After introduction of the plR2 plasmid into a fur mutant of E. coli, both the iron‐starved and the iron‐replete bacteria expressed the HMWP2. Repressibility of irp2 by iron was restored by introduction of a plasmid carrying the fur gene. These results demonstrate that the irp2 promoter is controlled by the Fur repressor in E. coli. Mutagenesis of the chromosomal irp2 gene of Yersinia pseudotuberculosis was obtained by homologous recombination with a 1 kb fragment of this gene cloned on the suicide plasmid pJM703.1. Inactivation of irp2 resulted in the non‐expression of both HMWPs, while introduction of plasmid plR2 into the mutant strain led to the synthesis of the HMWP2 only. Therefore, It is probable that the genes coding for the HMWPs constitute an operon where irp2 is upstream of irp1. When comparing the virulence of the wild‐type strain and of its irp2 mutant derivative, we found that the 50% lethality (LD50) for mice of the mutant strain was increased, whatever the route of infection, but more markedly when injected parenterally. Accordingly, these data demonstrate that a mutation in the irp2 gene alters the pathogenicity of Y. pseudotuberculosis. Since the introduction of the irp2 gene into the mutant strain did not restore its virulence, it is likely that both HMWPs are required for the expression of the high‐pathogenicity phenotype.
Porins and specific channels of bacterial outer membranes
Authors: Nikaido H.
Published: October 2006

Porins and specific channels both produce water‐filled pores that allow the transmembrane diffusion of small solutes, but the latter contain specific ligandbinding sites within the channels. Recent structural studies show that many or most of these proteins exist as βbarrels with the βstrands traversing the thickness of the outer membrane. The channels often have diameters in the range of 1 nm, and thus the penetration rates of solutes through porin channels are likely to be affected strongly by what appear to be minor differences in the size, shape, hydrophobicity or charge of the solute molecule. With the specific channels, the presence of binding sites can accelerate very significantly the diffusion of some ligands when they are present at low concentrations. Thus these simple channels can sometimes achieve a surprising degree of real or apparent specificity. Recent data tend to favour the idea that these proteins are first exported into the periplasm, and then inserted into the outer membrane. Although lipopolysaccharides seem to play a significant role in the final assembly of the trimeric porins, the details of the targeting process still remain to be elucidated.
Evolutionary origins of bacterial bioluminescence
Authors: O'Kane Dennis J. , Prasher Douglas C.
Published: October 2006

In bacteria, most genes required for the bioluminescence phenotype are contained in lux operons. Sequence alignments of several lux gene products show the existence of at least two groups of paralogous products. The α‐ and β‐subunits of bacterial luciferase and the non‐fluorescent flavoprotein are paralogous, and two antennae proteins (lumazine protein and yellow fluorescence protein) are paralogous with riboflavin synthetase. Models describing the evolution of these paralogous proteins are suggested, as well as a postulate for the identity of the gene encoding a protobioluminescent luciferase.
Role of horizontal genetic exchange in the antigenic variation of the class 1 outer membrane protein of Neisseria meningitidis
Authors: Feavers I. M. , Heath A. B. , Bygraves J. A. , Maiden M. C. J.
Published: October 2006

The nucleotide sequences of the genes encoding the class 1 outer membrane protein of Neisseria meningitidis (PorA) from 15 meningococcal isolates have been examined. These strains, isolated over a number of years, represented a variety of serological types, clonal groups, and geographical locations. Analysis of the aligned nucleotide sequences showed that the known serological relationships between these proteins were not necessarily reflected throughout the nucleotide sequences of their genes. The uneven distribution of base substitutions, revealed by a comparison of the informative bases, suggested that these genes possessed a mosaic structure. This structure probably resulted from the horizontal transfer of DNA between strains and would have contributed to both the generation and the spread of novel antigenic variants of the protein. In addition, the nucleotide differences between porA genes from different strains were not consistent with the nucleotide sequence divergence of the whole chromosome, as indicated by pulsed‐field get electrophoresis (PFGE) fingerprinting techniques: some strains with divergent PFGE fingerprints shared porA genes with extensive regions of nucleotide sequence identity and, conversely, some strains with similar chromosome structures possessed porA genes with different nucleotide sequences and serological properties. This suggested that entire genes had been exchanged between strains. Given that the meningococcal class 1 OMP is a major component in novel vaccines, some of which are currently undergoing field trials, the potential of horizontal genetic exchange to generate antigenic diversity has implications for the design of such vaccines.
Expression of chitin synthase genes during yeast and hyphal growth phases of Candida albicans
Authors: Chen‐Wu J. L. , Zwicker J. , Bowen A. R. , Robbins P. W.
Published: October 2006

Chitin, the β1,4‐linked polymer of N‐acetylglucosamine, is a fibrous polysaccharide that in many yeasts helps to maintain the structure of the mother‐bud junction and in filamentous fungi is often the major supporting component of the cell wall. We have previously described a Candida albicans chitin synthase, CHS1. The DNA and derived protein sequences of a second gene, CHS2, are presented and compared with previously published gene sequences. Northern blot analysis shows that strikingly different levels of synthase 1 and 2 expression occur during yeast and hyphal phases of Candida growth.
Molecular analysis and expression of a Borrelia burgdorferi gene encoding a 22kDa protein (pC) in Escherichia coli
Authors: Fuchs R. , Jauris S. , Lottspeich F. , Preac‐Mursic V. , Wilske B. , Soutschek E.
Published: October 2006

We describe the cloning and expression of the pc gene which encodes a major immunodominant protein of Borrelia burgdorferi, the causative agent of Lyme borreliosis. The pC protein was purified from lysates of B. burgdorferi strain PKo. After tryptic digestion of the pC protein the resulting oligopeptides were applied to a gas‐phase sequenator. Thus partial amino acid sequences were obtained. The deduced oligonucleotides were used as hybridization probes. After Southern blotting a reactive band in the 3 kb range of PstI‐digested genomic DNA was detected. The insertion of these fragments into pUC vectors finally resulted in pc‐positive Escherichia coli clones. The gene (encoding a protein with 212 amino acids) was expressed in E. coli with varying deletions at the 5′ end. A sequence comparison with other outer membrane proteins of B. burgdorferi indicates a processing of pC that is similar to that of lipoproteins.
Protein‐specific features of the general secretion pathway in yeast: the secretion of acid phosphatase
Authors: Haguenauer‐Tsapis Rosine
Published: October 2006

The major phosphate‐repressible acid phosphatase (APase) of Saccharomyces cerevisiae, a cell wall glycoprotein, has been extensively used as a reporter protein to analyse successive steps in the yeast secretory pathway. In contrast to other yeast secretory proteins, APase can still be translocated into the endoplasmic reticulum (ER) even when it is made without its signal peptide. This property illustrates the permissiveness of targeting to the ER in yeast. Studies on APase‐containing hybrid proteins have provided some of the evidence that specific soluble factors must interact with secretory proteins prior to their translocation across the ER membrane. A systematic analysis of mutations affecting the sequence of the APase signal peptide cleavage site demonstrated that cleavage occurs only when the last amino acid of the signal sequence is small and neutral. This was one of the first studies to verify the requirements for signal peptidase cleavage that had previously only been predicted from statistical analysis. Studies performed either with inhibitors of glycosylation or with mutant APases demonstrated the critical role of core glycosylation for APase folding, which is essential for efficient transport beyond the ER. Following the fate of particular modified APases along the secretory pathway provided insights into some general properties of the secretory apparatus and illustrated the specific requirements for a given protein during its intracellular traffic.
Zygotic induction of plasmid ssb and psiB genes following conjugative transfer of Incl1 plasmid Collb‐P9
Authors: Jones A. Louise , Barth Peter T. , Wilkins Brian M.
Published: October 2006

The Incl1 conjugative plasmid Collb‐P9 carries a psiB gene that prevents induction of the SOS response in host bacteria. This locus is located 2.5 kb downstream of the ssb (single‐stranded DNA‐binding protein) gene in the leading region. This portion of Collb is strikingly similar to part of the leading region of the otherwise distinct F plasmid. Expression of psiB and ssb is increased when the host cell is exposed to an SOS‐inducing treatment or the Collb transfer system is derepressed. Moreover, expression of both genes on a derepressed plasmid is strongly enhanced in conjugatively infected recipient cells. Carriage of the psiB gene by Collb is shown to prevent a low level of SOS induction following conjugation. Plasmid ssb and psiB genes may function to promote installation of the replicon in the new cell.
Involvement of FtsZ in coupling of nucleoid separation with septation
Authors: Tétart Françoise , Albigot Renaud , Confer Annie , Mulder Egbert , Bouché Jean‐Pierre
Published: October 2006

The cell‐cycle parameters of an Escherichia coli strain expressing essential division gene ftsZ at one‐fifth of its normal level, because of antisense regulation by DicF RNA, have been analysed. Inhibition of FtsZ expression affects neither the generation time nor the replication initiation mass, the C period, or the constriction period, but it does dramatically retard the initiation of constriction relative to replication termination. Separation of the nucleoids is equally postponed, indicating that division is not coupled to termination of replication, but to partitioning. The severe inhibition of nucleoid separation by DicF RNA, and its suppression by overproduction of FtsZ, suggest a role for FtsZ in the control of separation, and consequently in the coupling of separation and division. We suggest that the normal pattern of nucleoid separation previously found in cells deficient in ftsZ function was a consequence of the loss of a negative effect exerted by FtsZ on separation. In agreement with this view, we find that nucleoid separation is temporarily inhibited after arrest of FtsZ synthesis, but is later resumed as FtsZ is further diluted into the elongating filaments.
Identification of replication and stability functions in the complete nucleotide sequence of plasmid pUH24 from the cyanobacterium Synechococcus sp. PCC 7942
Authors: Van der Plas Jan , Oosterhoff‐Teertstra Renske , Borrias Mies , Weisbeek Peter
Published: October 2006

The complete nucleotide sequence is presented for pUH24, the small plasmid of Synechococcus sp. PCC 7942. pUH24 consists of 7835bp and has a G+C content of 59%. The distribution of translation start and stop codons in the sequence allows 36 open reading frames that potentially encode polypeptides of 50 or more amino acids. We postulate that eight of these open reading frames are actual coding sequences. A region has been identified, by experiment, that contains two functions, designated pmaA and pmaB, involved in the segregational stability of the plasmid. The minimal region of pUH24 fully capable of supporting autonomous replication consists of a 3.6kb DNA fragment, which is almost entirely occupied by two overlapping genes most likely coding for essential replication proteins (repA and repB).
Protein A–calmodulin fusions: a novel approach for investigating calmodulin function in yeast
Authors: Stirling Douglas A. , Petrie Alison , Pulford David J. , Paterson David T. W. , Stark Michael J. R.
Published: October 2006

A novel gene fusion approach which may be of more general use has been developed for investigating the function of calmodulin in the budding yeast Saccharomyces cerevisiae. By fusing a portion of the Staphylococcus aureus spa gene (encoding protein A) to CMD1, the S. cerevisiae gene encoding calmodulin, we have generated a yeast calmodulin with an affinity tag able to bind immunoglobulins. The chimaeric protein A–calmodulin (ProtA–CaM) polypeptide functions in vivo and shows Ca2+‐dependent binding to calmodulin target proteins. The spa–CMD1 fusion has been used (i) to prepare (by affinity chromatography) a fraction of yeast proteins which interact with calmodulin, (ii) to isolate genes encoding calmodulin target proteins by direct screening of an expression library, and (iii) to visualize calmodulin‐binding proteins in crude extracts by Western blot analysis.
Characterization of naturally elaborated blebs from serum‐susceptible and serum‐resistant strains of Neisseria gonorrhoeae
Authors: Pettit Robin K. , Judd Ralph C.
Published: October 2006

Outer‐membrane blebs from two serum‐susceptible and two serum‐resistant strains of Neisseria gonorrhoeae were characterized. In general, bleb surfaces resembled cell surfaces, but there were qualitative and quantitative protein differences in blebs released by serum‐susceptible and serum‐resistant strains. Relative to blebs from serum‐resistant strains, blebs from serum‐susceptible strains expressed reduced amounts of major outer‐membrane proteins I and III, and little if any 68 000 Dalton outer‐membrane protein.
An enzyme with type IV prepilin peptidase activity is required to process components of the general extracellular protein secretion pathway of Klebsiella oxytoca
Authors: Pugsley Anthony P. , Dupuy Bruno
Published: October 2006

The last gene (pulO) of the pulC–O pullulanase secretion gene operon of Klebsiella oxytoca codes for a protein that is 52% identical to the product of the pilD/xcpA gene required for extracellular protein secretion and type IV pilus biogenesis in Pseudomonas aeruginosa. The PilD/XcpA protein is known to remove the first six amino acids of the signal sequence of the type IV pilin precursor by cleaving after the glycine residue in the conserved sequence GF(M)XXXE (where X represents hydrophobic amino acids). This prepilin peptidase cleavage site is present in the products of four genes in the pulC–O operon (PulG, PulH, Pull and PulJ proteins). It is shown here that PulO processes the pulG gene product in vivo. Processing was maximal within 15 seconds, but experiments in which the expression of pulO was uncoupled from that of the other genes in the secretion operon suggest that processing can also occur post‐translationally. The products of two pulG derivatives with internal in‐frame deletions were also processed by PulO, but the three PulG–PhoA hybrids, two PulJ–PhoA hybrids and the single PulH–PhoA hybrid tested did not appear to be processed. Sucrose gradient fraction‐ation experiments showed that both precursor and mature forms of PulG appear to be associated with low‐density, outer membrane vesicles prepared by osmotic lysis of sphaeroplasts. Neither the xcpA gene nor the Bacillus subtilis gene comC, which is also homologous to pulO and codes for a protein with type IV prepilin peptidase activity, can correct the pullulanase secretion defect in an Escherichia coli strain carrying all of the genes required for secretion except pulO. Furthermore, neither XcpA nor ComC is able to process prePulG protein in vivo.
A leptomycin B resistance gene of Schizosaccharomyces pombe encodes a protein similar to the mammalian P‐glycoproteins
Authors: Nishi Kazunori , Yoshida Minoru , Nishimura Marie , Nishikawa Mitsuo , Nishiyama Makoto , Horinouchi Sueharu , Beppu Teruhiko
Published: October 2006

Screening for leptomycin B (LMB)‐resistant transformants in a gene library constructed in Schizosaccharomyces pombe with the chromosomal DNA of an LMB‐resistant mutant of S. pombe and with multicopy plasmid pDB248′ as the vector led to the isolation of a gene, named pmd1+, encoding a 1362‐amino‐acid protein. This protein showed great similarity in amino acid sequence to the mammalian P‐glycoprotein encoded by the multidrug resistance gene, mdr, and the Saccharomyces cerevisiae a‐factor transporter encoded by STE6. In addition, computer analyses predicted that the protein encoded by pmd1+ formed an intramolecular duplicated structure and each of the halves contained six transmembrane regions as well as two ATP‐binding domains, as observed with the P‐glycoproteins and the STE6 product. Consistent with this was that S. pombe cells containing the pmd1+ gene on a multicopy plasmid showed resistance not only to LMB but also to several cytotoxic agents. The pmd1 null mutants derived by gene disruption were viable and hypersensitive to these agents. All these data suggest that the pmd1+ gene encodes a protein that is a structural and functional counterpart of mammalian mdr proteins.
Overproduction of a selenocysteine‐containing polypeptide in Escherichia coli: the fdhF gene product
Authors: Chen Giafen T. , Axley Milton J. , Hacia Joseph , Inouye Masayori
Published: October 2006

The fdhF gene of Escherichia coli codes for the selenocysteine‐including protein subunit of formate dehydrogenase H. The protein subunit consists of 715 amino acid residues containing a single selenocysteine residue at position 140 which is encoded by a UGA codon. The decoding of this opal termination codon occurs under anaerobic growth conditions by means of a specific tRNA, i.e. the selC gene product. The ability of E. coli cells to overproduce a seleno‐polypeptide was examined using the fdhF gene as a model system. Surprisingly, E. coli was able to synthesize the fdhF gene product at the level of approximately 12% of the total cellular protein. This was achieved by cloning fdhF in a multicopy plasmid together with a synthetic selC gene under the Ipp promoter. FdhF production was absolutely dependent upon the addition of selenium to the culture medium and was almost completely blocked in the presence of oxygen. The product was specifically labelled with 75Se, proving that it consisted of a selenoprotein. The product was purified to homogeneity and shown to exhibit the catalytic properties characteristic of formate dehydrogenase H.
A novel sensor‐regulator protein that belongs to the homologous family of signal‐transduction proteins involved in adaptive responses in Escherichia coli
Authors: Nagasawa S. , Tokishita S. , Aiba H. , Mizuno T.
Published: October 2006

Expression of the Escherichia coli outer membrane porins, OmpC and OmpF, is regulated in response to changes in the medium osmolarity through the functions of the regulatory factors, EnvZ and OmpR. A 3.0 kilobase pair DNA fragment cloned from E. coli is able phenotypically to suppress the defect in ompC and ompF expression caused by an envZ deletion mutation, provided that a certain gene located in this fragment is expressed on a high copy‐number plasmid. Nucleotide sequencing revealed that the putative gene encodes a protein of 102452 Da. The deduced amino acid sequence of the protein shows a high degree of homology to those of both EnvZ and OmpR, i.e. it contains both a sensory kinase domain’ and a ‘response regulator domain’ in its primary amino acid sequence. The protein identified in this study is probably a novel member of the homologous family of proteins involved in bacterial adaptive responses. Hence, the gene encoding this novel sensor‐regulator protein was designated as barA (bacterial adaptive responses) and mapped at 60 min on the E. coli genetic map. The BarA protein in isolated membranes was demonstrated in vitro to undergo phosphorylation in the presence of ATP.
Rapid generation of directed and unmarked deletions in Xanthomonas
Authors: Kamoun Sophien , Tola Elisabetta , Kamdar Haresh , Kado Clarence I.
Published: October 2006

We have devised a rapid four‐step procedure for the generation of directed and unmarked chromosomal deletions in bacteria, based on the use of a novel cloning vector containing the Bacillus subtilis sacB gene that encodes levansucrase and confers sucrose sensitivity, which can be used for counter‐selection. Using this technique, we describe the construction of a 6.5 kb directed and unmarked deletion in a phytopathogenicity region of the chromosome in Xanthomonas campestris. This procedure allows rapid and easy transfer of a wide variety of mutant allelic DNA to the bacterial chromosome, and should be adaptable to various bacteria besides Xanthomonas spp.
Molecular basis of host epithelial cell recognition by Trichomonas vaginalis
Authors: Arroyo R. , Engbring J. , Alderete J. F.
Published: October 2006

Parasitism of host epithelial cells by Trichomonas vaginalis is a highly specific event. Four trichomonad surface proteins (adhesins) with molecular masses of 65000 daltons (65kDa; AP65), 51 kDa (AP51), 33kDa (AP33), and 23 kDa (AP23) mediate the interaction of T. vaginalis with epithelial cells. Fresh isolates, when compared with long‐term‐grown isolates, had greater amounts of adhesins, which corresponded with increased levels of cytoadherence. Anti‐adhesin antibodies reacted by immunobiol only with the respective protein and detected, by indirect immunofluore‐scence, each adhesin on the parasite surface. These antibodies inhibited the binding of live parasites to epithelial cells and protected epithelial cells from contact‐dependent cytotoxicity. The pretreatment of epithelial cells with a preparation of purified adhesins also blocked trichomonal cytoadherence. Moreover, HeLa cells possessed molecules which recognized and bound to adhesins on nitrocellulose blots.
Analysis of an Escherichia coli mutant strain resistant to the cell‐killing function encoded by the gef gene family
Authors: Poulsen Lars K. , Larsen Nethe W. , Molin Søren , Andersson Poul
Published: October 2006

The chromosomal genes gef and relF from Escherichia coli and the plasmid‐encoded genes hok, flmA, srnB, and pndA constitute the gef qene family, which encodes a cell‐killing function. In order to investigate the mechanism of cell killing we have isolated an E. coli mutant strain that is resistant to the overexpression of the toxic proteins encoded by the gef gene family. This phenotype requires at least two mutations, one of which has been mapped to 55.2 minutes. This mutation was sequenced and shown to represent a single base substitution in an open reading frame (ORF178) encoding a putative membrane protein having a molecular mass of 20.1 kDa. ORF178 and an upstream frame, ORF190, probably constitute an operon.
Isolation and characterization of conditional adherent and non‐type 1 fimbriated Salmonella typhimurium mutants
Authors: Lockman Hank A. , Curtiss Roy
Published: October 2006

Mutations in the genes encoding the type 1 fimbriae of Salmonella typhimurium were isolated by selecting for the deletion of Tn10 inserted adjacent to the chromosomal fim+ genes and screening for the loss of mannose‐sensitive haemagglutination (HA) activity. S. typhimurium strains with Tn10 insertions in ahp were hypersensitive to peroxides, and tetracycline‐sensitive derivatives of ahp::Tn10 mutants displayed two fim mutant phenotypes. The predominant class of fim mutants did not synthesize type 1 fimbriae. A second type of fim mutant synthesized type 1 fimbriae and exhibited a conditional lipoic acid requirement for HA. A fim–lip conditional mutant synthesized type 1 fimbriae when grown in Mueller‐Hinton broth but the haemagglutinating activity of the fimbriae was dependent upon the addition of lipoic acid to the growth medium. Independentiy isolated lip mutations did not demonstrate a similar pleiotropic effect on HA. Western blots of fimbriae extracted from a fim–lip conditional mutant that was grown under permissive and restrictive conditions indicated the presence of 33 and 36.6 kDa proteins in HA+ fimbriae that were absent in HA‐ fimbriae. The HA+ phenotype of both conditional and non‐fimbriafed mutants was restored by transformation with cloned genes encoding S. typhimurium type 1 fimbriae.
Organization and function of the mcrBC genes of Escherichia coli K‐12
Authors: Raleigh E. A.
Published: October 2006

Many natural DNA sequences are restricted in Escherichia coli K‐12, not only by the classic Type I restriction system EcoK, but also by one of three modification‐specific restriction systems found in K‐12. The McrBC system is the best studied of these. We infer from the base composition of the mcrBC genes that they were imported from an evolutionarily distant source. The genes are located in a hypervariable cluster of restriction genes that may play a significant role in generation of species identity in enteric bacteria. Restriction activity requires the products of two genes for activity both in vivo and in vitro. The mcrB gene elaborates two protein products, only one of which is required for activity in vitro, but both of which contain a conserved amino acid sequence motif identified as a possible GTP‐binding site. The mcrC gene product contains a leucine heptad repeat that could play a role in protein‐protein interactions. McrBC activity in vivo and in vitro depends on the presence of modified cytosine in a specific sequence context; three different modifications are recognized. The in vitro activity of this novel multi‐subunit restriction enzyme displays an absolute requirement for GTP as a cofactor.
Characterization of cat messenger RNA decay suggests that turnover occurs by endonucleolytic cleavage in a 3′ to 5′ direction
Authors: Meyer Barbara J. , Schottel Janet L.
Published: October 2006

Turnover of the chloramphenicol acetyltransferase (cat) messenger RNA in Escherichia coli was investigated by analysis of cellular mRNA decay intermediates and the transcript sequence. Analysis of the sequence has revealed the presence of a repetitive extragenic palindromic (REP) element at the 3′ end of the transcript as well as several 5′‐UUAU‐3′ sequences, two elements which have roles in modulating turnover of other E. coli mRNAs. For cat mRNA, however, removal of the REP sequence has no effect on the half‐life of the transcript, indicating that the REP sequence does not stabilize the upstream region of this message. Results from mapping of the mRNA decay products by several techniques suggest that the message is instead subject to endonucleolytic attack at five sites 5′ of the REP element. The sequence UUAU is present at three of these five sites. It also appears that the cat mRNA is sequentially cleaved in a 3′ to 5′ direction during turnover of this mRNA in vivo. A model for cat mRNA turnover is discussed.
Isolation and characterization of the major vegetative RNA polymerase of Streptomyces coelicolor A3(2); renaturation of a sigma subunit using GroEL
Authors: Brown Kelly L. , Wood Steven , Buttner Mark J.
Published: October 2006

The promoter region of the agarase gene (dagA) of Streptomyces coelicolor A3(2) is complex; it consists of four distinct promoters with different ‐10 and ‐35 regions. We report the isolation of a form of RNA polymerase that mediates transcription in vitro from the dag Ap4 promoter. The core components of this RNA polymerase are associated with a polypeptide of c. 66 kDa; holoenzyme reconstitution experiments show that the 66 kDa polypeptide functions as a sigma factor that directs transcription from the dag Ap4 and Bacillus subtilis veg promoters in vitro. Alignment of the DNA sequences of these two promoters shows that they have bases in common in the ‐10 and ‐35 regions and that these sequences are similar to those observed for the major RNA polymerases of other bacteria. N‐terminal amino acid sequence analysis of the 66 kDa polypeptide revealed it to be the product of the hrdB gene. Previous experiments showed that the predicted amino acid sequence of the hrdB gene product is very similar to the major sigma factors of other bacteria and suggested that disruption of the hrdB gene is lethal. These observations together lead to the conclusion that we have isolated the major RNA polymerase of Streptomyces coelicolor A3(2).

We have developed an improved protocol for the renaturation of sigma factors that have been Isolated by preparative sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS‐PAGE). This method involves renaturing the polypeptide in the presence of the bacterial chaperonin GroEL. We expect this protocol to find general application for renaturation of other polypeptides that have been subjected to SDS‐PAGE.
Biochemical activities of the ParA partition protein of the P1 plasmid
Authors: Davis M. A. , Martin K. A. , Austin S. J.
Published: October 2006

The unit‐copy P1 plasmid depends for stability on a plasmld‐encoded partition region called par, consisting of the parA and parB genes and the parS site. ParA is absolutely required for partition, but its partition‐critical role is not known. Purified ParA protein is shown to possess an ATPase activity in vitro which is specifically stimulated by purified ParB protein and by DNA. ParA is responsible for regulation of expression of parA and parB, and purified ParA has an ATP‐dependent, site‐specific DNA binding activity which recognizes a sequence that overlaps the parA promoter. The rote of the ATP‐dependence of the binding activity, as well as other possible functions of the ATPase activity in partition, is discussed.
High‐frequency rearrangements in the chromosome of Mycoplasma pulmonis correlate with phenotypic switching
Authors: Bhugra B. , Dybvig K.
Published: October 2006

Mycoplasma pulmonis is a murine pathogen that causes chronic respiratory disease in laboratory rats and mice. Several examples of high‐frequency phenotypic switching have been reported for M. pulmonis, the molecular basis of which is unknown. We report here that during growth the M. pulmonis chromosome undergoes DNA rearrangements at a high frequency. Some of the rearrangements we examined correlated with changes in the susceptibility of the cells to mycoplasma virus P1, an example of phenotypic switching involving changes in surface antigen structure. Other rearrangements, unrelated to phenotypic switching, involved a DNA element present in the chromosome in multiple copies. The high level of DNA recombination that occurred in M. pulmonis indicates that this may be one of the most variable genomes studied to date. High levels of DNA recombination may contribute to the unusually high rate of evolution that mycoplasmas are thought to be undergoing. Understanding the molecular basis for this phenomenon may provide an insight into the chronic nature of many mycoplasmal infections.
Mutants of Rhodobacter sphaeroides lacking one or more pigment‐protein complexes and complementation with reaction‐centre, LH1, and LH2 genes
Authors: Jones M. R. , Fowler G. J. S. , Gibson L. C. D. , Grief G. G. , Olsen J. D. , Crielaard W. , Hunter C. N.
Published: October 2006

The photosynthetic apparatus of Rhodobacter sphaeroides is comprised of three types of pigment‐protein complex: the photochemical reaction centre and its attendant LH1 and LH2 light‐harvesting complexes. To augment existing deletion/insertion mutants in the genes coding for these complexes we have constructed two further mutants, one of which is a novel double mutant which is devoid of all three types of complex. We have also constructed vectors for the expression of either LH1, LH2 or reaction‐centre genes. The resulting system allows each pigment‐protein complex to be studied either as part of an intact photosystem or as the sole complex in the cell. In this way we have demonstrated that reaction centres can assemble independently of either light‐harvesting complex in R sphaeroides. In addition, the isolation of derivatives of the deletion/insertion mutants exhibiting spontaneous mutations in carotenoid biosynthesis provides an avenue for examining the role of carotenoids in the assembly of the photo‐synthetic apparatus. We show that the LH1 complex is assembled regardless of the carotenoid background, and that the type of carotenoid present modifies the absorbance of the LH1 bacteriochlorophylls.
Identification and characterization of a novel Bacillus thuringiensis δ‐endotoxin entomocidal to coleopteran and lepidopteran larvae
Authors: Tailor Ravi , Tippett Jan , Gibb Graham , Pells Stephen , Jordan Linda , Ely Susan
Published: October 2006

A new class of Bacillus thuringiensisδ‐endotoxins, or insecticidal control proteins (ICPs), is defined by an apparently cryptic protein with a unique primary structure and novel entomocidal specificity for certain coleopteran and lepidopteran species. The discovery of a new group of ICPs will extend the use of this natural insecticide in integrated pest‐management systems.
Morphological and cytoskeletal changes in epithelial cells occur immediately upon interaction with Salmonella typhimurium grown under low‐oxygen conditions
Authors: Francis C. L. , Starnbach M. N. , Falkow S.
Published: October 2006

Salmonella typhimurium grown under oxygen‐limiting conditions were found to enter into, elicit actin filament rearrangement in, and effect morphological changes upon HEp‐2 cells within 15 min after infection. Video microscopy revealed that host cell morphological changes associated with entry began within 1 min of productive adherence. Polarized Caco‐2 cell morphology was affected 40 s after infection with low‐oxygen‐grown S. typhimurium. Stationary‐phase S. typhimurium did not elicit these phenomena within this time‐period even when adherence was enhanced with the afimbrial adhesin, AFA‐I. Thus, environmental cues regulate S. typhimurium invasion factors, allowing for immediate entry into host cells. Additionally, actin filament rearrangement and morphological changes in the eukaryotic host cell are essential for entry and occur within minutes of infection.
Isolation, organization and expression of the Pseudomonas aeruginosa threonine genes
Authors: Clepet C. , Borne F. , Krishnapillai V. , Baird C. , Patte J. C. , Cami B.
Published: October 2006

Three genes from Pseudomonas aeruginosa involved in threonine biosynthesis, hom, thrB and thrC, encoding homoserine dehydrogenase (HDH), homoserine kinase (HK) and threonine synthase (TS), respectively, have been cloned and sequenced. The hom and thrC genes lie at the thr locus of the P. aeruginosa chromosome map (31 min) and are likely to be organized in a bicistronic operon. The encoded proteins are quite similar to the Hom and TS proteins from other bacterial species. The thrB gene was located by pulsed‐field gel electrophoresis experiments at 10 min on the chromosome map. The product of this gene does not share any similarity with other known ThrB proteins. No phenotype could be detected when the chromosomal thrB gene was inactivated by an insertion. Therefore the existence of isozymes for this activity is postulated. HDH activity was feedback inhibited by threonine; the expression of all three genes was constitutive. The overall organization of these three genes appears to differ from that in other bacterial species.
DNA sequence determination and functional characterization of the OCT‐plasmid‐encoded alkJKL genes of Pseudomonas oleovorans
Authors: van Beilen Jan B. , Eggink Gerrit , Enequist Hans , Bos Rolf , Witholt Bernard
Published: October 2006

The alkBFGHJKL and alkST operons encode enzymes that allow Pseudomonas putida (oleovorans) to metabolize alkanes. In this paper we report the nucleotide sequence of a 4592 bp region of the alkBFGHJKL operon encoding the AlkJ, AlkK and AlkL polypeptides.

The alkJ gene encodes a protein of 59 kilodaltons. The predicted amino acid sequence shows significant homology with four flavin proteins: choline dehydrogenase, a glucose dehydrogenase and two oxidases. AlkJ is membrane‐bound and converts aliphatic medium‐chain‐length alcohols into aldehydes. The properties of AlkJ suggest that it is linked to the electron transfer chain. AlkJ is necessary for growth on alkanes only in P. putida alcohol dehydrogenase (AlcA) mutants.

AlkK is homologous to a range of proteins which act by an ATP‐dependent covalent binding of AMP to their substrate. This list includes the acetate, coumarate and long‐chain fatty acid CoA ligases. The alkK gene complements a fadD mutation in Escherichia coli, which shows that it indeed encodes an acyl‐CoA synthetase. AlkK is a 60 kilodalton protein located in the cytoplasm.

AlkL is homologous to OmpW, a Vibrio cholerae outer membrane protein of unknown function, and a hypothetical polypeptide encoded by ytt4 in E. coli. AlkL, OmpW and Ytt4 all have a signal peptide and end with a sequence characteristic of outer membrane proteins. The alkL gene product was found in the outer membrane of E. coli W3110 containing the alk‐genes. The alkL gene can be deleted without a clear effect on growth rate. Its function remains unknown.

The G+C content of the alkJKL genes is 45%, identical to that of the alkBFGH genes, and significantly lower than the G+C content of the OCT‐plasmid and the P. putida chromosome.
Molecular rearrangement of lactose plasmid DNA associated with high‐frequency transfer and cell aggregation in Lactococcus Iactis 712
Authors: Gasson Michael J. , Swindell Simon , Maeda Sumio , Dodd Helen M.
Published: October 2006

High‐frequency conjugation of the lactose plasmid pLP712 is associated with a constitutive cell aggregation phenotype and is facilitated by cointegration with a sex factor. Analysis of 23 independently derived enlarged lactose plasmids revealed that the sex factor DNA present in cointegrates varied in size. This suggested that more than simple cointegration with a sex factor plasmid was involved. Further analysis led to the discovery of a chromosomally located sex factor that could excise and be lost or exist as labile plasmid DNA. Cointegration with this sex factor was shown to be promoted by transposition of a copy of ISSI present on the lactose plasmid, and models are presented to account for the complex and variable structures of the resulting enlarged lactose plasmids.
Ribosomal association of the yeast SAL4 (SUP45) gene product: implications for its role in translation fidelity and termination
Authors: Stansfield Ian , Grant Christopher M. , Akhmaloka  , Tuite Mick F.
Published: October 2006

The SAL4 gene of the yeast Saccharomyces cerevisiae encodes a novel translation factor (Sal4p) involved in maintaining translational fidelity. Using a polyclonal antibody raised against a Sal4p‐β‐galac‐tosidase fusion protein, Sal4p was shown to be almost exclusively associated with the ribosomal fraction. Even when the ribosomes were treated with 0.8 M KCl, only low levels of Sal4p were detected in the post‐ribosomal supernatant, suggesting a very strong affinity between Sal4p and the ribosome. Analysis of the distribution of Sal4p in the ribosomal population revealed that it was principally associated with 40S subunits, monosomes and polysomes. Incubation in high salt concentrations (0.8 M KCl) suggested that the affinity of Sal4p for the 40S subunit was lower than that for monosomes or polysomes. The Sal4p:ribosome association was only maintained when ribosomes were prepared in the presence of the translation elongation inhibitor cycloheximide; in uninhibited cells much lower levels of Sal4p were detectable in the ‘run‐off’ polysomes. In view of these data, and given the stoichiometry of Sal4p to individual ribosomal proteins (estimated at less than 1:20), we suggest that Sal4p plays an ancillary role in translation termination.
Tandem DctD‐binding sites of the Rhizobium meliloti dctA upstream activating sequence are essential for optimal function despite a 50‐ to 100‐fold difference in affinity for DctD
Authors: Ledebur H. , Nixon B. T.
Published: October 2006

The Rhizobium melilot genes dctB and dctD positively regulate the expression of dctA, which encodes a C4‐dicarboxylate transport protein. Here we characterize an element (UAS) located upstream of dctA that has tandem binding sites for the dctD gene product (DctD). At relatively low concentrations of active DctD, the element activated dctA transcription, but at relatively high concentrations of DctD it was inhibitory. The UAS failed to function when placed further upstream of dctA. Both DctD‐binding sites were required for optimal UAS function, despite a 50‐ to 100‐fold difference in binding affinities. Moving the promoter distal binding site 5 bp further upstream was functionally equivalent to its deletion. Based on these data, we hypothesize that the α54 ‐dependent activator DctD binds co‐operatively to the R. meliloti dctA UAS, and that occupancy of both sites is required for maximal activation of dctA.