bluuebunny/crossref_metadata_2025 huggingface dataset:
A small (2.4 Mb) Bacillus cereus chromosome corresponds to a conserved region of a larger (5.3 Mb) Bacillus cereus chromosome
Authors: Carlson Cathrine Rein , Kolsto Anne‐Brit
Published: October 2006

We have determined the sizes of the chromosomes of six Bacillus cereus strains (range 2.4–4.3 Mb) and constructed a physical map of the smallest B. cereus chromosome (2.4 Mb). This map was compared to those of the chromosomes of four B. cereus strains and one B. thuringiensis strain previously determined to be 5.4‐6.3 Mb. Of more than 50 probes, 30 were localized to the same half of the larger B. cereus and B. thuringiensis chromosomes. All 30 were also present on the small chromosome. Twenty of the probes present on the other half of the larger chromosomes were either present on extrachromosomal DNA, or absent from the B. cereus strain carrying the small chromosome. We propose that the genome of B. cereus and B. thuringiensis has one constant part and another less stable part which is more easily mobilized into other genetic elements. This part of the genome is localized to one region of the chromosome and may be subject to deletions or more frequent relocations between the chromosome and episomal elements of varying sizes up to the order of megabases.
The energetics of bacterial growth: a reassessment
Authors: Neijssel Oense M. , de Mattos M. Joost Teixeira
Published: October 2006

The growth yield of microbial cultures can be used to estimate the efficiency of energy generation during a fermentation or respiration, in the past, the assessment of this efficiency in organisms carrying out a respiration has been the subject of many heated debates. This has partly been caused by the complexity of microbial respiratory chains. Strains of Escherichia coli specifically modified in their respiratory chain have been used recently to re‐evaluate the energetic efficiency of the bacterial respiration using chemostat cultures. The different strains indeed show different growth efficiencies. The physiological significance of energetically less‐efficient branches of the respiratory chain is discussed.
The terminal oxidases of Paracoccus denitrificans
Authors: de Gier Jan‐Willem L. , Lübben Mathias , Reijnders Willem N. M. , Tipker Corinne A. , Slotboom Dirk‐Jan , van Spanning Rob J. M. , Stouthamer Adriaan H. , van der Oost John
Published: October 2006

Three distinct types of terminal oxidases participate in the aerobic respiratory pathways of Paracoccus denitrificans. Two alternative genes encoding sub unit I of the aa3‐type cytochrome c oxidase have been isolated before, namely ctaDI and ctaDII. Each of these genes can be expressed separately to complement a double mutant (ActaDI, ActaDII), indicating that they are isoforms of subunit I of the aa3‐type oxidase. The genomic locus of a quinol oxidase has been isolated: cyoABC. Thisprotohaem‐containing oxidase, called cytochrome bb3, is the oniy quinoi oxidase expressed under the conditions used, in a triple oxidase mutant (ActaDI, ActaDII, cyoB::KmR) an alternative cyto‐chrome c oxidase has been characterized; this cbb3‐type oxidase has been partially purified. Both cytochrome aa3 and cytochrome bb3 are redox‐driven proton pumps. The proton‐pumping capacity of cytochrome cbb3 has been analysed; arguments for and against the active transport of protons by this novel oxidase complex are discussed.
A novel, small endoglucanase gene, egl5, from Trichoderma reesei isolated by expression in yeast
Authors: Saloheimo Anu , Henrissat Bernard , Hoffrén Anna‐Marja , Teleman Olle , Penttilä Merja
Published: October 2006

A method is presented for the isolation of genes encoding hydrolytic enzymes without any knowledge of the corresponding proteins. cDNA made from the organism of interest is cloned into a yeast vector to construct an expression library in the yeast Saccharomyces cerevisiae. Colonies producing hydrolytic enzymes are screened by activity plate assays. In this work, we constructed a yeast expression library from the filamentous fungus Trichoderma reesel and isolated a new β‐1,4‐endoglucanase gene on plates containing β‐glucan. This gene, eg15, codes for a previously unknown small protein of 242 amino acids. Despite its small size, the protein contains two conservative domains found in Trichoderma cellulases, namely the cellulose‐binding domain (CBD) and the iinker region that connects the CBD to the catalytic core domain. Molecular modelling of the EGV CBD revealed some interesting structural differences compared to the CBD of the major celluiase CBHI from T. reesei. The catalytic core of EGV is unusually small for a ceiiulase and represents a new family of ceilulases (Family K) and of glycosyl hydrolases (Famlly 45) together with the endoglucanase B of Pseudomonas fluorescens and the endoglucanase V of Humicola insolens on the basis of hydrophobic ciuster anaiysis.
Separate pathways for excision and processing of 16S and 23S rRNA from the primary rRNA operon transcript from the hyperthermophilic archaebacterium Sulfolobus acldocaldarius: similarities to eukaryotic rRNA processing
Authors: Durovic Peter , Dennis Patrick P.
Published: October 2006

In the hyperthermophilic archaebacterium Sulfolobus acidocaldarius, the mature 16S and 23S rRNA are generated by processing of a 5000‐nucleotide transcript. Analysis of intermediates that accumulate in vivo indicates that the transcript contains 11 separate processing sites. The processing and maturation of 23S rRNA appears to follow the typical archaebacterial pathway, utilizing a bulge‐helix‐bulge motif within the 23S processing helix as the substrate for an excision endonuclease. The precursor 23S rRNA that is released is trimmed at its 5′ and 3′ ends to generate the mature 23S rRNA found in 50S ribosomal subunits. The pathway for processing and maturation of 16S rRNA is distinctive and does not use the bulge‐helix‐bulge motif in the 165 processing stem, instead, the transcript is cleaved at several novel positions in the 5′ leader and in the 3′ intercistronic sequence. The excised precursor 16S is trimmed at the 5′ end but an extra 60 nucieotides of what is normally spacer sequence is retained at the 3′ end. The elongated 16S rRNA is present in active 30S subunits. An in vitro processing system for the 16S rRNA has been established. The RNA substrate containing the entire 144‐nucieotide 5′ leader and the first 72 nucleotides of 16S sequence is cleaved at the same positions observed in vivo by an endonuclease activity present in cell extract. These resuits demonstrate (i) that the 16S processing helix is neither utilized nor required for leader processing, and (ii) that complete maturation to the 5′ end of 16S rRNA can occur in the absence of concomitant ribosome assembly and in the absence of all but the first 72 nucleotides of the 16S rRNA sequence. The endonuclease activity responsible for cleavage of the 5′ leader substrate is sensitive to nuclease digestion, suggesting that it contains an essential RNA component. The cleavage sites appear to be located within regions of irregular secondary structure and have a consensus sequence of GAUUCC.
Multiple signalling systems controlling expression of luminescence in Vibrio harveyi: sequence and function of genes encoding a second sensory pathway
Authors: Bassler Bonnie L. , Wright Miriam , Silverman Michael R.
Published: October 2006

Density‐dependent expression of luminescence in Vibrio harveyl is regulated by the concentration of extracellular signal molecules (autoinducers) in the culture medium. One signal‐response system is encoded by the luxL,M,N locus. The luxL and luxM genes are required for the production of an autoinducer (probably β‐hydroxybutryl homoserine lactone), and the luxN gene is required for the response to that autoinducer. Analysis of the phenotypes of LuxL,M and N mutants indicated that an additional signal‐response system also controls density sensing. We report here the identification, cloning and analysis of luxP and luxQ, which encode functions required for a second density‐sensing system. Mutants with defects in luxP and luxQ are defective in response to a second autoinducer substance. LuxQ, like LuxN, is similar to members of the family of two‐component, signal transduction proteins and contains both a histidine protein kinase and a response regulator domain. Analysis of signalling mutant phenotypes indicates that there are at least two separate signal‐response pathways which converge to regulate expression of luminescence in V. harveyl.
traJ sense RNA initiates at two different proMolers in R100‐1 and forms two stable hybrids with antisense finP RNA
Authors: Dempsey Walter B.
Published: October 2006

RNase protection experiments show that the sizes of the two R100 finP molecules are 74 and 135 nucleotides. In an RNase III mutant, finP transcripts form stable double‐stranded hybrids of 108bp and 68 bp with traJ transcripts. RNase protection experiments also show that most R100‐1 transcripts originating in traM cross the traM‐traJ intergenic region and end inside the untranslated leader region of traJ. Some extend into the traJ open reading frame. These findings mean that the antisense finP RNA, thought to regulate traJ translation, must regulate traJ transcripts from both J and M proMolers.
Analysis of five tyiosin biosynthetic genes from the tyllBA region of the Streptomyces fradiae genome
Authors: Merson‐Davies Louise A. , Cundiiffe Eric
Published: October 2006

The tyllBA region of the tylosin biosynthetic gene cluster of Streptomyces fradiae contains at least five open reading frames (ORFs). ORF1 {tyll) encodes a cytochrome P450 and mutations in this gene affect macrolide ring hydroxylation. The product of 0RF2 (tylB) belongs to a widespread family of proteins whose functions are speculative, although tylB mutants are defective in the biosynthesis or addition of mycaminose during tylosin production. ORFs 3 and 4 (tylA1 and tylA2) encode δTDP‐giucose synthase and δTDP‐glucose dehydratase, respectively, enzymes responsible for the first two steps common to the biosynthesis of all three deoxyhexose sugars of tylosin via the common intermediate, δTDP‐4‐keto, 6‐deoxygiucose. ORF5 encodes a thioesterase similar to one encoded in the erythromycin gene cluster of Saccharopolyspora erythraea.
A copper‐transporting P‐type ATPase found in the thylakoid membrane of the cyanobacterium Synechococcus species PCC7942
Authors: Kanamaru Kengo , Kashiwagi Seiji , Mizuno Takeshi
Published: October 2006

P‐type ATPases constitute a large family of cation pumps that play crucial physiological roles in many organisms, including bacteria, plants and mammals. They are postulated to play important roles in a variety of environmental adaptation systems. Recently, we cloned two distinct putative P‐type ATPase genes (pacS and pacL) from a photosynthetic cyanobacterium, Synechococcus species PCC7942. in this study, one of the gene products (named PacS) was found to possess a putative metal‐binding motif (Gly‐Met‐X‐Cys‐X‐X‐Cys) in its N‐terminal portion. Thus we supposed that this ATPase may function as a metal pump. Indeed, the results of Northern blotting analysis showed that pacS‐mRNA specifically increases upon addition of copper or silver to the growth medium. The results of Western blotting analysis confirmed the view that PacS accumulates in copper‐treated Synechococcus cells. Thus we concluded that the expression of PacS ATPase is regulated in response to the change in concentration of external metals, namely copper and silver. Consistent with this, an insertional inactivation mutant of pacS exhibited hypersensitivity in terms of growth to these potentially toxic metals, it was also revealed that PacS was mainly located in the thylakoid membrane, in which the photosynthetic reactions take place. This P‐type ATPase in the thylakoid membrane is implicated as a copper‐transporting system that may be involved in copper‐homeostasis crucial to the photosynthetic thylakoid function.
Identification of a second oligopeptide transport system in Bacillus subtilis and determination of its role in sporulation
Authors: Koide Akiko , Hoch James A.
Published: October 2006

Summary

Sporulation in Bacillus subtilis depends on an intact oligopeptide transport system, the Opp system. Mutants in opp sporulate poorly but second‐site revertants can be found that restore sporulation and pep‐tide transport. These second‐site mutations were found in a second oligopeptide transport system, app, in which the peptide‐binding protein, AppA, is mutant owing to a frame‐shift mutation, and the revertants restore the original frame. The AppA mutation is present in the 168 strain of B. subtilis. The app operon consists of five genes in the order appD‐appF‐appA‐appB‐appC, with the locus designations corresponding to their homologue in the opp operon. Homology between the app and opp proteins ranges from 54% identity for AppF and OppF, to 22% identity for AppA and OppA. Both the App and Opp permease systems can transport tetra‐ and pentapeptides, but tripeptides are not transported by the App system. Strains of the genotype app+opp− are resistant to the tripeptide antibiotic bialaphos. The repaired App system can substitute completely for the Opp system in both sporulation and competence for genetic transformation. The pheno‐types raised some speculation about the subunit configuration of the Opp system.
EGTA induces the synthesis in Escherichia coli of three proteins that cross‐react with calmodulin antibodies
Authors: Laoudj Dalila , Andersen Catherine L. , Bras Ana , Goldberg Martin , Jacq Annick , Holland I. Barry
Published: October 2006

Escherichia coli mutants, (verA, dilA) specifically resistant to the Ca2+ channel inhibitors verapamil and diltiazem, respectively, are hypersensitive to EGTA and BAPTA. We have shown, using 1‐D and 2‐D gel electrophoresis, that the synthesis of at least 25 polypeptides in the mutants was enhanced by treatment with Ca2+ chelators and the synthesis of at least 11 polypeptides was repressed. This pattern of induction was not observed in heat‐ or SDS‐treated cells and therefore does not appear to be a general stress response. The majority of the induced proteins are low molecular weight, extremely heat stable and acidic, characteristic properties of calmodulin. Moreover, of the major induced species, three with apparent molecular masses of 12, 18, and 34kDa all cross‐reacted with polyclonal and monoclonal antibodies to eukaryote calmodulins or calerythrin, a heat‐resistant Ca2+‐binding protein from Saccharo‐polyspora erythraea. The verA, dilA mutants. In being hypersensitive to EGTA and to the Ca2+ ionophore A23187 + Ca2+, may be defective in the regulation of the level of free intracellular Ca2+.
A dominant mutation in the bacteriophage lambda S gene causes premature lysis and an absolute defective plating phenotype
Authors: Johnson‐Boaz Rebecca , Chang Chung‐Yu , Young Ry
Published: October 2006

The S and R genes of the bacteriophage λ are required for lysis of the host. R encodes ‘endolysin’, a soluble transglycosylase which accumulates in the cytoplasm during late protein synthesis. S encodes a ‘holin’, a small membrane protein which, at a precisely scheduled time, terminates the vegetative cycle by forming a lethal lesion in the membrane through which gpR gains access to the peptidoglycan. A missense allele of S, Ala52Gly, causes lysis to occur prematurely at about 19–20 min after induction of a lysogen, compared to 45min for the wild type. This allele has a severe plaque‐forming defect which appears to be entirely a consequence of the early lysis and resultant severe reduction in particle burst size. The early‐lysis phenotype is dominant and is aggravated, in terms of an even more reduced burst size, at both 30°C and 42°C. The mutation maps in the middle of a putative membrane‐spanning helical domain of S, near the sites of other S− mutations with recessive non‐lytic phenotypes. The mutation has no effect on S‐protein accumulation or on the ratio of S107 and S105 products in the membrane. The mutation appears to affect the intrinsic timing function by which the S protein controls the lysis schedule.
Domain structure and conserved epitopes of Sfb protein, the fibronectin‐binding adhesin of Streptococcus pyogenes
Authors: Talay Susanne R. , Valentin‐Weigand Peter , Timmis Kenneth N. , Chhatwal Gursharan S.
Published: October 2006

Streptococcus pyogenes expresses a fibronectin‐binding surface protein (Sfb protein) which mediates adherence to human epithelial cells. The nucleotide sequence of the sfb gene was determined and the primary sequence of the Sfb protein was analysed. The protein consists of 638 amino acids and comprises five structurally distinct domains. The protein starts with an N‐terminal signal peptide followed by an aromatic domain. The central part of the protein is formed by four proline‐rich repeats which are flanked by non‐repetitive spacer sequences. A second repeat region, consisting of four repeats that are distinct from the proline repeats and have been shown to form the fibronectin‐binding domain, is located in the Cterminal part of the protein. The protein ends with a typical cell wall and membrane anchor region. Comparative sequence analysis of the N‐terminal aromatic domain revealed similarities with carbohydrate‐binding sites of other proteins. The proline repeat region of the Sfb protein shares characteristic features with proline‐rich repeats of functionally distinct surface proteins from pathogenic Gram‐positive cocci. Immunoelectron microscopy revealed an even distribution of the fibronectin‐binding domain of Sfb protein on the surface of streptococcal cells. Analyses of 38 sfb genes originating from different S. pyogenes isolates revealed primary sequence variability in regions coding for the N‐termini of mature Sfb proteins, whereas sequences coding for the central and C‐terminal repeats were highly conserved. The repeat sequences are postulated to act as target sites for intragenic recombination events that result in variable numbers of repeats within the different sfb genes. A model of the Sfb protein is presented.
Vitamin B12 repression of the cob operon in Salmonella typhimurium: translational control of the cbiA gene
Authors: Richter‐Dahlfors A. A. , Ravnum S. , Andersson D. I.
Published: October 2006

Expression of the cob operon is repressed by B12 via a post‐transcriptional control mechanism which requires sequence elements within the leader region of the mRNA and the first gene of the operon, the cbiA gene. Here we show that B12 repression of cbiA gene expression occurs at the level of translation initiation through sequestration of the ribosomal binding site (rbs) in an RNA hairpin. Analysis of mutations that destabilize or restabilize the secondary structure demonstrates that folding of the hairpin is essential for repression. The existence of the hairpin was confirmed by a secondary structure analysis of RNA from the wild type and three mutants. Deletions that remove the upstream part of the leader confer a drastic reduction in translation efficiency. This low‐level translation is caused by the hairpin, as indicated by the finding that suppressor mutations that destabilize the hairpin restore efficient translation. Thus, the native upstream RNA functions as a translation enhancer and acts to relieve the hairpin's inhibitory effect on translation initiation. The inhibitory effect of the hairpin was confirmed by a ribosomal toeprinting analysis. We propose that the translational control of the cbiA gene mediates repression of the entire cob operon.
The Salmonella typhimurium invasion genes invF and invG encode homologues of the AraC and PulD family of proteins
Authors: Kaniga Koné , Bossio Juan C. , Galán Jorge E.
Published: October 2006

We have identified two novel Salmonella typhimurium genes, invF and invG, which are required for the efficient entry of these organisms into cultured epithelial cells. invF and invG are located immediately upstream of invE, a previously identified gene also required for Salmonella entry. Non‐polar mutations in these genes rendered S. typhimurium severely deficient for entry into cultured epithelial cells. The nucleotide sequences of invF and invG indicated that these genes encode polypeptides with predicted molecular weights of 24373 and 62275, respectively. Proteins of similar sizes were observed when invF and invG were expressed in a bacteriophage T7 RNA polymerase‐based expression system. Comparison of the predicted sequence of InvF with translated sequences in the existing databases indicated that this protein is homologous to members of the AraC family of prokaryotic transcription regulators. However, mutations in invF did not significantly affect the expression of other members of the inv locus. InvG was found to be homologous to members of the PuID family of specialized translocases. This homology suggests that InvG may be necessary for the export of invasion‐related determinants or involved in the assembly of a supramolecular structure that promotes entry.
Mutational analysis of the arginine repressor of Escherichia coli
Authors: Tian Guoling , Maas Werner K.
Published: October 2006

Arginine biosynthesis in Escherichia coli is negatively regulated by a hexameric repressor protein, encoded by the gene argR and the corepressor arginine. By hydroxylamine mutagenesis two types of argR mutants were isolated and mapped. The first type is transdominant. In heterodiploids, these mutant polypeptides reduce the activity of the wild‐type repressor, presumably by forming heteropolymers. Four mutant repressor proteins were purified. Two of these map in the N‐terminal half of the protein. Gel retardation experiments showed that they bind poorly to DNA, but they could be precipitated by l‐arginine at the same concentration as the wild‐type repressor. The other two mutant repressors map in the C‐terminal half of the protein. They are poorly precipitated by L‐arginine and they bind poorly to DNA. In addition, one of these mutants appears to exist as a dimer. The second type of argR mutant repressor consists of super‐repressors. Such mutants behave as arginine auxotrophs as a result of hyper‐repression of arginine biosynthetic enzymes. They map at many locations throughout the argR gene. Three arginine super‐repressor proteins were purified, in comparison with the wild‐type repressor, two of them were shown to have a higher DNA‐binding affinity in the absence of bound arginine, while the third was shown to have a higher DNA‐binding affinity when bound to arginine.
Partial suppression of an Escherichia coli TonB transmembrane domain mutation (ΔV17) by a missense mutation in ExbB
Authors: Larsen Ray A. , Thomas Michael G. , Wood Gwendolyn E. , Postle Kathleen
Published: October 2006

Active transport of vitamin B12 and Fe(III)‐siderophore complexes across the outer membrane of Escherichia coli appears to be dependent upon the ability of the TonB protein to couple cytoplasmic membrane‐generated protonmotive force to outer membrane receptors. TonB is supported in this role by an auxiliary protein, ExbB, which, in addition to stabilizing TonB against the activities of endogenous envelope proteases, directly contributes to the energy transduction process. The topological partitioning of TonB and ExbB to either side of the cytoplasmic membrane restricts the sites of interaction between these proteins primarily to their transmembrane domains. In this study, deletion of valine 17 within the amino‐terminal transmembrane anchor of TonB resulted in complete loss of TonB activity, as well as loss of detectable in vivo crosslinking into a 59 kDa complex believed to contain ExbB. The ΔV17 mutation had no effect on TonB export. The loss of crosslinking appeared to reflect conformational changes in the TonB/ExbB pair rather than loss of interaction since ExbB was still required for some stabilization of TonBΔV17. Molecular modeling suggested that the ΔV17 mutation caused a significant change in the predicted conserved face of the TonB amino‐terminal membrane anchor. TonBΔV17 was unable to achieve the 23 kDa proteinase K‐resistant form in lysed sphaeroplasts that is characteristic of active TonB. Wild‐type TonB also failed to achieve the proteinase K‐resistant configuration when ExbB was absent. Taken together these results suggested that the ΔV17 mutation interrupted productive TonB–ExbB interactions. The apparent ability to crosslink to ExbB as well as a limited ability to transduce energy were restored by a second mutation (A39E) in or near the first predicted transmembrane domain of the ExbB protein. Consistent with the weak suppression, a 23 kDa proteinase K‐resistant form of TonBΔV17 was not observed in the presence of ExbBA39E. Neither the ExbBA39E allele nor the absence of ExbB affected TonB or TonBΔV17 export. Unlike the tonBΔV17 mutation, the exbBA39E mutation did not greatly alter a modelled ExbB transmembrane domain structure. Furthermore, the suppressor ExbBA39E functioned normally with wild‐type TonB, suggesting that the suppressor was not allele specific. Contrary to expectations, the TonBδV17, ExbBA39E pair resulted in a TonB with a greatly reduced half‐life (≅ 10 min). These results together with protease susceptibility studies suggest that ExbB functions by modulating the conformation of TonB.
Fluctuations in glycolytic mRNA levels during morphogenesis in Candida albicans reflect underlying changes in growth and are not a response to cellular dimorphism
Authors: Swoboda R. K. , Bertram G. , Delbrück S. , Ernst J. F. , Gow N. A. R. , Gooday G. W. , Brown A. J. P.
Published: October 2006

The levels of pyruvate kinase (PYK), alcohol dehydrogenase (ADH1), phosphoglycerate kinase (PGK1) and phosphoglycerate mutase (GPM1) mRNAs were measured during batch growth and during the yeast‐to‐hyphal transition in Candida albicans. The four mRNAs behaved in a similar fashion. PYK1, ADH1, PGK1 and GPM1 mRNA levels were shown to increase dramatically during the exponential growth phase of the yeast form, and then to decrease to relatively low levels in the stationary phase. The dimorphic transition was induced using two sets of conditions: (i) an increase in temperature (from 25°C to 37°C) combined with the addition of serum to the medium; and (ii) an increase in temperature (from 25°C to 37°C) and an increase in pH of the growth medium (from pH 4.5 to pH 6.5). Additional cultures were analysed to control for the addition of serum, and for changes in temperature or pH. Immediately following dilution of late‐exponential cells into fresh media the levels of all four glycolytic mRNAs decreased rapidly in contrast to the ACT1 mRNA control, the level of which increased under most conditions. The recovery of glycolytic mRNA levels depended on the culture conditions, but there was no direct correlation with the formation of germ tubes, with the addition of serum to the medium, the Increase in culture temperature, the medium pH, or the glucose concentration. This indicates that the changes in glycolytic gene expression that accompany the dimorphic transition in C. albicans reflect the underlying physiological status of the cells during morphogenesis and not alterations to cell shape.
The pigmentation locus of Yersinia pestis KIM6+ is flanked by an insertion sequence and includes the structural genes for pesticin sensitivity and HMWP2
Authors: Fetherston Jacqueline D. , Perry Robert D.
Published: October 2006

The pigmentation (Pgm+) phenotype of Yersinia pestis includes a number of different characteristics which appear to be associated with a 102 kb segment of chromosomal DNA known as the pgm locus. In Y. pestis KIM6+, the pgm locus is flanked by direct copies of a repeated element that probably plays a role in the spontaneous deletion of this region. We have sequenced the ends of these elements and shown that they have features in common with bacterial insertion sequences, in addition we show that a clone, pSDR498, from the pgm locus of KIM6+ restores pesticin sensitivity and the iron‐regulated expression of three polypeptides, 240 kDa, 190 kDa, and 68 kDa in size, to Pgm− cells. In vitro transcription/translation assays and Escherichia coli minicells were used to analyse the products encoded by various subciones of pSDR498. Pesticin sensitivity mapped to a 5.9 kb fragment that encodes a 68 kDa protein derived from a 72 kDa precursor. Synthesis of the 190 kDa protein was restored by a 19.2 kb clone, indicating that the structural gene for this protein also resides within the pgm locus of Y. pestis KIM6+. Finally, a survey of our pgm− strains indicates that 97% have also deleted the sequences encoding the 190 kDa protein and pesticin sensitivity.
Molecular characterization of upstream regulatory sequences controlling the photoinduced expression of the albino‐3 gene of Neurospora crassa
Authors: Carattoli Alessandra , Cogoni Carlo , Morelli Glorgio , Macino Giuseppe
Published: October 2006

In the filamentous fungus Neurospora crassa the biosynthesis of carotenoids is regulated by blue light, principally through transcriptional activation of some key genes in the carotenogenic enzymatic pathway. Here we report the characterization of the photoinducible promoter of the albino‐3 (al‐3) gene, encoding GGPP synthase. We have modified the 5’non‐coding sequence of the cloned al‐3 gene by deletion and site‐directed mutagenesis, and we have tested the residual photoinducibility of the different constructs by transformation and subsequent analysis of gene expression in dark‐grown and light‐induced mycelia. The results indicate that a promoter region between positions −226 and −55 contains all the necessary information for blue light photoinduction. Multiple regulatory elements are involved in the regulated expression of the al‐3 gene. One (termed the APE element) is important for the light‐induction switch on of the gene and a second belongs to the CCAAT boxes family. The putative APE element is also found in the promoter of other N. crassa photoinducible genes.
Residue threonine‐149 of the Salmonella typhimurium CysB transcription activator: mutations causing constitutive expression of positively regulated genes of the cysteine regulon
Authors: Colyer Tracey E. , Kredich Nicholas M.
Published: October 2006

In both Salmonella typhimurium and Escherichia coli, CysB is a LysR family transcriptional activator, which regulates genes of the cysteine regulon. Transcription activation of cys genes also requires an inducer, W‐acetyl‐L‐serine, and cysB mutants that do not require Inducer are termed constitutive, i.e. cysBc. After finding that two independently isolated cysBc mutants are substituted at amino acid residue threonine‐149 (T149), we isolated the other 17 single‐amino‐acid substitutions by site‐directed mutagenesis. Of the 19 mutant alleles, 11 supported normal growth on sulphate, and nine of these were cysBc. Four other mutants were‘leaky’cysB+, and four were cysB−. Insertions of up to 14 amino acids were also tolerated at T149, and two of three such mutants were cysBc. An allele containing a TAG translation terminator at codon 149 had no detectable function in a δcysB strain, but gave a constitutive phenotype when introduced into either wild‐type S. typhimurium or the E. coll strain NK1, which contains a cysB− mutation in a predicted helix‐turn‐helix region that interferes with specific binding of CysB to DNA and with autoregulation ot cysB. The peptide encoded by the T149ter allele is proposed to interact with the wild‐type CysB peptide or with the NK1 mutant peptide to form hetero‐oligomers that do not require N‐acetyl‐L‐serine for cys gene activation.
The novel lectin‐like protein CHB1 is encoded by a chitin‐inducible Streptomyces olivaceoviridis gene and binds specifically to crystalline α‐chitin of fungi and other organisms
Authors: Schnellmann Jürgen , Zeltins Andris , Blaak Harald , Schrempf Hildgund
Published: October 2006

The chb1 gene, which encodes the unique lectin‐like α‐chitin‐binding protein CHB1 of Streptomyces olivaceoviridis, was cloned. Transformants of Streptomyces lividans harbouring the plasmid pCHB10 overproduced the extracellular CHB1 protein; the protein showed neither enzymatic nor antifungal activity. Biochemical analyses and immunofluorescence microscopy indicated that CHB1 binds strongly to α‐chitin, but neither to chitosan and β‐chitin, nor to various types of cellulose. Within hyphae of fungi, the relative location of crystalline chitin was visualized with fluor‐escein‐labelled CHB1. These studies suggest that the new protein could serve as a tool to identify α‐chitin within different organisms. The chb1 gene consists of a reading frame of 603 bp and its transcription occurred only if the Streptomyces strain was cultivated with chitin as the sole carbon source. The deduced mature CHB1 protein (18.7 kDa) shows no apparent similarity to any known protein. Within a region containing 100 residues of the deduced CHB1 protein, four tryptophan and two asparagine residues as well as one glycine and one cysteine residue were identified, the relative positions of which are analogous to those of several cellulose‐binding domains of bacterial glycohydrolases. The results of spectroscopical studies suggest a possible involvement of tryptophan residues in the interaction of CHB1 with α‐chitin.
Structural identification of metabolites produced by the NodB and NodC proteins of Rhizobium leguminosarum
Authors: Spaink Herman P. , Wijfjes André H. M. , der van Drift Koen M. G. M. , Haverkamp Johan , Thomas‐Oates Jane E. , Lugtenberg Ben J. J.
Published: October 2006

The Rhizobium nodulation genes nodABC are involved in the synthesis of lipo‐chitin oligosac‐charides. We have analysed the metabolites which are produced in vivo and in vitro by Rhizobium strains which express the single nodA, nodB and nodC genes or combinations of the three. In vivo radioactive labelling experiments, in which D‐[1‐14C]‐glucosamine was used as a precursor, followed by mass spectrometric analysis of the purified radiolabelled metabolic products, showed that Rhizobium strains that only express the combination of the nodB and nodC genes do not produce lipo‐chitin oligosaccharides but instead produce chitin oligomers (mainly pentamers) which are devoid of the N‐acetyl group on the non‐reducing terminal sugar residue (designated NodBC metabolites). Using the same procedure we have shown that when the nodL gene is expressed in addition to the nodBC genes the majority of metabolites contain an additional O‐acetyl substituent on the non‐reducing terminal sugar residue (designated NodBCL metabolites). The NodBC and NodBCL metabolites purified after in vivo labelling were compared with the radiolabelled metabolites produced in vitro by Rhizobium bacterial cell lysates to which UDP‐N‐acetyl‐D‐[U‐14C]‐glucosamine was added using thin‐layer chromatography. The results show that the lysates of strains which expressed the nodBC or nodBCL genes can also produce NodBC and NodBCL metabolites. The same results were obtained when the NodB and NodC proteins were produced separately in two different strains. On the basis of these and other recent results, we propose that NodB is a chitin oilgosaccharide deacetylase, NodC an N‐acetylglucosaminyltransferase and, by default, NodA is involved in lipie attachment.
Threonine dehydratases of Corynebacterium glutamicum with altered allosteric control: their generation and biochemical and structural analysis
Authors: Möckel Bettina , Eggeling Lothar , Sahm Hermann
Published: October 2006

Threonine dehydratase is the key enzyme in L‐isoleucine synthesis, since it is allosterically feedback‐inhibited by L‐isoleucine. With the aim of obtaining regulatorily altered mutants of the threonine dehydratase of Corynebacterium glutamicum, amino acids were specifically exchanged and a new biological system of mutant selection was developed, based on the intoxication of Escherichia coli by ketobutyrate, which is the dehydratase reaction product. A collection of 19 mutant enzymes was generated and genetically and biochemically characterized comprising a whole range of regulatorily and catalytically altered enzymes. Of particular interest is the mutant Val‐323‐Ala, which is characterized by the fact that the L‐isoleucine inhibition is entirely abolished so that the enzyme is always present in a relaxed, high‐activity state. Correspondingly, the Hill coefficient is 1.4, in contrast to the value of 3.4 characteristic of the wild‐type enzyme. Another peculiar mutant generated is the double mutant His‐278‐Arg‐Leu‐351‐Ser. Here, again, L‐isoleucine no longer inhibits catalytic activity, but the effector still promotes major structural changes of the protein, as ascertained from the L‐isoleucine‐dependent loss of pyridoxal‐5 ‐phosphate from this mutant enzyme. Further enzymes obtained are reduced in L‐isoleucine inhibition to a varying degree. Detailed studies on the structure of the enzyme revealed a partially very high similarity of the secondary structure to the mechanistically identical β‐subunit of the tryptophan synthase. This provides further indications concerning the localization of the regulatory and catalytic domain of threonine dehydratase.
DNA polymerase III of Mycoplasma pulmonis: isolation and characterization of the enzyme and its structural gene, polC
Authors: Barnes Marjorie H. , Tarantino Paul M. , Spacciapoli Peter , Brown Neal C. , Yu Huilan , Dybvig Kevin
Published: October 2006

Mycoplasmas have originated from Gram‐positive bacteria via rapid degenerative evolution. The results of previous investigations of mycoplasmal DNA polymerases suggest that the process of evolution has wrought a major simplification of the typical Gram‐positive bacterial DNA polymerase profile, reducing it from three exonuclease (exo)‐positive enzymes to a single exo‐negative species. The objective of this work was to rigorousiy investigate this suggestion, focusing on the evolutionary fate of DNA polymerase III (Pol III), the enzyme which Gram‐positive bacteria specifically require for replicative DNA synthesis. The approach used Mycoplasma pulmonis as the model organism and exploited structural gene cloning, enzymology, and Pol III‐specific inhibitors of the HPUra class as investigative tools. Our results indicate that M. pulmonis has strongly conserved a single copy of a structural gene homologous to polC, the Gram‐positive bacterial gene encoding Pol III M. pulmonis was found to possess a DNA polymerase that displays the size, primary structure, exonuclease activity, and level of HPUra sensitivity expected of a prototypical Gram‐positive Pol III. The high level of sensitivity of M. pulmonis growth to Gram‐positive Pol III‐selective inhibitors of the HPUra type strongly suggests that Mycoplasma has conserved not only the basic structure of Pol III, but also its essential replicative function. Evidence for a second, HPUra‐resistant polymerase activity in M. pulmonis is also described, indicating that the DNA polymerase composition of Mycoplasma is complex and closer to that of Gram‐positive bacteria than previously thought.
The 100 kDa haem:haemopexin‐binding protein of Haemophilus Influenzae: structure and localization
Authors: Cope Leslie D. , Thomas Sharon E. , Latimer Jo L. , Slaughter Clive A. , Müller‐Eberhard Ursula , Hansen Eric J.
Published: October 2006

All Haemophilus influenzae strains have an absolute requirement for exogenously supplied haem for aerobic growth. A majority of strains of H. influenzae type b (Hib) produce a 100 kDa protein which binds haem: haemopexin complexes. This 100 kDa haem:haemopexin binding protein, designated HxuA, was originally detected on the Hib cell surface. Monoclonal antibody (mAb)‐based analyses revealed that the HxuA protein was also present in soluble form in Hib culture supernatants. This soluble HxuA protein exhibited haem:haemopexin‐binding activity in a direct binding assay. Nucleotide sequence analysis of the hxuA gene from Hib strain DL42, together with N‐terminal amino acid analysis of HxuA protein purified from Hib culture supernatant, revealed that this protein was synthesized as a 101 kDa precursor with a leader peptide that was removed to yield a 99kDa protein. Southern blot analysis of chromosomal DNA from four Hib and four non‐typeable H. influenzae (NTHI) strains detected the presence of a single band in each strain that hybridized a Hib hxuA gene probe. Subsequent analysis of these NTHI strains showed that all four strains released into culture supernatant a haem:haemopexin‐binding protein that migrated in SDS‐PAGE at a rate similar or identical to that of the Hib HxuA protein. A Hib hxuA mutant was used to screen an NTHI genomic DNA library and an NTHI gene was cloned that complemented the mutation in this Hib strain. Nucleotide sequence analysis of this NTHI gene revealed that it encoded a protein with 87% identity to the Hib HxuA protein. The expression of HxuA by both Hib and NTHI strains indicates that this particular haem acquisition system is conserved among H. influenzae strains.
Investigation of the role of the disulphide bond in the activity and structure of staphylococcal enterotoxin C1
Authors: Hovde Carolyn J. , Marr James C. , Hoffmann Marcy L. , Hackett Sean P. , Chi Young‐in , Crum Kimberlee K. , Stevens Dennis L. , Stauffacher Cynthia V. , Bohach Gregory A.
Published: October 2006

The goal of this study was to Investigate the role of the disulphide bond of staphylococcal enterotoxin C1 (SEC1) in the structure and activity of the toxin. Mutants unable to form a disulphide bond were generated by substituting alanine or serine for cysteine at positions 93 and/or 110. Although we did not directly investigate the residues between the disulphide linkage, tryptic lability showed that significant native structure in the cystine loop is preserved in the absence of covalent bonding between residues 93 and 110. Since no correlation was observed between the behaviour of these mutants with regard to toxin stability, emesis and T cell proliferation, we conclude that SEC1 ‐induced emesis and T cell proliferation are dependent on separate regions of the molecule. The disulphide bond itself is not an absolute requirement for either activity. However, conformation within or adjacent to the loop is important for emesis. Although mutants with alanine substitutions were not emetic, those with serine substitutions retained this activity, suggesting that the disulphide linkage stabilizes a crucial conformation but can be replaced by residues which hydrogen bond.
Structural analysis and molybdenum‐dependent expression of the pAO1‐encoded nicotine dehydrogenase genes of Arthrobacter nicotinovorans
Authors: Grether‐Beck Susanne , Igloi Gabor L. , Pust Stefan , Schilz Emil , Decker Karl , Brandsch Roderich
Published: October 2006

The genes of nicotine dehydrogenase (NDH) were identified, cloned and sequenced from the catabolic plasmid pA01 of Arthrobacter nicotinovorans. In immediate proximity to this gene cluster is the beginning of the 6‐hydroxy‐L‐niotine oxidase (6‐HLNO) gene. NDH is composed of three subunits (A, B and C) of Mr 30011, 14924 and 87677. It belongs to a family of bacterial hydroxylases with a similar subunit structure; they have molybdopterin dinucleotide, FAD and Fe‐S clusters as cofactors. Here the first complete primary structure of a bacterial hydroxylase is provided. Sequence alignments of each of the NDH subunits show similarities to the sequences of eukaryotic xanthine dehydrogenase (XDH) but not to other known molybdenum‐containing bacterial enzymes. Based on alignment with XDH it is inferred that the smallest subunit (NDHB) carries an iron‐sulphur cluster, that the middle‐sized subunit (NDHA) binds FAD, and that the largest NDH subunit (NDHC) corresponds to the molybdopterin‐binding domain of XDH. Expression of both the ndh and the 6‐hlno genes required the presence of nicotine and molybdenum in the culture medium. Tungsten inhibited enzyme activity but not the synthesis of the enzyme protein. The enzyme was found in A. nicotinovorans cells in a soluble form and in a membrane‐associated form. In the presence of tungsten the fraction of membrane‐associated NDH increased.
The pilE gene product of Pseudomonas aeruginosa, required for pilus biogenesis, shares amino acid sequence identity with the N‐termini of type 4 prepilin proteins
Authors: Russell Mary A. , Darztns Aldis
Published: October 2006

A new locus required for type 4 pilus biogenesis by Pseudomonas aeruginosa has been identified. A pilE mutant, designated MJ‐6, was broadly resistant to pili‐specific phages and unable to translocate across solid surfaces by the pilus‐dependent mechanism of twitching motility (Twt−). Immunoblot analysis demonstrated that MJ‐6 was devoid of pili (Pil−) but was unaffected in the production of unassembled pilin pools. Genetic studies aimed at localizing the pilE mutation on the P. aeruginosa PAO chromosome demonstrated a strong co‐linkage between MJ‐6 phage resistance and the proB marker located at 71 min. Cloning of the pilE gene was facilitated by the isolation and identification of a proB+‐containing plasmid from a PAO1 cosmid library. Upon introduction of the PA01 proB+ cosmid clone into MJ‐6, sensitivity to pili‐specific phage, twitching motility and pilus production were restored. The nucleotide sequence of a 1 kb Eco RV‐Clal fragment containing the pilE region revealed a single complete open reading frame with characteristic P. aeruginosa codon bias. PilE, a protein with a molecular weight of 15278, showed significant sequence identity to the pilin precursors of P. aeruginosa and to other type 4 prepilin proteins. The region of highest homology was localized to the N‐terminal 40 amino acid residues. The putative PilE N‐terminus contained a seven‐residue basic leader sequence followed by a consensus cleavage site for prepilin pep‐tidase and a largely hydrophobic region which contained tyrosine residues (Tyr‐24 and Tyr‐27) previously implicated in maintaining pilin subunit‐subunit interactions. The requirement of PilE in pilus biogenesis was confirmed by demonstrating that chromosomal pilE insertion mutants were pilus‐ and twitching‐motility deficient.
TcpA pilin sequences and colonization requirements for O1 and O139 Vibrio cholerae
Authors: Rhine Janet A. , Taylor Ronald K.
Published: October 2006

The distribution, characterization and function of the tcpA gene was investigated in Vibrio cholerae O1 strains of the El Tor biotype and in a newly emergent non‐O1 strain classified as serogroup O139. The V. cholerae tcpA gene from the classical biotype strain O395 was used as a probe to identify a clone carrying the tcpA gene from the El Tor biotype strain E7946. The sequence of the E7946 tcpA gene revealed that the mature El Tor TcpA pilin has the same number of residues as, and is 82% identical to, TcpA of classical biotype strain O395. The majority of differences in primary structure are either conservative or clustered in a manner such that compensatory changes retain regional amino acid size, polarity and charge. In a functional analysis, the cloned gene was used to construct an El Tor mutant strain containing an insertion in tcpA. This strain exhibited a colonization defect in the infant mouse cholera model similar in magnitude to that previously described for classical biotype tcpA mutants, thus establishing an equivalent role for TCP in intestinal colonization by El Tor biotype strains. The tcpA analysis was further extended to both a prototype El Tor strain from the Peru epidemic and to the first non‐O1 strain known to cause epidemic cholera, an O139 V. cholerae isolate from the current widespread Asian epidemic. These strains were shown to carry tcpA with a sequence identical to E7946. These results provide further evidence that the newly emergent non‐O1 serogroup O139 strain represents a derivative of an El Tor biotype strain and, despite its different LPS structure, shares common TCP‐associated antigens. Therefore, there appear to be only two related sequences associated with TCP pilin required for colonization by all strains responsible for epidemic cholera, one primary sequence associated with classical strains and one for El Tor strains and the recent O139 derivative. A diagnostic correlation between the presence of tcpA and the V. cholerae to colonize and cause clinical is now extended to strains of both O1 and non‐O1 serotypes.