bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification and localization of an iron-regulated 35 kDa protein of Pasteurella haemolytica serotype A2
Authors: Lainson F. A. , Harkins D. C. , Wilson C. F. , Sutherland A. D. , Murray J. E. , Donachie W. , Baird G. D.
Published: June 2009

Iodination of intact Pasteurella haemolytica serotype A2 cells labelled a sub-set of total cellular proteins. Comparison of the autoradiographic patterns obtained from iodinated cells grown on complete medium and on iron-depleted medium showed that expression of three proteins, of 100,70 and 35 kDa, respectively, was increased by growth under iron-depleted conditions. Of these proteins, that of 35 kDa had not been reported previously. Like the 100 and 70 kDa proteins, the 35 kDa protein was expressed in natural infections, since it was recognized by antiserum from sheep that had recovered from an experimental infection with P. haemolytica A2. The 35 kDa protein was partially purified by reverse-phase HPLC and was found to be antigenic in both sheep and mice. A monoclonal antibody that was specific for the 35 kDa protein was used to identify the cellular location of the protein by immunoblotting of cell fractions enriched for particular cellular components. This demonstrated that the 35 kDa protein was located mainly in the periplasm.
State of penicillin-binding proteins and requirements for their bactericidal interaction with β-lactam antibiotics in Serratia marcescens highly resistant to extended-spectrum β-lactams
Authors: Gunkel Anna-Greta , Hechler Ulrike , Martin Hans H.
Published: June 2009

The quantities of penicillin-binding proteins (PBPs), and sensitivity to extended-spectrum β-lactams, were measured in isogenic strains of Serratia marcescens with high (HR) and low (LR) resistance to extended-spectrum β-lactam antibiotics and with constitutively overproduced chromosomal β-lactamase in the periplasm. The binding of structurally different β-lactams to PBPs in growing resistant bacteria was determined quantitatively. In S. marcescens HR, the amounts of PBPs 3 and 6 were, respectively, 1·5 and 2 times those in strain LR and in sensitive reference strains. Sensitivities of the essential PBPs in S. marcescens LR and HR to the tested β-lactams were identical. Only a single target, PBP 3, was highly sensitive to cefotaxime, ceftazidime and aztreonam. In contrast, three PBPs (2, 1A and 3) were highly sensitive to imipenem. In growing S. marcescens HR and LR, all antibiotics, even at fractions of their minimal growth inhibitory concentrations (MICs), bound extensively to those PBPs which were highly sensitive to them. Thus, overproduced β-lactamase did not prevent PBP-β-lactam interaction. Only at or above their (high) MICs did cefotaxime, ceftazidime and aztreonam bind to multiple targets. Growth inhibition of the otherwise highly resistant S. marcescens HR at the lower MIC of imipenem was correlated with the binding of this antibiotic to multiple, highly sensitive targets in the bacteria. Killing of the bacteria by inactivation of multiple targets was suggested. This assumption was supported by the synergistic killing of HR bacteria by combinations of the PBP-2-specific mecillinam with PBP-3-specific β-lactams.
The bialaphos biosynthetic genes of Streptomyces viridochromogenes: cloning, heterospecific expression, and comparison with the genes of Streptomyces hygroscopicus
Authors: Hara Osamu , Murakami Takeshi , Imai Satoshi , Anzai Hiroyuki , Itoh Reiko , Kumada Yoichi , Takano Eriko , Satoh Eriko , Satoh Atsuyuki , Nagaoka Kozo , Thompson Charles
Published: June 2009

The bialaphos resistance gene, bar, was used as a selectable marker to isolate the bialaphos production genes (bap) from the Streptomyces viridochromogenes genome. The S. viridochromogenes bar gene was cloned on overlapping restriction fragments using pIJ680 and pIJ702 in the bialaphos-sensitive host, S. lividans. Although the restriction endonuclease cleavage map of these fragments was not similar to the bap cluster of S. hygroscopicus, the presence and location of bar and four other bap genes as well as a gene required for the transcriptional activation of the cluster (brpA) was demonstrated by heterologous cloning experiments using a series of previously characterized bialaphos-nonproducing S. hygroscopicus mutants. Since recombination-deficient mutants of streptomycetes have not been isolated, restored function provided by cloned homologous DNA results from both recombination (marker rescue) and complementation in trans. In contrast to our previously reported homologous cloning experiments where we were able to define the position of mutant alleles by recombination, in these heterologous cloning experiments we observed little if any recombination between plasmid-cloned genes and the chromosome. As a result, this approach allowed us to define the location and orientation of functional genes using a genetic complementation test. The organization of the clustered S. viridochromogenes bap genes was indistinguishable from the corresponding S. hygroscopicus mutant alleles. The fact that the S. viridochromogenes transcriptional regulatory gene, brpA, functioned in S. hygroscopicus implied that some transcriptional regulatory signals may also be interchangeable. In these two Streptomyces species, which have considerable nucleotide sequence divergence, the complex biochemical and genetic organization of the bialaphos biosynthetic pathway is conserved.
A molecular analysis of the 53.3 minute region of the Escherichia coli linkage map
Authors: Andrews Simon C. , Harrison Pauline M. , Guest John R.
Published: June 2009

The coding characteristics of four plasmids expressing a protein (BCP) which comigrates with bacterioferritin were examined and the nucleotide sequence of a common 1985 bp segment from the 53 min region of the Escherkhia coli linkage map was determined. Three open reading-frames (orf1, orf2 and orf3) were detected, and orf2 (bcp, 156 amino acid codons) appeared to encode the bacterioferritin comigratory protein, BCP. The translation product of orf3 (205 amino acid codons) resembled the iron-sulphur protein component (DMS B subunit) of the anaerobic dimethylsulphoxide reductase complex of E. coli.
Use of green fluorescent protein for detection of cell-specific gene expression and subcellular protein localization during sporulation in Bacillus subtilis
Authors: Lewis Peter J. , Errington Jeffery
Published: June 2009

Wild-type and mutant forms of the gene encoding green fluorescent protein (GFP) from Aequorea victoria have been introduced into Bacillus subtilis as translational fusions to the prespore-specific and mother-cell-specific genes dacF and spoIVA. In both cases, the protein was readily detected by fluorescence microscopy, and its synthesis was correctly localized. The S65T substitution, which improves the quantum yield and rate of development of fluorescence, also produced a spectral shift that allowed the protein to be colocalized with DNA, after staining with 4′,6-diamidino-2-phenylindole. Three different translational fusions to the N-terminal region of GFP all produced active protein. Moreover, a full-length spoIVA-GFP fusion showed proper targeting to the surface of the spore, albeit at low temperature and in the presence of wild-type spoIVA protein. A mutation in the gfp gene which changes the light emitted by the protein from green to blue was found not to be useful because of the intrinsic autofluorescence of B. subtilis in the blue part of the spectrum.
A new, broad-substrate-specificity aminopeptidase from the dairy organism
Lactobacillus helveticus
SBT 2171
Authors: Sasaki Masahiro , Bosman Boukje W. , Tan Paris S. T.
Published: June 2009

An aminopeptidase with a very broad substrate specificity was purified to homogeneity from
Lactobacillus helveticus
SBT 2171 by FPLC. The enzyme was purified 144-fold from a cell-free extract with a yield of 16%. The purified enzyme appeared as a single band on an SDS-PAGE gel. It had a molecular mass of 95 kDa and an isoelectric point of 4.9. The enzyme hydrolysed a large range of naphthylamide- and nitroanilide-substituted amino acids, as well as several di-, tri- and oligopeptides. It also exhibited significant prolineiminopeptidase-like activity, since it hydrolysed several proline-containing peptides. Prolyl-p-nitroanilide was hydrolysed with a low affinity (Michaelis-Menten constant 0.6 mM) and a V
max of 2.5 μmol min-1 (mg protein)-1 while lysyl-p-nitroanilide was hydrolysed with a high affinity [K
m 0.003 mM; V
max 37.5 μmol min-1 (mg protein)-1]. The aminopeptidase activity, which was optimal between pH 6.0 and 8.0 and at 50 °, was very stable at 30 ° for more than 7 d. The activity lost by treatment with the thiol-blocking reagents could be restored with ß-mercaptoethanol, while Co2+ and Mn2+ restored the activity of the EDTA-treated enzyme. Immunological experiments with antibodies raised against the aminopeptidases from Lactococcus lactis and Lb. helveticus clearly showed that both aminopeptidases are at least immunologically different from each other.
Degradation of host protease inhibitors and activation of plasminogen by proteolytic enzymes from Porphyromonas gingivalis and Treponema denticola
Authors: Grenier Daniel
Published: June 2009

Bacterial proteases may participate in the pathogenesis of periodontal diseases through their action on host proteins. In the present study, the ability of selected periodontopathogens, as well as two proteases isolated from Porphyromonas gingivalis and Treponema denticola, to degrade host protease inhibitors was evaluated. The activation of human plasminogen by the two bacterial proteases was also investigated. Proteolytic breakdown of host protease inhibitors (α-1-antitrypsin, antichymotrypsin, α2-macroglobulin, antithrombin III, antiplasmin and cystatin C) was evaluated by SDS-PAGE. The 80 kDa trypsin-like protease of P. gingivalis completely digested the six protease inhibitors under investigation, whereas the 95 kDa chymotrypsin-like protease of T. denticola was slightly less active, more particularly on α2-macroglobulin and cystatin C. When whole cells from a number of oral bacterial species were tested, the most significant degradation was obtained with P. gingivalis, T. denticola, Prevotella intermedia, Prevotella nigrescens and Capnocytophaga spp. Peptostreptococcus micros and Propionibacterium acnes had only some degradative activity on selected inhibitors, whereas three bacterial species, Actinobacillus actinomycetemcomitans, Bacteroides forsythus and Fusobacterium nucleatum, had no effect on the protease inhibitors. The 80 kDa protease of P. gingivalis demonstrated strong plasminogen activation, whereas no such activity was associated with the 95 kDa protease of T. denticola. This study indicates the high potential of some periodontal pathogens to destroy protease inhibitors and activate plasminogen. This may result in an uncontrolled degradation of periodontal tissues and a rapid progression of the disease.
High Affinity Iron Acquisition in Rhizobium Leguminosarum Requires the cycHJKL Operon and the feuPQ Gene Products, which belong to the Family of Two-Component Transcriptional Regulators
Authors: Yeoman Kay H. , Delgado Maria-Jesus , Wexler Margaret , Downie J. Allan , Johnston Andrew W. B.
Published: June 2009

The cycHJKL operon of Rhizobium leguminosarum has previously been shown to be involved in the maturation of cytochrome c, possibly by its involvement in the covalent attachment of haem to the apoprotein. Mutations in the cycHJKL genes abolish symbiotic nitrogen fixation. Here, we show that cyc mutants are pleiotropically defective. They have lost a high affinity iron acquisition system due to their failure to make or to export siderophores. They also accumulate protoporphyrin IX, the immediate precursor of haem. A model to account for these phenotypes is presented. Immediately upstream of cycH is a gene, lipA, which is predicted to encode an outer-membrane lipoprotein. Further upstream of lipA, there are two other genes, whose products are similar in sequence to the widespread family of two-component transcriptional regulators. These two genes, feuP and feuQ, did not affect the transcription of lipA, or of the cycHJKL operon. However, a mutation in feuQ also led to the loss of the high affinity iron uptake system, although siderophores were still produced.
Expulsion of Uracil and Thymine from the Yeast Saccharomyces Cerevisiae: Contrasting Responses to Changes in the Proton Electrochemical Gradient
Authors: Eddy A. A.
Published: June 2009

The outflow of uracil from the yeast Saccharomyces cerevisiae is known to be relatively fast in certain circumstances, to be retarded by proton conductors and to occur in strains lacking a uracil proton symport. In the present work, it was shown that uracil exit from washed yeast cells is an active process, creating a uracil gradient of the order of -80 mV relative to the surrounding medium. Glucose accelerated uracil exit, while retarding its entry. DNP or sodium azide each lowered the gradient to about -30 mV, simultaneously increasing the rate of uracil entry. They also lowered cellular ATP content. Manipulation of the external ionic conditions governing Δμ;H+ at the plasma membrane had no detectable effect on uracil transport in yeast preparations thoroughly depleted of ATP. It was concluded that uracil exit is probably not driven by the proton gradient but may utilize ATP directly. It is known that thymine is not normally absorbed by yeast. However, thymine expulsion was here observed during deamination of the substrate 5-methylcytosine in the presence of glucose. In the absence of glucose, or following ATP depletion, thymine uptake from the medium only occurred when Δμ;H+ was dissipated, either by DNP or azide, or by manipulation of the external ionic environment. The yeast expelled absorbed thymine when Δμ;H+ was restored to the physiological range. The properties of the system corresponded to those of an H+/thymine antiport that is distinct from the mechanism expelling uracil.
Colonial Opacity Variations among the Choleragenic Vibrios
Authors: Finkelstein Richard A. , Boesman-Finkelstein Mary , Sengupta Dilip K. , Page William J. , Stanley C. Michael , Phillips Thomas E.
Published: June 2009

Cultures ofVibrio choleraeO1, biotype El Tor, from the current epidemic of cholera in the Western Hemisphere, and of the newV. choleraeserogroup O139, from the current outbreak in India and Bangladesh, revealed marked colonial heterogeneity when received by the authors. By comparison with reference colony types, using a stereoscope and transmitted oblique illumination, colonies of approximately 10 different degrees of opacity could be distinguished. In contrast, strains freshly isolated from patients and rapidly and carefully preserved were more homogeneous although still differentiable by this technique. These (and older) observations prompted the questions: (1) why is aV. choleraecolony opaque or translucent? and (2) what benefit is it to the vibrios to vary their colonial appearance? The observed changes in colonial opacity, which are reversible, are sometimes (rarely) accompanied by changes in virulence for infant rabbits and, more frequently, by other phenotypic variations including the ability to produce poly-β-hydroxybutyrate inclusion bodies on glycerol-containing medium, the degree of encapsulation in O139, changes in outer-membrane proteins, alteration in lipopolysaccharide structure, changes in expression of glycolytic pathways, and differences in ability to survive under adverse conditions. Colonial variations in choleragenic vibrios are phenotypically multifactorial. The genetic mechanisms(s) underlying the observed phenotypic changes remain to be defined.
Mycotic acid composition of Corynebacterium glutamicum and its cell surface mutants: effects of growth with glycine and isonicotinic acid hydrazide
Authors: Jang Ki-Hyo , Pierotti David , Kemp Gregory W. , Best Geoffrey R. , Britz Margaret L.
Published: June 2009

Auxotrophic mutants of Corynebacterium glutamicum strain ATCC 13059 (parent of AS019, a rifampicin-resistant variant), which were morphologically distinct from the parent and formed protoplasts more readily, had been isolated previously. Mutants MLB130-133 and MLB194 were more sensitive to growth inhibition by isonicotinic acid hydrazide (INH) and glycine, which caused branching and budding. Fatty acid and mycolic acid (MA) profiles were determined after growth in LBG (Luria broth plus glucose), LBG-glycine (LBG- and LBG-INH (LBG-I). The fatty acid profiles of all strains were similar, except that mutant MLB133 showed some increase in stearic acid (C18:0), normally a minor component, late in the growth cycle and oleic acid proportionately decreased. All strains had five major types of MAs (C32:0, C34:0, C34:1, C36:1, C36:2) but the relative proportion of each varied with the strain, age of culture and medium composition. Mutants MLB133 and MLB194 showed slightly higher levels of non-covalentiy bound MAs than the parent and normally showed a higher proportion of longer-chained, unsaturated MAs. The proportion of extracellular MAs increased with culture age for these mutants. Typically, by late stationary phase, mycolic acids in culture fluids increased to 6.5% of the total MAs for MLB194 and 7.9% for MLB133 compared with 3.5% for the parent strain grown in LBG. The main effect of glycine (2%, w/v) addition was to increase the proportion of mycolic acids found in extracellular fluids (16.1 % for AS019 and 31% for MLB133). The most significant effects of INH were seen when strains were cultured in LBG with 8 mg INH ml−1. When harvested at late stationary phase, strains MLB133 and MLB194 had 18.8% and 21.2% extracellular mycolic acids respectively, with a significant increase in the relative proportion of unsaturated mycolic acids. This effect was not as marked for AS019, which also showed a similar decrease in C32:0 relative to increases in the proportion of C34:1 and C36:2 plus a corresponding increase in the overall proportion of unsaturated mycolic acids and increased extracellular mycolates (8.5%). These results suggest that the mutations in strains MLB133 and MLB194 are associated with synthesis of specific mycolic acids (e.g. C32:0) and attachment of mycolic acids to the cell surface, both of which are likely target sites for glycine and INH action for cell-surface modifications. In addition to previously reported targeting of the peptidoglycan cross-linking, these results show that glycine affects mycolic acid attachment to the cell surface of C. glutamicum.
Dipeptidyl aminopeptidase processing and biosynthesis of alkaline extracellular protease from Yarrowia lipolytica
Authors: Matoba Sam , Morano Kevin A. , Klionsky Daniel J. , Kim Keunsung , Ogrydziak David M.
Published: June 2009

Alkaline extracellular protease (AEP) fromYarrowia lipolyticais synthesized as a precursor with a 157 aa prepro-region. Signal peptide cleavage was shown to occur after Ala15byN-terminal amino acid radiosequencing of the largest intracellular AEP precursor. AEP proteolytic activity was not required for AEP processing. After a change of the putative active site Ser to Ala, inactive AEP with the same mobility on SDS-PAGE as wild-type mature AEP was secreted. The role of dipeptidyl aminopeptidase (DPAPase) activity in AEP processing was also investigated. Mutations early in the -X-Ala- and -X-Pro- dipeptide stretch (Pro17to Met which should prevent DPAPase processing and Ala19to Val which should allow removal of only the first dipeptide) did not prevent synthesis of active mature AEP nor did use of the DPAPase inhibitor Pro-boroPro. Deletion of the entire dipeptide stretch (Ala16to Pro33) resulted in intracellular accumulation of an AEP precursor, which surprisingly was not glycosylated, and little or no secretion of AEP-related polypeptides. Expression of AEP in wild-type anddpp1 dap2 Saccharomyces cerevisiaestrains (lacking both the Golgi and vacuolar DPAPases) resulted in secretion of only mature AEP and no AEP precursors. Transit times and levels of AEP secretion were similar for both strains. These results indicate that theKEX2-like cleavage after Lys156-Arg157, which yields mature active AEP can occur in the absence of DPAPase processing and that DPAPase processing is not necessary for secretion of mature active AEP.
The Bacillus subtilis genome from gerBC (311°) to licR (334°)
Authors: Presecan E. , Moszer I. , Boursier L. , Ramos H.Cruz , de la Fuente V. , Hullo M.-F. , Lelong C. , Schleich S. , Sekowska A. , Song B. H. , Villani G. , Kunst F. , Danchin A. , Glaser P.
Published: June 2009

As part of the international project to sequence theBacillus subtilisgenome, the DNA region located betweengerBC(311°) andlicR(334°) was assigned to the Institut Pasteur. In this paper, the cloning and sequencing of 176 kb of DNA and the analysis of the sequence of the entire 271 kb region (6·5% of theB. subtilischromosome) is described; 273 putative coding sequences were identified. Although the complete genome sequences of seven other organisms (five bacteria, one archaeon and the yeastSaccharomyces cerevisiae) are available in public databases, 65 genes from this region of theB. subtilischromosome encode proteins without significant similarities to other known protein sequences. Among the 208 other genes, 115 have paralogues in the currently knownB. subtilisDNA sequences and the products of 178 genes were found to display similarities to protein sequences from public databases for which a function is known. Classification of these genes shows a high proportion of them to be involved in the adaptation to various growth conditions (non-essential cell wall constituents, catabolic and bioenergetic pathways); a small number of the genes are essential or encode anabolic enzymes.
Sequence and RFLP analysis of the elongation factor Tu gene used in differentiation and classification of phytoplasmas
Authors: Schneider Bernd , Gibb Karen S.
Published: June 2009

Primers designed from sequences of the gene encoding the elongation factor Tu (tuf gene) of several culturabie mollicutes amplified most of the tuf gene from phytoplasmas of the aster yellows, stolbur and X-disease groups. About 85% of the tuf gene from two aster yellows strains and a tomato stolbur phytoplasma was sequenced. The nucleotide sequence similarity between these related phytoplasmas was between 87·8 and 97·0%, whereas the homology with other mollicutes was 66·3-72·7%. The similarity of the deduced amino acid sequence was significantly higher, ranging from 96·0 to 99·4% among the phytoplasmas and 78·5% to 83·3% between phytoplasmas and the culturabie mollicutes examined. From the nucleotide sequences of the phytoplasma strains, two pairs of primers were designed; one amplified the phytoplasmas of most phylogenetic groups that were established, the other was specific for the aster yellows and stolbur groups. The phytoplasmas of the various groups that were amplified could be distinguished by RFLP analysis using Sau3AI, AluI and Hpall. The aster yellows group could be divided into five Sau3AI RFLP groups. These results showed that the tuf gene has the potential to be used to differentiate and classify phytoplasmas. Southern blot analysis revealed that the tuf gene is present as a single copy.
Streptomyces lividans groES, groEL1 and groEL2 genes
Authors: de León Patricia , Marco Sergio , Isiegas Carolina , Marina Anabel , Carrascosa José L. , Mellado Rafael P.
Published: June 2009

The Streptomyces lividans groES/ELI operon and groEL2 gene were cloned and their respective DNA sequences determined. The sequenced DNA comprised the genes and their respective regulatory regions in both cases. Transcription of both groES/EL1 and groEL2 seemed to be subjected to temporal control at 30 °C. At 45 °C the amount of the groEL2 transcript increased considerably in comparison to that of groES/EL1. Among the proteins synthesized under heat shock by S. lividans, a fraction enriched in GroEL2 showed the presence of a ring-shaped structure that resembles that of other chaperonins and was active in a rhodanase folding assay.
Characterization of the initiator tRNA gene locus and identification of a strong promoter from Mycobacterium tuberculosis
Authors: Vasanthakrishna M. , Kumar N. Vinay , Varshney U.
Published: June 2009

An initiator tRNA gene, metA, and a closely linked fragment of a second initiator-tRNA-like sequence, metB, from Mycobacterium tuberculosis H37Ra have been cloned and characterized. The promoter region of metA shows the presence of conserved sequence elements, TAGCCT and TTGGCG, with resemblance to -10 and -35 promoter regions. The deduced sequence of the mature tRNA contains the three unique features of the eubacterial initiator tRNAs represented by (i) a C:U mismatch at position 1:72, (ii) three consecutive base pairs, 29-31G:C39-41 in the anticodon stem, and (iii) a purine:pyrimidine (A:U) base pair at position 11:24 in the dihydrouridine stem. A putative hairpin structure consisting of an 11 bp stem and a three-base loop found in the 3′ flanking region is followed by a stretch of T residues and may serve as a transcription terminator. Analysis of the expression of metA and of its promoter using chloramphenicol acetyltransferase fusion constructs in Mycobacterium smegmatis shows that metA is a functional gene driven by a strong promoter. Furthermore, the overexpressed transcripts are fully processed and formylated in vivo. The metB clone shows the presence of sequences corresponding to those downstream of position 30 of the tRNA. However, the CCA sequence at the 3′ end has been mutated to CCG. Interestingly, the 3′ flanking sequences of both the genes are rich in GCT repeats. The metB locus also harbours a repeat element, IS6110. A method to prepare total RNA from mycobacteria (under acidic conditions) to analyse in vivo status of tRNAs is described.
A novel 27 kDa lipoprotein antigen from Mycobacterium bovis
Authors: Bigi F. , Espitia C. , Alito A. , Zumarraga M. , Romano M. I. , Cravero S. , Cataldi A.
Published: June 2009

A novelMycobacterium bovisantigen was identified from an expression library using sera from naturally infected cattle. TheEscherichia colirecombinant clone expressed a 27 kDa protein, named P27. A rabbit serum against the recombinant antigen recognized a protein of 27 kDa in cellular extracts fromM. bovisandM. tuberculosis.No protein was recognized in the culture supernatant. Sequence analysis indicated that P27 has a molecular mass of 24 kDa, showing a characteristic signal sequence for lipoprotein modification (a signal peptidase type II site). The gene is identical to a gene identified in theM. tuberculosisgenome sequencing project. Cellular fractionation experiments suggested that P27 is an integral membrane protein. The antigen was recognized by individual sera and peripheral blood mononuclear cells (PBMC) from diseased cattle. PCR experiments with specific primers directed to the P27 structural gene indicated that it is only present in theM. tuberculosisspecies complex. In conclusion, a novel immunogenic lipoprotein inM. bovis/M.tuberculosis has been identified. The results presented here and elsewhere suggest that mycobacterial lipoproteins should be considered in the design of new recombinant vaccines and diagnostic methods.
Enzymological and physiological consequences of restructuring the lipoyl domain content of the pyruvate dehydrogenase complex of Escherichia coli
Authors: Guest John R. , Attwood Margaret M. , Machado Rosane S. , Matqi Khalil Y. , Shaw John E. , Turner Sarah L.
Published: June 2009

The core-forming lipoate acetyltransferase (E2p) subunits of the pyruvate dehydrogenase (PDH) complex of Escherichia coli contain three tandemly repeated lipoyl domains although one lipoyl domain is apparently sufficient for full catalytic activity in vitro. Plasmids containing IPTG-inducible aceEF-IpdA operons which express multilip-PDH complexes bearing one N-terminal lipoyl domain and up to seven unlipoylated (mutant) domains per E2p chain, were constructed. Each plasmid restored the nutritional lesion of a strain lacking the PDH complex and expressed a sedimentable PDH complex, although the catalytic activities declined significantly as the number of unlipoylated domains increased above four per E2p chain. It was concluded that the extra domains protrude from the 24-meric E2p core without affecting assembly of the E1p and E3 subunits, and that the lipoyl cofactor bound to the outermost domain can participate successfully at each of the three types of active site in the assembled complex. Physiological studies with two series of isogenic strains expressing multilip-PDH complexes from modified chromosomal pdh operons (pdhR-aceEF-IpdA) showed that three lipoyl domains per E2p chain is optimal and that only the outermost domain need be lipoylated for optimal activity. It is concluded that the reason for retaining three lipoyl domains is to extend the reach of the outermost lipoyl cofactor rather than to provide extra cofactors for catalysis.
The recA gene from Clostridium perfringens is induced by methyl methanesulphonate and contains an upstream Cheo box
Authors: Johnston Joanne L. , Sloan Joan , Fyfe Janet A. M. , Davies John K. , Rood Julian I.
Published: June 2009

The recA gene from Clostridium perfringens was cloned using degenerate oligonucleotide primers designed from conserved regions of RecA proteins from other bacteria. The 1089 bp gene encoded a putative RecA protein with 69% amino acid sequence similarity to the RecA protein from Bacillus subtilis. The C. perfringens recA gene was induced by exposure to methyl methanesulphonate and complemented a recA mutant of Escherichia coli. A Cheo box was identified in the region upstream of the gene. Since this SOS-like operator site is conserved in many DNA-damage-inducible recA gene regions from Gram-positive bacteria, the results suggest that the regulation of the C. perfringens recA gene also involves the binding of a LexA-like protein to this site.
Influence of Bacillus subtilis phoR on cell wall anionic polymers
Authors: Müler Jörg P. , An Zhidong , Merad Tarek , Hancock Ian C. , Harwood Colin R.
Published: June 2009

In Bacillus subtilis the Pho regulon is controlled by a sensor and regulator protein pair, PhoR and PhoP, that respond to phosphate concentrations. To facilitate studies of the Pho regulon, a strain with an altered PhoR protein was isolated by in vitro mutagenesis. The mutation in this strain (phoR12) leads to the production of a PhoR sensor kinase that, unlike the wild-type, is functionally active in phosphate-replete conditions. The lesion in phoR12 was shown to be a single base change that results in an Arg to Ser substitution in a region of PhoR that is highly conserved in histidine sensor kinases. While a phoR-negative mutant was unable to induce the synthesis of cell wall teichuronic acid under phosphate-limited conditions, the phoR12 mutant showed a relative increase in teichuronic acid and a decrease in teichoic acid, even under phosphate-replete conditions. The latter suggests that some or all of the genes required for teichuronic acid synthesis are members of the Pho regulon.
The Bacillus subtilis L-arabinose (ara) operon: nucleotide sequence, genetic organization and expression
Authors: S-Nogueira Isabel , Nogueira Teresa V. , Soares Snia , de Lencastre Hermnia
Published: June 2009

The Bacillus subtilis L-arabinose metabolic genes araA, araB and araD, encoding L-arabinose isomerase, L-ribulokinase and L-ribulose-5-phosphate 4-epimerase, respectively, have been cloned previously and the products of araB and araD were shown to be functionally homologous to their Escherichia coli counterparts by complementation experiments. Here we report that araA, araB and araD, whose inactivation leads to an Ara- phenotype, are the first three ORFs of a nine cistron transcriptional unit with a total length of 11 kb. This operon, called ara, is located at about 256 on the B. subtilis genetic map and contains six new genes named araL, araM, araN, araP, araQ and abfA. Expression of the ara operon is directed by a strong sA-like promoter identified within a 150 bp DNA fragment upstream from the translation start site of araA. Analysis of the sequence of the ara operon showed that the putative products of araN, araP and araQ are homologous to bacterial components of binding-protein-dependent transport systems and abfA most probably encodes an a-L-arabinofuranosidase. The functions of araL and araM are unknown. An in vitro-constructed insertion-deletion mutation in the region downstream from araD allowed us to demonstrate that araL, araM, araN, araP, araQ and abfA are not essential for L-arabinose utilization. Studies with strains bearing transcriptional fusions of the operon to the E. coli lacZ gene revealed that expression from the ara promoter is induced by L-arabinose and repressed by glucose.
The trp RNA-binding attenuation protein (TRAP) regulates the steady-state levels of transcripts of the Bacillus subtilis folate operon
Authors: de Saizieu Antoine , Vankan Pierre , Vockler Cassandra , van Loon Adolphus P. G. M.
Published: June 2009

The Bacillus subtilis folate operon contains nine genes. The first six genes are involved in the biosynthesis of folic acid and tryptophan and have been characterized previously. The 3-region of the folate operon contains three additional ORFs: orf3, potentially encoding a DNA-binding protein of 68 amino acids, orf4, encoding a protein of 338 amino acids with homology to the Orf1 of the E. coli fis operon, and a putative lysyl-tRNA synthetase gene (lysS). Four transcripts were identified which encode the first two, eight or all nine proteins or only the last protein LysS. The folate operon contains two promoters, one upstream of the first gene and the second preceding lysS. Transcription of the entire folate operon starts 33 bp upstream of the ATG codon of pab, the first gene of the operon. The mtrB-encoded trp RNA-binding attenuation protein (TRAP) dramatically reduces the steady-state levels of the folate operon transcripts encoding the first eight and all nine proteins, but only has a relatively small effect on the steady-state level of the 2.1 kb transcript encoding the first two genes of the operon, pab and trpG. In addition, transcription of the folate operon is regulated in a growth-phase-dependent manner. Transcripts were present in very low levels after mid-exponential phase, but were dramatically increased directly after transfer of the cells to fresh medium. These results indicate that transcription of the folate operon is regulated by TRAP and also depends on the growth phase of the culture.
Expression of a Butyrivibrio fibrisolvens E14 gene (cinB) encoding an enzyme with cinnamoyl ester hydrolase activity is negatively regulated by the product of an adjacent gene (cinR)
Authors: Dalrymple Brian P. , Swadling Yolande
Published: June 2009

A second cinnamoyl ester hydrolase (CEH) encoding gene (cinB) has been characterized from the ruminal bacterium Butyrivibrio fibrisolvens E14. CinB is more similar to CinA (previously named Cinl) (28% amino acid identity), the first CEH described from B. fibrisolvens E14, than either of the enzymes are to any other member of the family of hydrolases to which they belong. Upstream of cinB, and in the opposite orientation, is a gene (cinR) encoding a protein with substantial similarity to members of the MarR family of negative regulators of bacterial gene expression. By alignment of these sequences, a possible helix-turn-helix DNA-binding domain has been identified. CinR was expressed at a high level in Escherichia coli using the lac promoter. In E. coli CinR repressed the expression of CinB, but had no effect on the expression of CinA. In gel mobility-shift assays, CinR bound specifically to the cinR-cinB intergenic region. Two identical 16 nucleotide inverted repeats adjacent to the putative PcinR and PcinB promoters are likely binding sites for CinR. The addition of FAXX (O-[5-O-(trans-feruloyl)-α-L-arabinofuranosyl]-(1,3)-O-ß-D-xylopyranosyl-(1,4)-D-xylopyranose) and Fara [5-O-(trans-feruloyl)-arabinofuranose], but not xylobiose, ferulic acid and a number of other soluble components of hemicellulose, inhibited the binding of CinR to DNA.
Characterization and overexpression of the Aspergillus niger gene encoding the cAMP-dependent protein kinase catalytic subunit
Authors: Bencina Mojca , Panneman Henk , Ruijter George J. G. , Legiša Matic , Visser Jaap
Published: June 2009

The genepkaCencoding the catalytic subunit of cAMP-dependent protein kinase has been isolated from the industrially important filamentous fungusAspergillus niger.A probe for screeningA. nigerphage libraries was generated by a polymerase chain reaction using degenerate primers. cDNA and genomic DNA clones were isolated and sequenced. An open reading frame of 1440 bp, interrupted by three short introns, encodes a polypeptide of 480 amino acids with a calculated molecular mass of 53813 Da. The cAMP-dependent protein kinase catalytic subunit (PKA-C) fromA. nigerhas a 126 amino acid extension at the N-terminus compared to the PKA-C of higher eukaryotes that - except for the first 15 amino acids, which are homologous to theMagnaporthe griseaPKA-C - shows no significant similarity to the N-terminal extension of PKA-C of other lower eukaryotes. The catalytic core of PKA-C of A.nigershows extensive homology with the PKA-C isolated from all other eukaryotes. Low-stringency hybridization did not reveal any otherpkaChomologue inA. niger.The clonedpkaCwas used for transformation ofA. niger,leading to increased levels ofpkaCmRNA and PKA-C activity. Transformants overexpressingpkaCwere phenotypically different with respect to growth, showing a more compact colony morphology, accompanied by a more dense sporulation, especially on media containing trehalose and glycerol. A number of transformants also showed a strongly reduced or complete absence of sporulation. This phenotype was quickly lost upon propagation of the strains.
A 23 911 bp region of the Bacillus subtilis genome comprising genes located upstream and downstream of the Iev operon
Authors: Parro Víctor , Román Marta San , Galindo Inmaculada , Purnelle Bénédicte , Bolotin Alexander , Sorokin Alexei , Mellado Rafael P.
Published: June 2009

Within the framework of the European project to sequence the whole Bacillus subtilis 168 genome, a 23 911 bp long chromosomal DNA fragment located around 233° on the B. subtilis genetic map was cloned and sequenced. From the generated sequencing data and the results of the homology search, the primary structure of this region was determined. In addition to the whole Iev operon, the region contains putative genes for an amino acid permease, two different alcohol dehydrogenases, a chitosanase, a protein belonging to the LysR family of transcriptional regulators, a protein related to the MerR transcriptional regulator, up to four proteins related to the product of the spoF gene, and genes coding for nine more inferred proteins of unknown function.
Variation within serovars of Neisseria gonorrhoeae detected by structural analysis of outer-membrane protein PIB and by pulsed-field gel electrophoresis
Authors: Cooke Susan J. , de la Paz Helen , La Poh Chit , Ison Catherine A. , Heckels John E.
Published: June 2009

Outer-membrane protein PI is the antigen responsible for serovar specificity of Neisseria gonorrhoeae and is a potential vaccine target. In order to investigate possible hidden variation within a serovar, the sequence of the por genes encoding protein PIB have been obtained from a series of strains, including isolates known to be epidemiologically linked. The inferred amino acid sequences of the PIB molecules of isolates from known sexual contacts were identical, but non-related isolates showed significant heterogeneity in PIB sequence. These differences were not confined to the two variable regions (Var1 and Var2) which have previously been identified, but were largely, although not exclusively, located in regions predicted to form one of eight surface-exposed loops. The isolates were subjected to pulsed-field gel electrophoresis of restriction digests of chromosomal DNA, which also demonstrated identity between linked strains but revealed diversity within a serovar. The deduced amino acid sequences of PIB were also used to synthesize peptides for epitope-mapping experiments. These revealed that some mAbs, used to define serovar specificity, recognized linear epitopes located in loops 5 and 6, while others appeared to recognize conformational epitopes elsewhere in the molecule. The occurrence of the sequence differences within a serovar, which are not detected by the serotyping reagents, reveals that PIB represents a potential source of information which should permit considerably more detailed epidemiological studies than are currently possible and focuses attention on more conserved regions of the protein as potential targets for vaccination.
Homologous regions of the Salmonella enteritidis virulence plasmid and the chromosome of Salmonella typhi encode thiol: disulphide oxidoreductases belonging to the DsbA thioredoxin family
Authors: Zehr Jonathan P. , Mellon Mark T. , Hiorns William D.
Published: June 2009

DNA sequences of a fragment of nifH from diverse cyanobacteria were amplified, cloned and sequenced to determine the evolutionary relationship of nitrogenase within the cyanobacteria as a group, and to provide a basis for the identification of uncultivated strains of cyanobacteria in the environment. Analysis of 30 nitrogenase DNA and deduced amino acid sequences from cyanobacteria representing five major taxonomic subdivisions showed great variation in phylogenetic distances between the sequences. Sequences from heterocystous cyanobacteria formed a coherent cluster, in which branching forms did not form a clade distinct from the non-branching forms. Nitrogenase sequences from the unicellular cyanobacteria Gloeothece and Synechococcus sp. RF-1 formed a cluster, as did sequences from the genera Xenococcus and Myxosarcina. The nifH sequences of filamentous nonheterocystous cyanobacteria were not closely related to each other, forming deep branches with respect to the heterocystous cyanobacterial nifH sequences. The phylogeny of nifH based on amino acid sequences was consistent with taxonomic relationships among the strains; for example, a sequence obtained from a natural assemblage believed to be dominated by ‘Lyngbya’ clustered with nifH from Lyngbya lagerheimii. Results also indicate that the phylogeny of nifH among the cyanobacteria is largely consistent with the phylogeny of 16S rRNA, and furthermore that the nifH sequence can be used to identify uncultivated strains of nitrogen-fixing cyanobacteria.
Brucella abortus strain 2308 putative glucose and galactose transporter gene: cloning and characterization
Authors: Essenberg Richard C. , Candler Chris , Nida S. Kay
Published: June 2009

The gene for the putative transporter for glucose and galactose from Brucella abortus strain 2308 was isolated by functional complementation of Escherichia coli strains lacking either glucose or galactose transport systems. The same two plasmid clones were isolated from each screen. These clones restored glucose and galactose transport to the respective E. coli strains. The sequence of the 1806 bp overlap between these two plasmids was determined. A 1242 bp ORF whose disruption eliminated complementation of both E. coli strains showed 36% identity with the E. coli fucP gene encoding a fucose transporter. These two transporters are members of the major facilitator superfamily, in which they represent a previously undescribed family. In addition, an incomplete gene similar to E. coli hisG was found. One of the plasmids complemented E. coli hisG mutants.
Cloning of a protopectinase gene of Trichosporon penicillatum and its expression in Saccharomyces cerevisiae
Authors: Iguch Ken-ichi , Hirano Haruka , Kishida Masao , Kawasaki Haruhiko , Sakai Takuo
Published: June 2009

A protopectinase (PPase)-encoding gene, PSE3, from Trichosporon penicillatum was cloned by colony hybridization using two oligonucleotide probes synthesized from the N-terminal amino acid sequences of native PPase SE1 and one peptide from a lysyl endopeptidase digest. Nucleotide sequencing revealed that PSE3 contains an ORF encoding a 367 amino acid protein. Mature PPase SE3 is composed of 340 amino acids and the N-terminus of the ORF appeared to correspond to a signal peptide and a propeptide processed by a KEX2-like proteinase. The deduced amino acid sequence of PSE3 was 65.4, 56.7, 58.1, 61.8 and 48.9% homologous to the polygalacturonases of Aspergillus oryzae, Aspergillus niger, Aspergillus tubigensis, Cochliobolus carbonum and Fusarium moniliforme, respectively. One domain, which might interact with polygalacturonic acid, is highly conserved not only in fungal polygalacturonases but also in bacterial and plant polygalacturonases. PSE3 was expressed in Saccharomyces cerevisiae, but three forms (the mature form, a glycosylated form and an uncharacterized processed form) of PPase SE3 were present among the PSE3 products.
Near-UV-induced absorbance change and photochemical decomposition of ergosterol in the plasma membrane of the yeast Saccharomyces cerevisiae
Authors: Arami Shin-ichiro , Hada Megumi , Tada Mikiro
Published: June 2009

When cells of the yeast Saccharomyces cerevisiae were exposed to near-UV (300-400 nm), their absorption spectra changed slightly within the range 220-300 nm with increasing dosage. Difference spectra, calculated by subtracting the curve recorded in cells exposed to near-UV from the curve of unexposed cells, decreased with increasing dosage over a broad band with peaks at 272, 282 and 295 nm and a shoulder at 265 nm. These peaks were in agreement with the absorption maxima of ergosterol, which is one of the major components of the plasma membrane of yeast. Near-UV radiation induced a simultaneous decrease in absorption spectra and reduction of ergosterol content in the plasma membrane. Photochemical decomposition of ergosterol by near-UV radiation was revealed in vivo, although ergosterol is generally known to be photoconverted to previtamin D2 industrially by UV radiation in vitro. In order to remove photosensitizers, liposomes were prepared from phospholipids and glycolipids, with or without ergosterol from purified yeast plasma membranes. Liposomal ergosterol in the orientated state was photochemically decomposed by near-UV radiation but ergosterol in the disorientated state in a homogeneous solution was not. Near-UV radiation also induced a decrease in activity of membrane-bound ATPase. Dose-response curves for the reduction of ATPase activity were similar to that for decomposition of ergosterol, suggesting that near-UV caused membrane function damage.
β-Glucosylated proteins in the cell wall of the black yeast Exophiala (Wangiella) dermatitidis
Authors: Montijn Roy C. , Van Wolven Patricia , De Hoog Sybren , Klis Frans M.
Published: June 2009

Wild-type cells of the pathogenic black yeast Exophiala (Wangiella) dermatitidis grown in a low-pH ascorbate medium became less melanized and less resistant to Zymolyase. This was accompanied by increased staining with fluorescently labelled concanavalin A. The sugar composition of wild-type and mutant cell walls was, except for the presence of galactose, similar to that of Saccharomyces cerevisiae. Digestion of mutant cell walls with laminarinase released galactomannoproteins. In addition, the released cell wall proteins contained glucose and reacted with affinity-purified 1,6-β-glucan antiserum, indicating that they are linked to 1,6-β-glucan. It is proposed that 1,6-β-glucosylated cell wall proteins generally occur among ascomycetes.