bluuebunny/crossref_metadata_2025 huggingface dataset:
Classification and identification of bacteria by Fourier-transform infrared spectroscopy
Authors: Helm D. , Labischinski H. , Schallehn Gisela , Naumann D.
Published: June 2009

This study describes a computer-based technique for classifying and identifying bacterial samples using Fourier-transform infrared spectroscopy (FT-IR) patterns. Classification schemes were tested for selected series of bacterial strains and species from a variety of different genera. Dissimilarities between bacterial IR spectra were calculated using modified correlation coefficients. Dissimilarity matrices were used for cluster analysis, which yielded dendrograms broadly equated with conventional taxonomic classification schemes. Analyses were performed with selected strains of the taxa Staphylococcus, Streptococcus, Clostridium, Legionella and Escherichia coli in particular, and with a database containing 139 bacterial reference spectra. The latter covered a wide range of Gram-negative and Gram-positive bacteria. Unknown specimens could be identified when included in an established cluster analysis. Thirty-six clinical isolates of Staphylococcus aureus and 24 of Streptococcus faecalis were tested and all were assigned to the correct species cluster. It is concluded that: (1) FT-IR patterns can be used to type bacteria; (2) FT-IR provides data which can be treated such that classifications are similar and/or complementary to conventional classification schemes; and (3) FT-IR can be used as an easy and safe method for the rapid identification of clinical isolates.
Purification and characterization of S layer proteins from Clostridium difficile GAI 0714
Authors: Takeoka Aya , Takumi Kenji , Koga Tetsuro , Kawata Tomio
Published: June 2009

The Slayer of Clostridium difficile GAI 0714 was shown to be composed of two proteins, of 32 kDa and 45 kDa, as determined by SDS-PAGE. The two proteins were extracted with 8 M-urea (pH 8·3) from a cell wall preparation and purified by DEAE-Sepharose CL-6B chromatography followed by HPLC gel filtration. When solubilized in 0·1 M-urea, both proteins appeared to exhibit dimeric forms, with respective molecular masses of about 61 kDa and 99 kDa, upon HPLC. Although the amino acid compositions of the two proteins differed from each other, both proteins had a high content of acidic amino acids, very low contents of histidine and methionine, and no cysteine. The 32 kDa protein exhibited multiple isoelectric forms (pI 3·7-3·9), whereas the 45 kDa protein had a single form (pI 3·3). Radioiodination and immunogold labelling revealed that both proteins were exposed evenly over the entire cell surface. Based on immunodiffusion analysis using monospecific antiserum raised to the individual proteins, there was no antigenic relationship between the two proteins. Furthermore, immunoblot analysis showed that the antigenicity of the 32 kDa protein appeared to be strain specific, whereas that of the 45 kDa protein appeared to be group specific.
recA-dependence of the response of Pseudomonas aeruginosa to UVA and UVB irradiation
Authors: Kidambi Saranga P. , Booth Melissa G. , Kokjohn Tyler A. , Miller Robert V.
Published: June 2009

The responses of the autochthonous soil and aquatic organism, Pseudomonas aeruginosa to UV radiation wavelengths (UVA, 320-400 nm, and UVB, 280-320 nm) has been investigated in this study. P. aeruginosa recA mutants were found to be more sensitive to both UVA and UVB radiation than were their isogenic RecA+ parents. Introduction of a low-copy-number plasmid containing the cloned wild-type P. aeruginosa recA gene restored UVA and UVB resistance to recA mutants. The concentration of RecA protein increased twofold 120 min after exposure to either UVA or UVB radiation, suggesting induction of expression of the recA gene by these wavelengths. In this study, we found that a functional RecA protein is required for activation of D3 prophage in lysogenic cells following exposure to UVB radiation. Prophage were not induced by exposure of their hosts to UVA radiation. Induction of damage-inducible (din) genes in response to UVA or UVB irradiation was also shown to be RecA dependent. These data indicate that the recA gene plays a role in the response of P. aeruginosa to exposure to wavelengths of UV radiation found in the solar spectrum.
Dissimilatory iron(III) reduction by Rhodobacter capsulatus
Authors: Dobbin Paul S. , Warren Louise H. , Cook Nicola J. , McEwan Alastair G. , Powell Anne K. , Richardson David J.
Published: June 2009

The photosynthetic proteobacterium
Rhodobacter capsulatus
was shown to be capable of dissimilatory Fe(III) reduction. Activity was expressed during anaerobic phototrophic and microaerobic growth with malate as the carbon source, but not during equivalent aerobic growth. A variety of Fe(III) complexes were demonstrated to act as substrates for intact cells and membrane fractions of strain N22DNAR+ using a ferrozine assay for Fe(II) formation. Rates of reduction appeared to be influenced by the reduction potentials of the Fe(III) complexes. However, Fe(III) complexed by citrate, which is readily reduced by Shewanella putrefaciens, was a poor substrate for dissimilation by R. capsulatus. The Fe(III)-reducing activity of R. capsulatus was located solely in the membrane fraction. The reduction of Fe(III) complexes by intact cells was inhibited by 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO), suggesting the involvement of ubiquinol: cytochrome c oxidoreductases in the electron transport chain. Lack of sensitivity to myxothiazol plus data from mutant strains implies that the cytochrome bc
1 complex and cytochrome c
2 are not obligatory for dissimilation of Fe(III)(maltol)3. Alternative pathways of electron transfer to Fe(III) must hence operate in R. capsulatus. Using strain N22DNAR+, the reduction rate of Fe(III) complexed by nitrilotriacetic acid (NTA) was elevated compared to that of Fe(III)(maltol)3, and moreover was sensitive to myxothiazol. However, these differences were not observed in the absence of the electron donor malate. The governing factor for the reduction rate of Fe(III)(maltol)3 thus appears to be the limited Fe(III)-reducing activity, whilst the reduction rate of Fe(III) complexed by NTA is controlled by the flux of electrons through the respiratory chain. The use of mutant strains confirmed that the role of the cytochrome bc
1 complex in Fe(III) reduction becomes apparent only with the superior substrate. The energy-conserving nature of Fe(III) reduction by R. capsulatus was demonstrated by electrochromic measurements, with the endogenous carotenoid pigments being employed as indicators of membrane potential generation in intact cells. Using Fe(III)EDTA as electron acceptor, periods of membrane potential generation were directly proportional to the quantity of complex added, and were extended in the presence of HQNO. Fe(III)-dependent carotenoid bandshifts were abolished by addition of the protonophoric uncoupler carbonyl cyanide p-trifluoromethoxy-phenylhydrazone.
Acidimicrobium ferrooxidans gen. nov., sp. nov.: mixed-culture ferrous iron oxidation with Sulfobacillus species
Authors: Clark Darren A. , Norris Paul R.
Published: June 2009

A new species of ferrous-iron-oxidizing, moderately thermophilic, acidophilic bacteria, Acidimicrobium ferrooxidans, has been described. Two isolates of the species differed only in the tendency of one, previously known as strain TH3, to grow in filaments. The chromosomal DNA base composition is between 67 and 69 mol% G + C. The capacity of this species to fix CO2 from air was greater than that of iron-oxidizing thermoacidophiles of the genus Sulfobacillus, which required an enhanced CO2 concentration for optimum autotrophic growth. Under air, ferrous iron oxidation in mixed cultures of A. ferrooxidans with either Sulfobacillus thermosulfidooxidans or Sulfobacillus acidophilus was more extensive than in pure cultures of these three strains. The greater part of ferrous iron oxidation in mixed cultures probably resulted from activity of the Sulfobacillus species, which possess a greater tolerance of ferric iron, and which presumably grew mixotrophically utilizing organic compounds from A. ferrooxidans.
Primary metabolite kinetics of bacteriocin biosynthesis by Lactobacillus amylovorus and evidence for stimulation of bacteriocin production under unfavourable growth conditions
Authors: De Vuyst Luc , Callewaert Raf , Crabbé Kurt
Published: June 2009

To optimize bacteriocin production processes, the relationships between growth, bacteriocin production and factors affecting the occurrence and intensity of the activity peak during the growth cycle must be understood. Amylovorin L471, a bacteriocin produced by
Lactobacillus amylovorus
DCE 471, displays primary metabolite kinetics with a peak activity during the midexponential phase. Because of this growth association, only conditions favouring a drastic increase in biomass improve the volumetric bacteriocin titre. Specific bacteriocin production is enhanced under unfavourable growth conditions such as low temperatures (30°), and the presence of potentially toxic compounds such as ethanol (1.0%, v/v) and oxygen (80%, v/v, air saturation). Whereas volumetric biomass formation and growth-associated bacteriocin production are dependent on the amount of glucose and nitrogen supplied, slow growth rates stimulate specific bacteriocin production. Bacteriocin inactivation can be ascribed to protein aggregation and adsorption phenomena. It may be overcome by switching the pH to 2.0 during the fermentation run after having reached the peak activity. Thus, manipulation of the cell environment can stimulate bacteriocin production. The latter can be induced by unfavourable growth conditions, so-called stress factors. The specific growth rate seems to play an important role in the control of bacteriocin production.
Direct evidence for the involvement of extracellular proteins in the adhesion of Azospirillum brasilense
Authors: Dufrěne Yves F. , Vermeiren Hans , Vanderleyden Jos , Rouxhet Paul G.
Published: June 2009

Adhesion of Azospirillum brasilense to glass and polystyrene was investigated by bringing the cells into contact with the support by sedimentation. Adhesion depended on time and temperature: lower adhesion densities were observed when the contact time was only 2 h or 6 h, as compared to 24 h, or when the test was performed at 4 -C, as compared to 30 °. The influence of cell physiology was further demonstrated by the effect of tetracycline, which inhibited adhesion. Scanning electron microscopy showed that cells produced extracellular material when left in contact with a support for 24 h. The surface elemental composition of cells and of polystyrene supports after cell adhesion and subsequent detachment was determined by X-ray photoelectron spectroscopy; this provided information on the relative concentrations of proteins and polysaccharides at the surface. The protein concentration at the surface of a cell sediment increased as a function of time at 30 °, correlating with an increase of adhesion density. A similar correlation between protein concentration and adhesion density was found when comparing exponentialphase cells with stationary-phase cells. The surface composition of polystyrene supports examined after cell detachment was found to be rich in proteins, indicating that proteins are the major constituent at the support surface. Lowering the contact time, or performing adhesion under unfavourable metabolic conditions (4 °) or in the presence of tetracycline, resulted in a decrease in protein concentration at the support surface, which was correlated with a decrease in adhesion density. The correlation between protein concentration at the cell surface or at the support surface and adhesion density, under different experimental conditions, provides a direct demonstration of the involvement of extracellular proteins in the adhesion of A. brasilense to inert surfaces.
The Azotobacter vinelandii gene algJ encodes an outer-membrane protein presumably involved in export of alginate
Authors: Rehm Bernd H. A.
Published: June 2009

The algJ gene from Azotobacter vinelandii was cloned using a labelled RNA probe representing the coding region of the algE gene from Pseudomonas aeruginosa. DNA sequencing revealed an ORF of 1452 bp encoding a protein of 484 amino acid residues with a calculated molecular mass of 54611 Da. An RNA probe corresponding to algE was also used for Southern hybridization of chromosomal DNA, which showed that algE-related DNA sequences are also present in the alginate-producing phytopathogen species Pseudomonas marginalis and Pseudomonas syringae pv. glycinea. The coding region of algJ was subcloned in the expression vector pT7-7, leading to a corresponding gene product with an apparent molecular mass of 54 kDa which could be identified in the outer membrane (OM) of
Escherichia coli
BL21(DE3). Additionally, a cross-reacting protein with the same molecular mass was also found in the OM of A. vinelandii using an anti-AlgE antiserum. The derived amino acid sequence of AlgJ shared approximately 52% identity with AlgE from P. aeruginosa. The hydrophilicity profile as well as the amphipathicity of regions in the amino acid sequence of AlgJ showed significant similarities to AlgE. Based on these data, a topological model of AlgJ was created with the aid of known structures of outer-membrane proteins. This model presents AlgJ as a ß-barrel containing 18 ß-strands inserted in the OM.
Rickettsia prowazekii sigma factor σ73 can be overexpressed in Escherichia coli and promotes RNA polymerase binding and transcription
Authors: Aniskovitch Lyudmila P. , Winkler Herbert H.
Published: June 2009

The sigma factor σ73 of the obligate intracytoplasmic bacterium Rickettsia prowazekii was overexpressed and purified from
Escherichia coli
. The rickettsial rpoD gene encoding σ73 was cloned into a Ndel-BamHI-cleaved pET15b vector under control of T7 transcription and translation signals. The recombinant plasmid encoded a 75 kDa fusion protein that was overproduced in E. coli BL21(DE3) and purified from inclusion bodies after solubilization with guanidine hydrochloride and using His. Bind metal chelation resin. The N-terminal His. Tag sequence of the 75 kDa fusion protein was removed by thrombin treatment to obtain R. prowazekii σ73T. The R. prowazekii σ73T as well as the 75 kDa fusion protein had the ability to bind to core DNA-dependent RNA polymerase of both R. prowazekii and E. coli and to stimulate their interaction with a rickettsial promoter.
Stress tolerance and pathogenic potential of a mannitol mutant of Cryptococcus neoformans
Authors: Chaturvedi Vishnu , Flynn Timothy , Niehaus Walter G. , Wong Brian
Published: June 2009

Cryptococcus neoformans produces large amounts of the acyclic hexitol mannitol in culture and infected animals, but the functional and pathogenic significance of mannitol production by this fungus is not known. We exposed C. neoformans H99 (Cn H99) to UV irradiation (1 × LD50) and screened survivors for mannitol production. A mutant, Cn MLP (Mannitol Low Producer), synthesized less mannitol from glucose (2.7 vs 8.2 nmol per 108 cells min−1 at 37 °) and contained less intracellular mannitol (1 vs 11 μmol per 106 cells at 37 °) than did Cn H99. Cn MLP and Cn H99 were similar with respect to carbon assimilation patterns, rates of glucose consumption, growth rates at 30 °, urease and phenoloxidase activities, morphology, capsule formation, mating type, electrophoretic karyotype, rapid amplification of polymorphic DNA (RAPD) patterns and antifungal susceptibility. However, Cn MLP was more susceptible than was Cn H99 to growth inhibition and killing by heat and high NaCl concentrations. Also, the LD50 values in mice injected intravenously were 3.7 × 106 c.f.u. for Cn MLP compared to 6.9 × 102 c.f.u. for Cn H99. Moreover, 500 c.f.u. Cn H99 intravenously killed 12 of 12 mice by 60 d, whereas all mice given the same inoculum of Cn MLP survived. Classical genetic studies were undertaken to determine if these differences were due to a single mutation, but the basidiospores were nonviable. These results suggest that the abilities of C. neoformans to produce and accumulate mannitol may influence its tolerance to heat and osmotic stresses and its pathogenicity in mice.
xyIUW, Two Genes at the Start of the upper Pathway Operon of TOL Plasmid Pwwo, Appear to Play no Essential Part in Determining its Catabolic Phenotype
Authors: Williams Peter A. , Shaw Linda M. , Pitt Christopher W. , Vrecl Milka
Published: June 2009

The upper pathway operon of the toluene catabolic pathway of TOL plasmid pWW0 was shown to carry two open reading frames between the start of transcription and xylC (encoding benzaldehyde dehydrogenase), the first previously reported gene of the operon. These were designated xyIUW: xyIU encoded a protein of 131 amino acid residues (Mr
14244) which bore no relationship with any protein in the databases, and xyIW encoded a protein of 348 residues (Mr
36992) which was strongly homologous to other long-chain Zn-containing alcohol dehydrogenases. Extracts of Escherichia coli carrying xyIUW in expression vector pTrc99A contained a novel protein corresponding to XyIW, but no NAD+ -dependent dehydrogenase activity against benzyl alcohol, mandelate or benzylamine. A mini-Tn5 transposon carrying the meta pathway operon was constructed and from it two strains of Pseudomonas putida were constructed with the normally plasmid-encoded catabolic operons integrated into the chromosome. Three derivatives of plasmid pKNG101 containing modified xyIUW genes were constructed, two of which had frameshifts in xyIU and xyIW, respectively, and a third with a deletion from the 3′ end of xyIU into the 5′ end of xyIW. The wild-type genes of the two Pseudomonas strains were substituted by the mutant alleles by reverse genetics. The ability of the constructed mutant strains to utilize the aromatic substrates of the TOL pathway was not significantly affected.
An iron-Regulated Outer-Membrane Protein Specific to Bordetella Bronchiseptica and Homologous to Ferric Siderophore Receptors
Authors: Beall Bernard , Hoenes Theresa
Published: June 2009

ThebfrA (Bordetella bronchisepticaferric iron repressed outer-membrane protein) gene was cloned fromBordetella bronchisepticaby screening a library of TnphoAinsertion mutants for iron-repressed fusions tophoA.ThebfrAgene encoded an 80 kDa outer-membrane protein with a high level of amino acid sequence identity to several bacterial proteins belonging to the family of Ton B-dependent outer-membrane receptors. BfrA was especially homologous to Cir ofEscherichia coli, IrgA ofVibrio choleraeand to three previously characterized ferric enterobactin receptors. DNA hybridization results indicated thatbfrAwas not present in otherBordetellaspecies. Expression of thebfrAgene was induced by low iron availability from a promoter overlapped by a sequence resembling a consensus Fur-binding sequence, andbfrAexpression was derepressed in aB. bronchiseptica furmutant. Utilization of theBordetellasiderophore alcaligin and the exogenous siderophore enterobactin was unaffected inbfrAmutants. Upon attempting to find the specificity of BfrA, 2,3-dihydroxybenzoylserine (DHBS) was shown to be utilized in abfeA (Bordetellaferric enterobactin receptor gene)-dependent manner byB. bronchisepticaandB. pertussis.In addition, the hydroxamate siderophores ferrichrome and desferrioxamine B, and the iron source haemin were shown to be utilized independently ofbfeAandbfrAinB. bronchisepticaandB. pertussis.
Enterobactin Synthase Polypeptides of Escherichia Coli are Present in an Osmotic-Shock-Sensitive Cytoplasmic Locality
Authors: Hantash Feras M. , Ammerlaan Marcus , Earhart Charles F.
Published: June 2009

The terminal reactions in the synthesis of the siderophore enterobactin (Ent) by Escherichia coli require the EntD, E, F and B/G polypeptides. The idea that these molecules form a complex (Ent synthase) that is membrane-associated was re-evaluated. In vitro results provided no evidence in support of the proposal: (i) Ent synthase activity occurred normally under conditions where membrane was either absent or disrupted by high concentrations of neutral detergents, and (ii) immunoprecipitation experiments conducted on extracts engaged in Ent synthesis failed to detect any association among the Ent polypeptides. However, Western blot analyses showed that EntE, F and B/G were released from cells by osmotic shock and freeze/thaw treatment but not by conversion of cells to spheroplasts. These results demonstrated that EntE, F and B/G belong to the Beacham group D class of proteins. The shockability of a given group D Ent protein was unaffected by the absence of either EntB/G or EntD and, for EntB/G, the N-terminus was sufficient for release by osmotic shock. The behaviour of group D proteins is generally attributed to their association (partial, loose or transient) with cytoplasmic membrane; therefore, the results are indirect evidence that Ent synthase interacts with membrane in vivo. At the very least, the data indicate that EntE, F and B/G are compartmentalized in E. coli and, because other biosynthetic enzymes for siderophores and surfactants are related to these Ent proteins, suggest that this entire protein class may be sequestered in vivo.
Use of Morphology Index Histograms to Quantify Populations of the Fungal Pathogen Paracoccidioides Brasiliensis
Authors: San-Blas Gioconda , Padr�n Ra�l , Alamo Lorenzo , San-Blas Felipe
Published: June 2009

To quantify the dimorphic process in wild and mutant strains of Paracoccidioides brasiliensis, we defined a morphology index (Mi) in terms of the maximum cell length (I), maximum cell diameter (d), and septal diameter (s), according to the equation Mi = 2.13 + 1.13 log10(Is/d
2), whose intercept and slope were such that Mi was around 1 for yeast (spherical) cells or 4 for hyphal (elongated) cells. This discriminatory power was used to quantify morphological population mixtures through Mi histograms. During the temperature-induced dimorphic transition (either way), mean Mi (Mi) varied linearly with time, suggesting a continuity in the process. Also, in wild strains and mutants thereof we found an inverse relationship between Mi and content of both cell wall chitin and 1,3-a-glucan.
Monoclonal Antibodies Against Streptococcus Pneumoniae Detect Epitopes on Eubacterial Ribosomal Proteins L7/L12 and on Streptococcal Elongation Factor Ts
Authors: Kolberg Jan , Høiby E. Arne , Lopez Rodrigo , Sletten Knut
Published: June 2009

Two monoclonal antibodies (mAbs) designated 144,H-3 (lgG2a) and 218,C-5 (lgM) were produced after immunization of mice with two different heattreated and sonicated pneumococcal strains. Western blotting, with solubilized proteins from different bacterial genera and from mammalian lymphocytes, showed that both mAbs reacted with a protein of approximately 12 kDa in all 66 strains of eubacteria examined, representing 27 different species. The 12 kDa protein was isolated by immunoaffinity chromatography. Subsequent preparative Western blotting enabled N-terminal amino acid sequence analysis by microsequencing. A high degree of amino acid sequence similarity with eubacterial ribosomal proteins L7/L12 was demonstrated. One of the mAbs (144,H-3) also cross-reacted in Western blotting with a 43 kDa protein, but only from streptococci. The 43 kDa protein carrying the common streptococcal epitope was isolated and sequenced in the N-terminal region. A high degree of amino acid sequence identity was found to elongation factor Ts from Escherichia coli.
Thioredoxin is Essential for Rhodobacter Sphaeroides Growth by Aerobic and Anaerobic Respiration
Authors: Pasternak Cécile , Assemat Karine , Clément-Métral Jenny D. , Klug Gabriele
Published: June 2009

To investigate the biological role of thioredoxin in the facultative photosynthetic bacterium Rhodobacter sphaeroides, attempts were made to construct a thioredoxin-deficient mutant by site-specific mutagenesis, using the Tn903 kanamycin resistance gene for selection. In situ and Southern hybridization analyses have demonstrated that the TrxA-mutation is lethal for R. sphaeroides growth under anaerobic conditions with DMSO as terminal electron acceptor and under aerobic conditions. In addition, the DNA region upstream of the trxA initiation codon is essential for aerobic growth of R. sphaeroides. An ORF of unknown function was identified in this region and is suggested to encode a product essential for aerobic metabolism of R. sphaeroides. The mechanism of thioredoxin action was also analysed by using the procedure for gene replacement to introduce a Cys33 to Ser mutation into the trxA chromosomal copy. The strain carrying this mutation produced a thioredoxin impaired in its protein-disulfide reductase activity and was also not viable. These data suggest that the physiological function of R. sphaeroides thioredoxin is redox-dependent. Thioredoxin purified from R. sphaeroides was shown to have a glutathione-disulfide oxidoreductase activity typical of glutaredoxins. This unexpected finding suggests that R. sphaeroides thioredoxin, in contrast to Escherichia coli thioredoxin, has the potential to act in GSH-dependent processes. Thus, the fundamental role of R. sphaeroides thioredoxin in cell growth probably originates from the multiple functions it can serve in vivo.
Binding of colicins A and E1 to purified ToIA domains
Authors: Derouiche Rahmona , Zeder-Lutz Gabrielle , Bénédetti Hélène , Gavioli Marthe , Rigal Alain , Lazdunski Claude , LloubèAs Roland
Published: June 2009

Colicins are divided into two groups according to the proteins required for their import into sensitive bacteria. The Tol and TonB pathways are involved in import of group A and group B colicins respectively. Because previous analyses have shown that colicin E1 and colicin A (two group A colicins) interactin vitrowith the C-terminal domain of TolA (TolAIII) while colicin B (group B colicin) does not, attention was focused on these interactions with purified proteins. TolA has been described as a three-domain protein with an N-terminal inner-membrane anchor and a long periplasmic region formed by two domains (TolAII and TolAIII). TolAIII, TolAII and TolAII-III soluble domains with an N-terminal hexa-histidine extension were purified. The interactions of colicins with the purified TolA domains were analysed by overlay Western blotting, which indicated that both N-terminal domains of colicins A and E1 interacted with TolAIII, while a gel shift procedure detected no interaction with colicin E1. The binding kinetic values of the N-terminal domains of colicins A and E1 to TolAIII were estimated by surface plasmon resonance and were shown to be similar.
Characterization of the rate-limiting step of the secretion of Bacillus subtilis α-amylase overproduced during the exponential phase of growth
Authors: Leloup Laurence , Haddaoui El Arbi , Chambert Régis , Petit-Glatron Marie-FranÇoise
Published: June 2009

The Bacillus subtilis α-amylase gene, amyE, was expressed under the regulated control of sacR, the levansucrase leader region. The gene fusion including the complete amyE coding sequence with the signal peptide sequence was integrated into the chromosome of a degU32(Hy) strain deleted of the sacB DNA fragment. In this genetic context, α-amylase is produced in the culture supernatant at a high level (2% of total protein) during the exponential phase of growth upon induction by sucrose. Pulse-chase experiments showed that the rate-limiting step (t
1/2 = 120 s) of the secretion process is the release of a cell-associated precursor form whose signal peptide has been cleaved. The efficiency of this ultimate step of secretion decreased dramatically in the presence of a metal chelator (EDTA) or when the cells were converted to protoplasts. The hypothesis that this step is tightly coupled with the folding process of α-amylase occurring within the cell wall environment was substantiated by in vitro folding studies. The unfolding-folding transition, monitored by the resistance to proteolysis, was achieved within the same time range (t
1/2 = 60 s) and required the presence of calcium. This metal requirement could possibly be satisfied in vivo by the integrity of the cell wail. The t
1/2 of the α-amylase release step is double that of levansucrase, although their folding rates are similar. This perhaps indicates that the passage through the cell wall may depend on parietal properties (e.g. metal ion binding and porosity) and on certain intrinsic properties of the protein (molecular mass and folding properties).
Sequence analysis and characterization of øO1205, a temperate bacteriophage infecting Streptococcus thermophilus CNRZ1205
Authors: Stanley Elizabeth , Fitzgerald Gerald F. , Marrec Claire Le , Fayard Blandine , van Sinderen Douwe
Published: June 2009

The complete nucleotide sequence of øO1205, a temperate bacteriophage infecting Streptococcus thermophilus strain CNRZ1205, was determined. The phage genome has a unit length of 43075 bp and appears to be packaged by the so-called headful mechanism. The genomic organization and structure of øO1205 resemble those of several temperate lactococcal phages that display a life-cycle-specific organization, where ORFs believed to be involved in the lysogenic life-cycle are clustered and arranged in an orientation opposite to the ORFs supposedly involved in the lytic life-cycle. Database searches revealed putative functions for several identified ORFs and further indicated that øO1205 is genetically related to a particular group of lactococcal phages. Three genes encoding the major structural proteins were identified on the øO1205 genome. The phage attachment site attP, the bacterial attachment site attB, and the two phage/chromosome junctions attL and attR were identified and found to contain a 40 bp common core sequence.
Phospholipase D activity is required for dimorphic transition in Candida albicans
Authors: McLain Nealoo , Dolan Joseph W.
Published: June 2009

Candida albicans is an opportunistic pathogen that causes significant morbidity and mortality in immunocompromised patients. In this report, the presence of a phospholipase D (PLD) activity in C. albicans, designated CaPLD1, is demonstrated. This is the first description of PLD activity in this organism. CaPLD1 activity was stimulated by inducers of dimorphic transition. Furthermore, transition was stimulated by the addition of exogenous PLD to cells. The addition of 1-propanol to the medium, which resulted in the production of phosphatidylpropanol by CaPLD1 at the expense of the usual product phosphatide acid, delayed the yeast to hypha transition. These results suggest that CaPLD1 may be an important regulator of dimorphic transition in C. albicans.
Differentiation of Streptomyces coelicolor A3(2) under nitrate-limited conditions
Authors: Karandikar Atul , Sharples George P. , Hobbs Glyn
Published: June 2009

The life cycle of Streptomyces coelicolor during development on solid medium has been studied from a physiological perspective. A biphasic growth pattern was demonstrated, evidenced by a continuous transition from an initial exponential growth period into a slower phase of biomass accretion. The switch between the two phases coincided with the exhaustion of nitrate from the medium. The depletion of nitrate from the medium coincided with the initiation of aerial mycelium formation within the cultures and the development of hydrophobic surface properties. During secondary growth, cultures remained metabolically active, continuing to accumulate DNA, despite a cessation in the levels of RMA and cell protein accretion. In addition, the accumulation of glycogen and lipid contributed to the observed accretion of biomass in this phase. The depletion of nitrate also marked an increase in the production of α-ketoglutarate by the culture and a coincident decrease in medium pH. Latter stages of the secondary growth phase saw the development of spores within the culture, this in turn was associated with a decrease in cellular glycogen. This supported previous observations that glycogen degradation and spore maturation were intimately associated.
Increased and controlled expression of the Rickettsia prowazekii ATP/ADP translocase and analysis of cysteine-less mutant translocase
Authors: Dunbar Sherry A. , Winkler Herbert H.
Published: June 2009

Detailed molecular analysis of theRickettsia prowazekiiATP/ADP translocase, an obligate exchange transport system that is specific for ATP and ADP, has been extremely difficult due to limited quantities of material available from these obligate intracytoplasmic bacteria and by the toxicity and poor expression in recombinantEscherichia coliexpression systems. In this study, a stable and controllable system for the increased expression of the rickettsial ATP/ADP translocase was developed inE. coliwhere the expression of translocase from the bacteriophage T7 promoter in the pET11a vector led to a 26-fold increase in ATP transport activity and a 34-fold increase in translocase protein as compared to the expression with the native rickettsial promoter inE. coli.When compared toR. prowazekii,ATP transport activity was increased sixfold and membrane translocase was increased threefold. Approximately 24% of the translocase protein produced was localized in an inclusion body fraction. This expression system was then used to determine whether the two cysteine residues in the ATP/ADP translocase were essential for activity or expression. The translocase was modified by oligonucleotide-directed site-specific mutagenesis such that the two cysteines were converted to alanines. The ATP transport properties and ATP/ADP translocase production kinetics, translocase protein concentration and subcellular localization were indistinguishable in the wild-type and mutant strains, proving that cysteines play no functional role in theR. prowazekiiATP/ADP translocase and providing a system suitable for cysteine-scanning mutagenesis.
Acetate acts as a protonophore and differentially affects bead movement and cell migration of the gliding bacterium Cytophaga johnsonae (Flavobacterium johnsoniae)
Authors: Dzink-Fox JoAnn L. , Leadbetter Edward R. , Godchaux Walter
Published: June 2009

Cells of Cytophaga johnsonae (now Flavobacterium johnsoniae) are able to translocate on solid surfaces but are unable to swim in liquid media. Organelles that may be involved in this gliding motility have not been detected, and the mechanism(s) responsible remains unknown. The movement of latex beads attached to the cell surface is considered by some to be a manifestation of the gliding machinery. In this study, acetate (in nutrient-level quantity, 45 mom) was found to inhibit bead movement on cell surfaces, whilst formation and movement of groups of cells (rafts) and typical colony spread were not affected; generation time (in liquid culture) was only slightly increased. Since acetate is a weak acid and is recognized as a protonophore, various electron-transport-associated features were assessed in an effort to understand the differential effects of acetate on bead movement and cell motility. Selected protonophores and electron transport inhibitors were tested to compare their effects on cell translocation and metabolic activities with those of acetate. Although O2 consumption was not significantly affected in the presence of acetate and the protonmotive force decreased only minimally, ATP levels were markedly decreased. Arsenate and cyanide were also shown to inhibit bead movement but did not inhibit either movement of rafts of cells or colony spreading. Cyanide lowered O2 consumption, while arsenate did not; both compounds effected substantial decreases in cellular ATP content, but little or no decrease in protonmotive force. The inhibitory effects of these compounds on bead movement over cell surfaces contrasted with the continued ability of cells to form rafts, to glide and to form spreading colonies and led to the conclusion that bead movement is not a complete correlate of the gliding machinery of C. johnsonae. In addition, it seems likely that bead movement is more affected by the level of cellular ATP than it is by the protonmotive force, which has been assumed to provide the energy (derived from the transmembrane gradients) for the gliding machinery.
Phylogenetic diversity of a bacterial community determined from Siberian tundra soil DNA
Authors: Zhou Jizhong , Davey Mary Ellen , Figueras Jordi B. , Rivkina Elizaveta , Gilichinsky David , Tiedje James M.
Published: June 2009

Genomic DNA was isolated from the active layer of tundra soil collected from the Kolyma lowland. Northeast Eurasia, near the Arctic Ocean coast. The SSU (small subunit) rRNA genes were amplified with eubacterial primers from the bulk genomic community DNA and cloned into plasmid vectors. Forty-three SSU rDNA clones were obtained, and all of them had different RFLP patterns. Phylogenetic analysis based on partial sequences (about 300 bp) established with the maximum likelihood method revealed the presence of three major and several minor groups that fell into 11 of the established lines of bacteria, and one sequence that could not be assigned to any of the described groups. Most of the clones belonged to the alpha (20.9%) and delta (25.6%) subdivisions of the Proteobacteria, with lesser proportions in the beta (9.3%) and gamma (4.7%) subdivisions, groups typically isolated from soil by culture methods. Fewer than 12% of the clones belonged to Gram-positive bacteria, and 16% of the clones were related to Fibrobacter. The majority of the clones (70%) had sequences that were 5-15% different from those in the current databases, and 7% of the clones had sequences that differed by more than 20% from those in the database. The results suggest that these tundra-derived clones are very diverse in phylogeny, and that many probably reflect new genera or families. Hence, most of the tundra soil bacterial community has never been isolated and thus the physiology and function of its dominant members appears to be unknown.
WO-2, a stable aneuploid derivative of Candida albicans strain WO-1, can switch from white to opaque and form hyphae
Authors: Magee B. B. , Magee P. T.
Published: June 2009

Candida albicans strain WO-2 was isolated as a spontaneous derivative of the white-opaque switching strain WO-1. The electrophoretic karyotype of WO-2 lacks two bands which are found in the parent. These bands correspond to one homologue of chromosome 7 and to a translocation product containing parts of chromosomes 6 and 5. Probing a blot of the karyotype demonstrated that the genetic material in these bands had been lost, yielding an aneuploid strain. UV-irradiation experiments showed that auxotrophs due to mutation in genes located in this region predominated, supporting the conclusion that WO-2 is partially haploid. WO-2 contained about 10% of its genome in the haploid state, and it grew with a doubling time of about twice that of its parent. However, it was able to undergo both the yeast-to-hyphal transition and the white-opaque transition. Hence, these processes do not require perfect diploidy.
Cloning, analysis and one-step disruption of the ARG5,6 gene of Candida albicans
Authors: Negredo A. , Monteoliva L. , Gil C. , Pla J. , Nombela C.
Published: June 2009

The ARG5,6 gene from the dimorphic fungus Candida albicans was cloned by functional complementation of the arginine auxotrophy present in strain EL2 (Arg-) using a gene library constructed in the double autonomously replicating sequence vector pRM1. Sequence analysis revealed a putative 857 amino acid polypeptide (95 kDa) which showed high homology (63% protein identity) to the Saccharomyces cerevisiae ARG5,6 gene. Similarly to the S. cerevisiae gene, the C. albicans ARG5,6 gene is responsible for both the acetylglutamate kinase and acetylglutamyl-phosphate reductase activities, the second and third steps of arginine biosynthesis at the mitochondria. The C. albicans ARG5,6 gene complemented the arg6 mutation present in S. cerevisiae (strain D160-4D) on a yeast episomal plasmid using its own regulatory signals. A set of non-integrative high-efficiency plasmid vectors based on this gene marker was constructed and a null C. albicans arg5,6d strain was obtained using the common URA3-blaster strategy. In addition, we generated an arg5,6d null mutant in a single transformation event, thus improving the basic strategy for generating gene deletions in C. albicans.
A new insertion sequence IS1452 from Acetobacter pasteurianus
Authors: Kondo Koichi , Horinouchi Sueharu
Published: June 2009

A new insertion sequence element, IS1452, was found to be associated with inactivation of the alcohol dehydrogenase by insertion in the adhS gene encoding subunit III of the three-component membrane-bound alcohol dehydrogenase complex in Acetobacter pasteurianus. Cloning and sequencing analyses of the mutated subunit III gene locus revealed that IS 1452 was inserted at or near the ribosome-binding sequence of adhS. Analysis of transcription using the chloramphenicol acetyltransferase gene as the reporter indicated that IS1452 abolished transcription of adhS by separating its promoter from the subunit III structural gene. IS1452 was 1411 bp in length and had a terminal inverted repeat of 21 bp. IS1452 contained one long ORF of 416 amino acids rich in basic amino acids. This protein showed homology with a putative transposase, Tra1, of IS701 isolated from the cyanobacterium Calothrix species PCC 7601. Like IS701, IS7452 was found to generate a 4 bp direct repeat at the site of insertion upon transposition. The target site specificity was rather strict, and a CTA(A or G) sequence appeared to be preferentially recognized. Transposition of IS1452 was replicative, since it was accompanied by an increase in the copy number of IS1452. Several strains belonging to the genus Acetobacter also contained IS1452 at varying copy numbers from one to more than ten. These observations suggest that IS1452 is one of the insertion sequences that are responsible for genetic instability leading to deficiencies in various physiological properties in acetic acid bacteria.
Gain-of-function mutation of sapB that affects formation of alkaline phosphatase by Bacillus subtilis in sporulation conditions
Authors: Whalen Michael B. , Piggot Patrick J.
Published: June 2009

The sapB locus was defined by mutations that render sporulation alkaline phosphatase formation independent of sf and sE without affecting the temporal control of formation. The sapB locus has been cloned and sequenced. The deduced polypeptide is 232 amino acids long, with a molecular mass of 26 kDa. It is very similar to four sequences in the database, none of which has a known function. Analysis of the transcription of sapB indicates that it is induced during late exponential phase, and that maximum expression is reached during the first hour of stationary phase, both under sporulation and non-sporulation conditions. The defining mutations of the locus, sapB2 and sapB10, have been sequenced and found to contain the same change, a G → A transition resulting in an Ala111 Thr switch. This mutation apparently results in a gain-of-function, as sapB null mutants are indistinguishable from sap
+ strains in terms of their APase production during sporulation.
Missense mutations in the 3' end of the Escherichia coli dnaG gene do not abolish primase activity but do confer the chromosome-segregation-defective (par) phenotype
Authors: Versalovic James , Lupski James R.
Published: June 2009

Isogenic dnaG strains of Escherichia coli with the parB and dnaG2903 alleles in the MG1655 chromosomal background displayed the classic par phenotype at the nonpermissive temperature of 42 �. These strains synthesized DNA at 42 �, but remained chromosome segregation defective as determined by cytology. A strain with the dnaG2903 allele was tested for its ability to support DNA replication of a primase-dependent G4oric-containing M13 phage derivative by quantitative competitive PCR (QC-PCR). The dnaG2903 strain converted the single-stranded DNA into double-stranded replicative form DNA at 42 �. These results indicate that DnaG2903 retains primase activity at the restrictive temperature. Nucleoids remained unsegregated in the central region of cell filaments at 42 �. The observed suppression of cell filamentation in dnaG sfiA or dnaG lexA double mutants suggests that the SOS response is induced at the restrictive temperature in parB and dnaG2903 strains but fails to account entirely for the cell filamentation phenotype. ParB and DnaG2903 presumably can synthesize primer RNA for DNA replication, but may be defective in their interactions with DNA replication proteins, cell cycle regulatory factors, or the chromosome segregation apparatus itself.
A novel gene, algK, from the alginate biosynthetic cluster of Pseudomonas aeruginosa
Authors: Aarons Simon J. , Sutherland Ian W. , Chakrabarty A. M. , Gallagher Maurice P.
Published: June 2009

Colonization of the cystic fibrosis lung byPseudomonas aeruginosais greatly facilitated by the production of an exopolysaccharide called alginate. Many of the enzymes involved in alginate biosynthesis are clustered in an operon at 34 min on theP. aeruginosachromosome. This paper reports the nucleotide sequence of a previously uncharacterized gene,algK,which lies between thealg44andalgEgenes of the operon. DNA sequencing data foralgKpredicted a protein product of approximately 52.5 kDa which contains a putative 27 amino acid N-terminal signal sequence and a consensus cleavage and lipid attachment site for signal peptidase II. Expression ofalgKusing either T7 ortacpromoter expression systems, andin vivolabelling studies with [35S]methionine, indicated thatalgKencodes a polypeptide of approximately 53 kDa which is processed to a mature protein of approximately 50 kDa when expressed inEscherichia coliorP. aeruginosa,in agreement with the nucleotide sequence analysis. Results from an AlgK-β-lactamase fusion survey support this interpretation and also provide evidence that mature AlgK is entirely periplasmic and is probably membrane-anchored.
Bacterial capsules: no barrier against Bdellovibrio
Authors: Koval Susan F. , Bayer Manfred E.
Published: June 2009

Bdellovibrio bacteriovorus 109J attached to both capsulated and noncapsulated Escherichia coli K29 cells. Electron microscopy revealed penetration of the thick polysaccharide capsule without any major disintegration of the neighbouring capsular matrix. The capsule remained intact during bdelloplast formation and lysis was unaffected by capsulation of the prey cell. This study shows that, in contrast to its effect on bacteriophage penetration and its protective activities against immune defence mechanisms, the capsule of E. coli does not serve as a barrier against invasion by B. bacteriovorus.
Cra-mediated regulation of Escherichia coli adenylate cyclase
Authors: Crasnier-Mednansky Martine , Park Maxwell C. , Studley William K. , Saier Milton H.
Published: June 2009

In Escherichia coli, expression of certain genes and operons, including the fructose operon, is controlled by Cra, the pleiotropic catabolite repressor/activator protein formerly known as FruR. In this study we have demonstrated that cra mutant strains synthesize 10-fold less cAMP than isogenic wild-type strains, specifically when grown in fructose-containing minimal media. The glucose-specific IIA protein (IIAglc) of the phosphotransferase system, which activates adenylate cyclase when phosphorylated, is largely dephosphorylated in cra but not wild-type strains growing under these conditions. Dephosphorylation of IIAglcin cra strains apparently results from enhanced fructose operon transcription and fructose uptake. These conclusions were supported by showing that fructose-grown cra strains possess 2·5-fold higher fructose-1-phosphate kinase activity than fructose-grown wild-type strains. Moreover, artificially increasing fructose operon expression in cells transporting fructose dramatically decreased the activity of adenylate cyclase. The results establish that Cra indirectly regulates the activity of adenylate cyclase by controlling the expression of the fructose operon in cells growing with fructose as the sole carbon source.