bluuebunny/crossref_metadata_2025 huggingface dataset:
Production and consumption of nitric oxide by denitrifyingFlexibacter canadensis
Authors: Wu Qitu , Knowles Roger , Chan Yiu-Kwok
Published: October 2009

Production and consumption of nitric oxide (NO) by Flexibacter canadensis cells under anaerobic conditions was investigated using a chemiluminescence NO analyzer. Net NO production from nitrite in the presence of carbonyl cyanide m-chlorophenylhydrazone (CCCP) was pH dependent, increased in the pH range from 4.5 to 6.5, and sharply decreased at pH >6.5. CCCP inhibited NO consumption but only at pH values ≤6.5. This can explain why CCCP stimulation of NO production depends on the pH. Denitrification of nitrite at high concentrations (≥5 mM) also resulted in net NO accumulation. Diethyldithiocarbamate, a copper chelating agent, prevented not only net production of NO during the reduction of nitrite in the presence of CCCP, but also production of nitrous oxide (N2O) from nitrite in the presence of C2H2. This suggests that F. canadensis may possess a copper-type nitrite reductase. However, cytochrome cd1- and copper-containing nitrite reductase DNA probes from Pseudomonas species did not hybridize with the total DNA of F. canadensis, indicating that the nitrite reductase of F. canadensis may possess unique properties. In addition to diethyldithiocarbamate, sulfide, carbon monoxide, azide, cyanide, hydroxylamine and Triton X-100 prevented net NO production from nitrite in the presence of CCCP, and also inhibited NO consumption. C2H2, an inhibitor of N2O reductase, did not affect NO production or consumption.Key words: nitrite reductase, nitric oxide (NO), carbonyl cyanide m-chlorophenylhydrazone (CCCP), Flexibacter canadensis.
Investigation of the lignin-degrading activity of Serratia marcescens: biochemical screening and ultrastructural evidence
Authors: Rhoads Tim L. , Mikell Jr. Alfred T. , Eley Michael H.
Published: October 2009

Forty-one morphologically distinct bacterial isolates were developed from six lignin-containing environments. Each isolate was initially screened for potential lignin-degrading activity using relative growth on a lignocellulosic substrate and relative decolorization of a polymeric dye. Screened isolates were then tested for the ability to oxidize various lignin-related monomers, and the dimers anisoin and veratrylglycerol-β-guaiacyl ether. Although most of the isolates oxidized the monomers, only two successfully oxidized the dimers. The dimer-degrading isolates were tested for extracellular activity against the β-O-4 dimer veratryl-glycerol-β-guaiacyl ether. No activity was detected for the isolates. Phanerochaete chrysosporium Burds used as a positive control demonstrated a high degree of activity in each assay. Extensive ultrastructural studies of lignocellulose alteration by the dimer-degrading isolates were conducted via light and transmission electron microscopy. These studies indicate that one of the isolates, identified as Serratia marcescens, is capable of degrading highly lignified secondary cell wall components. This activity is localized, apparently requiring direct contact between cells and substrate, which could be facilitated by an associated glycocalix. The results of the dimer degradation assays concur with the characterization of the responsible enzyme system as being membrane associated.Key words: lignin, β-O-4 dimer, bacteria, Serratia, ultrastructure.
Characterization by arbitrary primer polymerase chain reaction of polychlorinated biphenyl (PCB)-degrading strains of Comamonas testosteroni isolated from PCB-contaminated soil
Authors: Joshi Bindu , Walia Satish
Published: October 2009

In this study, we isolated and characterized biphenyl (BP) and polychlorinated biphenyl (PCB) degrading bacterial strains found in PCB-contaminated soil from an auto manufacturing plant located in Syracuse, New York. Twenty-one BP and PCB-degrading bacteria were randomly selected to form a representative sample of the bacterial population present at the site. Of the 21 bacteria, 13 were identified as Comamonas testosteroni, constituting about 60% of the bacterial population examined. Other PCB degraders identified were Acidovorax facilis, Alcaligenes xylosoxydans, Bacillus sphericus, Hydrogenophaga pseudoflava, Pseudomonas avanae, and Rhodococcus fascians. Owing to the abundance of C. testosteroni at this site, only these isolates were further characterized for their PCB congener degradation profile, 2,3-dihydroxybiphenyl 1,2-dioxygenase activity, and genetic relatedness by polymerase chain reaction (PCR) analysis. The PCB congener degradation pattern revealed a high degree of variability among the C. testosteroni isolates. The majority of the C. testosteroni isolates tested could degrade more than 95% of the PCB congeners up to pentachiorinated biphenyl. Only four isolates could degrade more than 80% of hexachlorobiphenyl. All 12 isolates of C. testosteroni tested were able to attack 2,3,4,5,6,3′,4′-heptachlorobiphenyl, indicating involvement of biphenyl 2,3-dioxygenase, while 2,3,5,6,2′,3′,6′-heptach!orobiphenyl was attacked by 6 strains, suggesting an oxidation reaction mediated by 3,4-dioxygenase. 2,3-Dihydroxybiphenyl 1,2-dioxygenase activity was also found to vary among the C. testosteroni isolates tested in this study. Eleven strains showed 2,3-dihydroxybiphenyl 1,2-dioxygenase activity specific for 2,3-dihydroxybiphenyl, whereas isolate BW169 could metabolize both 2,3-dihydroxybiphenyl and 4-methylcatechol, and isolate BW74 had the ability to metabolize all three substrates (2,3-dihydroxybiphenyl, 4-chlorocatechol, and 4-methylcatechol). Further molecular characterization of these isolates was done by a PCR-based assay, random amplified polymorphic DNA (RAPD) analysis. Amplifications of common DNA fragments of 450 dp using primer OPJ6 and 650 bp using primer OPJ14 were identified in the C. testosteroni isolates, although a distinct RAPD pattern was obtained for all of the isolates using primer OPJ14, and a combination of the two primers. Therefore, the results presented in this study demonstrate that RAPD can be used to fingerprint the C. testosteroni isolates.Key words: comamonas testosteroni, polychlorinated biphenyl, arbitrary primer-PCR.
Occurrence of cop-like copper resistance genes among bacteria isolated from a water distribution system
Authors: Lin Chuzhao , Olson Betty H.
Published: October 2009

The occurrence of cop-like copper resistance determinants homologous to the cop genes of Pseudomonas syringae among bacteria isolated from a water distribution system experiencing copper corrosion was investigated in this study. It was found that at least 49% of the copper-resistant bacteria and less than 15% of the copper-sensitive isolates possessed a cop homolog. The occurrence of this determinant in the copper-resistant population correlated with the degree of copper tolerance exhibited by the bacteria. The effect of organic substances present in the culture media on the empirical degree of bacterial copper tolerance is also discussed.Key words: copper resistance genes, water distribution system, cop.
Bacterial succession within a biofilm in water supply lines of dental air–water syringes
Authors: Tall B. D. , Williams H. N. , George K. S. , Gray R. T. , Walch M.
Published: October 2009

Biofilms have been implicated as reservoirs for bacterial contamination of water delivered by dental air–water syringes. A 6-month study was done of bacterial colonization and biofilm formation in plastic water supply lines connected to dental air–water syringes. Changes in biofilm flora were observed by both scanning electron microscopy and bacteriologic culture. By day 7, many rod- and spiral-shaped bacteria had colonized the ridged surface of the luminal wall of the tubing, as revealed by scanning electron microscopy. By day 30, individual microcolonies were embedded in extracellular polymeric material. By day 120, these microcolonies had begun to coalesce, and by day 180 the biofilm had developed into a multilayered, heterogenous mixture of microcolonies. The mean aerobic plate counts of colony-forming units of planktonic and biofilm populations were, in log10 values, 5.9 ± 0.54/mL and 4.2 ± 0.82/cm2, respectively. Early colonizers were predominantly Pseudomonas spp., but included Pasteurella, Moraxella, Ochrohactrum, and Aeromonas spp. Flavobacterium and Acinetobacter spp. were observed later. Many of these organisms are opportunistic pathogens. These results demonstrate the longitudinal dynamics of biofilm formation.Key words: dental equipment, air–water syringes, biofilms.
ThentrBCgenes ofAzospirillum brasilenseare part of anifR3-like–ntrB–ntrCoperon and are negatively regulated
Authors: Machado H. B. , Yates M. G. , Funayama S. , Rigo L. U. , Steffens M. B. R. , Souza E. M. , Pedrosa F. O.
Published: October 2009

A cosmid able to complement the Nif−and nitrate-dependent growth phenotypes of the Azospirillum brasilense mutant FP9 was isolated from a genomic library of the wild-type strain FP2. A 6-kb DNA region was sequenced and showed two open reading frames (ORFs) identified as the ntrB and ntrC genes. An ORF1 located upstream from the ntrB gene and coding for a 36-kDa polypeptide showed similarity to the nifR3 gene of Rhodobacter capsulatus and the ORF1 of Rhizobium leguminosarum, both located upstream from the ntrB gene in a complex operon. Two other unidentified ORFs (ORF5 and partial ORF4) coding for hydrophobic polypeptides were also observed. ΔORF1-ntrBC, ORF1, ntrB, and ntrC mutants obtained by recombination of suicide plasmids containing an insertion of a promoterless lacZ kanamycin cassette showed decreased nitrogenase activities and were unable to grow on nitrate as the sole N source. These phenotypes were restored by complementation with plasmids containing the ntrC gene. Analysis of lacZ transcriptional fusions suggested that the ORF1-ntrBC operon in Azospirillum brasilense is expressed from a promoter located upstream from the ORF1 and that it is negatively regulated by the ntrC gene product.Key words: Azospirillum brasilense, ntrB, ntrC, nifR3-like, nitrogen fixation.
Monochloroacetate dehalogenase activities of bacterial strains isolated from soil
Authors: Diez Amalia , Alvarez Maria J. , Prieto M. Isabel , Bautista José M. , Garrido-Pertierra Amando
Published: October 2009

Seven bacterial strains capable of utilizing monochloroacetate (MCA) at a concentration of 50 mM as the sole carbon source were isolated from soil and displayed MCA dehalogenase activity. Three of them were identified as Pseudomonas spp., and the remaining four strains as Alcaligenes sp., Agrobacterium sp., Arthrobacter sp., and Azotobacter sp. This latter is the first reported example of a bacterium fixing atmospheric nitrogen under aerobic conditions that also uses a chloro-organic compound as sole source of carbon and energy. MCA dehalogenase activity in these strains was found to be inducible under different growth conditions. Crude extracts from all seven isolated strains also displayed dehalogenating activity with a relatively wide range of halogenated organic compounds (aliphatic acids, ketones, alcohols, alkanes, and aromatics), which, depending on the strain, were dehalogenated to different extents. The estimated Kmvalues for MCA were used to classify the dehalogenase activities into three groups: high affinity (30–40 μM) in Alcaligenes and Agrobacterium species, medium affinity (100–180 μM) in Pseudomonas and Azotobacter species, and low affinity (100 mM) in Arthrobacter sp. Both the optimal pH range for MCA dehalogenase activity (between pH 8 and 10) and the pH profile of stability (in the neutral–basic range) were found to be similar in all strains, whereas the thermal stability profiles were variable.Key words: dehalogenase, halohydrolase, monochloroacetate, soil.
Plasmid-borne determinants of pigmentation, exopolysaccharide production, and virulence in Xanthomonas campestris pv. malvacearum
Authors: Chakrabarty P. K. , Raj Sheo , Meshram M. K. , Mahadevan A. , Gabriel D. W.
Published: October 2009

Three plasmids of 55.0, 31.2, and 7.4 kb, designated as pXCM18-I, pXCM18-II, and pXCM18-III, respectively, were identified and isolated from strain HR13B/2 of Xanthomonas campestris pv. malvacearum. The bacterium was cured of all three plasmids in high frequency (3.1%) with heat (42 °C). The cured strains were nonmucoid and light yellow and were greatly impaired in exopolysaccharide production and virulence. Transformation of a cured strain using a mixture of purified plasmid DNA from HR13B/2 resulted in colonies carrying all six possible combinations of the three plasmids. Of the transformants, two morphologically distinct colony types were observed. The first type of transformants were mucoid and deep yellow, not distinguished from wild-type colonies in morphology, and fully complemented for exopolysaccharide production. These transformants invariably carried pXCM18-II. The second type of transformants were not distinguished from the untransformed strain in morphology but were found to be transformed after screening for plasmid content. These transformants carried either pXCM18-I, pXCM18-III, or both, but never pXCM18-II. In pathogenicity tests, each of the three plasmids restored virulence significantly, and additively, with plasmid pXCM18-I playing a predominant role.Key words: Xanthomonas campestris pv. malvacearum, cotton, plasmid, pigmentation, exopolysaccharide, virulence.
Fixed bacterial biomass estimated by potential exoproteolytic activity
Authors: Laurent Patrick , Servais Pierre
Published: October 2009

A method is proposed to estimate the bacterial biomass fixed on solid supports colonized in water without detachment of the cells from the supports. The method consists of measuring an enzymatic activity of the fixed bacteria: the potential exoproteolytic activity, which has been shown to be proportional to the bacterial biomass.Key words: fixed bacterial biomass, support colonization, exoproteolytic activity.
Killer activity of yeasts isolated from the water environment
Authors: Vadkertiová Renáta , Sláviková Elena
Published: October 2009

The killer activity of 46 strains belonging to 12 yeast and yeast-like species isolated from water or sediment samples was studied. Only two strains of the genus Cryptococcus did not show killer activity. Killer activity of yeast-like species Aureobasidium pullulans, Hyphopichia burtonii and Geotrichum candidum, and yeast species Candida krusei and Candida lambica was low. Sporobolomyces salmonicolor, Cryptococcus laurentii and Cryptococcus albidus had better activity against basidiomycetous than ascomycetous species. Hansenula anomala strains showed good activity against Geotrichum candidum strains, Cryptococcus albidus, and Sporobolomyces salmonicolor. Rhodotorula species showed activity against the majority of both ascomycetous and basidiomycetous species.Key words: yeasts, killer activity, sediment and water samples.
Regulation of valine catabolism by ammonium inStreptomyces ambofaciens, producer of spiramycin
Authors: Lounès Anissa , Lebrihi Ahmed , Benslimane Chouki , Lefebvre Gérard , Germain Pierre
Published: October 2009

In Streptomyces ambofaciens, valine favored spiramycin biosynthesis by supplying aglycone precursors. The kinetics of valine consumption and isobutyrate production showed that isobutyrate accumulated in the cell during the growth phase, was excreted in the stationary phase, and then was reassimilated during spiramycin production. When valine was in excess, its deamination led to high ammonium excretion and to a significant drop in spiramycin production. We demonstrated that ammonium ions were the cause of the negative effect. Addition of a chelator agent, Ca3(PO4)2, improved spiramycin production by sixfold. In contrast, addition of ammonium, between 0 and 48 h, severely reduced spiramycin production. The negative effect of ammonium was reversed by addition of a catabolic intermediate of valine, isobutyrate. In addition to stimulating the specific growth rate, ammonium ions slowed down valine catabolism: the specific valine uptake rate, excretion, and reassimilation of isobutyrate were lowered by the pulse of ammonium. Our study showed that in addition to valine dehydrogenase, which provided the nitrogen necessary to the cell, ammonium ions repressed ketoisovalerate dehydrogenase, which introduced valine as carbon, energy, and aglycone precursor sources. However, valine dehydrogenase and ketoisovalerate dehydrogenase did not constitute the principal enzymatic targets of the negative effect of ammonium in spiramycin production.Key words: spiramycin, Streptomyces ambofaciens, valine catabolism, ammonium.
Detection of Rhizobium meliloti cells in field soil and nodules by polymerase chain reaction
Authors: Watson R. J. , Haitas-Crockett C. , Martin T. , Heys R.
Published: October 2009

A genetically marked Rhizobium meliloti strain, R692, was prepared by insertion of a 1.7-kb DNA segment from Tn903 between the nifHDK and fixABC genes in the nod megaplasmid. This DNA was used as a marker, detectable by polymerase chain reaction (PCR), for the specific identification of bacteria in soil samples and alfalfa nodules. This detection technique was tested by applying different titres of the marked strain to field plots seeded with alfalfa. Samples of soil and nodules were assayed for the presence of the marker DNA fragment by PCR using primers specific to the marker sequence. The experiments revealed that the bacteria could be detected directly in soil containing about 103–104 bacteria/g, but greater sensitivity was prevented by potent PCR inhibitors present in the samples. The titre of the bacteria in the soil decreased rapidly after inoculation, dropping about 10-fold per week. Tests of vertical location of the bacteria in soil cores showed that the bacteria were initially dispersed to a depth of 18 cm, and subsequently retained viability in the top 2–8 cm. As few as 10 marked R. meliloti per gram of soil resulted in its establishment at detectable levels in nodules. Application of about 104–105 bacteria/g soil was sufficient to give the maximum number of nodules per plant and resulted in 70–90% occupancy by the marked strain. Limited movement of the inoculant was detected by analysis of nodules from plants adjacent to the sites where the bacteria were applied, probably by movement in water. The experiments demonstrated the advantages of PCR for the monitoring of marked microorganisms in the environment.Key words: genetically engineered microorganism, PCR inhibitor, nitrogen fixation, nif and fix genes, genetic marker.
In vitro cellulolytic activity of the plant pathogenClavibacter michiganensissubsp.sepedonicus
Authors: Baer Debra , Gudmestad Neil C.
Published: October 2009

The activity of four Clavibacter michiganensis subsp. sepedonicus strains against various cellulose substrates was investigated. Sixty-seven Clavibacter michiganensis subsp. sepedonicus strains grew well on media amended with carboxymethylcellulose, 64 strains produced zones of hydrolysis. Endoglucanase activity was optimal at 37 °C and pH 6.0 against carboxymethylcellulose incorporated in plate assays. Zymogram and sodium dodecyl sulfate – polyacrylamide gel electrophoresis revealed the presence of a protein band corresponding to the cellulolytic activity in the molecular weight (MW) range of approximately 28 000. Protein bands in the same range were detected in five Clavibacter michiganensis subsp. sepedonicus strains. Studies on crude enzyme extracts of Clavibacter michiganensis subsp. sepedonicus strain N-1-1 revealed that p-nitrophenyl β-D-cellobioside (pNPC) was hydrolyzed, with optimal activity at 37 °C and pH 7.0.Key words: cellulase, endo-1,4-β-glucanase (EC 3.2.1.4), Corynebacterium sepedonicum, Solanum tuberosum.
Electron spectroscopic imaging of encapsidated DNA in vaccinia virus
Authors: Harauz G. , Evans D. H. , Beniac D. R. , Arsenault A. L. , Rutherford B. , Ottensmeyer F. P.
Published: October 2009

We have used electron spectroscopic imaging to locate the phosphorus in vaccinia DNA in situ in unstained, ultrathin sections of virions. The phosphorus of the DNA backbone appeared to form a halo on the core periphery surrounding a phosphorus-impoverished central element. These results constrain models for how DNA could be packaged into mature vaccinia particles.Key words: vaccinia, electron spectroscopic imaging, DNA.
Investigation by NMR of metabolic routes to bacterial γ-poly(glutamic acid) using13C-labeled citrate and glutamate as media carbon sources
Authors: Cromwick Anne-Marie , Gross Richard A.
Published: October 2009

Bacillus licheniformis ATCC 9945A produces γ-poly(glutamic acid) (γ-PGA) when using glutamate, citrate, and glycerol in combination as media carbon sources. Also, various aspects of B. licheniformis cellular physiology are affected by the concentration of Mn(II) salts in culture media. Thus, the metabolism of carbon sources into γ-PGA as a function of MnSO4 concentration was studied by enriching the media with either L-[1,2-13C]glutamic acid (NH2—2CH(1COOH)—(CH2)2-COOH) or [1,5-13C]citric acid (HOO1C—CH2—C(OH)(COOH)—CH2—5COOH) at two media MnSO4concentrations (615 and 0 μM). Enrichment factors (EF) from13C-NMR spectra were calculated from the ratio of peak intensities from13C-enriched γ-PGA divided by the ratio of peak intensities for nonenriched γ-PGA. EF values were than used to determine the percentage of repeat units that were formed from glutamate and citrate. The percentage of repeat units formed from provided glutamate at the 0 and 615 μM Mn(II) media concentrations was 89 ± 14 and 67 ± 11%, respectively. These respective products have 51 ± 9 and 39 ± 11% of their repeat units formed with apparent retention of the glutamate carbon skeleton. Also, enrichment of γ-PGA repeat units at C-1 was found to be lower than C-2 at both Mn(II) levels. Thus, provided glutamate was used to a large extent for polymer formation with both retention of the carbon skeleton as well as decarboxylation at C-1. Provided citrate was also used as a source of carbon to form repeat units. At the 0 and 615 μM media Mn(II) levels, 9 ± 4 and 19 ± 5% of repeat units were formed from citrate. It is believed that citrate is metabolized to γ-PGA by entry into the citric acid cycle and formation of α-ketoglutarate. Analysis of products from cultivations where both glutamate and citrate were13C enriched indicated that citrate and glutamate carbon source metabolism to γ-PGA occurs via independent pathways to common monomer precursors without multiple recycling of these carbon sources through catabolic and anabolic pathways.Key words: γ-poly(glutamic acid), metabolism, manganese, Bacillus licheniformis, nuclear magnetic resonance.
Comparison of 16S ribosomal RNA genes inClavibacter michiganensissubspecies with other coryneform bacteria
Authors: Li Xiang , Boer Solke H. De
Published: October 2009

Nearly complete sequences (97–99%) of the 16S rRNA genes were determined for type strains of Clavibacter michiganensis subsp. michiganensis, Clavibacter michiganensis subsp. insidiosus, Clavibacter michiganensis subsp. sepedonicus, and Clavibacter michiganensis subsp. nebraskensis. The four subspecies had less than 1% dissimilarity in their 16S rRNA genes. Comparative studies indicated that the C. michiganensis subsp. shared relatively high homology with the 16S rRNA gene of Clavibacter xyli. Further comparison with representatives of other Gram-positive coryneform and related bacteria with high G + C% values showed that this group of bacteria was subdivided into three clusters. One cluster consisted of the Clavibacter michiganensis subsp., Clavibacter xyli, Arthrobacter globiformis, Arthrobacter simplex, and Frankia sp.; another cluster consisted of members of the corynebacteria–mycobacteria–nocardia (CMN) group of Mycobacteriaceae including Tsukamurella paurometabolum; and Propionibacterium freudenreichii alone formed a unique cluster, which was remote from other coryneform bacteria analyzed. The three clusters may reflect a systematic rank higher than the genus level among these bacteria.Key words: Clavibacter, coryneform bacteria, phylogeny, 16S rRNA analysis.
The taxonomic significance of the growth response to Na+by strains ofVibrio
Authors: Goudie E. Dwayne , Gow John A.
Published: October 2009

Eighty regional Vibrio strains were studied for their growth responses at 13 Na+concentrations. Using a chemically defined plating medium, together with a multipoint inoculation technique, approximately 45% of the strains showed a specific growth requirement for Na+. The remaining strains grew, with a lag period, on the basal medium that contained about 2 mM background Na+. Based on the growth responses to Na+, a numerical analysis was used to explore differences between the strains. A dendrogram was produced in which the strains were grouped into four major clusters. At an equivalent level of similarity the cluster composition was not significantly different from that shown in a second dendrogram that was based on standard tests recommended in the 9th edition of Bergey's Manual of Determinative Bacteriology. The study showed that, over a range of concentrations, the growth response to Na+was taxonomically significant for Vibrio strains.Key words: Vibrio, marine bacteria, Na+requirement, growth response.
Serogroup conversion of Vibrio cholerae
Authors: Colwell R. R. , Huq A. , Chowdhury M. A. R. , Xu B. , Brayton P. R.
Published: October 2009

Vibrio cholerae serogroup O1 can be detected in the environment in a viable but nonculturable form, whereas V. cholerae non-O1 cells can be readily cultured during interepidemic periods in geographical regions where cholera is endemic. In the present study, pure cultures of V. cholerae non-O1 cells contained 01 cells when examined by immune-fluorescence microscopy. Laboratory microcosms were used to examine the outgrowth of the O1 cells in cultures of non-O1 V. cholerae. One O1 cell per 106 non-O1 cells could be detected by direct fluorescent-monoclonal antibody staining but only after incubation of the non-O1 culture for 48 h. Individual O1 cells were not detected in cultures incubated less than 48 h. Hybridization study, using a polymerase chain reaction (PCR) amplified fragment of the O-antigen of V. cholerae O1 as a probe, revealed the existence of a homologous gene in a microcosm sample of V. cholerae non-O1 containing serogroup-converted cells. The mechanism by which O1 cells can occur in cultures of non-O1 V. cholerae most likely resulted from spontaneous mutation of gene(s) encoding the O-somatic properties and (or) chemical, physical, or biological changes in the environment inducing expression or repression of the controlling gene(s). These findings have important implications for the epidemiology of cholera and the environmental source(s) of toxin producing V. cholerae O1.Key words: serogroup conversion, Vibrio cholerae O1, Vibrio cholerae non-O1, cholera.
Antimicrobial actions of fluoride for oral bacteria
Authors: Marquis Robert E.
Published: October 2009

Fluoride is widely used as a highly effective anticaries agent. Although it is felt that its anticaries action is related mainly to effects on mineral phases of teeth and on the process of remineralization, fluoride also has important effects on the bacteria of dental plaque, which are responsible for the acidification of plaque that results in demineralization. The results of recent studies have shown that fluoride can affect bacterial metabolism through a set of actions with fundamentally different mechanisms. It can act directly as an enzyme inhibitor, for example for the glycolytic enzyme enolase, which is inhibited in a quasi-irreversible manner. Direct action seems also to occur in inhibition of heme-based peroxidases with binding of fluoride to heme. The flavin-based peroxidases of many oral bacteria are insensitive to fluoride. Another mode of action involves formation of metal–fluoride complexes, most commonly AlF4−. These complexes are responsible for fluoride inhibition of proton-translocating F-ATPases and are thought to act by mimicking phosphate to form complexes with ADP at reaction centers of the enzymes. However, the actions of fluoride that are most pertinent to reducing the cariogenicity of dental plaque are those related to its weak-acid character. Fluoride acts to enhance membrane permeabilities to protons and compromises the functioning of F-ATPases in exporting protons, thereby inducing cytoplasmic acidification and acid inhibition of glycolytic enzymes. Basically, fluoride acts to reduce the acid tolerance of the bacteria. It is most effective at acid pH values. In the acidic conditions of cariogenic plaque, fluoride at levels as low as 0.1 mM can cause complete arrest of glycolysis by intact cells of Streptococcus mutans. Overall, the anticaries actions of fluoride appear to be complex, involving effects both on bacteria and on mineral phases. The antibacterial actions of fluoride appear themselves to be complex but to be dominated by weak-acid effects.Key words: fluoride, oral bacteria, dental caries, glycolysis.
Accumulation of Golgi-specific mannosyltransferases inCandida albicanscells grown in the presence of brefeldin A
Authors: Kossaczká Z. , Drgoňová J. , Farkaš V. , Podobová B. , Betina V.
Published: October 2009

The fungal metabolite brefeldin A (BFA) is known for its ability to block the secretory process in eukaryotic cells by interfering in the endoplasmic reticulum (ER) to Golgi membrane traffic, causing the disassembly of Golgi apparatus and redistribution of Golgi enzymes into the ER. In sensitive yeasts, underglycosylated forms of secretory proteins accumulate in the cytoplasm in the presence of BFA. We investigated whether the incomplete glycosylation of mannoproteins could be due to repression of the synthesis of Golgi-located terminal mannosyltransferases and whether the underglycosylated mannoproteins can be incorporated into the cell walls in Candida albicans. However, we found that the microsomal membranes isolated from the yeast cells grown in the presence of 14 μg∙mL−1of BFA had on average three times higher overall specific activity of mannan synthase than membranes from control cells. The increase in specific activity of mannan synthase was mainly due to accumulation of Golgi-specific mannosyltransferases responsible for elongation of the O-glycosidically linked mannooligosaccharides and for the synthesis of the N-glycosidically linked mannan outer chain. As a consequence, the mannans synthesized in vitro from GDP-[U-14C]mannose by the membranes from cells grown in the presence of BFA had longer O-glycosidically linked oligosaccharides and longer side-chains in the N-glycosidically linked polymannose part of the molecule than mannans synthesized by membranes from the control cells. Contrary to results obtained in vitro, the structural features of cell wall mannans isolated from intact BFA-grown and from control cells were almost indistinguishable.Key words: brefeldin A, Golgi apparatus, mannoproteins, mannosyltransferase, Candida albicans.
Improving the growth response ofBorreliaspecies
Authors: Padgett Penelope J. , Golden Mary J. , Pekala David J.
Published: October 2009

Borrelia species are microaerophilic spirochetes, i.e., they are unable to grow in the presence of atomospheric oxygen (21%) or under anaerobic conditions. Compounds that have been shown to quench toxic products of oxygen (i.e., superoxide radicals, hydrogen peroxide, hydroxyl radicals, and singlet oxygen) were added to Borrelia growth media (Kelly's medium). Among the compounds tested were catalase, superoxide dismutase, peroxidase, dithiothreitol (DTT), formate, fumarate, dimethyl sulfoxide (DMSO), manganese, activated charcoal (Norit A), mannitol, and a combination of ferric chloride and potassium (meta)bisulfite (iron-bisulfite). Of these, the compounds that were the most effective in enhancing the growth of the species of Borrelia tested were iron-bisulfite, DTT, and DMSO. DTT possesses the ability to break down hydrogen peroxide, and iron-bisulfite has been reported to have superoxide dismutase-like activity in artificial media. Iron-bisulfite, DMSO, sodium formate, DTT, and combinations of these compounds have been incorporated into solid and biphasic Kelley's medium to determine their effect on microaerobic and aerobic growth on plates and in biphasic medium. Iron-bisulfite was the most effective compound tested under these conditions.Key words: Borrelia, bisulfite, spirochetes, microaerobe.
Comparative study of two trehalase activities fromFusarium oxysporumvar.lini
Authors: Amaral Fernando Costa , Brandäo Rogelio Lopes , Nicoli Jacques Robert , Ortiz Claudio Henrique Dias
Published: October 2009

Acid and neutral trehalase activities (optimum pH of 4.6 and 6.8, respectively) from Fusarium oxysporum var. lini were studied separately through partial isolation by ammonium sulfate precipitation followed by ion-exchange chromatography on DEAE-Sephacel for neutral enzyme, or using some of their differential properties. Acid activity was unaffected by 1 mM of Ca2+, Mg2+, Mn2+, Ba2+, or EDTA. Contrarily, the neutral enzyme was activated by Ca2+with an apparent Kaof 0.15 mM; was inhibited by EDTA, Zn2+, Hg2+, or Mg2+-ATP; and showed an increase in activity by the raise of buffer ionic strength or by the addition of 100 mM KCl. Acid and neutral enzymes have, respectively, an apparent optimum temperature of 45 and 30 °C, an apparent Kmfor trehalose of 0.43 and 8.45 mM, and an apparent Mrof 160 000 and 100 000 (by glycerol gradient ultracentrifugation). Acid trehalase was specifically inhibited by acetate buffer and more stable at 50 °C than the neutral enzyme. Neutral enzyme exhibited a pi of 6.2 by isoelectric focusing. Contrary to neutral trehalases from other fungi, the enzyme from Fusarium oxysporum var. lini was not activated in crude extract by treatment with Mg2+-ATP in the presence of cAMP and not inactivated by alkaline phosphatase from Escherichia coli.Key words: acid trehalase, neutral trehalase, Fusarium oxysporum.
Water activity, temperature, and pH effects on growth of Fusarium moniliforme and Fusarium proliferatum isolates from maize
Authors: Marin Sonia , Sanchis Vicente , Magan Naresh
Published: October 2009

The effects of water activity (aw, 0.994–0.90 ≡ 0.4–14.0 (–)MPa water potential), temperature (4–45 °C), and pH (3.6, 5.5, 7.0), and their interactions on growth of isolates of Fusarium moniliforme and Fusarium proliferatum were determined in vitro on a maize extract agar medium. Growth of two isolates of F. moniliforme and four isolates of F. proliferatum were significantly influenced by water activity regardless of solute type used (NaCl, glycerol, or glucose). However, at steady-state aw levels, growth was optimum at 0.994–0.98 aw and reduced significantly at 0.92 aw. Further detailed studies with one isolate of F. moniliforme (25N) and two isolates of F. proliferatum (73N, 13 IN) showed that growth occurred over the range of 0.994–0.90 aw in the temperature range 20–35 °C with slight differences between species. Growth did occur at 4 °C and 0.994–0.96 aw, but no growth was recorded at 40 and 45 °C regardless of aw. Profiles of aw × temperature relations for growth of these two species were constructed from these data for the first time. Optimum pH and temperature for growth was 5.5 and 25 °C for both isolates of F. proliferatum, and pH 7.0 and 30 °C for the isolate of F. moniliforme. However, for the latter isolate at <0.98 aw, optimum pH and temperature for growth changed. The effects of pH, temperature, and aw for single, two-way and three-way interactions were all found to be statistically significant for these three isolates. The ecological significance of this information for understanding these important fumonisin-producing fungi is discussed.Key words: water activity, temperature, fumonisin-producing, Fusarium moniliforme, Fusarium proliferatum, maize.
Microbial ecology of simultaneous thermophilic microbial leaching and digestion of sewage sludge
Authors: Shooner Frédéric , Tyagi Rajeshwar D.
Published: October 2009

The microbial population encountered during a simultaneous thermophilic microbial leaching and digestion process at 50 °C, based on microbial sulfur oxidation, was investigated. The cell count of the sulfuric acid producer Thiobacillus thermosulfatus increased, followed by a decrease. In the absence of sulfur (control: conventional thermophilic digestion), Thiobacillus thermosulfatus population decreased under the detection limit. Acidophilic and neutrophilic heterotrophic populations increased during the leaching process, and the final acidophilic population count was higher than the neutrophilic population. During the thermophilic digestion (control), the final neutrophilic population count was higher than the acidophilic. Six heterotrophic bacterial strains were isolated and partially characterized. Bacillus was the most predominant genus. The type of bacterial populations in thermophilic microbial leaching and digestion, as well as the thermophilic digestion process (control), were the same, while only the relative concentrations changed. In both processes, the bacterial indicators decreased under the detection limit after 12 h. Mesophilic heterotrophic population was more affected by the thermophilic microbial leaching process than by thermophilic digestion. Sludge mineralization was probably more influenced by the final cell concentration rather than the presence of an individual species or mixed population.Key words: Thiobacillus thermosulfatus, thermophilic metal leaching, thermophilic sludge digestion, indicator microorganisms.
A cloned cell line ofSpodopteta exiguahas a highly increased susceptibility to theSpodoptera exiguanuclear polyhedrosis virus
Authors: Hara Koyu , Funakoshi Masako , Kawarabata Takeshi
Published: October 2009

A cloned cell line designated Se301, derived from a continuous Se3FH cell line of the beet army worm Spodoptera exigua, showed 10 times greater sensitivity when tested with the plaque assay to a S. exigua nuclear polyhedrosis virus (SeNPV) as compared with the parent cell line. Nearly 100% of Se301 cells were infected with the plaque purified isolate SeNPV-I1 or the wild isolate SeNPV-IW. Plaques were first detected 3–4 days in the Se301 cell line, which was 1–2 days faster than the Se3FH cell line. The size of plaques formed in the Se301 cell line was markedly larger than that of the parental cell line. At 12 h post infection, the production of the extracellular viruses (ECVs) in the Se301 cell line infected with either SeNPV-I1 or SeNPV-IW was 20–40 times greater than that in the Se3FH cell line. In contrast, Se301 cells infected with SeNPV-I1 or SeNPV-IW produced polyhedral inclusion bodies (PIBs) at lower levels compared with Se3FH cells. The isolate SeNPV-I1 consistently produced more ECVs and PIBs in both the Se301 and Se3FH cell lines than the isolate SeNPV-IW.Key words: Spodoptera exigua, nuclear polyhedrosis virus, cloned cell line, increased virus sensitivity, virus production.
Detection of additional restriction fragment length polymorphisms among the weakly virulent (nonaggressive) and highly virulent (aggressive) isolates of Leptosphaeria maculans
Authors: Patterson N. A. , Kapoor M.
Published: October 2009

Isolates of Leptosphaeria maculans were analyzed for their genetic relatedness based on DNA restriction fragment length polymorphisms (RFLPs), employing as Southern hybridization probes a combination of heat shock responsive genes (hsp70 and hsp80 from Neurospora crassa), the cutinase gene of Magnaporthe grisea, and cloned genomic DNA sequences from a virulent strain. Southern hybridization analysis revealed a high frequency of DNA polymorphism. Restriction fragments generated by each enzyme-probe combination resulted in distinct banding patterns, clearly separating the isolates into two groups. The cutinase gene probe did not reveal any polymorphisms. Although the majority of the probes used displayed RFLP profiles unique to each group, a nonaggressive isolate, LmA, showed additional genetic characteristics in common with the virulent pathotype.Key words: Leptosphaeria, genomic libraries, RFLP.
Formation of novel poly(hydroxyalkanoates) from long-chain fatty acids
Authors: Eggink Gerrit , Waard Pieter de , Huijberts Gern N. M.
Published: October 2009

Poly(hydroxyalkanoates) (PHAs) were isolated from Pseudomonas aeruginosa 44T1 cultivated on euphorbia oil and castor oil. With the aid of 2-D proton NMR spectra and proton-detected multiple bond coherence NMR spectra the structures of the PHAs were determined. In addition to the usual PHA constituents (C6–C143-hydroxy fatty acids), PHAs formed from euphorbia oil contained Δ8,9-epoxy-3-hydroxy-5c-tetradecenoate, and probably Δ6,7-epoxy-3-hydroxydodecanoate and Δ4,5-epoxy-3-hydroxydecanoate. These novel constituents account for approximately 15% of the total amount of monomers and are clearly generated via β-oxidation of vernolic acid (Δ12,13-epoxy-9c-octadecenoic acid), the main component of euphorbia oil. In PHAs formed from castor oil, 7% of the monomers found were derived from ricinoleic acid (12-hydroxy-9c-octadecenoic acid). The presence of 3,8-dihydroxy-5c tetradecenoate was clearly demonstrated. Furthermore, NMR analysis strongly suggested the presence of 3,6-dihydroxydodecanoate, 6-hydroxy-3c-dodecenoate, and 4-hydroxydecanoate.Key words: poly(hydroxyalkanoates), pseudomonads, ricinoleic acid, vernolic acid.
Methylobacterium rhodesianumMB 126 possesses two stereospecific crotonyl-CoA hydratases
Authors: Mothes Gisela , Babel Wolfgang
Published: October 2009

Two distinct crotonyl-CoA hydratases of Methylobacterium rhodesianum MB 126 were separated by column chromatography on DEAE-Sepharose CL-6B. The two enzymes were further purified by chromatography on red 120 agarose and Mono Q. Enzyme A was specific for L(+)-hydroxybutyryl-CoA. It had an apparent molecular weight of 160 000 with two identical subunits of 43 000 and 34 000. The apparent Kmvalues for L(+)-hydroxybutyryl-CoA and crotonyl-CoA were 83 and 90 μM, respectively. Enzyme B was specific for D(−)-hydroxybutyryl-CoA. It had an apparent molecular weight of 39 000 with identical subunits of 12 500. It showed sigmoidal kinetics for crotonyl-CoA, and the Hill coefficient was about 2.5. The apparent Kmvalue for D(−)-hydroxybutyryl-CoA was 0.5 mM. The possible contribution of a sequence including β-ketothiolase, NADH-linked L(+)-specific acetoacetyl-CoA reductase, and two stereospecific crotonyl-CoA hydratases to PHB synthesis in methylotrophic serine-pathway bacteria is discussed.Key words: poly(β-hydroxybutyrate), PHB, crotonyl-CoA hydratase, methylotroph, Methylobacterium rhodesianum.
Polyhydroxyalkanoate analysis inAzospirillum brasilense
Authors: Itzigsohn Robin , Yarden Oded , Okon Yaacov
Published: October 2009

The considerable industrial interest in the qualitative and quantitative production of polyhydroxyalkanoates in microorganisms has led to the characterization of those synthesized in the nitrogen-fixing bacteria Azospirillum brasilense and Azotobacter paspali. In contrast to some other bacterial species, Azospirillum brasilense does not produce copolymers of hydroxyalkanoates when grown under the different carbon sources assayed, namely n-alkanoic acids, hydroxyalkanoates, and sugars with varying C:N ratios. Rather, only homopolymers of polyhydroxybutyrate were detected, comprising up to 70% of the cell dry mass. No copolymers were detected in Azotobacter paspali. Quantitative analyses of poly(β-hydroxybutyrate) are also presented.Key words: Azospirillum spp., Azotobacter paspali, polyhydroxyalkanoate analysis, PHA, PHB.
Poly(β-hydroxyalkanoate) accumulation in bacterioplankton from Lake Cisó (Spain)
Authors: Mas-Castellà J. , Guerrero R.
Published: October 2009

The specific poly(β-hydroxyalkanoate) (PHA) content of the bacterioplankton from Lake Cisó (Spain) was measured at different depths. Phototrophic bacteria reach large populations in this lake and the PHA concentration changed seasonally according to their biomass. During summer stratification of the lake, phototrophic bacteria formed a metalimnetic peak. Bacterial counts were maximal at a depth of 1.5–2.0 m, whereas the PHA specific content reached a maximal value at 3.0–3.5 m. Since the limiting factor in this ecosystem is light availability, we assume that cells beneath the dense metalimnetic peak have unbalanced growth conditions and thus accumulate large amounts of PHA.Key words: bacterioplankton, PHA accumulation, Lake Cisó, light limitation.
The ordered macromolecular surface of polyester inclusion bodies inPseudomonas oleovotans
Authors: Stuart Elizabeth S. , Fuller R. Clinton , Lenz Robert W.
Published: October 2009

Intracellular inclusion bodies of poly(β-hydroxyalkanoates) (PHAs) have been studied in various microorganisms since Lemoigne's discovery of PHAs in 1925. Recently, the research in several laboratories, including our own, has addressed the role of proteins, lipids, and water associated with these accumulations. Our research has examined the boundary of polymer inclusion bodies. Electron microscopy demonstrated that the polymer is encompassed by two paracrystalline arrays. SDS-PAGE, Western blot, or immunogold labelling demonstrated that both contain a 43-kDa protein as a major component. Immunogold labelling also demonstrated that 55- and 59-kDa proteins are located, exclusively, on the array associated with the accumulating polymer. Results from microelemental analysis and preliminary thin-layer chromatography of released lipids were consistent with the suggestion that phospholipids also had a role in this organized assembly. A model has been suggested, aimed at focusing attention on this organized boundary region. It is consistent with maintenance of the amorphous state of the polymer both intracellularly and after isolation, provides sites for biosynthetic and degradative enzymes, and accounts for the polyester, protein, and lipid components known to be present. Interestingly, the anti-43-kDa antibody also recognized a 43-kDa species released from the outer surface of this microbe. The research presented here and the model developed from it, suggest that microbial synthesis, containment, and degradation of polyester are carried out in association with a highly organized and complex intracellular assembly that may provide, within the bacterial cytosol, a unique microenvironment for biochemical activities.Key words: polyester, inclusion granule, Pseudomonas oleovorans.
The role of β-oxidation of short-chain alkanoates in polyhydroxyalkanoate copolymer synthesis inAzotobacter vinelandiiUWD
Authors: Page William J. , Manchak Janet
Published: October 2009

Valerate and other short-chain, uneven-length fatty acids promoted the formation of the polyhydroxyalkanoate (PHA) copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) in Azotobacter vinelandii UWD growing in glucose medium. The uptake of valerate was inducible, being repressed by acetate but not by glucose. A likely route that would direct valerate into PHA synthesis involved the β-oxidation pathway. The short-chain fatty acids butyrate, valerate, trans.-2-pentenoate, crotonate, hexanoate, heptanoate, and octanoate induced the coordinate production of the β-oxidation enzymes enoyl-CoA hydratase (EGH) and L-(+)-3-hydroxybutyryl-CoA dehydrogenase (HAD).trans-3-Pentenoate was the best inducer of these activities, which suggested that the isomerase of the β-oxidation complex also was present. However, 3-hydroxyacyl-CoA epimerase activity of the β-oxidation complex was not detected. 3-Ketoacyl-CoA thiolase activity was constitutive in A. vinelandii and appeared to associate only loosely with the 73 000 Da ECH–HAD complex. Thus, 3-ketoacyl-CoA, the end product of HAD activity, could be directed into PHA synthesis through acetoacetyl-CoA reductase generating the 3-hydroxyvalerate subunit of the polymer. When valerate was the sole carbon source, the incorporation of valerate into the polymer was normal, but most of the valerate was directed into metabolism and very little PHA was formed. When glucose also was present, the β-oxidation of short-chain alkanoates inhibited the specific activity of acetoacetyl-CoA reductase and 3-ketothiolase and the PHA yield. A model for PHA synthesis was developed that suggests that the use of fatty acids to promote PHA copolymer formation in A. vinelandii will inevitably result in decreased PHA yield.Key words: β-oxidation, poly(β-hydroxyalkanoate) synthesis, short chain fatty acids, regulation.