bluuebunny/crossref_metadata_2025 huggingface dataset:
Accumulation of Mannosylglycerate and Di-myo-Inositol-Phosphate by Pyrococcus furiosus in Response to Salinity and Temperature
Authors: Martins L O , Santos H
Published: January 2020

(sup13)C and (sup1)H nuclear magnetic resonance spectroscopy was used to identify and quantify organic solutes accumulated by the hyperthermophilic archaeon Pyrococcus furiosus in response to temperature and salinity. Di-myo-inositol-phosphate and 2-O-(beta)-mannosylglycerate were the major organic solutes accumulated in these cells. The total intracellular organic solutes increased significantly in response either to an increase in temperature or to an increase in salinity, but (beta)-mannosylglycerate accumulated mainly at high salinities, whereas the concentration of di-myo-inositol-phosphate increased dramatically at supraoptimal growth temperatures. Glutamate was present at concentrations detectable by nuclear magnetic resonance only in cells grown in low-salinity media. The intracellular levels of K(sup+) are clearly dependent on the salinity of the medium, and the concentrations of this cation are high enough to counterbalance the negative charges of (beta)-mannosylglycerate and di-myo-inositol-phosphate in the cell. The results presented here together with those previously reported for Pyrococcus woesei (S. Scholz, J. Sonnenbichler, W. Schafer, and R. Hensel, FEBS Lett. 306:239-242, 1992) strongly support a role for di-myo-inositol-phosphate in thermoprotection.
Determination of effective transport coefficients for bacterial migration in sand columns
Authors: Barton J W , Ford R M
Published: January 2020

A well-characterized experimental system was designed to evaluate the effect of porous media on macroscopic transport coefficients which are used to characterize the migration of bacterial populations. Bacterial density profiles of Pseudomonas putida PRS2000 were determined in the presence and absence of a chemical attractant (3-chlorobenzoate) gradient within sand columns having a narrow distribution of particle diameters. These experimental profiles were compared with theoretical predictions to evaluate the macroscopic transport coefficients. The effective random motility coefficient, used to quantify migration due to a random process in a porous medium, decreased nearly 20-fold as grain size in the columns decreased from 800 to 80 (mu)m. The effective random motility coefficient (mu)(infeff) was related to the random motility coefficient (mu), measured in a bulk aqueous system, according to (mu)(infeff) = ((epsilon)/(tau))(mu) with porosity (epsilon) and tortuosity (tau). Over the times and distances examined in these experiments, bacterial density profiles were unaffected by the presence of an attractant gradient. Theoretical profiles with the aqueous phase value of the chemotactic sensitivity coefficient (used to quantify migration due to a directed process) were consistent with this result and suggested that any chemotactic effect on bacterial migration was below the detection limits of our assay.
Impact of Field Release of Genetically Modified Pseudomonas fluorescens on Indigenous Microbial Populations of Wheat
Authors: De Leij F , Sutton E J , Whipps J M , Fenlon J S , Lynch J M
Published: January 2020

In a field release experiment, an isolate of Pseudomonas fluorescens, which was chromosomally modified with two reporter gene cassettes (lacZY and Kan(supr)-xylE), was applied to spring wheat as a seed coating and subsequently as a foliar spray. The wild-type strain was isolated from the phylloplane of sugar beet but was found to be a common colonizer of both the rizosphere and phylloplane of wheat as well. The impact on the indigenous microbial populations resulting from release of this genetically modified microorganism (GMM) was compared with the impact of the unmodified, wild-type strain and a nontreated control until 1 month after harvest of the crop. The release of the P. fluorescens GMM and the unmodified, wild-type strain resulted in significant but transient perturbations of some of the culturable components of the indigenous microbial communities that inhabited the rhizosphere and phylloplane of wheat, but no significant perturbations of the indigenous culturable microbial populations in nonrhizosphere soil were found. Fast-growing organisms that did not produce resting structures (for example, fluorescent pseudomonads and yeasts) seemed to be most sensitive to perturbation. In terms of hazard and risk to the environment, the observed microbial perturbations that resulted from this GMM release may be considered minor for several reasons. First, the recombinant P. fluorescens strain caused changes that were, in general, not significantly different from those caused by the unmodified wild-type strain; second, perturbations resulting from bacterial inoculations were mainly small; and third, the release of bacteria had no obvious effects on plant growth and plant health.
Permanent presence of grazing-resistant bacteria in a hypertrophic lake
Authors: Sommaruga R , Psenner R
Published: January 2020

The size structure of planktonic bacteria from a hypertrophic lake was investigated at 5- to 15-day intervals by means of a semiautomatic image analysis system during 1 year. Characteristic of this bacterial assemblage was the permanent presence of large filamentous bacteria and small cocci with cell sizes of <0.01 (mu)m(sup3). These filamentous bacteria, sometimes longer than 200 (mu)m and with cell volumes of up to 276 (mu)m(sup3), are larger than nanoflagellates (<20 (mu)m) and, even, metazoans living in the lake. Although they account for only 4 to 16% of bacterial abundance, their contribution to total bacterial biovolume was between 45 and 86%. An analysis of the food web structure indicates that this particular bacterial size structure may be the consequence of a strong bacterivory pressure by nanoflagellates and the absence of other larger bacterivores. The persistence of bacterial forms resistant to grazing has important consequences for the carbon flow within the microbial food web.
Reexamination of tetrodotoxin production by bacteria
Authors: Matsumura K
Published: January 2020

Vibrio alginolyticus has been reported as a good producer of tetrodotoxin (TTX), but the toxin extracted from this bacterium did not react to the monoclonal antibody against TTX. Surprisingly, chromatographic analyses detected high TTX peaks for polypeptone and yeast extracts used as medium materials, which were, as expected, all negative by the mouse bioassay. These results may require us to revise the bacterial production of TTX.
Screening of tropical wood-rotting mushrooms for copper biosorption
Authors: Muraleedharan T R , Iyengar L , Venkobachar C
Published: January 2020

Fruiting bodies (mushrooms) of nine nonedible macrofungi were screened for copper(II) uptake potential. The maximum uptake potentials (Q(infmax)s) derived from equilibrium studies indicated that all nine species exhibited higher Q(infmax)s at pH 4.0 than that of Filtrasorb-400, a generally used adsorbent for metal removal. Wide variation in Q(infmax) was observed among the species and ranged from 0.048 to 0.383 mmol per g of sorbent. The uptake capacity of Ganoderma lucidum, which exhibited the highest Q(infmax), was higher than those of other microbial biosorbents reported in the literature.
Cloning and characterization of styrene catabolism genes from Pseudomonas fluorescens ST
Authors: Marconi A M , Beltrametti F , Bestetti G , Solinas F , Ruzzi M , Galli E , Zennaro E
Published: January 2020

A gene bank from Pseudomonas fluorescens ST was constructed in the broad-host-range cosmid pLAFR3 and mobilized into Pseudomonas putida PaW340. Identification of recombinant cosmids containing the styrene catabolism genes was performed by screening transconjugants for growth on styrene and epoxystyrene. Transposon mutagenesis and subcloning of one of the selected genome fragments have led to the identification of three enzymatic activities: a monooxygenase activity encoded by a 3-kb PstI-EcoRI fragment and an epoxystyrene isomerase activity and an epoxystyrene reductase activity encoded by a 2.3-kb BamHI fragment. Escherichia coli clones containing the 3-kb PstI-EcoRI fragment were able to transform styrene into epoxystyrene, and those containing the 2.3-kb BamHI fragment converted epoxystyrene into phenylacetaldehyde or, only in the presence of glucose, into 2-phenylethanol. The three genes appear to be clustered and are probably encoded by the same DNA strand. In E. coli, expression of the epoxystyrene reductase gene was under the control of its own promoter, whereas the expression of the other two genes was dependent on the presence of an external vector promoter.
Enhanced Glycerol Content in Wines Made with Immobilized Candida stellata Cells
Authors: Ciani M , Ferraro L
Published: January 2020

Screening tests carried out for 10 strains of Candida stellata confirmed high levels of glycerol production, although a low fermentation rate and reduced ethanol content were observed. To overcome the poor competition with Saccharomyces cerevisiae, fermentation tests with immobilized C. stellata cells, alone or in combination with S. cerevisiae, have been carried out. The immobilization of C. stellata cells consistently reduced the fermentation length when compared with that obtained with free cells, immobilized cells exhibiting about a 30-and a 2-fold improvement in fermentation rate compared with rates for C. stellata and S. cerevisiae free cells, respectively. Moreover, immobilized C. stellata cells produced a twofold increase in ethanol content and a strong reduction in acetaldehyde and acetoin production in comparison with levels for free cells. The evaluation of different combinations of C. stellata immobilized cells and S. cerevisiae showed interesting results with regard to analytical profiles for practical application in wine making. In fact, analytical profiles of combinations showed, apart from a high glycerol content, a reduction in the amounts of acetic acid and higher alcohols and a consistent increase in succinic acid content in comparison with values for the S. cerevisiae control strain. Sequential fermentation first with immobilized C. stellata cells and then after 3 days with an added inoculum of S. cerevisiae free cells was the best combination, producing 15.10 g of glycerol per liter, i.e., 136% more than the S. cerevisiae control strain produced. Fermentation with immobilized C. stellata cells could be an interesting process by which to enhance glycerol content in wine.
Phylogenetic position of yeastlike endosymbionts of anobiid beetles
Authors: Noda H , Kodama K
Published: January 2020

The Anobiid beetles Stegobium paniceum and Lasioderma serricorne possess the intracellular yeastlike symbionts Symbiotaphrina buchneri and Symbiotaphrina kochii, respectively, in the mycetome between the foregut and midgut. The nucleotide sequences of the small-subunit rRNA-encoding genes of the symbionts were determined for phylogenetic analysis. Five group I introns were found in the small-subunit rRNA-encoding genes of S. buchneri, but S. kochii lacked introns. The phylogenetic position of both symbionts was close to the class Discomycetes, which is a paraphyletic group. The two symbionts formed a monophyletic group distinct from the other major lineages. Both appear to have parted from other filamentous fungi during the early radiation of the euascomycetes and to have later become obligatory partners of the beetles.
Isolation, DNA sequence analysis, and mutagenesis of a proline dehydrogenase gene (putA) from Bradyrhizobium japonicum
Authors: Straub P F , Reynolds P H , Althomsons S , Mett V , Zhu Y , Shearer G , Kohl D H
Published: January 2020

We report here the cloning and sequencing of the gene for proline dehydrogenase (putA) of Bradyrhizobium japonicum. An open reading frame coding for 1,016 amino acids was identified. The B. japonicum gene codes for a bifunctional protein with proline dehydrogenase and pyrroline-5-carboxylate (P5C) dehydrogenase activities, as it does in Escherichia coli and Salmonella typhimurium. Comparison of the sequences of these proteins with other proline and P5C dehydrogenase sequences identified proline dehydrogenase and P5C dehydrogenase catalytic domains. Within the proline dehydrogenation domain, several areas of high identity were observed between B. japonicum, E. coli, S. typhimurium, Saccharomyces cerevisiae put1, and Drosophila melanogaster slgA. Within the P5C dehydrogenase domain, several areas of high identity were observed between B. japonicum, E. coli, S. typhimurium, Bacillus subtilis ipa76d, and S. cerevisiae put2. A consensus catalytic site for semialdehyde dehydrogenase was observed in the P5C dehydrogenase domain. This suggests that the substrate for this domain may be the open-chain gamma-glutamylsemialdehyde, not its cyclized form, P5C. Unlike the gene isolated from E. coli, S. typhimurium, and K. pneumoniae, the B. japonicum putA gene does not appear to be part of an operon with the proline porter gene (putP). Additionally, the B. japonicum gene lacks the putative C-terminal regulatory domain present in the E. coli and S. typhimurium genes. The gene was disrupted by insertion of antibiotic resistance gene cassettes, which were then recombined into the bacterial chromosome. Symbiotically active mutant strains that were devoid of putA activity were isolated. With this proline dehydrogenase clone, we will test the hypothesis that putA in symbiotic nitrogen-fixing B. japonicum bacteroids is transcriptionally regulated by drought and other stresses.
Rapid Anaerobic Benzene Oxidation with a Variety of Chelated Fe(III) Forms
Authors: Lovley D R , Woodward J C , Chapelle F H
Published: January 2020

Fe(III) chelated to such compounds as EDTA, N-methyliminodiacetic acid, ethanol diglycine, humic acids, and phosphates stimulated benzene oxidation coupled to Fe(III) reduction in anaerobic sediments from a petroleum-contaminated aquifer as effectively as or more effectively than nitrilotriacetic acid did in a previously demonstrated stimulation experiment. These results indicate that many forms of chelated Fe(III) might be applicable to aquifer remediation.
Isolation of a species-specific mitochondrial DNA sequence for identification of Tilletia indica, the Karnal bunt of wheat fungus
Authors: Ferreira M A , Tooley P W , Hatziloukas E , Castro C , Schaad N W
Published: January 2020

Mitochondrial DNA (mtDNA) from five isolates of Tilletia indica was isolated and digested with several restriction enzymes. A 2.3-kb EcoRI fragment was chosen, cloned, and shown to hybridize with total DNA restricted with EcoRI from T. indica and not from a morphologically similar smut fungus, Tilletia barclayana. The clone was partially sequenced, and primers were designed and tested under high-stringency conditions in PCR assays. The primer pair Ti1/Ti4 amplified a 2.3-kb fragment from total DNA of 17 T. indica isolates from India, Pakistan, and Mexico. DNA from 25 isolates of other smut fungi (T. barclayana, Tilletia foetida, Tilletia caries, Tilletia fusca, and Tilletia controversa) did not produce any bands, as detected by ethidium bromide-stained agarose gels and Southern hybridizations. The sensitivity of the assay was determined and increased by using a single nested primer in a second round of amplification, so that 1 pg of total mycelial DNA could be detected. The results indicated that the primers which originated from a cloned mtDNA sequence can be used to differentiate T. indica from other Tilletia species and have the potential to identify teliospores contaminating wheat seeds.
Biological degradation of tannins in sericea lespedeza (Lespedeza cuneata) by the white rot fungi Ceriporiopsis subvermispora and Cyathus stercoreus analyzed by solid-state 13C nuclear magnetic resonance spectroscopy
Authors: Gamble G R , Akin D E , Makkar H P , Becker K
Published: January 2020

Leaves of sericea lespedeza exhibit a high proportion of condensed tannin, resulting in poor forage quality. The white rot fungi Ceriporiopsis subvermispora and Cyathus sterocoreus are known to preferentially degrade lignin in a variety of plants and were evaluated for their ability to degrade condensed tannin from sericea leaves with the aim of improving digestibility. Relative levels of condensed tannin, cutin, pectin, and cellulose were monitored as a function of fungal treatment by solid-state cross-polarization and magic angle spinning 13C nuclear magnetic resonance spectroscopy. Total soluble phenolics, soluble tannins, and soluble and insoluble proanthocyanidin levels in fungus-treated and control samples were measured by established chemical techniques. Results indicate that both species of fungus preferentially degrade condensed tannin and that C. subvermispora is markedly superior to C. stercoreus in this capacity.
Tailing of thermal inactivation curve of Aspergillus niger spores
Authors: Fujikawa H , Itoh T
Published: January 2020

The nonlinear thermal inactivation of Aspergillus niger spores in phosphate-citrate buffer was studied. The thermal inactivation pattern of the spore consisted of a shoulder, an accelerated decline, and a tail at various constant temperatures around 60 degrees C. The pattern fitted a thermotolerant subpopulation model. In the model, we postulated that some spores in the initial population had become thermotolerant at a certain ratio during heating. The model parameters including the rate coefficients, the time lag, and the existence ratio of thermotolerant cells were analyzed at various temperatures. The tailing was not observed at an initial concentration below 10(3) cells per ml. Cells cultured from thermotolerant cells showed an inactivation pattern similar to that of the original cells. Also, cells at the second heating showed the same thermotolerance as or were slightly more thermosensitive than the original cells. Intermittent heating was found to be effective to inactivate cells at a high concentration.
Competition and coexistence of sulfate-reducing and methanogenic populations in anaerobic biofilms
Authors: Raskin L , Rittmann B E , Stahl D A
Published: January 2020

The microbial population structure and function of natural anaerobic communities maintained in laboratory fixed-bed biofilm reactors were tracked before and after a major perturbation, which involved the addition of sulfate to the influent of a reactor that had previously been fed only glucose (methanogenic), while sulfate was withheld from a reactor that had been fed both glucose and sulfate (sulfidogenic). The population structure, determined by using phylogenetically based oligonucleotide probes for methanogens and sulfate-reducing bacteria, was linked to the functional performance of the biofilm reactors. Before the perturbation, the methanogenic reactor contained up to 25% methanogens as well as 15% sulfate-reducing bacteria, even though sulfate was not present in the influent of this reactor. Methanobacteriales and Desulfovibrio spp. were the most abundant methanogens and sulfate-reducing bacteria, respectively. The presence of sulfate-reducing bacteria (primarily Desulfovibrio spp. and Desulfobacterium spp.) in the absence of sulfate may be explained by their ability to function as proton-reducing acetogens and/or fermenters. Sulfate reduction began immediately following the addition of sulfate consistent with the presence of significant levels of sulfate-reducing bacteria in the methanogenic reactor, and levels of sulfate-reducing bacteria increased to a new steady-state level of 30 to 40%; coincidentally, effluent acetate concentrations decreased. Notably, some sulfate-reducing bacteria (Desulfococcus/Desulfosarcina/Desulfobotulus group) were more competitive without sulfate. Methane production decreased immediately following the addition of sulfate; this was later followed by a decrease in the relative concentration of methanogens, which reached a new steady-state level of approximately 8%. The changeover to sulfate-free medium in the sulfidogenic reactor did not cause a rapid shift to methanogenesis. Methane production and a substantial increase in the levels of methanogens were observed only after approximately 50 days following the perturbation.
Direct selection of cloned DNA in Bacillus subtilis based on sucrose-induced lethality
Authors: Bramucci M G , Nagarajan V
Published: January 2020

Expression of the Bacillus subtilis or Bacillus amyloliquefaciens sacB gene in the presence of sucrose is lethal for a variety of bacteria. Sucrose-induced lethality can be used to select for inactivation of sacB by insertion of heterologous DNA in sensitive bacteria. This procedure has not been applicable to B. subtilis heretofore because expression of wild-type sacB is not detrimental to B. subtilis. The W29 mutation in the B. amyloliquefaciens sacB gene interferes with processing of the levansucrase signal peptide. The W29 mutation does not affect growth of B. subtilis in media lacking sucrose. However, this mutation inhibited growth of B. subtilis in media containing sucrose. Inactivation of the fructose polymerase activity encoded by sacB indicated that levan production was essential for sucrose-induced lethality. As a result, it was possible to select for cloned DNA in B. subtilis by insertional inactivation of the mutant sacB gene located on a multicopy plasmid vector in medium containing sucrose.
Hypochlorous acid activates the heat shock and soxRS systems of Escherichia coli
Authors: Dukan S , Dadon S , Smulski D R , Belkin S
Published: January 2020

A series of plasmids, containing fusions of different stress promoters to lux reporter genes, was used in an attempt to monitor the defense circuits activated upon exposure of Escherichia coli to sublethal doses of free chlorine. A significant level of activation was exhibited by promoters of three heat shock genes (grpE, dnaK, and lon), in an rpoH-dependent manner. The promoter of micF, a gene under the control of the soxRS regulon, was also strongly induced, but not in a soxR mutant. This induction was not affected by sodA and sodB mutations, implying that it did not involve oxygen radical activity. Free-chlorine activation of both heat shock and soxRS regulons required an exposure of less then I s in duration. The oxyR or the SOS regulons were apparently not induced by free chlorine (as judged by lack of activation of katG and recA, respectively), and neither was the universal stress (uspA) protein.
Siderophore-Mediated Aluminum Uptake by Bacillus megaterium ATCC 19213
Authors: Hu X , Boyer G L
Published: January 2020

The bacterium Bacillus megaterium ATCC 19213 is known to produce two hydroxamate siderophores, schizokinen and N-deoxyschizokinen, under iron-limited conditions. In addition to their high affinity for ferric ions, these siderophores chelate aluminum. Aluminum was absorbed by B. megaterium ATCC 19213 through the siderophore transport receptor, providing an extra pathway for aluminum accumulation into iron-deficient bacteria. At low concentrations of the metal, siderophore-mediated uptake was the dominant process for aluminum accumulation. At high concentrations of aluminum, passive transport dominated and siderophore production slowed the passive transport of aluminum into the cell. Siderophore production was affected by the aluminum content in the media. High concentrations of aluminum increased production of siderophores in iron-limited cultures, and this production continued into stationary phase. Aluminum did not stimulate siderophore production in iron-replete cultures. The production of siderophores markedly affected aluminum uptake. This has direct implications on the toxicity of heavy metals under iron-deficient conditions.
Expression of the antimicrobial peptide carnobacteriocin B2 by a signal peptide-dependent general secretory pathway
Authors: McCormick J K , Worobo R W , Stiles M E
Published: January 2020

Carnobacteriocin B2 is a well-characterized class II bacteriocin produced by a 61-kb plasmid from Carnobacterium piscicola LV17. Export of this bacteriocin is dependent on specific ABC (ATP-binding cassette) secretion proteins. Divergicin A is a strongly hydrophobic narrow-spectrum bacteriocin produced by a 3.4-kb plasmid from Carnobacterium divergens LV13. Predivergicin A contains a signal peptide and utilizes the general secretary pathway for export (R. W. Worobo, M. J. van Belkum, M. Sailer, K. L. Roy, J. C. Vederas, and M. E. Stiles, J. Bacteriol. 177:3143-3149, 1995). Fusion of the carnobacteriocin B2 structural gene (devoid of its natural leader peptide) behind the signal peptide of divergicin A in the expression vector pMG36e permitted production and export of active carnobacteriocin B2 in the absence of the specific secretion genes. N-terminal sequencing of purified carnobacteriocin B2 established that correct processing of the prebacteriocin occurred beyond the Ala-Ser-Ala cleavage site of the signal peptide. Carnobacteriocin B2 was produced by the wild-type strain of C. divergens, LV13, and in C. piscicola LV17C, the nonbacteriocinogenic, plasmidless variant of the original carnobacteriocin B2 producer strain. The corresponding immunity gene was included immediately downstream of the structural gene. Both of the host strains are sensitive to the bacteriocin, and both acquired immunity when they were transformed with the construct. C. divergens LV13 containing the divergicin A signal peptide-carnobacteriocin B2 fusion construct produces both divergicin A and carnobacteriocin B2 and demonstrates the first example of multiple-bacteriocin expression via the general secretory pathway. The small amount of genetic material required for independent bacteriocin expression has implications for the development of a food-grade multiple-bacteriocin expression vector for use in lactic acid bacteria.
Expression of the Escherichia coli pmi gene, encoding phosphomannose-isomerase in Zymomonas mobilis, leads to utilization of mannose as a novel growth substrate, which can be used as a selective marker
Authors: Weisser P , Krämer R , Sprenger G A
Published: January 2020

Wild-type Zymomonas mobilis can utilize only three substrates (sucrose, glucose, and fructose) as sole carbon sources, which are largely converted into ethanol and carbon dioxide. Here, we show that although D-mannose is not used as a growth substrate, it is taken up via the glucose uniport system (glucose facilitator protein) with a Vmax similar to that of glucose. Moreover, D-mannose was phosphorylated by a side activity of the resident fructokinase to mannose-6-phosphate. Fructokinase was purified to homogeneity from an frk-recombinant Z. mobilis strain showing a specific activity of 205 +/- 25 U of protein mg-1 with fructose (K(m), 0.75 +/- 0.06 mM) and 17 +/- 2 U mg-1 (relative activity, 8.5%) with mannose (K(m), 0.65 +/- 0.08 mM). However, no phosphomannoseisomerase activity could be detected for Z. mobilis, and this appeared to be the reason for the lack of growth on mannose. Therefore, we introduced the Escherichia coli gene pmi (manA) in Z. mobilis under the control of a lacIq-Ptac system on a broad-host-range plasmid (pZY507; Cmr). Subsequently, in pmi-recombinant cells of Z. mobilis, phosphomannoseisomerase was expressed in a range of from 3 U (without isopropyl-beta-D-thiogalactopyranoside [IPTG]) to 20 U mg-1 of protein in crude extracts (after IPTG induction). Recombinant cells of different Z. mobilis strains utilized mannose (4%) as the sole carbon source with a growth rate of 0.07 h-1, provided that they contained fructokinase activity. When the frk gene was additionally expressed from the same vector, fructokinase activities of as much as 9.7 U mg-1 and growth rates of as much as 0.25 h-1 were detected, compared with 0.34 h-1 on fructose for wild-type Z. mobilis. Selection for growth on mannose was used to monitor plasmid transfer of pZY507pmi from E. coli to Z. mobilis strains and could replace the previous selection for antibiotic resistance.
Autoradiographic method for isolation of diverse microbial species with unique catabolic traits
Authors: Dunbar J , Wong D , Yarus M J , Forney L J
Published: January 2020

A novel autoradiographic method for isolation of bacteria with unique catabolic traits was developed to overcome many of the limitations of traditional selective enrichment techniques. The method consists of five steps. (i) An environmental sample is directly plated (without enrichment) on a microporous filter atop a solid medium that allows cultivation of diverse kinds of microorganisms. (ii) Once colonies form, two replicas of the filter are prepared and the colonies are regrown. (iii) The replica filters are starved 24 to 72 h to deplete intracellular carbon reserves and then (iv) placed on Na(inf2)(sup35)SO(inf4)-containing solid media with and without a test compound. (v) Following an incubation period, the replica filters are exposed to film in order to identify colonies that incorporate more (sup35)S into cell biomass in the presence of the test compound than in its absence, providing presumptive evidence for metabolism of the compound. The colonies identified in this manner can be recovered from the master filter. To demonstrate this technique, bacteria capable of degrading benzoate were isolated from a single soil slurry by traditional enrichment as well as by autoradiography. From the enrichment culture, a single isolate able to degrade benzoate was obtained. In contrast, 18 distinct strains were obtained by purifying 19 putative benzoate-degrading colonies identified by autoradiography. Each of the 18 strains was able to completely transform the substrate, as determined by high-performance liquid chromatography analyses. The doubling times of a subset of the isolates grown in benzoate medium ranged from 1.4 to 17.1 h, whereas the doubling time of the isolate obtained by enrichment was 2.0 h. These data demonstrate that the method described here can be used to obtain a collection of diverse organisms able to metabolize a specific compound.
Distribution of Symbiotic Genotypes in Rhizobium leguminosarum biovar viciae Populations Isolated Directly from Soils
Authors: Louvrier P , Laguerre G , Amarger N
Published: January 2020

The distribution of symbiotic (Sym) plasmid types across background genotypes was investigated in two field populations of Rhizobium leguminosarum biovar viciae isolated directly from soils. PCR-based methods were used to characterize the background genotypes and the Sym gene types. Identical Sym gene types were associated with a variable range of background genotypes, while the same background genotype could harbor distinct Sym gene types. Random distributions of Sym gene types in the background genotypes were observed in the two soil populations. These results suggest that Sym plasmid transfer is less restricted than previously thought on the basis of the analysis of strains isolated from legume nodules.
Multiple Serotypes of the Moderate Thermophile Thiobacillus caldus, a Limitation of Immunological Assays for Biomining Microorganisms
Authors: Hallberg K B , Lindstrom E B
Published: January 2020

Phylogenetic and phenotypic analysis indicates that a moderately thermophilic isolate, C-SH12, from Australia belongs to the species Thiobacillus caldus. Antiserum generated against whole cells of T. caldus KU recognized protein antigens common to cell lysates of the three T. caldus strains KU, BC13, and C-SH12 but did not recognize whole cells of isolate C-SH12. Differences in the lipopolysaccharide (LPS) of strain C-SH12 and those of the other two T. caldus strains were found, and the anti-KU antiserum did not recognize the LPS from strain C-SH12. These data indicate that this T. caldus isolate belongs to a serotype different from that of strains KU and BC13.
Isolation of saprophytic basidiomycetes from soil
Authors: Thorn R G , Reddy C A , Harris D , Paul E A
Published: January 2020

A method with the combined advantages of soil particle washing, selective inhibitors, and an indicator substrate was developed to isolate saprophytic basidiomycetes from soil. Organic particles were washed from soil and plated on a medium containing lignin, guaiacol, and benomyl, which reduced mold growth and allowed detection of basidiomycetes producing laccase or peroxidase. The 64 soil samples yielded 67 basidiomycete isolates, representing 51 groups on the basis of morphology and physiology. This method should facilitate investigations into the biodiversity of soil basidiomycetes and yield organisms that are useful in bioremediation of soils contaminated with pesticides or other recalcitrant aromatic compounds.
Complete microbial degradation of both enantiomers of the chiral herbicide mecoprop [(RS)-2-(4-chloro-2-methylphenoxy)propionic acid] in an enantioselective manner by Sphingomonas herbicidovorans sp. nov
Authors: Zipper C , Nickel K , Angst W , Kohler H P
Published: January 2020

Sphingomonas herbicidovorans MH (previously designated Flavobacterium sp. strain MH) was able to utilize the chiral herbicide (RS)-2-(4-chloro-2-methylphenoxy)propionic acid (mecoprop) as the sole carbon and energy source. When strain MH was offered racemic mecoprop as the growth substrate, it could degrade both the (R) and the (S) enantiomer to completion, as shown by biomass formation, substrate consumption, and stoichiometric chloride release. However, the (S) enantiomer disappeared much faster from the culture medium than the (R) enantiomer. These results suggest the involvement of specific enzymes for the degradation of each enantiomer. This view was substantiated by the fact that resting cells of strain MH grown on (S)-mecoprop were able to degrade the (S) but not the (R) enantiomer of mecoprop. Accordingly, resting cells of strain MH grown on (R)-mecoprop preferentially metabolized the (R) enantiomer. Nevertheless, such cells could transform (S)-mecoprop at low rates. Oxygen uptake rates with resting cells confirmed the above view, as oxygen consumption was strongly dependent on the growth substrate. Cells grown on (R)-mecoprop showed oxygen uptake rates more than two times higher upon incubation with the (R) than upon incubation with the (S) enantiomer and vice versa.
Biodegradation of dimethylsilanediol in soils
Authors: Sabourin C L , Carpenter J C , Leib T K , Spivack J L
Published: January 2020

The biodegradation potential of [14C]dimethylsilanediol, the monomer unit of polydimethylsiloxane, in soils was investigated. Dimethylsilanediol was found to be biodegraded in all of the tested soils, as monitored by the production of 14CO2. When 2-propanol was added to the soil as a carbon source in addition to [14C]dimethylsilanediol, the production of 14CO2 increased. A method for the selection of primary substrates that support cometabolic degradation of a target compound was developed. By this method, the activity observed in the soils was successfully transferred to liquid culture. A fungus, Fusarium oxysporum Schlechtendahl, and a bacterium, an Arthrobacter species, were isolated from two different soils, and both microorganisms were able to cometabolize [14C]dimethylsilanediol to 14CO2 in liquid culture. In addition, the Arthrobacter sp. that was isolated grew on dimethylsulfone, and we believe that this is the first reported instance of a microorganism using dimethylsulfone as its primary carbon source. Previous evidence has shown that polydimethylsiloxane is hydrolyzed in soil to the monomer, dimethylsilanediol. Now, biodegradation of dimethylsilanediol in soil has been demonstrated.
Development of an arabinose-fermenting Zymomonas mobilis strain by metabolic pathway engineering
Authors: Deanda K , Zhang M , Eddy C , Picataggio S
Published: January 2020

The substrate fermentation range of the ethanologenic bacterium Zymomonas mobilis was expanded to include the pentose sugar, L-arabinose, which is commonly found in agricultural residues and other lignocellulosic biomass. Five genes, encoding L-arabinose isomerase (araA), L-ribulokinase (araB), L-ribulose-5-phosphate-4-epimerase (araD), transaldolase (talB), and transketolase (tktA), were isolated from Escherichia coli and introduced into Z. mobilis under the control of constitutive promoters that permitted their expression even in the presence of glucose. The engineered strain grew on and produced ethanol from L-arabinose as a sole C source at 98% of the maximum theoretical ethanol yield, based on the amount of consumed sugar. This indicates that arabinose was metabolized almost exclusively to ethanol as the sole fermentation product, with little by-product formation. Although no diauxic growth pattern was evident, the microorganism preferentially utilized glucose before arabinose, apparently reflecting the specificity of the indigenous facilitated diffusion transport system. This microorganism may be useful, along with the previously developed xylose-fermenting Z. mobilis (M. Zhang, C. Eddy, K. Deanda, M. Finkelstein, and S. Picataggio, Science 267:240-243, 1995), in a mixed culture for efficient fermentation of the predominant hexose and pentose sugars in agricultural residues and other lignocellulosic feedstocks to ethanol.
A novel porous nylon biocarrier for immobilized bacteria
Authors: Heitkamp M A , Stewart W P
Published: January 2020

A highly porous nylon biocarrier was developed to support immobilized bacteria in bioreactors used to treat liquid wastes. Porosity analyses and scanning electron microscopy showed microbial colonization of accessible pores typically in the range of 100 to 1,200 (mu)m, with some as large as 3.9 mm. A bench-scale packed-bed reactor achieved a p-nitrophenol (PNP) removal rate of 5.95 kg of PNP m(sup-3) day(sup-1) for wastes containing 1,200 mg of PNP liter(sup-1). Complete mixing of the biocarrier bed to remove excess surface biomass was routinely achieved with simple air injection. These porous polymer biocarriers are promising as microbial supports in liquid-waste treatment and bioremediation applications.
DNA recovery from soils of diverse composition
Authors: Zhou J , Bruns M A , Tiedje J M
Published: January 2020

A simple, rapid method for bacterial lysis and direct extraction of DNA from soils with minimal shearing was developed to address the risk of chimera formation from small template DNA during subsequent PCR. The method was based on lysis with a high-salt extraction buffer (1.5 M NaCl) and extended heating (2 to 3 h) of the soil suspension in the presence of sodium dodecyl sulfate (SDS), hexadecyltrimethylammonium bromide, and proteinase K. The extraction method required 6 h and was tested on eight soils differing in organic carbon, clay content, and pH, including ones from which DNA extraction is difficult. The DNA fragment size in crude extracts from all soils was > 23 kb. Preliminary trials indicated that DNA recovery from two soils seeded with gram-negative bacteria was 92 to 99%. When the method was tested on all eight unseeded soils, microscopic examination of indigenous bacteria in soil pellets before and after extraction showed variable cell lysis efficiency (26 to 92%). Crude DNA yields from the eight soils ranged from 2.5 to 26.9 micrograms of DNA g-1, and these were positively correlated with the organic carbon content in the soil (r = 0.73). DNA yields from gram-positive bacteria from pure cultures were two to six times higher when the high-salt-SDS-heat method was combined with mortar-and-pestle grinding and freeze-thawing, and most DNA recovered was of high molecular weight. Four methods for purifying crude DNA were also evaluated for percent recovery, fragment size, speed, enzyme restriction, PCR amplification, and DNA-DNA hybridization. In general, all methods produced DNA pure enough for PCR amplification. Since soil type and microbial community characteristics will influence DNA recovery, this study provides guidance for choosing appropriate extraction and purification methods on the basis of experimental goals.
Growth-Associated Production of Poly(3-Hydroxyvalerate) from n-Pentanol by a Methylotrophic Bacterium, Paracoccus denitrificans
Authors: Yamane T , Chen X , Ueda S
Published: January 2020

Paracoccus denitrificans accumulated a polyester in its cells during growth on n-pentanol. The composition of the polyester varied during the cultivation: the level of the 3-hydroxyvalerate unit in the polyester increased, and eventually a homopolymeric poly(3-hydroxyvalerate) [P(3HV)] accumulated to an amount 22 to 24% of the cell dry weight. Growth-associated polyester synthesis was considerably affected by n-pentanol when its concentration was controlled at several levels. Maximum accumulation of the polyester was obtained at 0.02% (vol/vol). Physical and mechanical characteristics of the P(3HV) were determined and compared with those of other homo- and copolyesters. The P(3HV) was dextrorotatory and had number-averaged and weight-averaged molecular masses of 128,000 and 888,000 Da, respectively, with a rate of polydispersity of 6.93. The level of tensile strength of the P(3HV) was lower, and its extension to break was higher than that of the poly(3-hydroxybutyrate) homopolyester.
Species-Specific Detection of Vibrio anguillarum in Marine Aquaculture Environments by Selective Culture and DNA Hybridization
Authors: Martinez-Picado J , Alsina M , Blanch A R , Cerda M , Jofre J
Published: January 2020

Methods for specific detection of Vibrio anguillarum in complex microbial communities within diverse marine aquaculture environments were evaluated. A system for the detection of culturable cells based on the combined use of a selective medium and a nonradioactively labeled oligodeoxynucleotide complementary to 16S rRNA was developed. Four hundred fourteen bacterial cultures were evaluated in order to assess the specificity of the method. When both the selective medium and the specific probe gave positive results, the cultures were always identified as V. anguillarum. The selectivity for colony hybridization was 1 V. anguillarum cell in 10,000 total bacterial cells in environmental samples. The utility of the method was also compared with detection by dot blot hybridization of either raw DNA purified from environmental samples or PCR-amplified DNA of 16S rRNA genes, using universal eubacterial primers. The post-PCR hybridization was more sensitive (8 x 10(sup2) cells) than direct hybridization of the whole purified DNA (10(sup6) cells). However, the selective medium-probe combined method was as sensitive as post-PCR hybridization, albeit more specific.
A Novel PhosphorImager-Based Technique for Monitoring the Microbial Reduction of Technetium
Authors: Lloyd J R , Macaskie L E
Published: January 2020

A novel PhosphorImager-based technique which can be used to quantify low concentrations of radionuclides is described. The technique offers several benefits, combining very high sensitivity with containment of the radioisotope in the solid state, thus minimizing disposal procedures. In this study, it was used in conjunction with paper chromatography to quantify different oxidation states of (sup99)Tc in solution. The technique was used to evaluate the potential of anaerobic cultures of Shewanella putrefaciens and Geobacter metallireducens (bacteria with known metal-reducing capabilities) to reduce highly soluble Tc(VII) to insoluble lower-valence species, facilitating its removal from solution. Both organisms reduced Tc(VII), but profiles of Tc species produced in culture supernatants were strain specific. S. putrefaciens produced Tc(V), Tc(IV), and one unidentified species, but no Tc was removed from solution. G. metallireducens removed 70% of the 250 (mu)M Tc added in solution, with trace amounts of Tc(V) and the unidentified species detected in culture supernatants. Possible uses for these organisms in the bioremediation of Tc-contaminated waters are discussed, and other uses of the PhosphorImager technique are highlighted.
Bias caused by template annealing in the amplification of mixtures of 16S rRNA genes by PCR
Authors: Suzuki M T , Giovannoni S J
Published: January 2020

The PCR is used widely for the study of rRNA genes amplified from mixed microbial populations. These studies resemble quantitative applications of PCR in that the templates are mixtures of homologs and the relative abundance of amplicons is thought to provide some measure of the gene ratios in the starting mixture. Although such studies have established the presence of novel rRNA genes in many natural ecosystems, inferences about gene abundance have been limited by uncertainties about the relative efficiency of gene amplification in the PCR. To address this question, three rRNA gene standards were prepared by PCR, mixed in known proportions, and amplified a second time by using primer pairs in which one primer was labeled with a fluorescent nucleotide derivative. The PCR products were digested with restriction endonucleases, and the frequencies of genes in the products were determined by electrophoresis on an Applied Biosystems 373A automated DNA sequencer in Genescan mode. Mixtures of two templates amplified with the 519F-1406R primer pair yielded products in the predicted proportions. A second primer pair (27F-338R) resulted in strong bias towards 1:1 mixtures of genes in final products, regardless of the initial proportions of the templates. This bias was strongly dependent on the number of cycles of replication. The results fit a kinetic model in which the reannealing of genes progressively inhibits the formation of template-primer hybrids.
Development of a Sensitive Chemiluminometric Assay for the Detection of (beta)-Galactosidase in Permeabilized Coliform Bacteria and Comparison with Fluorometry and Colorimetry
Authors: Van Poucke S O , Nelis H J
Published: January 2020

Volume 61, no. 12, p. 4505, Introduction, column 1, line 13, and column 2, line 5: reference 19, which refers to a toxicity test based on the inhibition of (beta)-glucuronidase, not (beta)-galactosidase, in E. coli, should not be cited. [This corrects the article on p. 4505 in vol. 61.].
Relationship between Symptom Development and Actual Sites of Infection in Leaves of Anthurium Inoculated with a Bioluminescent Strain of Xanthomonas campestris pv. dieffenbachiae
Authors: Fukui R , Fukui H , McElhaney R , Nelson S C , Alvarez A M
Published: January 2020

The infection process of bacterial blight of anthurium was monitored with a bioluminescent strain of Xanthomonas campestris pv. dieffenbachiae. The relationship between symptom expression on infected leaves (assessed visually) and the extent of bacterial movement within tissues (evaluated by bioluminescence emission) varied among anthurium cultivars. In several cultivars previously considered susceptible on the basis of symptom development alone, bacterial invasion of leaves extended far beyond the visually affected areas. In other cultivars previously considered resistant, bacterial invasion was restricted to areas with visible symptoms. In three cultivars previously considered resistant, leaves were extensively invaded by the bacterium, and yet few or no symptoms were seen on infected leaves. The pathogen was consistently recovered from leaf sections emitting bioluminescence but not from sections emitting no light. At an early stage of infection, no significant differences in the percentages of infected areas as determined by visual assessment were observed in any of the cultivars. However, differences among cultivars were detected by bioluminescence as the disease progressed, because bacterial invasion was not always accompanied by symptom expression. In susceptible cultivars, the advancing border of infection was 5 to 10 cm inward from the margins of the visible symptoms and often reached to the leaf petiole even when symptoms were visible in <10% of the total leaf area. Comparisons of anthurium cultivars in which a nondestructive method was used to quantify the severity of leaf infection by a bioluminescent pathogen have enabled us to evaluate susceptibility and resistance to bacterial blight accurately. Such evaluations will be of importance in breeding resistant cultivars for disease control.