bluuebunny/crossref_metadata_2025 huggingface dataset:
Production and Excretion of Nod Metabolites by
Rhizobium leguminosarum
bv. trifolii Are Disrupted by the Same Environmental Factors That Reduce Nodulation in the Field
Authors: McKay Ian A. , Djordjevic Michael A.
Published: January 2020

Lipooligosaccharides (Nod metabolites) have been shown to be essential for the successful nodulation of legumes. In strains of
Rhizobium leguminosarum
bv. trifolii, Nod metabolites were detected predominantly within the cell and to a lesser extent in the periplasmic space and the growth medium. The production, and in particular the excretion, of Nod metabolites was restricted by a range of environmental conditions which are associated with poor nodulation in the field. Lowering the medium pH from 7.0 to 5.0, reducing the phosphate concentration from 1 mM to 5 μM KH
2
PO
4
, and lowering the incubation temperature from 28 to 18°C affected the number and relative concentrations of the Nod metabolites made. The form and concentration of the nitrogen source affected the relative concentrations of the Nod metabolites produced and excreted. KNO
3
concentrations of >10 mM did not affect cell growth rate but substantially reduced the number of Nod metabolites released. Environmental stresses differentially altered Nod metabolite production and excretion in the same strain carrying different introduced
nod
regions. Strain ANU845(pWLH1) produced and excreted comparatively fewer Nod metabolites at pH 5.0 and at 18°C than strain ANU845(pRI4003). The excretion but not the production of Nod metabolites by strain ANU845(pRtO32) was dependent on the presence of both
nodI
and
nodJ
. Tn
5
-induced
nodI
and
nodJ
mutants did not accumulate any metabolites either outside the cell or within the outer membrane or periplasmic space. Recognition that Nod metabolite accumulation is a complex system of production and excretion, with each component responding differently to changes in environmental conditions, has many consequences, both at the molecular level and in the field. The ability of different strains to produce and release Nod metabolites is likely to be a major determinant of nodule occupancy and should be considered when screening strains suitable for adverse environments.
Binding of small, acid-soluble spore proteins to DNA plays a significant role in the resistance of Bacillus subtilis spores to hydrogen peroxide
Authors: Setlow B , Setlow P
Published: January 2020

Dormant spores of Bacillus subtilis which lack the majority of the alpha/beta-type small, acid-soluble proteins (SASP) (termed alpha- beta- spores) that coat the DNA in wild-type spores are significantly more sensitive to hydrogen peroxide than are wild-type spores. Hydrogen peroxide treatment of alpha- beta- spores causes DNA strand breaks more readily than does comparable treatment of wild-type spores, and alpha- beta- spores, but not wild-type spores, which survive hydrogen peroxide treatment have acquired a significant number of mutations. The hydrogen peroxide resistance of wild-type spores appears to be acquired in at least two incremental steps during sporulation. The first increment is acquired at about the time of alpha/beta-type SASP synthesis, and the second increment is acquired approximately 2 h later, at about the time of dipicolinic acid accumulation. During sporulation of the alpha- beta- strain, only the second increment of hydrogen peroxide resistance is acquired. In contrast, sporulation mutants which accumulate alpha/beta-type SASP but progress no further in sporulation acquire only the first increment of hydrogen peroxide resistance. These findings strongly suggest that binding of alpha/beta-type SASP to DNA provides one increment of spore hydrogen peroxide resistance. Indeed, binding of alpha/beta-type SASP to DNA in vitro provides strong protection against cleavage of DNA by hydrogen peroxide.
Comparative in vivo efficiencies of hand-washing agents against hepatitis A virus (HM-175) and poliovirus type 1 (Sabin)
Authors: Mbithi J N , Springthorpe V S , Sattar S A
Published: January 2020

The abilities of 10 hygienic hand-washing agents and tap water (containing approximately 0.5 ppm of free chlorine) to eliminate strain HM-175 of hepatitis A virus (HAV) and poliovirus (PV) type 1 (Sabin) were compared by using finger pad and whole-hand protocols with three adult volunteers. A mixture of the two viruses was prepared in a 10% suspension of feces, and 10 microliters of the mixture was placed on each finger pad. The inoculum was allowed to dry for 20 min, and the contaminated area was exposed to a hand-washing agent for 10 s, rinsed in tap water, and dried with a paper towel. In the whole-hand protocol, the hands were contaminated with 0.5 ml of the virus mixture, exposed for 10 s to a hand-washing agent, washed, and dried as described above. Tryptose phosphate broth was used to elute any virus remaining on the finger pads or hands. One part of the eluate was assayed directly for PV with FRhK-4 cells, while the other part was first treated with a PV-neutralizing serum and then assayed for HAV with the same cell line. The results are reported as mean percentages of reduction in PFU compared with the amount of infectious virus detectable after initial drying.(ABSTRACT TRUNCATED AT 250 WORDS)
Induction and Regeneration of Autoplasts from
Clostridium thermohydrosulfuricum
JW102 and
Thermoanaerobacter ethanolicus
JW200
Authors: Peteranderl R. , Canganella F. , Holzenburg A. , Wiegel J.
Published: January 2020

Autolysis was induced to form stable, cell wall-free cells of
Clostridium thermohydrosulfuricum
JW102 and
Thermoanaerobacter ethanolicus
JW200, using a complex medium containing glycine (0.4% wt/vol) and/or sucrose or glycerol (10% wt/vol) at an optimum temperature of 64°C. Autoplasts of both bacteria were grown as L-phase colonies on solid medium; more than 50% of these colonies regenerated to the walled form during prolonged incubation. The removal of the cell wall was confirmed by electron microscopy.
Adaptation of Pseudomonas putida S12 to high concentrations of styrene and other organic solvents
Authors: Weber F J , Ooijkaas L P , Schemen R M , Hartmans S , de Bont J A
Published: January 2020

Pseudomonas putida S12 could adapt to grow on styrene in a two-phase styrene-water system. Acetate was toxic for P. putida S12, but cells were similarly able to adapt to higher acetate concentrations. Only by using these acetate-adapted cells was growth observed in the presence of supersaturating concentrations of toxic nonmetabolizable solvents such as toluene.
Survival of subsurface microorganisms exposed to UV radiation and hydrogen peroxide
Authors: Arrage A A , Phelps T J , Benoit R E , White D C
Published: January 2020

Aerobic and microaerophilic subsurface bacteria were screened for resistance to UV light. Contrary to the hypothesis that subsurface bacteria should be sensitive to UV light, the organisms studied exhibited resistance levels as efficient as those of surface bacteria. A total of 31% of the aerobic subsurface isolates were UV resistant, compared with 26% of the surface soil bacteria that were tested. Several aerobic, gram-positive, pigmented, subsurface isolates exhibited greater resistance to UV light than all of the reference bacterial strains tested except Deinococcus radiodurans. None of the microaerophilic, gram-negative, nonpigmented, subsurface isolates were UV resistant; however, these isolates exhibited levels of sensitivity similar to those of the gram-negative reference bacteria Escherichia coli B and Pseudomonas fluorescens. Photoreactivation activity was detected in three subsurface isolates, and strain UV3 exhibited a more efficient mechanism than E. coli B. The peroxide resistance of four subsurface isolates was also examined. The aerobic subsurface bacteria resistant to UV light tolerated higher levels of H2O2 than the microaerophilic organisms. The conservation of DNA repair pathways in subsurface microorganisms may be important in maintaining DNA integrity and in protecting the organisms against chemical insults, such as oxygen radicals, during periods of slow growth.
Bacterial strains from human feces that reduce CO2 to acetic acid
Authors: Wolin M J , Miller T L
Published: January 2020

We used dilutions of fecal suspensions from a human volunteer to enrich cultures for bacteria that reduce CO2 to acetate in the colon. The soluble enrichment substrates used were glucose, methanol, formate, and vanillate, which were used with a gas phase that contained 80% N2 and 20% CO2. The gaseous enrichment substrates used were 80% H2-20% CO2 and 50% CO-50% CO2. We isolated three different strains that produced acetate from CO2. One strain produced acetate from methanol, vanillate, H2-CO2, glucose, and other sugars. The other two strains did not form acetate from methanol or vanillate. Both of the latter strains formed acetate from glucose and other sugars, but only one of these strains formed acetate from H2-CO2. Both of these strains cometabolized formate. However, none of the enrichment cultures or pure cultures used CO or formate as a substrate for growth. The two strains that produced acetate from H2 and CO2 grew slowly when the gases alone were used as substrates, but they rapidly cometabolized H2 and CO2 when they were grown with organic substrates. The ability of all of the strains to produce acetate from CO2 and/or other one-carbon precursors was verified by determining the radioactivity of the methyl and carboxyl groups of the acetate formed after growth with 14CO2 or other radioactively labeled one-carbon precursors.
Utilization of xylooligosaccharides by selected ruminal bacteria
Authors: Cotta M A
Published: January 2020

The ability of ruminal bacteria to utilize xylooligosaccharides was examined. Xylooligosaccharides were prepared by partially hydrolyzing oat spelt xylan in phosphoric acid. This substrate solution was added (0.2%, wt/vol) to a complex medium containing yeast extract and Trypticase that was inoculated with individual species of ruminal bacteria, and growth and utilization were monitored over time. All of the xylanolytic bacteria examined were able to utilize this oligosaccharide mixture as a growth substrate. Butyrivibrio fibrisolvens, Eubacterium ruminantium, and Ruminococcus albus used xylooligosaccharides and whole, unhydrolyzed xylan to similar extents, while Prevotella ruminicola used twice as much xylooligosaccharides as xylan (76 versus 34%). Strains of Selenomonas ruminantium were the only nonxylanolytic species that were able to grow on xylooligosaccharides. The ability of individual S. ruminantium strains to utilize xylooligosaccharides was correlated with the presence of xylosidase and arabinosidases activities.
Reduction of uranium by cytochrome c3 of Desulfovibrio vulgaris
Authors: Lovley D R , Widman P K , Woodward J C , Phillips E J
Published: January 2020

The mechanism for U(VI) reduction by Desulfovibrio vulgaris (Hildenborough) was investigated. The H2-dependent U(VI) reductase activity in the soluble fraction of the cells was lost when the soluble fraction was passed over a cationic exchange column which extracted cytochrome c3. Addition of cytochrome c3 back to the soluble fraction that had been passed over the cationic exchange column restored the U(VI)-reducing capacity. Reduced cytochrome c3 was oxidized by U(VI), as was a c-type cytochrome(s) in whole-cell suspensions. When cytochrome c3 was combined with hydrogenase, its physiological electron donor, U(VI) was reduced in the presence of H2. Hydrogenase alone could not reduce U(VI). Rapid U(VI) reduction was followed by a subsequent slow precipitation of the U(IV) mineral uraninite. Cytochrome c3 reduced U(VI) in a uranium-contaminated surface water and groundwater. Cytochrome c3 provides the first enzyme model for the reduction and biomineralization of uranium in sedimentary environments. Furthermore, the finding that cytochrome c3 can catalyze the reductive precipitation of uranium may aid in the development of fixed-enzyme reactors and/or organisms with enhanced U(VI)-reducing capacity for the bioremediation of uranium-contaminated waters and waste streams.
Reductive dehalogenation of the trichloromethyl group of nitrapyrin by the ammonia-oxidizing bacterium Nitrosomonas europaea
Authors: Vannelli T , Hooper A B
Published: January 2020

Suspensions of Nitrosomonas europaea catalyzed the reductive dehalogenation of the commercial nitrification inhibitor nitrapyrin (2-chloro-6-trichloromethylpyridine). The product of the reaction was identified as 2-chloro-6-dichloromethylpyridine by its mass fragmentation and nuclear magnetic resonance spectra. A small amount of 2-chloro-6-dichloromethylpyridine accumulated during the conversion of nitrapyrin to 6-chloropicolinic acid in an aerated solution in the presence of ammonia (T. Vannelli and A.B. Hooper, Appl. Environ. Microbiol. 58:2321-2325, 1992). Nearly stoichiometric conversion of nitrapyrin to 2-chloro-6-dichloromethylpyridine occurred at very low oxygen concentrations and in the presence of hydrazine as a source of electrons. Under these conditions the turnover rate was 0.37 nmol of nitrapyrin per min per mg of protein. Two specific inhibitors of ammonia oxidation, acetylene and allylthiourea, inhibited the rate of the dehalogenation reaction by 80 and 84%, respectively. In the presence of D2O, all 2-chloro-6-dichloromethylpyridine produced in the reaction was deuterated at the methyl position. In an oxygenated solution and in the presence of ammonia or hydrazine, cells did not catalyze the oxidation of exogenously added 2-chloro-6-dichloromethylpyridine to 6-chloropicolinic acid. Thus, 2-chloro-6-dichloromethylpyridine is apparently not an intermediate in the aerobic production of 6-chloropicolinic acid from nitrapyrin.
Detection of Legionella species in sewage and ocean water by polymerase chain reaction, direct fluorescent-antibody, and plate culture methods
Authors: Palmer C J , Tsai Y L , Paszko-Kolva C , Mayer C , Sangermano L R
Published: January 2020

Legionella spp. are ubiquitous in most environmental water sources; however, sewage treatment plants have not been examined as potential environmental reservoirs for these bacteria. This study used polymerase chain reaction, direct fluorescent-antibody staining, and culture methods to examine raw and treated sewage, ocean-receiving waters, and nearshore coastal environments for the presence of Legionella pneumophila and other Legionella spp. The study concluded that Legionella spp. are present in all phases of sewage treatment and that population numbers do not significantly decline through the treatment process. Ocean-receiving waters located 5 miles offshore, where the treated sewage is discharged, were found to contain Legionella spp., but ocean water between the discharge site and coastal bathing beaches was negative. This suggests that the Legionella spp. from the ocean discharge site were not reaching the nearshore beach waters. A flood control channel and river that entered the ocean were found to contain Legionella spp., and a nearby beach swimming area was also found to be positive, suggesting that land runoff from the flood control channel and river were the source of the Legionella spp. in the beach water samples that tested positive.
Dichloromethane as the sole carbon source for an acetogenic mixed culture and isolation of a fermentative, dichloromethane-degrading bacterium
Authors: Braus-Stromeyer S A , Hermann R , Cook A M , Leisinger T
Published: January 2020

Dichloromethane (DCM) is utilized by the strictly anaerobic, acetogenic mixed culture DM as a sole source of carbon and energy for growth. Growth with DCM was linear, and cell suspensions of the culture degraded DCM with a specific activity of 0.47 mkat/kg of protein. A mass balance of 2 mol of chloride and 0.42 mol of acetate per mol of DCM was observed. The dehalogenation reaction showed similar specific activities under both anaerobic and aerobic conditions. Radioactivity from [14C]DCM in cell suspensions was recovered largely as 14CO2 (58%), [14C]acetate (23%), and [14C]formate (11%), which subsequently disappeared. This suggested that formate is a major intermediate in the pathway from DCM to acetate. Efforts to isolate from culture DM a pure culture capable of anaerobic growth with DCM were unsuccessful, although overall acetogenesis and the partial reactions are thermodynamically favorable. We then isolated bacterial strains DMA, a strictly anaerobic, gram-positive, endospore-forming rod, and DMB, a strictly anaerobic, gram-negative, endospore-forming homoacetogen, from culture DM. Both strain DMB and Methanospirillum hungatei utilized formate as a source of carbon and energy. Coculture of strain DMA with either M. hungatei or strain DMB in solid medium with DCM as the sole added source of carbon and energy was observed. These data support a tentative scheme for the acetogenic fermentation of DCM involving interspecies formate transfer from strain DMA to the acetogenic bacterium DMB or to the methanogen M. hungatei.
Ribotypes and plasmid contents of Vibrio anguillarum strains in relation to serovar
Authors: Olsen J E , Larsen J L
Published: January 2020

Eighty-six strains of the 10 major agglutination types of Vibrio anguillarum (serovars O1 to O10) and 6 nontypeable strains of V. anguillarum have been characterized by ribotyping with a probe complementary to 16S and 23S rRNA of Escherichia coli and by plasmid profile analysis. Forty-four different ribotypes were observed with the restriction enzyme HindIII. Ribotype similarity was compared by using the Dice coefficient (Sd), and three significantly different levels of homogeneity within the V. anguillarum serovars were observed (serovars O1, O3A, O7, and O9, Sds of > 90%; serovars O2B, O4, and O10, Sds of 80 to 90%; serovars O2A, O3B, O5, and O8, Sds between 46 and 70%). None of the ribotype patterns of V. anguillarum strains were observed among 20 other Vibrio strains typed for comparison. By cluster analysis, the V. anguillarum strains were divided into a main cluster containing 83 strains, while all strains of serovar O3B, one strain (each) of serovars O2A, O5, and O8, and a nontypeable strain were separated from this cluster by at least 15% difference in similarity coefficients. Plasmids were demonstrated in only six strains other than serovar O1. In serovar O1, a 67- to 70-kilobase-pair (kb) plasmid molecule was present in 17 of 19 strains tested; of the two remaining strains, one strain harbored two plasmids (45 and 6.5 kb) and one strain had no plasmids.
Derivation of DNA probes for enumeration of a specific strain of Lactobacillus acidophilus in piglet digestive tract samples
Authors: Rodtong S , Dobbinson S , Thode-Andersen S , McConnell M A , Tannock G W
Published: January 2020

Four DNA probes were derived that hybridized specifically to DNA from Lactobacillus acidophilus O. The probes were constructed by randomly cloning lactobacillus DNA in plasmid vector pBR322. Two of the probes (pSR1 and pSR2) were composed of vector and plasmid DNA inserts (3.6 and 1.6 kb, respectively); the others (pSR3 and pSR4) were composed of vector and chromosomally derived inserts (6.9 and 1.4 kb, respectively). The probes were used to enumerate, by colony hybridization, strain O in digestive tract samples collected from piglets inoculated 24 hours previously with a culture of the strain. The probes did not hybridize to DNA from lactobacilli inhabiting the digestive tract of uninoculated piglets. Strain O made up about 10% of the total lactobacillus population of the pars esophagea and about 20% of the population in other digestive tract samples.
Gene cloning, sequence analysis, purification, and characterization of a thermostable aminoacylase from Bacillus stearothermophilus
Authors: Sakanyan V , Desmarez L , Legrain C , Charlier D , Mett I , Kochikyan A , Savchenko A , Boyen A , Falmagne P , Pierard A
Published: January 2020

A genomic DNA fragment encoding aminoacylase activity of the eubacterium Bacillus stearothermophilus was cloned into Escherichia coli. Transformants expressing aminoacylase activity were selected by their ability to complement E. coli mutants defective in acetylornithine deacetylase activity, the enzyme that converts N-acetylornithine to ornithine in the arginine biosynthetic pathway. The 2.3-kb cloned fragment has been entirely sequenced. Analysis of the sequence revealed two open reading frames, one of which encoded the aminoacylase. B. stearothermophilus aminoacylase, produced in E. coli, was purified to near homogeneity in three steps, one of which took advantage of the intrinsic thermostability of the enzyme. The enzyme exists as homotetramer of 43-kDa subunits as shown by cross-linking experiments. The deacetylating capacity of purified aminoacylase varies considerably depending on the nature of the amino acid residue in the substrate. The enzyme hydrolyzes N-acyl derivatives of aromatic amino acids most efficiently. Comparison of the predicted amino acid sequence of B. stearothermophilus aminoacylase with those of eubacterial acetylornithine deacylase, succinyldiaminopimelate desuccinylase, carboxypeptidase G2, and eukaryotic aminoacylase I suggests a common origin for these enzymes.
Isolation and Characterization of Actinomycete Antagonists of a Fungal Root Pathogen
Authors: Crawford Don L. , Lynch James M. , Whipps John M. , Ousley Margaret A.
Published: January 2020

By use of selective media, 267 actinomycete strains were isolated from four rhizosphere-associated and four non-rhizosphere-associated British soils. Organic media with low nutrient concentrations were found to be best for isolating diverse actinomycetes while avoiding contamination and overgrowth of isolation media by eubacteria and fungi. While all isolates grew well at pHs 6.5 to 8.0, a few were unable to grow at pH 6.0 and a significant number failed to grow at pH 5.5. Eighty-two selected isolates were screened for in vitro antagonism towards
Pythium ultimum
by use of a Difco cornmeal agar assay procedure. Five isolates were very strong antagonists of the fungus, four were strong antagonists, and ten others were weakly antagonistic. The remaining isolates showed no antagonism by this assay. Additional studies showed that several of the
P. ultimum
antagonists also strongly inhibited growth of other root-pathogenic fungi. Twelve isolates showing antifungal activity in the in vitro assay were also tested for their effects on the germination and short-term growth of lettuce plants in glasshouse pot studies in the absence of pathogens. None of the actinomycetes prevented seed germination, although half of the isolates retarded seed germination and outgrowth of the plants by 1 to 3 days. During 18-day growth experiments, biomass yields of some actinomycete-inoculated plants were reduced in comparison with untreated control plants, although all plants appeared healthy and well rooted. None of the actinomycetes significantly enhanced plant growth over these short-term experiments. For some, but not all, actinomycetes, some correlations between delayed seed germination and reduced 18-day plant biomass yields were seen. For others, plant biomass yields were not reduced despite an actinomycete-associated delay in seed germination and plant outgrowth. Preliminary glasshouse experiments indicated that some of the actinomycetes protect germinating lettuce seeds against damping-off caused by
P. ultimum.
Expression of
cryIVA
and
cryIVB
Genes, Independently or in Combination, in a Crystal-Negative Strain of
Bacillus thuringiensis
subsp.
israelensis
Authors: Delécluse Armelle , Poncet Sandrine , Klier André , Rapoport Georges
Published: January 2020

The
cryIVA
and
cryIVB
genes, encoding the 125- and 135-kDa proteins, respectively, of
Bacillus thuringiensis
subsp.
israelensis
, were cloned either alone or together into a shuttle vector and expressed in a nontoxic strain of
B. thuringiensis
subsp.
israelensis
. The CryIVB protein was produced at a high level during sporulation and accumulated as inclusions; in contrast, the CryIVA polypeptide did not form such structures unless it was cloned on a higher-copy-number plasmid. Transcriptional fusions between the
cryIVA
or
cryIVB
gene promoter and the
lacZ
gene were constructed. The poor synthesis of CryIVA was not due to a poor efficiency of transcription from the
cryIVA
gene promoter. Mosquitocidal assays performed with purified inclusions showed that CryIVA was toxic for larvae of the species
Aedes aegypti, Anopheles stephensi
, and
Culex pipiens
, whereas CryIVB displayed activity only toward
Aedes aegypti
and
Anopheles stephensi
. The activity of inclusions containing both polypeptides was higher than that of single-peptide inclusions but was not as high as that of the native crystals, which contain at least four polypeptides.
Biodegradation and transformation of 4,4'- and 2,4-dihalodiphenyl ethers by Sphingomonas sp. strain SS33
Authors: Schmidt S , Fortnagel P , Wittich R M
Published: January 2020

The bacterium Sphingomonas sp. strain SS33, obtained from parent diphenyl ether-mineralizing strain SS3 (S. Schmidt, R.-M. Wittich, D. Erdmann, H. Wilkes, W. Francke, and P. Fortnagel, Appl. Environ. Microbiol. 58:2744-2750, 1992) after several weeks of adaptation on 4,4'-difluorodiphenyl ether as the new target compound, also utilized 4,4'-dichlorodiphenyl ether for growth. Intermediary halocatechols were also mineralized via the ortho pathway by type I enzymes. 4,4'-Dibromodiphenyl ether was not used as a carbon source although transformation by resting cells yielded mononuclear haloaromatic compounds, such as 4-bromophenol and 4-bromocatechol. The same was true for the conversion of 2,4-dichlorodiphenyl ether, which yielded the respective (halo-) phenols and (halo-) catechols.
Physicochemical Cell Surface and Adhesive Properties of Coryneform Bacteria Related to the Presence and Chain Length of Mycolic Acids
Authors: Bendinger Bernd , Rijnaarts Huub H. M. , Altendorf Karlheinz , Zehnder Alexander J. B.
Published: January 2020

The presence and chain length of mycolic acids of bacteria of the genera
Corynebacterium, Rhodococcus, Gordona, Mycobacterium
, and
Arthrobacter
and of coryneform bacteria containing a type B peptidoglycan were related to the cell surface hydrophobicity of the bacteria, which in turn was related to adhesion of the cells to defined surfaces such as Teflon and glass. The origin of the overall negative charge of these bacteria is discussed.
Differential expression of conserved protease genes in crucifer-attacking pathovars of Xanthomonas campestris
Authors: Dow J M , Fan M J , Newman M A , Daniels M J
Published: January 2020

Strains of Xanthomonas campestris pathovars armoraciae and raphani, which cause leaf spotting diseases in brassicas, produce a major extracellular protease in liquid culture which was partially purified. The protease (PRT 3) was a zinc-requiring metalloenzyme and was readily distinguishable from the two previously characterized proteases (PRT 1 and PRT 2) of X. campestris pv. campestris by the pattern of degradation of beta-casein and sensitivity to inhibitors. PRT 3 was produced at a low level in the vascular brassica pathogen X. campestris pv. campestris (five strains tested), in which PRT 1 and PRT 2 predominate. In contrast, expression of PRT 1, a serine protease, could not be detected in the six tested strains of the leaf spotting mesophyll pathogens. However, all these strains had DNA fragments which hybridized to a prtA probe and which probably carry a functional prtA (the structural gene for PRT 1). The structural gene for PRT 3 (prtC) was cloned by screening a genomic library of X. campestris pv. raphani in a protease-deficient X. campestris pv. campestris strain. Subcloning and Tn5 mutagenesis located the structural gene to 1.2 kb of DNA. DNA fragments which hybridized to the structural gene were found in all strains of the crucifer-attacking X. campestris pathovars tested as well as in a number of other pathovars. Experiments in which the pattern of protease production of the pathovars was manipulated by introduction of cloned genes into heterologous pathovars suggested that no determinative relationship exists between the pattern of protease gene expression and the (vascular or mesophyllic) mode of pathogenesis.
Occurrence of Killer Yeasts in Spontaneous Wine Fermentations from the Tuscany Region of Italy
Authors: Vagnoli Paola , Musmanno Rosa Anna , Cresti Stefania , Di Maggio Tiziana , Coratza Grazietta
Published: January 2020

The occurrence of killer yeasts in an area of Tuscany (central Italy) was studied. Killer yeasts were found in 88% of spontaneous wine fermentations from 18 wineries. The incidence of killers varied with respect to fermentation stage and vintage period, increasing from the first vintage to successive ones and from the commencement to the end of fermentation. At the end of fermentation, the proportion of killer strains relative to total yeast population was below 25% in 15 cases, above 75% in 6 cases, from 25 to 50% in 5 cases, and from 50 to 75% in 3 cases. Karyotype analysis also showed a mixed killer population in the fermentations in which the killers dominated.
Selective Adhesion of
Thiobacillus ferrooxidans
to Pyrite
Authors: Ohmura Naoya , Kitamura Keiko , Saiki Hiroshi
Published: January 2020

Bacterial adhesion to mineral surfaces plays an important role not only in bacterial survival in natural ecosystems, but also in mining industry applications. Selective adhesion was investigated with
Thiobacillus ferrooxidans
by using four minerals, pyrite, quartz, chalcopyrite, and galena.
Escherichia coli
was used as a control bacterium. Contact angles were used as indicators of hydrophobicity, which was an important factor in the interaction between minerals and bacteria. The contact angle of
E. coli
in a 0.5% sodium chloride solution was 31°, and the contact angle of
T. ferrooxidans
in a pH 2.0 sulfuric acid solution was 23°.
E. coli
tended to adhere to more hydrophobic minerals by hydrophobic interaction, while
T. ferrooxidans
selectively adhered to iron-containing minerals, such as pyrite and chalcopyrite. Ferrous ion inhibited the selective adhesion of
T. ferrooxidans
to pyrite competitively, while ferric ion scarcely inhibited such adhesion. When selective adhesion was quenched by ferrous ion completely, adhesion of
T. ferrooxidans
was controlled by hydrophilic interactions. Adhesion of
E. coli
to pyrite exhibited a liner relationship on langmuir isotherm plots, but adhesion of
T. ferrooxidans
did not.
T. ferrooxidans
recognized the reduced iron in minerals and selectively adhered to pyrite and chalcopyrite by a strong interaction other than the physical interaction.
Adhesion of human bifidobacterial strains to cultured human intestinal epithelial cells and inhibition of enteropathogen-cell interactions
Authors: Bernet M F , Brassart D , Neeser J R , Servin A L
Published: January 2020

Thirteen human bifidobacterial strains were tested for their abilities to adhere to human enterocyte-like Caco-2 cells in culture. The adhering strains were also tested for binding to the mucus produced by the human mucus-secreting HT29-MTX cell line in culture. A high level of calcium-independent adherence was observed for Bifidobacterium breve 4, for Bifidobacterium infantis 1, and for three fresh human isolates from adults. As observed by scanning electron microscopy, adhesion occurs to the apical brush border of the enterocytic Caco-2 cells and to the mucus secreted by the HT29-MTX mucus-secreting cells. The bacteria interacted with the well-defined apical microvilli of Caco-2 cells without cell damage. The adhesion to Caco-2 cells of bifidobacteria did not require calcium and was mediated by a proteinaceous adhesion-promoting factor which was present both in the bacterial whole cells and in the spent supernatant of bifidobacterium culture. This adhesion-promoting factor appeared species specific, as are the adhesion-promoting factors of lactobacilli. We investigated the inhibitory effect of adhering human bifidobacterial strains against intestinal cell monolayer colonization by a variety of diarrheagenic bacteria. B. breve 4, B. infantis 1, and fresh human isolates were shown to inhibit cell association of enterotoxigenic, enteropathogenic, diffusely adhering Escherichia coli and Salmonella typhimurium strains to enterocytic Caco-2 cells in a concentration-dependent manner. Moreover, B. breve 4 and B. infantis 1 strains inhibited, dose dependently, Caco-2 cell invasion by enteropathogenic E. coli, Yersinia pseudotuberculosis, and S. typhimurium strains.
Outer Membrane Proteins and Lipopolysaccharides in Pathovars of
Xanthomonas campestris
Authors: Ojanen Tuula , Helander Ilkka M. , Haahtela Kielo , Korhonen Timo K. , Laakso Tuula
Published: January 2020

Variations in the outer membrane proteins (OMPs) and lipopolysaccharides (LPSs) of 54 isolates belonging to 16 different pathovars of
Xanthomonas campestris
were characterized. OMP samples prepared by sarcosyl extraction of cell walls and LPS samples prepared by proteinase K treatment of sonicated cells were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of 4 M urea. In general, the OMP and LPS profiles within each pathovar were very similar but different from the profiles of other pathovars. Heterogeneity in OMP and LPS profiles was observed within
X. campestris
pv. campestris,
X. campestris
pv. translucens, and
X. campestris
pv. vesicatoria. LPSs were isolated from six
X. campestris
pathovars, which fell into two major groups on the basis of O antigenicity. The O antigens of
X. campestris
pv. begoniae,
X. campestris
pv. graminis, and
X. campestris
pv. translucens cross-reacted with each other; the other group consisted of
X. campestris
pv. campestris,
X. campestris
pv. pelargonii, and
X. campestris
pv. vesicatoria. A chemical analysis revealed a significant difference between the compositions of the neutral sugars of the LPSs of those two groups; the LPSs of the first group contained xylose and a 6-deoxy-3-
O
-methyl hexose, whereas the LPSs of the other group lacked both sugars.
Effect of Organic Matter Decomposition Level on Bacterial Species Diversity and Composition in Relationship to Pythium Damping-Off Severity
Authors: Boehm M. J. , Madden L. V. , Hoitink H. A. J.
Published: January 2020

Rhizosphere bacteria were isolated from root tip segments of cucumber seedlings grown in a suppressive, slightly decomposed light-colored peat mix, a conducive, more decomposed dark-colored peat mix, and a suppressive dark peat mix amended with composted hardwood bark. The bacteria were identified by a gas chromatographic fatty acid methyl ester analysis. The total number of taxa recovered from a single root tip segment ranged from 9 to 18. No single taxon predominated on all root tip segments harvested from any of the mixes. The highest relative population density reached by a given taxon on any root tip segment was 45%. Hill's first and second diversity numbers, the modified Hill's ratio, and Hurlbert's rarefaction method, which were used as measures of species diversity, indicated that the organic matter decomposition level of the potting mixes did not affect bacterial species diversity. Bray-Curtis polar ordination and Dice resemblance functions, however, indicated that the organic matter decomposition level of a mix significantly influenced the composition of bacterial species in the rhizosphere.
Pseudomonas
spp. and other taxa capable of inducing suppression of pythium damping-off predominated in the suppressive mixes. These organisms were absent from the conducive mix, in which
Arthrobacter
and
Bacillus
spp. predominated. Although effective bacterial biocontrol agents were isolated from both the suppressive mixes and the conducive mix, the majority were isolated from the less decomposed suppressive mixes. Finally, the efficacy of strains was significantly greater in the slightly decomposed light peat mix than in the decomposed dark peat mix. Natural disease suppression within these mixes was associated with the organic matter decomposition level and the bacterial species compositions of the mixes.
Ozone inactivation of Cryptosporidium parvum in demand-free phosphate buffer determined by in vitro excystation and animal infectivity
Authors: Finch G R , Black E K , Gyürék L , Belosevic M
Published: January 2020

Inactivation of Cryptosporidium parvum oocysts by ozone was performed in ozone demand-free 0.05 M phosphate buffer (pH 6.9) in bench-scale batch reactors at 7 and 22 degrees C. Ozone was added to each trial from a concentrated stock solution for contact times ranging from 5 to 15 min. The viability of the control and treated oocysts was determined by using in vitro excystation and infection in neonatal CD-1 mice. It was found that excystation consistently underestimated inactivation when compared with animal infectivity (P < or = 0.05). As inactivations increased, the difference between excystation and infectivity also increased. The inactivation kinetics of C. parvum by ozone deviated from the simple first-order Chick-Watson model and was better described by a nonlinear Hom model. The use of the Hom model for predicting inactivation resulted in a family of unique concentration and time values for each inactivation level rather than the simple CT product of the Chick-Watson model.
Virulence of an Escherichia coli O157:H7 sorbitol-positive mutant
Authors: Fratamico P M , Buchanan R L , Cooke P H
Published: January 2020

Virulence and pathogenicity of an Escherichia coli O157:H7 sorbitol-positive mutant were investigated with an infant rabbit animal model as well as a battery of in vitro assays. Total cell lysate protein profiles, outer membrane protein profiles, plasmid profiles, and levels of cytotoxic activity against Vero cells were similar in the wild-type and mutant strains. Both adhered to intestinal epithelial cells in culture and reacted with fluorescein isothiocyanate-labeled antiserum against E. coli O157:H7. The mutant appeared to be similar to the wild type in all respects except in its ability to ferment sorbitol. [14C]sorbitol uptake and sorbitol-6-phosphate dehydrogenase activities were notably increased in the mutant strain. Diarrhea developed in rabbits administered the wild-type strain and in those fed the sorbitol-positive mutant. There was greater bacterial attachment and mucosal damage in the cecum and large intestine than in the small intestine. Scanning electron microscopy revealed bacteria adhering as single cells and as aggregates closely associated with mucus. Mucosal lesions consisted of areas of tissue necrosis with sloughing of epithelial cells. By transmission electron microscopy, electron-dense necrotic epithelial cells were visible in areas where bacteria were present, and epithelial cell debris containing bacteria was observed between the villar luminal surfaces. Light microscopy of epithelial cells of intestinal sections of infected rabbits revealed noticeable vacuolation and spherical, pyknotic nuclei. These data indicate that the sorbitol-negative phenotype is not associated with the pathogenicity of E. coli O157:H7.
Alteration of growth yield by overexpression of phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxykinase in Escherichia coli
Authors: Chao Y P , Liao J C
Published: January 2020

Phosphoenolpyruvate and oxaloacetate are key intermediates at the junction between catabolism and biosynthesis. Alteration of carbon flow at these branch points will affect the growth yield and the formation of products. We attempted to modulate the metabolic flow between phosphoenolpyruvate and oxaloacetate by overexpressing phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxykinase from a multicopy plasmid under the control of the tac promoter. It was found that overexpression of phosphoenolpyruvate carboxylase decreased the rates of glucose consumption and organic acid excretion, but the growth and respiration rates remained unchanged. Consequently, the growth yield on glucose was improved. This result indicates that the wild-type level of phosphoenolpyruvate carboxylase is not optimal for the most efficient glucose utilization in batch cultures. On the other hand, overexpression of phosphoenolpyruvate carboxykinase increased glucose consumption and decreased oxygen consumption relative to those levels required for growth. Therefore, the growth yield on glucose was reduced because of a higher rate of fermentation product excretion. These data provide useful insights into the regulation of central metabolism and facilitate further manipulation of pathways for metabolite production.
Microbial Delignification with White Rot Fungi Improves Forage Digestibility
Authors: Akin D. E. , Sethuraman A. , Morrison W. H. , Martin S. A. , Eriksson K.-E. L.
Published: January 2020

Three wild-type white rot fungi and two cellulase-less mutants developed from
Phanerochaete chrysosporium
K-3 (formerly
Sporotrichum pulverulentum
) were tested for their ability to delignify grass cell walls and improve biodegradation by rumen microorganisms. Fungal-treated and control stems of Bermuda grass were analyzed for their content of ester- and ether-linked aromatics by using alkali extraction and gas chromatography, for in vitro dry weight digestion and production of volatile fatty acids in in vitro fermentations with mixed ruminal microorganisms, for loss of lignin and other aromatics from specific cell wall types by using microspectrophotometry, and for structural changes before and after in vitro degradation by rumen microorganisms by using transmission electron microscopy.
P. chrysosporium
K-3 and
Ceriporiopsis subvermispora
FP 90031-sp produced the greatest losses in lignin and improved the biodegradation of Bermuda grass over that of untreated control substrate. However,
C. subvermispora
removed the most lignin and significantly improved biodegradation over all other treatments.
Phellinus pini
RAB-83-19 and cellulase-less mutants 3113 and 85118 developed from
P. chrysosporium
K-3 did not improve the biodegradation of Bermuda grass lignocellulose. Results indicated that
C. subvermispora
extensively removed ester-linked
p
-coumaric and ferulic acids and also removed the greatest amount of non-ester-linked aromatics from plant cell walls. Microscopic observations further indicated that
C. subvermispora
removed esters from parenchyma cell walls as well as esters and lignin from the more recalcitrant cell walls (i.e., sclerenchyma and vascular tissues).
C. subvermispora
improved in vitro digestion and volatile fatty acid production by ruminal microorganisms by about 80%, while dry matter loss due to fungi was about 20% greater than loss in untreated control stems. The chemical and structural studies used identified sites of specific fungal attack and suggested mechanisms whereby improvement occurred.
Kinetics of the persistence of chromosomal DNA from genetically engineered Escherichia coli introduced into soil
Authors: Recorbet G , Picard C , Normand P , Simonet P
Published: January 2020

Investigations to quantify bacterial survival and DNA persistence of a genetically engineered population of Escherichia coli introduced into soil microcosms were carried out. The survival of E. coli was monitored by plate counting and immunofluorescence methods, whereas the persistence of the DNA was evaluated by using a most-probable-number-polymerase chain reaction method. Whereas the E. coli population density declined below the plate-counting-technique detection threshold (10(2) CFU.g-1) after 15 days, 10(3) extracellular and 5 x 10(5) total DNA target sequences were still detected after 40 days. Additionally, the E. coli cell counts fell below the detection limit of the immunofluorescence method (10(5) cells.g-1) before the end of the experiment. Colony hybridizations did not reveal gene transfer to the indigenous microflora. These results confirm the persistence of residual E. coli target sequences that could not be detected by the classical cell counting method and offer promising applications for the environmental detection of microorganisms, either engineered, pathogenic, or released for beneficial effects.
Glutamine Involvement in Nitrogen Control of Gibberellic Acid Production in
Gibberella fujikuroi
Authors: Muñoz Gastón A. , Agosin Eduardo
Published: January 2020

When the fungus
Gibberella fujikuroi
ATCC 12616 was grown in fermentor cultures, both intracellular kaurene biosynthetic activities and extracellular GA
3
accumulation reached high levels when exogenous nitrogen was depleted in the culture. Similar patterns were exhibited by several nonrelated enzymatic activities, such as formamidase and urease, suggesting that all are subject to nitrogen regulation. The behavior of the enzymes involved in nitrogen assimilation (glutamine synthetase, glutamate dehydrogenase, and glutamate synthase) during fungal growth in different nitrogen sources suggests that glutamine is the final product of nitrogen assimilation in
G. fujikuroi.
When ammonium or glutamine was added to hormone-producing cultures, extracellular GA
3
did not accumulate. However, when the conversion of ammonium into glutamine was inhibited by L-methionine-DL-sulfoximine, only glutamine maintained this effect. These results suggest that glutamine may well be the metabolite effector in nitrogen repression of GA
3
synthesis, as well as in other nonrelated enzymatic activities in
G. fujikuroi.
Isolation and Characterization of a Novel Atrazine Metabolite Produced by the Fungus
Pleurotus pulmonarius
, 2-Chloro-4-Ethylamino-6-(1-Hydroxyisopropyl)Amino-1,3,5-Triazine
Authors: Masaphy Segula , Levanon Dan , Vaya Jacob , Henis Yigal
Published: January 2020

The white rot fungus
Pleurotus pulmonarius
exhibited metabolism of atrazine (2-chloro-4-ethylamino-6-isopropylamino-1,3,5-triazine) in liquid culture, producing the dealkylated metabolites desethylatrazine, desisopropylatrazine, and desethyl-desisopropylatrazine. A fourth, unknown metabolite was also produced. It was isolated and was identified as 2-chloro-4-ethylamino-6-(1-hydroxyisopropyl)amino-1,3,5-triazine by gas chromatography-mass spectrometry, Fourier transformed infrared spectroscopy, and
1
H nuclear magnetic resonance analysis. The structure of this metabolite was confirmed by chemical synthesis of the compound and comparison with the fungally produced metabolite.
Abiotic Reduction of 4-Chloronitrobenzene to 4-Chloroaniline in a Dissimilatory Iron-Reducing Enrichment Culture
Authors: Heijman Cornelis G. , Holliger Christof , Glaus Martin A. , Schwarzenbach René P. , Zeyer Josef
Published: January 2020

4-chloronitrobenzene (4-Cl-NB) was rapidly reduced to 4-chloroaniline with half-lives of minutes in a dissimilatory Fe(III)-reducing enrichment culture. The initial pseudo-first-order rate constants at 25°C ranged from 0.11 to 0.19 per minute. The linear Arrhenius correlation in a temperature range of 6 to 85°C and the unchanged reactivity after pasteurization indicated that the nitroreduction occurred abiotically. A fine-grained black solid which was identified as poorly crystalline magnetite (Fe
3
O
4
) by X-ray diffraction accumulated in the enrichments. Magnetite produced by the Fe(III)-reducing bacterium
Geobacter metallireducens
GS-15 and synthetic magnetite also reduced 4-Cl-NB. These results suggest that the reduction of 4-Cl-NB by the enrichment material was a surface-mediated reaction by dissimilatory formed Fe(II) associated with magnetite.
Effects of Preservatives on Viability of
Cryptosporidium parvum
Oocysts
Authors: Campbell A. T. , Robertson L. J. , Smith H. V.
Published: January 2020

Potassium dichromate and formalin reduced the viability of
Cryptosporidium parvum
oocysts as assessed by inclusion or exclusion of 4′,6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI) and excystation. Some formalin-treated oocysts containing dead sporozoites excluded PI; that this fluorogenic assay relies not solely upon exclusion of PI but also upon highlighting of sporozoite nuclei by DAPI is reiterated.
Common soybean inoculant strains in Brazil are members of Bradyrhizobium elkanii
Authors: Rumjanek N G , Dobert R C , van Berkum P , Triplett E W
Published: January 2020

The Brazilian inoculant strains 29W and 587 were found to be members of Bradyrhizobium elkanii primarily on the basis of 16S rRNA gene sequences identical to that of B. elkanii USDA76 and on the basis of reactivity with antibodies against serogroups 76 and 31, respectively. The agronomic consequences of using strains of B. elkanii as soybean inoculants are discussed.