bluuebunny/crossref_metadata_2025 huggingface dataset:
Colonization of broiler chickens by waterborne Campylobacter jejuni
Authors: Pearson A D , Greenwood M , Healing T D , Rollins D , Shahamat M , Donaldson J , Colwell R R
Published: January 2020

Chickens on a broiler farm in southern England were found to be colonized with Campylobacter jejuni of a single serotype, Lior 1 Penner 4. The farm was the sole supplier of a local slaughterhouse associated with a campylobacter outbreak in 1984 caused by this serotype. The serotype persisted on the farm for at least 18 months after the outbreak; its prevalence in the human population served by the farm remained high until it disappeared from the farm in 1986. The possible sources and routes of transmission of C. jejuni to the broilers on the farm were investigated. The results showed that vertical transmission, feed, litter, small mammals, and environmental or airborne cross-contamination between sheds or successive crops could be excluded as persistent sources of C. jejuni. The predominant source of C. jejuni on the farm was shown to be the water supply. Direct microscopy and fluorescent antibody methods revealed presumptive campylobacters throughout the farm's water system. Campylobacter-free chickens raised in an animal house and given water from the farm supply became colonized with the serotype of C. jejuni endemic on the farm (Lior 1 Penner 4). An intervention program based on water chlorination, shed drinking system cleaning and disinfection, and withdrawal of furazolidone from feed reduced the proportion of birds colonized with campylobacter from 81 to 7% and was associated with a 1,000- to 10,000-fold reduction in campylobacters recoverable from the carcasses. Two months after the end of the intervention program colonization of the birds returned to high levels (84%), indicating that there was a temporal association between intervention and reduced colonization with C. jejuni. Investigations continue to establish the general applicability of these findings.
Isolation and Diversity of Actinomycetes in the Chesapeake Bay
Authors: Takizawa Masayuki , Colwell Rita R. , Hill Russell T.
Published: January 2020

Chesapeake Bay was investigated as a source of actinomycetes to screen for production of novel bioactive compounds. The presence of relatively large populations of actinoplanetes (chemotype II/D actinomycetes) in Chesapeake Bay sediment samples indicates that it is an eminently suitable ecosystem from which to isolate actinomycetes for screening programs. Actinomycetes were isolated from sediment samples collected in Chesapeake Bay with an isolation medium containing nalidixic acid, which proved to be more effective than heat pretreatment of samples. Actinomycete counts ranged from a high of 1.4 × 10
5
to a low of 1.8 × 10
2
CFU/ml of sediment. Actinomycetes constituted 0.15 to 8.63% of the culturable microbial community. The majority of isolates from the eight stations studied were actinoplanetes (i.e., chemotype II/D), and 249 of these isolates were obtained in a total of 298 actinomycete isolates. Antimicrobial activity profiles indicated that diverse populations of actinoplanetes were present at each station. DNA hybridization studies showed considerable diversity among isolates between stations, but indicated that actinoplanete strains making up populations at nearby stations were more similar to each other than to populations sampled at distant stations. The diversity of actinoplanetes and the ease with which these organisms were isolated from Chesapeake Bay sediments make this a useful source of these actinomycetes.
Production of polyclonal antibodies to the trichothecene mycotoxin 4,15-diacetylnivalenol with the carrier-adjuvant cholera toxin
Authors: Abouzied M M , Azcona-Olivera J I , Yoshizawa T , Pestka J J
Published: January 2020

The trichothecene mycotoxin 4,15-diacetylnivalenol (DNIV) was conjugated to cholera toxin (DNIV-CT) for use as an immunogen and as an adjuvant for specific antibody production. Repeated intravenous injection of 7.5 micrograms of the conjugate was effective at generating specific antibodies to DNIV in rabbits as determined by enzyme-linked immunosorbent assay (ELISA). When small amounts (1 to 10 micrograms per animal) of DNIV-CT were used to immunize mice, polyclonal antibodies were observed as early as 4 weeks of immunization. The relative affinity of the antibodies to DNIV increased with the immunogen dose in mice. Antibodies were not detectable in either rabbits or mice that were injected with DNIV conjugated to the carrier protein bovine serum albumin or when DNIV-CT was blocked with glutaraldehyde. Competitive ELISA of mouse and rabbit serum revealed that the antibodies were most specific for DNIV but reacted to a small extent with fusarenone-X, deoxynivalenol, and nivalenol. No reactivity was observed with 3- or 15-acetyldeoxynivalenol. The results suggest that specific polyclonal antibodies can be prepared against a trichothecene when CT is used as an adjuvant and carrier protein. DNIV antibodies will be useful for monitoring the compound in food in conjunction with other trichothecene antibodies, detection of DNIV-producing cultures, and investigation of 8-ketotrichothecene biosynthesis.
H
2
-CO
2
-Dependent Anaerobic O-Demethylation Activity in Subsurface Sediments and by an Isolated Bacterium
Authors: Liu Shi , Suflita Joseph M.
Published: January 2020

The ability of microorganisms in sediments from the Atlantic Coastal Plain to biodegrade methoxylated aromatic compounds was examined. O-demethylation activity was detected in deep (121- and 406-m) sediments, as well as in the surface soil. A syringate-demethylating consortium, containing at least three types of bacteria, was enriched from a deep-sediment sample in a medium containing syringate as the sole organic carbon source and with a N
2
-CO
2
atmosphere. An isolate which demethylated syringate was obtained from the enrichment on an agar medium incubated under a H
2
-CO
2
but not a N
2
-CO
2
or N
2
atmosphere. O demethylation of syringate of this isolate was dependent on the presence of both H
2
and CO
2
in the gas phase. The metabolism of syringate occurred in a sequential manner: methylgallate accumulated transiently before it was converted to gallate. Mass balance analysis suggests that the stoichiometry of the reaction in this isolate proceeds in accordance with the following generalized equation: C
7
H
3
O
3
(OCH
3
)

n

-
+
n
HCO
3
-
+
n
H
2
→ C
7
H
3
O
3
(OH)

n

-
+
n
CH
3
COO
-
+
n
H
2
O.
Further studies on the feasibility of one-day Salmonella detection by enzyme-linked immunosorbent assay
Authors: Wyatt G M , Langley M N , Lee H A , Morgan M R
Published: January 2020

A model system previously developed for the rapid detection of Salmonella typhimurium in foods was improved and extended to many other Salmonella serotypes. The original protocol, which consisted of an overnight nonselective culture followed by a specific enzyme-linked immunosorbent assay (ELISA), was modified and improved. A sandwich ELISA which used polyclonal antibodies for the capture stage and a cocktail of monoclonal antibodies for the detector stage was developed. The assay recognized a wide range of Salmonella serotypes; S. enteritidis, the most important serotype in the United Kingdom had a detection limit in the ELISA of about 4 x 10(2) cells ml-1. The cultural stage prior to the ELISA was either a single nonselective broth (incubated for 28 h) or a preenrichment broth (incubated for 7 h) plus a selective broth (incubated for 21 h). Antibodies which bind to cells grown in the unfavorable conditions of a selective medium were selected. It was concluded that, in the future, the shortened protocols for the detection of Salmonella spp. in foods described here will be of considerable value.
Bromoalkane-degrading Pseudomonas strains
Authors: Shochat E , Hermoni I , Cohen Z , Abeliovich A , Belkin S
Published: January 2020

Two Pseudomonas isolates, named ES-1 and ES-2, were shown to possess a wide degradative spectrum for haloalkanes in general and bromoalkanes in particular but did not degrade nonsubstituted alkanes. The utilization of water-insoluble haloalkanes, such as 1-bromooctane, appeared to consist of three phases: (i) extracellular emulsification by a constitutively excreted, broad-spectrum surface-active agent, (ii) dehalogenation by an inducible hydrolytic dehalogenase (possibly periplasmic), and (iii) intracellular degradation of the residual carbon skeleton. Several observations suggest the existence of more than one dehalogenase in strain ES-2.
Complete Oxidation of Propionate, Valerate, Succinate, and Other Organic Compounds by Newly Isolated Types of Marine, Anaerobic, Mesophilic, Gram-Negative, Sulfur-Reducing Eubacteria
Authors: Finster Kai , Bak Friedhelm
Published: January 2020

Anaerobic enrichment cultures with either propionate, succinate, lactate, or valerate and elemental sulfur and inocula from shallow marine or deep-sea sediments were dominated by rod-shaped motile bacteria after three transfers. By application of deep-agar dilutions, five eubacterial strains were obtained in pure culture and designated Kyprop, Gyprop, Kysw2, Gylac, and Kyval. All strains were gram negative and grew by complete oxidation of the electron donors and concomitant stoichiometric reduction of elemental sulfur to hydrogen sulfide. The isolates used acetate, propionate, succinate, lactate, pyruvate, oxaloacetate, maleate, glutamate, alanine, aspartate, and yeast extract. All isolates, except strain Gylac, used citrate as an electron donor but valerate was oxidized only by strain Kyval. Fumarate and malate were degraded by all strains without an additional electron donor or acceptor. Kyprop, Gyprop, and Gylac utilized elemental sulfur as the sole inorganic electron acceptor, while Kysw2 and Kyval also utilized nitrate, dimethyl sulfoxide, or Fe(III)-citrate as an electron acceptor.
Evidence for the existence of independent chloromethane- and S-adenosylmethionine-utilizing systems for methylation in Phanerochaete chrysosporium
Authors: Coulter C , Hamilton J T , Harper D B
Published: January 2020

O methylation of acetovanillone at 4 position by C2H3Cl and S-adenosyl[methyl-2H3]methionine was monitored in whole mycelia of Phanerochaete chrysosporium in the presence and absence of S-adenosylhomocysteine. Both the amount of the methylation product, 3,4-dimethoxyacetophenone, and the percent C2H3 incorporation into the 4-methoxyl group of the compound were determined. The results strongly suggest the presence of biochemically distinct systems for O methylation of acetovanillone utilizing S-adenosylmethionine and chloromethane, respectively, as the methyl donor. The S-adenosylmethionine-dependent enzyme is induced early in the growth cycle, with activity attaining an initial maximum after 55 h of incubation. Methylation by this enzyme is totally suppressed by 1 mM S-adenosylhomocysteine over almost the entire growth cycle. S-Adenosylmethionine-dependent O-methyltransferase activity is detectable in cell extracts, and the purification and characterization of the enzyme are described elsewhere (C. Coulter, J. T. Kennedy, W. C. McRoberts, and D. B. Harper, Appl. Environ. Microbiol. 59:706-711, 1993). The chloromethane-utilizing methylation system is absent in early growth but attains peak activity in the mid-growth phase after 72 h of incubation. The system is not significantly inhibited by S-adenosylhomocysteine at any stage of growth. No chloromethane-dependent O-methyltransferase activity is detectable in cell extract, suggesting that the enzyme is membrane bound and/or part of a multienzyme complex. Although the biochemical role of the chloromethane-dependent methylation system in metabolism is not known, one possible function could be the regeneration of veratryl alcohol degraded by the attack of lignin peroxidase.
Specific detection of Salmonella spp. by multiplex polymerase chain reaction
Authors: Way J S , Josephson K L , Pillai S D , Abbaszadegan M , Gerba C P , Pepper I L
Published: January 2020

Three sets of oligonucleotide primers were used in the polymerase chain reaction (PCR) assay to detect Salmonella species. phoP primers specific to the phoP/phoQ loci of coliform pathogenic bacteria such as Salmonella, Shigella, Escherichia coli, and Citrobacter species served as presumptive indicators of enteric bacteria. In addition to the phoP primers, the Hin and the H-1i primers, which targeted a 236-bp region of hin/H2 and a 173-bp region of the H-1i flagellin gene, respectively, were used. Both Hin and H-1i primers are specific to motile Salmonella species and are not present in Shigella, E. coli, or Citrobacter species. Thus, by multiplex PCR amplification, Salmonella species including Salmonella typhi, Salmonella typhimurium, Salmonella paratyphi A, and Salmonella enteritidis can be specifically detected. Optimal reaction conditions have been described to demonstrate this specific, sensitive detection of Salmonella species. By using agarose gel electrophoresis for detection of the PCR-amplified products, the sensitivity of detection was 10(2) CFU after 25 cycles of PCR and 1 (10(0)) CFU after a 50-cycle double PCR. The efficacy of these primers was demonstrated on environmental isolates which had previously been confirmed as Salmonella species by the use of conventional cultural techniques. In addition, positive amplifications resulted from Salmonella species in environmental samples including soil and water.
Role of D-ribose as a cometabolite in D-xylose metabolism by Saccharomyces cerevisiae
Authors: van Zyl C , Prior B A , Kilian S G , Brandt E V
Published: January 2020

The influence of D-ribose as a cosubstrate on the uptake and metabolism of the non-growth substrate D-xylose by Saccharomyces cerevisiae ATCC 26602 was investigated. Xylose was taken up by means of low- and high-affinity glucose transport systems. In cells exposed for 2 days to a mixture of xylose and ribose, only the high-affinity system could be detected. Glucose strongly inhibited the transport of xylose by both systems. Starvation or exposure to either xylose or ribose resulted in inactivation of xylose transport, which did not occur in the presence of a mixture of ribose and xylose. A constitutive non-glucose-repressible NADPH2-dependent xylose reductase with a specific activity of ca. 5 mU/mg of protein that converted xylose to xylitol was present in a glucose-grown culture. No activity converting xylitol to xylulose or vice versa was found in crude extracts. Both xylose and ribose were converted to their corresponding polyols, xylitol and ribitol, as indicated by 13C nuclear magnetic resonance spectroscopy. Furthermore, ethanol was detected, and this implied that pathways for the complete catabolism of xylose and ribose exist. However, the NADPH2 required for the conversion of xylose to xylitol is apparently not supplied by the pentose phosphate pathway since the ethanol produced from D-[1-13C]xylose was labelled only in the C-2 position. Acetic acid was produced from ribose and may assist in the conversion of xylose to xylitol by cycling NADPH2.
Biochemical Characterization of a Protease Involved in the Processing of a
Streptomyces reticuli
Cellulase (Avicelase)
Authors: Moormann Michael , Schlochtermeier André , Schrempf Hildgund
Published: January 2020

A 36-kDa protease from
Streptomyces reticuli
had recently been shown to be responsible for the in vivo and in vitro processing of the 82-kDa cellulase (Avicelase) Cel-1 from
S. reticuli
to a 42-kDa truncated enzyme. It was induced only in the presence of Avicel, hydroxyethylcellulose, and xylan. The addition of the nonionic detergent Tween 80 to the culture medium containing Avicel as the carbon source led to a 10-fold increase in extracellular proteolytic activity. The protease, which has an isoelectric point of 3.9, was purified to homogeneity from the culture filtrate by a combination of anion-exchange and hydrophobic-interaction chromatographies and was characterized biochemically. The enzyme hydrolyzed gelatin and the chromogenic substrates Azocoll, Azocasein, and Azoalbumin. Its highest activity was determined between pH 7.0 and 7.7 and at 55°C. The proteolytic activity was inhibited by 1,10-phenanthroline and EDTA; however, no metal ions were detected to be associated with the protein. The protease was stable in the presence of 1 M urea and 0.01 M sodium dodecyl sulfate. The inhibitory effect of alpha-2-macroglobulin indicated an endo-mode of proteolytic cleavage. Studies with lectins and sugar analysis by mass spectroscopy indicated that the cellulase (Avicelase) Cel-1 was neither N nor O glycosylated. Its processing by the protease occurred at temperatures ranging from 30 to 55°C, pH 7.5, in the presence of 2 mM dithiothreitol.
Characteristics of Insertional Mutants of
Pseudomonas syringae
with Reduced Epiphytic Fitness
Authors: Lindow Steven E. , Andersen Gary , Beattie Gwyn A.
Published: January 2020

Random Tn
5
mutagenesis was used to identify genes ir.
Pseudomonas syringae
which contribute to epiphytic fitness. Mutants were selected on the basis of deficiencies in epiphytic growth or survival on plants rather than deficiencies in predetermined phenotypes exhibited in culture. A sample freezing procedure was used to measure the population sizes of 5,300 mutants of
P. syringae
exposed to alternating wet and dry conditions on bean leaves in growth chambers. Eighty-two mutants exhibited reduced population sizes. Of these mutants, over half exhibited a reduced ability to survive the stresses associated with dry leaves, while others grew more slowly or attained reduced stationary-phase population sizes on leaves. While some epiphytic fitness mutants were altered in phenotypes that could be measured in culture, many mutants were not altered in any in vitro phenotype examined. Only three of the epiphytic fitness mutants were auxotrophs, and none had catabolic deficiencies for any of 31 organic compounds tested. Other mutants that exhibited reductions in one or more of the following were identified: motility, osmotolerance, desiccation tolerance, growth rate in batch culture, and extracellular polysaccharide production. All of the mutants retained the abilities to produce disease symptoms on the compatible host plant, bean, to incite a hypersensitive response on the non-host plant, tobacco, and to produce a fluorescent pyoverdine siderophore.
Influence of the Endogenous Storage Lipid Poly-β-Hydroxybutyrate on the Reducing Power Availability during Cometabolism of Trichloroethylene and Naphthalene by Resting Methanotrophic Mixed Cultures
Authors: Henrysson Tomas , McCarty Perry L.
Published: January 2020

The role of the storage lipid poly-β-hydroxybutyrate (PHB) in trichloroethylene transformation by methanotrophic mixed cultures was investigated. Naphthalene oxidation rates were used to assay for soluble methane monooxygenase activity. The PHB content of methanotrophic cells grown in reactors varied diurnally as well as from day to day. A positive correlation between the amount of PHB in the cells and the naphthalene oxidation rate as well as between PHB and the trichloroethylene transformation rate and capacity was found. Addition of β-hydroxybutyrate increased the naphthalene oxidation rates significantly. PHB content in cells could be manipulated by incubation at different methane-to-nitrogen ratios. A positive correlation between the naphthalene oxidation rate and the PHB content after these incubations could be seen. Both the PHB content and the naphthalene oxidation rates decreased with time in resting methanotrophic cells exposed to oxygen. However, this decrease in the naphthalene oxidation rate cannot be explained by the decrease in the PHB content alone. Probably a deactivation of the methane monooxygenase itself is also involved.
The Bradyrhizobium japonicum serocluster 123 hyperreiterated DNA region, HRS1, has DNA and amino acid sequence homology to IS1380, an insertion sequence from Acetobacter pasteurianus
Authors: Judd A K , Sadowsky M J
Published: January 2020

We have sequenced and analyzed the hyperreiterated DNA region, HRS1, from Bradyrhizobium japonicum USDA 424. The 2.1-kb HRS1 fragment is closely linked to the B. japonicum common and genotype-specific nodulation genes in serogroup 123 and 127 strains. Southern hybridization analyses indicated that one copy of HRS1 is also located next to the fixRnifA locus in B. japonicum USDA 424. Nucleotide sequence analysis revealed the presence of a 4-bp target site duplication in HRS1 which is identical to a terminal repeat found in the B. japonicum USDA 110 repeated sequence RS alpha. Computer searches of the PIR (Protein Identification Resource) protein data base revealed a high degree of amino acid sequence homology between a putative 329-amino-acid polypeptide from HRS1 and a large polypeptide from IS1380, an insertion sequence from Acetobacter pasteurianus. RNA slot blot hybridizations suggest that transcripts showing homology to HRS1 are constitutively produced in strains USDA 424 (serogroup 127) and USDA 438 (serogroup 123).
Natural Transformation of
Acinetobacter calcoaceticus
by Plasmid DNA Adsorbed on Sand and Groundwater Aquifer Material
Authors: Chamier Bärbel , Lorenz Michael G. , Wackernagel Wilfried
Published: January 2020

It is known that plasmid DNA and linear duplex DNA molecules adsorb to chemically purified mineral grains of sand and to particles of several clay fractions. It seemed desirable to examine whether plasmid DNA would also adsorb to nonpurified mineral materials taken from the environment and, particularly, whether adsorbed plasmid DNA would be available for natural transformation of bacteria. Therefore, microcosms consisting of chemically pure sea sand plus buffered CaCl
2
solution were compared with microcosms consisting of material sampled directly from a groundwater aquifer (GWA) plus groundwater (GW) with respect to the natural transformation of
Acinetobacter calcoaceticus
by mineral-associated DNA. The GWA minerals were mostly sand with inorganic precipitates and organic material plus minor quantities of silt and clay (illite and kaolinite). The amount of plasmid DNA which adsorbed to GWA (in GW) was about 80% of the amount which adsorbed to purified sand (in buffered CaCl
2
solution). Plasmid DNA adsorbed on sand transformed
A. calcoaceticus
significantly less efficiently than did plasmid DNA in solution. In contrast, the transformation by sand-adsorbed chromosomal DNA was as high as that by DNA in solution. In GWA/GW microcosms, the efficiency of transformation by chromosomal DNA was similar to that in sand microcosms, whereas plasmid transformation was not detectable. However, plasmid transformants were found at a low frequency when GWA was loaded with both chromosomal and plasmid DNA. Reasons for the low transformation efficiency of plasmid DNA adsorbed to mineral surfaces are discussed. Control experiments showed that the amounts of plasmid and chromosomal DNA desorbing from sand during incubation with a cell-free filtrate of a competent cell suspension did not greatly contribute to transformation in sand microcosms, suggesting that transformation occurred by direct uptake of DNA from the mineral surfaces. Taken together, the observations suggest that plasmid DNA and chromosomal DNA fragments which are adsorbed on mineral surfaces in a sedimentary or soil habitat may be available (although with different efficiencies for the two DNA species) for transformation of a naturally competent gram-negative soil bacterium.
Copper Hypersensitivity and Uptake in
Pseudomonas syringae
Containing Cloned Components of the Copper Resistance Operon
Authors: Cha Jae-Soon , Cooksey Donald A.
Published: January 2020

Copper resistance in
Pseudomonas syringae
carrying the
copABCD
operon is associated with accumulation of copper in the periplasm and outer membrane, apparently as a function of the copper-binding activities of the
copABC
gene products. However, no specific function for
copD
has been determined. In this study,
P. syringae
cells containing
copCD
or
copBCD
cloned behind the
lac
promoter were hypersensitive to copper. An increased accumulation of copper was measured in cells containing several combinations of
cop
genes that included
copC
and
copD
. Our data suggest that CopC, a periplasmic copper-binding protein, and CopD, a probable inner membrane protein, may function together in copper uptake.
Siderophore Utilization by
Bradyrhizobium japonicum
Authors: Plessner Ora , Klapatch Taryn , Guerinot Mary Lou
Published: January 2020

Bradyrhizobium japonicum
USDA 110 and 61A152 can utilize the hydroxamate-type siderophores ferrichrome and rhodotorulate, in addition to ferric citrate, to overcome iron starvation. These strains can also utilize the pyoverdin-type siderophore pseudobactin St3. The ability to utilize another organism's siderophores may confer a selective advantage in the rhizosphere.
Selection of Xenobiotic-Degrading Microorganisms in a Biphasic Aqueous-Organic System
Authors: Ascon-Cabrera Miguel , Lebeault Jean-Michel
Published: January 2020

Microbial selection on mixtures of chlorinated and nonchlorinated compounds that are poorly soluble in water and/or toxic to growing microbial cells was examined in both biphasic aqueous-organic and monophasic aqueous systems. A biphasic system in which silicone oil was used as the organic phase permitted the acceleration of acclimation, leading to rapid selection and to an increase in xenobiotic compound degradation. In contrast, acclimation, selection, and degradation were very slow in the monophasic aqueous system. The variation in microbial growth rate with the degree of dispersion (i.e., dispersion at different silicone oil concentrations and agitation rates), and cell adhesion to the silicone oil indicate that the performance of the biphasic aqueous-organic system is dependent on the interfacial area between the two phases and that microbial activity is important at this interface. Therefore, the biphasic water-silicone oil system could be used for microbial selection in the presence of xenobiotic compounds that are toxic and have low water solubility.
Acetate Degradation at 70°C in Upflow Anaerobic Sludge Blanket Reactors and Temperature Response of Granules Grown at 70°C
Authors: Rintala Jukka , Lepistö Satu , Ahring Birgitte
Published: January 2020

Anaerobic acetate degradation at 70°C and at 55°C (as a reference) was studied by running laboratory upflow anaerobic sludge blanket (UASB) reactors inoculated with mesophilic granular sludge. In UASB reactors fed with acetate-containing media (3 g of chemical oxygen demand [COD] per liter, corresponding to 47 mM acetate) approximately 50 days was needed at 70°C and less than 15 days was needed at 55°C to achieve an effluent COD of 500 to 700 mg/liter. In the UASB reactors at both 70 and 55°C up to 90% of the COD was removed. Batch assays showed that sludges from two 70°C UASB reactors, one run at a low effluent acetate concentration and the other run at a high effluent acetate concentration, exhibited slightly different responses to temperatures in the range from 37 to 70°C. Both 70°C sludges, as well as the 55°C sludge, produced methane at temperatures of 37 to 73°C. The 55°C sludge exhibited shorter lag phases than the 70°C sludges and higher specific methane production rates between 37 and 65°C.
Erwinia chrysanthemi
EC16 Produces a Second Set of Plant-Inducible Pectate Lyase Isozymes
Authors: Kelemu Segenet , Collmer Alan
Published: January 2020

The enterobacterium
Erwinia chrysanthemi
causes soft-rot diseases involving extensive tissue maceration in a wide variety of plants and secretes multiple pectic enzymes that degrade plant cell walls and middle lamellae. An
E. chrysanthemi
mutant with directed deletions or insertions in genes
pehX, pelX, pelA, pelB, pelC
, and
pelE
, which encode exo-poly-α-
d
-galacturonosidase, exopolygalacturonate lyase, and four isozymes of pectate lyase, respectively, was constructed by the marker exchange of a cloned
pehX
::Tn
phoA
fragment into
E. chrysanthemi
CUCPB5010, a Δ(
pelA pelE
) Δ(
pelB pelC
)::28bp Δ(
pelX
)Δ4bp derivative of strain EC16. This mutant,
E. chrysanthemi
CUCPB5012, no longer caused pitting in a standard pectate semisolid agar medium used to detect pectolytic activity in bacteria. Nevertheless, the mutant still macerated leaves of chrysanthemum (
Chrysanthemum morifolium
), although with reduced virulence. The mutant was found to produce significant pectate lyase activity in rotting chrysanthemum tissue and in minimal media containing chrysanthemum extracts or cell walls as the sole carbon source. Activity-stained, ultra-thin-layer isoelectric focusing gels revealed the presence in these preparations of several pectate lyase isozymes with pIs ranging from highly acidic to highly alkaline. Sterile culture fluids containing these isozymes were able to macerate chrysanthemum leaf tissue. Unlike the products of the
pelA, pelB, pelC
, and
pelE
genes in
E. chrysanthemi
EC16, these plant-inducible pectate lyase isozymes were not produced in minimal medium containing pectate. The results suggest that
E. chrysanthemi
produces two sets of independently regulated pectate lyase isozymes that are capable of macerating plant tissues.
Plasmids pJP4 and r68.45 Can Be Transferred between Populations of Bradyrhizobia in Nonsterile Soil
Authors: Kinkle B. K. , Sadowsky M. J. , Schmidt E. L. , Koskinen W. C.
Published: January 2020

IncP plasmid r68.45, which carries several antibiotic resistance genes, and IncP plasmid pJP4, which contains genes for mercury resistance and 2,4-dichlorophenoxyacetic acid degradation, were evaluated for their ability to transfer to soil populations of rhizobia. Transfer of r68.45 was detected in nonsterile soil by using
Bradyrhizobium japonicum
USDA 123 as the plasmid donor and several
Bradyrhizobium
sp. strains as recipients. Plasmid transfer frequencies ranged up to 9.1 × 10
-5
in soil amended with 0.1% soybean meal and were highest after 7 days with strain 3G4b4-RS as the recipient. Transconjugants were detected in 7 of 500 soybean nodules tested, but the absence of both parental strains in these nodules suggests that plasmid transfer had occurred in the soil, in the rhizosphere, or on the root surface. Transfer of degradative plasmid pJP4 was also evaluated in nonsterile soil by using
B. japonicum
USDA 438 as the plasmid donor and several
Bradyrhizobium
sp. strains as recipients. Plasmid pJP4 was transferred only when strains USDA 110-ARS and 3G4b4-RS were the recipients. The plasmid transfer frequency was highest for strain 3G4b4-RS (up to 7.4 × 10
-6
). Mercury additions to soil, ranging from 10 to 50 μg/g of soil, did not affect population levels of parental strains or the plasmid transfer frequency.
The GUS gene fusion system (Escherichia coli beta-D-glucuronidase gene), a useful tool in studies of root colonization by Fusarium oxysporum
Authors: Couteaudier Y , Daboussi M J , Eparvier A , Langin T , Orcival J
Published: January 2020

The plant-pathogenic fungus Fusarium oxysporum was successfully transformed with the beta-D-glucuronidase gene from Escherichia coli (gusA) (GUS system) in combination with the gene for nitrate reductase (niaD) as the selectable marker. The frequency of cotransformation, as determined by GUS expression on plates containing medium supplemented with 5-bromo-4-chloro-3-indolyl glucuronide (GUS+), was very high (up to 75%). Southern hybridization analyses of GUS+ transformants revealed that single or multiple copies of the gusA gene were integrated into the genomes. High levels of GUS activity are expressed in some transformants, but activity in F. oxysporum does not appear to be correlated with the copy number of the gusA gene. Since the highest activity was found in a transformant with a single copy, it can be assumed that sequence elements of F. oxysporum integrated upstream of the gene can act as a promoter or enhancer. Expression of the gusA gene was also detected during growth of the fungus in plants, indicating that the GUS system can be used as a sensitive and easy reporter gene assay in F. oxysporum.
Characterization of a nitrophenol reductase from the phototrophic bacterium Rhodobacter capsulatus E1F1
Authors: Blasco R , Castillo F
Published: January 2020

The phototrophic bacterium Rhodobacter capsulatus E1F1 photoreduced 2,4-dinitrophenol to 2-amino-4-nitrophenol by a nitrophenol reductase activity which was induced in the presence of nitrophenols and was repressed in ammonium-grown cells. The enzyme was located in the cytosol, required NAD(P)H as an electron donor, and used several nitrophenol derivatives as alternative substrates. The nitrophenol reductase was purified to electrophoretic homogeneity by a simple method. The enzyme was composed of two 27-kDa subunits, was inhibited by metal chelators, mercurial compounds, and Cu2+, and contained flavin mononucleotide and possibly nonheme iron as prosthetic groups. Purified enzyme also exhibited NAD(P)H diaphorase activity which used tetrazolium salt as an electron acceptor.
Adhesion of Nongerminated
Botrytis cinerea
Conidia to Several Substrata
Authors: Doss Robert P. , Potter Sandra W. , Chastagner Gary A , Christian James K.
Published: January 2020

Conidia of the plant pathogenic fungus
Botrytis cinerea
adhered to tomato cuticle and to certain other substrata immediately upon hydration. This immediate adhesion occurred with both living and nonliving conidia. Adhesion was not consistently influenced by several lectins, sugars, or salts or by protease treatment, but it was strongly inhibited by ionic or nonionic detergents. With glass and oxidized polyethylene, substrata whose surface hydrophobicities could be conveniently varied, there was a direct relationship between water contact angle and percent adhesion. Immediate adhesion did not involve specific conidial attachment structures, although the surfaces of attached conidia were altered by contact with a substratum. Freshly harvested conidia were very hydrophobic, with more than 97% partitioning into the organic layer when subjected to a phase distribution test. Percent adhesion of germinated conidia was larger than that of nongerminated conidia. Evidence suggests that immediate adhesion of conidia of
B. cinerea
depends, at least in part, on hydrophobic interactions between the conidia and substratum.
Impact of Bacterial Biomass on Contaminant Sorption and Transport in a Subsurface Soil
Authors: Bellin C. A. , Rao P. S. C.
Published: January 2020

The objective of this study was to investigate the impact of bacterial biomass on the sorption and transport of three solutes (quinoline, naphthalene, and
45
Ca) in a subsurface soil. Miscible displacement techniques were employed to measure sorption and transport of the above compounds during steady, saturated water flow in sterile and/or bacterium-inoculated soil columns. The soil was inoculated with either a quinoline-degrading bacterium,
Pseudomonas
sp. 3N3A isolate, or its mutant isolate, B53, which does not degrade quinoline. In soil columns inoculated with the B53 and 3N3A isolates, quinoline sorption was reduced by about 60 and 20%, respectively. In contrast,
45
Ca sorption was minimally reduced, which indicated that biomass did not significantly alter the cation-exchange capacity of the soil. Biomass impacts on sorption were solute specific, even when the sorption mechanism for both quinoline and
45
Ca was similar. Thus, the differential response is attributed to biomass-induced changes in quinoline speciation; an increase in pH at the sorbent-water interface would result in a larger proportion of the neutral species and a decrease in sorption. Sorption of naphthalene was reduced by about 30%, which was attributed to accessibility of hydrophobic regions. Minimal biosorption of all solutes indicated negligible biofacilitated transport. Alteration of the soil surfaces upon addition of bacterial biomass reduced sorption of quinoline and naphthalene, thereby enhancing transport.
Biometric Study of Acetoin Production in
Hanseniaspora guilliermondii
and
Kloeckera apiculata
Authors: Romano Patrizia , Suzzi Giovanna , Zironi Roberto , Comi Giuseppe
Published: January 2020

Gas chromatographic analysis by direct injection of samples yielded quantitative data on acetoin content. Ninety-six strains of
Hanseniaspora guilliermondii
and
Kloeckera apiculata
were investigated for the ability to produce acetoin in synthetic medium and in must. High-level production of acetoin was found to be a characteristic of both species. In synthetic medium, the two species were not significantly different with respect to sugar utilization and ethanol or acetoin production. In grape must, the two species were significantly different (
P
= 0.001) in acetoin production and
K. apiculata
exhibited a significantly negative correlation between acetoin production and either sugar consumption or ethanol production. Use of selected apiculate yeasts in mixed cultures with
Saccharomyces cerevisiae
seems promising for optimization of wine bouquet.
Effects of Kraft Pulp and Lignin on
Trametes versicolor
Carbon Metabolism
Authors: Roy Brian P. , Archibald Frederick
Published: January 2020

The white rot basidiomycete
Trametes
(
Coriolus
)
versicolor
can substantially increase the brightness and decrease the lignin content of washed, unbleached hardwood kraft pulp (HWKP). Monokaryotic strain 52J was used to study how HWKP and the lignin in HWKP affect the carbon metabolism and secretions of
T. versicolor.
Earlier work indicated that a biobleaching culture supernatant contained all components necessary for HWKP biobleaching and delignification, but the supernatant needed frequent contact with the fungus to maintain these activities. Thus, labile small fungal metabolites may be the vital biobleaching system components renewed or replaced by the fungus. Nearly all of the CO
2
evolved by HWKP-containing cultures came from the added glucose, indicating that HWKP is not an important source of carbon or energy during biobleaching. Carbon dioxide appeared somewhat earlier in the absence of HWKP, but the culture partial O
2
pressure was little affected by the presence of pulp. The presence of HWKP in a culture markedly increased the culture's production of a number of acidic metabolites, including 2-phenyllactate, oxalate, adipate, glyoxylate, fumarate, mandelate, and glycolate. Although the total concentration of these pulp-induced metabolites was only 4.3 mM, these compounds functioned as effective manganese-complexing agents for the manganese peroxidase-mediated oxidation of phenol red, propelling the reaction at 2.4 times the rate of 50 mM sodium malonate, the standard chelator-buffer. The presence of HWKP in a culture also markedly stimulated fungal secretion of the enzymes manganese peroxidase, cellulase, and cellobiose-quinone oxidoreductase, but not laccase (phenol oxidase) or lignin peroxidase.
Biodegradation of trichloroethylene and toluene by indigenous microbial populations in soil
Authors: Fan S , Scow K M
Published: January 2020

The biodegradation of trichloroethylene (TCE) and toluene, incubated separately and in combination, by indigenous microbial populations was measured in three unsaturated soils incubated under aerobic conditions. Sorption and desorption of TCE (0.1 to 10 micrograms ml-1) and toluene (1.0 to 20 micrograms ml-1) were measured in two soils and followed a reversible linear isotherm. At a concentration of 1 micrograms ml-1, TCE was not degraded in the absence of toluene in any of the soils. In combination, both 1 microgram of TCE ml-1 and 20 micrograms of toluene ml-1 were degraded simultaneously after a lag period of approximately 60 to 80 h, and the period of degradation lasted from 70 to 90 h. Usually 60 to 75% of the initial 1 microgram of TCE ml-1 was degraded, whereas 100% of the toluene disappeared. A second addition of 20 micrograms of toluene ml-1 to a flask with residual TCE resulted in another 10 to 20% removal of the chemical. Initial rates of degradation of toluene and TCE were similar at 32, 25, and 18 degrees C; however, the lag period increased with decreasing temperature. There was little difference in degradation of toluene and TCE at soil moisture contents of 16, 25, and 30%, whereas there was no detectable degradation at 5 and 2.5% moisture. The addition of phenol, but not benzoate, stimulated the degradation of TCE in Rindge and Yolo silt loam soils, methanol and ethylene slightly stimulated TCE degradation in Rindge soil, glucose had no effect in either soil, and dissolved organic carbon extracted from soil strongly sorbed TCE but did not affect its rate of biodegradation.
Overproduction of lignin peroxidase by Phanerochaete chrysosporium (BKM-F-1767) under nonlimiting nutrient conditions
Authors: Dosoretz C G , Rothschild N , Hadar Y
Published: January 2020

The ligninolytic enzymes synthesized by Phanerochaete chrysosporium BKM-F-1767 immobilized on polyurethane foam were characterized under limiting, sufficient, and excess nutrient conditions. The fungus was grown in a nonimmersed liquid culture system under conditions close to those occurring in nature, with nitrogen concentrations ranging from 2.4 to 60 mM. This nonimmersed liquid culture system consisted of fungal mycelium immobilized on porous pieces of polyurethane foam saturated with liquid medium and highly exposed to gaseous oxygen. Lignin peroxidase (LIP) activity decreased to almost undetectable levels as the initial NH4+ levels were increased over the range from 2.4 to 14 mM and then increased with additional increases in initial NH4+ concentration. At 45 mM NH4+, LIP was overproduced, reaching levels of 800 U/liter. In addition, almost simultaneous secretion of LIP and secretion of manganese-dependent lignin peroxidase were observed on the third day of incubation. Manganese-dependent lignin peroxidase activity was maximal under nitrogen limitation conditions (2.4 mM NH4+) and then decreased to 40 to 50% of the maximal level in the presence of sufficient or excess initial NH4+ concentrations. Overproduction of LIP in the presence of a sufficient nitrogen level (24 mM NH4+) and excess nitrogen levels (45 to 60 mM NH4+) seemed to occur as a response to carbon starvation after rapid glucose depletion. The NH4+ in the extracellular fluid reappeared as soon as glucose was depleted, and an almost complete loss of CO2 was observed, suggesting that an alternative energy source was generated by self-proteolysis of cell proteins.(ABSTRACT TRUNCATED AT 250 WORDS)
Plasmid-mediated mineralization of naphthalene, phenanthrene, and anthracene
Authors: Sanseverino J , Applegate B M , King J M , Sayler G S
Published: January 2020

The well-characterized plasmid-encoded naphthalene degradation pathway in Pseudomonas putida PpG7(NAH7) was used to investigate the role of the NAH plasmid-encoded pathway in mineralizing phenanthrene and anthracene. Three Pseudomonas strains, designated 5R, DFC49, and DFC50, were recovered from a polynuclear aromatic hydrocarbon-degrading inoculum developed from a manufactured gas plant soil slurry reactor. Plasmids pKA1, pKA2, and pKA3, approximately 100 kb in size, were isolated from these strains and characterized. These plasmids have homologous regions of upper and lower NAH7 plasmid catabolic genes. By conjugation experiments, these plasmids, including NAH7, have been shown to encode the genotype for mineralization of [9-14C]phenanthrene and [U-14C]anthracene, as well as [1-14C]naphthalene. One strain, Pseudomonas fluorescens 5RL, which has the complete lower pathway inactivated by transposon insertion in nahG, accumulated a metabolite from phenanthrene and anthracene degradation. This is the first direct evidence to indicate that the NAH plasmid-encoded catabolic genes are involved in degradation of polynuclear aromatic hydrocarbons other than naphthalene.
Purification and preliminary characterization of permethrinase from a pyrethroid-transforming strain of Bacillus cereus
Authors: Maloney S E , Maule A , Smith A R
Published: January 2020

Bacillus cereus SM3 was isolated on a mineral salts medium with Tween 80 as the primary carbon source. It was able to hydrolyze second- and third-generation pyrethroids, thereby generating noninsecticidal products. The enzyme responsible for this hydrolytic reaction was named permethrinase for this study. This is the first instance in which pyrethroid detoxification has been achieved with a cell-free microbial enzyme system. Permethrinase was purified by ion-exchange chromatography and gel filtration chromatography. The molecular mass of native permethrinase was 61 +/- 3 kDa, as estimated by Sephadex G-100 gel filtration. This novel microbial esterase seems to be a carboxylesterase. Permethrinase activity had an optimum pH of 7.5 and a temperature optimum of 37 degrees C. No cofactors or coenzymes were required for permethrinase activity. The enzyme may be a serine esterase, as it seems to be sensitive to the organophosphorus compound tetraethylpyrophosphate at concentrations in the micromolar range. Addition of dithiothreitol afforded permethrinase protection against the inhibitory effects of the sulfydryl agents p-chloromercuribenzoate and N-ethylmaleimide. The enzyme was stable over a range of temperatures. Cell extracts of strain SM3 also contained another esterase, which was active towards beta-naphthylacetate, but this enzyme was distinct from permethrinase.
Isolation and characterization of the lantibiotic salivaricin A and its structural gene salA from Streptococcus salivarius 20P3
Authors: Ross K F , Ronson C W , Tagg J R
Published: January 2020

A bacteriocin-like inhibitory substance, salivaricin A, was purified from cultures of Streptococcus salivarius 20P3 and was shown by ion spray mass spectrometry to have a molecular mass of 2,315 +/- 1.1 Da. Amino acid composition analysis demonstrated the presence of lanthionine, indicating that salivaricin A may be a member of the lantibiotic class of antibiotic substances. The sequence of eight amino acids at the N terminus of the molecule was determined by Edman degradation, and mixed oligonucleotide probes based on part of this sequence (GSGWIA) were used to detect the salivaricin A structural gene. A 6.2-kb EcoRI fragment of chromosomal DNA from strain 20P3 that hybridized with the probes was cloned, and the hybridizing region was further localized to a 379-bp DraI-AluI fragment. Analysis of the nucleotide sequence of this fragment indicated that salivaricin A is synthesized as a 51-amino-acid prepeptide that is posttranslationally modified and cleaved to give a biologically active 22-residue peptide containing one lanthionine and two beta-methyllanthionine residues. The secondary structure of presalivaricin A was predicted to be similar to that of type A lantibiotics, with a hydrophilic alpha-helical leader sequence and a propeptide region with potential for beta-turn formation and a lack of alpha-helicity. The sequence around the cleavage site of presalivaricin A differed from that of other type A lantibiotics but was similar to that of several bacteriocin-like inhibitory substances produced by lactic acid bacteria.
Isolation and characterization of Selenomonas ruminantium strains capable of 2-deoxyribose utilization
Authors: Rasmussen M A
Published: January 2020

Microbes from ruminal contents of cattle were selectively enriched by using 2-deoxyribose (2DR) as a substrate for growth. Bacterial isolates growing on 2DR were gram-negative, curved, motile rods. The isolates grew on a broad range of substrates, including deoxyribose, glucose, ribose, mannitol, and lactate as well as ribonucleosides and deoxyribonucleosides. The strains also grew on rhamnose (6-deoxymannose) but not DNA. Organic acids produced from growth on hexoses and pentoses included acetate, propionate, lactate, and succinate. The isolates were identified as Selenomonas ruminantium subsp. lactilytica on the basis of morphology, substrate specificity, and other biochemical characteristics. Several characterized species of ruminal bacteria were also screened for growth on 2DR, with only one strain (S. ruminantium PC-18) found able to grow on 2DR. Ethanol was produced by 2DR when strains were grown on ribose or 2DR.