bluuebunny/crossref_metadata_2025 huggingface dataset:
Occurrence and distribution of Vibrio spp., Listonella spp., and Clostridium botulinum in the Seto Inland Sea of Japan
Authors: Venkateswaran K , Nakano H , Okabe T , Takayama K , Matsuda O , Hashimoto H
Published: January 2020

The distribution of Vibrio species in samples of surface water, bottom water (water 2 m above the sediment), and sediment from the Seto Inland Sea was studied. A simple technique using a membrane filter and short preenrichment in alkaline peptone water was developed to resuscitate the injured cells, followed by plating them onto TCBS agar. In addition, a survey was conducted to determine the incidence of Clostridium botulinum in sediment samples. Large populations of heterotrophs were found in surface water, whereas large numbers of total vibrios were found in bottom water. In samples from various water sampling regions, high counts of all bacterial populations were found in the inner regions having little exchange of seawater when compared with those of the open region of the inland sea. In the identification of 463 isolates, 23 Vibrio spp. and 2 Listonella spp. were observed. V. harveyi was prevalent among the members of the Vibrio genus. Vibrio species were categorized into six groups; an estimated 20% of these species were in the so-called "pathogenic to humans" group. In addition, a significant proportion of this group was hemolytic and found in the Bisan Seto region. V. vulnificus, V. fluvialis, and V. cholerae non-O1 predominated in the constricted area of the inland sea, which is eutrophic as a result of riverine influence. It was concluded that salinity indirectly governs the distribution of total vibrios and analysis of variance revealed that all bacterial populations were distributed homogeneously and the variance values were found to be significant in some water sampling regions.(ABSTRACT TRUNCATED AT 250 WORDS)
Identification of two distinct Bacillus circulans xylanases by molecular cloning of the genes and expression in Escherichia coli
Authors: Yang R C , MacKenzie C R , Bilous D , Narang S A
Published: January 2020

Two genes coding for xylanase synthesis in Bacillus circulans were cloned and expressed in Escherichia coli. After digestion of genomic DNA from Bacillus circulans with EcoRI and PstI, the fragments were ligated into the corresponding sites of pUC19 and transformed into Escherichia coli. Restriction enzyme mapping of the two inserts coding for xylanase activity indicated distinctly different nucleotide sequences. Cross-hybridization assays confirmed the absence of sequence homology between the two genes. In vitro transcription-translation assays indicated that the cloned genes encoded for proteins with molecular weights of 22,000 and 59,000. The gene products displayed different substrate specificities. The 22,000-dalton enzyme readily hybrolyzed aspeen, larchwood, and oat spelt xylans, whereas the second was unable to extensively depolymerize oat spelt xylan and resulted in very limited reducing sugar release from any of the xylan substrates tested. Both of the xylanases had isoelectric points of approximately 9.0.
Influence of Alternate Electron Acceptors on the Metabolic Fate of Hydroxybenzoate Isomers in Anoxic Aquifer Slurries
Authors: Kuhn Elmar P. , Suflita Joseph M. , Rivera Maria D. , Young L. Y.
Published: January 2020

The biodegradation of hydroxybenzoate isomers was investigated with samples obtained from two sites within a shallow anoxic aquifer. The metabolic fates of the substrates were compared in denitrifying, sulfate-reducing, and methanogenic incubations. Under the latter two conditions, phenol was detected as a major intermediate of
p
-hydroxybenzoate, but no metabolites were initially found with
m
- or
o
-hydroxybenzoate. However, benzoate accumulation was noted when metabolic inhibitors were used with these samples. About 9 to 17 days was required for >95% removal of the parent isomers under these conditions. When aquifer slurries were amended with nitrate, the equivalent removal of the hydroxybenzoates occurred within 4 days. In the denitrifying incubations, phenol was formed from all three hydroxybenzoates and accounted for about 30% of the initial substrate amendment. No benzoate was measured in these samples. All metabolites were identified by chromatographic mobility, mass spectral profiles, or both. Autoclaved controls were uniformly incapable of transforming the parent substrates. These results suggest that the anaerobic fate of hydroxybenzoate isomers depends on the relative substitution pattern and the prevailing ecological conditions. Furthermore, since these compounds are central metabolites formed during the breakdown of many aromatic chemicals, our findings may help provide guidelines for the reliable extrapolation of metabolic fate information from diverse anaerobic environments.
Genetic transformation of intact Lactococcus lactis subsp. lactis by high-voltage electroporation
Authors: McIntyre D A , Harlander S K
Published: January 2020

To apply recombinant DNA techniques for genetic manipulation of the industrially important lactococci, an efficient and reliable high-frequency transformation system must be available. High-voltage electric pulses have been demonstrated to enhance uptake of DNA into protoplasts and intact cells of numerous gram-negative and gram-positive microorganisms. The objective of this study was to develop a system for electroporating intact cells of Lactococcus lactis subsp. lactis LM0230 (previously designated Streptococcus lactis LM0230) with a commercially available electroporation unit (BTX Transfector 100; BTX, Inc., San Diego, Calif.). Parameters which influenced the efficiency of transformation included growth phase and final concentration of cells, ionic strength of the suspending medium, concentration of plasmid DNA, and the amplitude and duration of the pulse. Washed suspensions of intact cells suspended in deionized distilled water were subjected to one high-voltage electric pulse varying in voltage (300 to 900 V corresponding to field strengths of 5 to 17 kV/cm) and duration (100 microseconds to 1 s). Transformation efficiencies of 10(3) transformants per microgram of DNA were obtained when dense suspensions (final concentration, 5 x 10(10) CFU/ml) of stationary-phase cells were subjected to one pulse with a peak voltage of 900 V (field strength, 17 kV/cm) and a pulse duration of 5 ms in the presence of plasmid DNA. Dilution of porated cells in broth medium followed by an expression period of 2 h at 30 degrees C was beneficial in enhancing transformation efficiencies. Plasmids ranging in size from 9.8 to 30.0 kilobase pairs could be transformed by this procedure.
Physical Degradation of Lignified Stem Tissues by Ruminal Fungi
Authors: Akin D. E. , Lyon C. E. , Windham W. R. , Rigsby L. L.
Published: January 2020

Ruminal bacteria or fungi were selected by the addition of cycloheximide or streptomycin and penicillin, respectively, to ruminal fluid, and the weakening and degradation of lignified tissues in alfalfa and Bermuda grass stems by these treatments and whole ruminal fluid were evaluated in vitro. Dry weight loss in alfalfa was similar for whole ruminal fluid and streptomycin-penicillin treatment, whereas that with streptomycin-penicillin treatment was significantly higher (
P
≤ 0.05) than that with cycloheximide treatment. In Bermuda grass, dry weight loss was significantly higher with streptomycin-penicillin than that with whole ruminal fluid and cycloheximide treatment, which were equal. Both peak load (Newtons) and peak stress were less (
P
≤ 0.05) for streptomycin-penicillin treatment than with other treatments in both forages. Fungi colonized the lignified ring in alfalfa and tended to reduce the width of cell walls in this tissue, but a large number of fungal penetrations through cell walls was not observed. In contrast, fungal rhizoids frequently penetrated into and through cell walls in the lignified ring of Bermuda grass, often expanding the pit fields between the cells. Ruminal fungi disrupt lignified tissues in stems, and their activity results in a weakened residue more amendable to physical degradation. This weakening may allow plant digesta to be more easily broken apart during animal's rumination and thus facilitate digesta flow and fiber utilization.
Diel Variation in Population Size and Ice Nucleation Activity of
Pseudomonas syringae
on Snap Bean Leaflets
Authors: Hirano Susan S. , Upper Christen D.
Published: January 2020

The extent to which diel changes in the physical environment affect changes in population size and ice nucleation activity of
Pseudomonas syringae
on snap bean leaflets was determined under field conditions. To estimate bacterial population size and ice nucleation activity, bean leaflets were harvested at 2-h intervals during each of three 26-h periods. A tube nucleation test was used to assay individual leaflets for ice nuclei. Population sizes of
P. syringae
were determined by dilution plating of leaflet homogenates. The overall diel changes in
P. syringae
population sizes differed during each of the 26-h periods. In one 26-h period, there was a continuous increase in the logarithm of
P. syringae
population size despite intense solar radiation, absence of free moisture on leaf surfaces, and low relative humidity during the day. A mean doubling time of approximately 4.9 h was estimated for the 28-fold increase in
P. syringae
population size that occurred from 0900 to 0900 h during the 26-h period. However, doubling times of 3.3 and 1.9 h occurred briefly during this period from 1700 to 2300 h and from 0100 to 0700 h, respectively. Thus, growth rates of
P. syringae
in association with leaves in the field were of the same order of magnitude as optimal rates measured in the laboratory. The frequency with which leaflets bore ice nuclei active at −2.0, −2.2, and −2.5°C varied greatly within each 26-h period. These large diel changes were inversely correlated primarily with the diel changes in air temperature and reflected changes in nucleation frequency rather than changes in population size of
P. syringae.
Thus, the response of bacterial ice nucleation activity to the physical environment was distinct from the changes in population size of ice nucleation-active
P. syringae.
Effects of zinc, iron, cobalt, and manganese on Fusarium moniliforme NRRL 13616 growth and fusarin C biosynthesis in submerged cultures
Authors: Jackson M A , Slininger P J , Bothast R J
Published: January 2020

The influence of zinc, iron, cobalt, and manganese on submerged cultures of Fusarium moniliforme NRRL 13616 was assessed by measuring dry weight accumulation, fusarin C biosynthesis, and ammonia assimilation. Shake flask cultures were grown in a nitrogen-limited defined medium supplemented with various combinations of metal ions according to partial-factorial experimental designs. Zinc (26 to 3,200 ppb [26 to 3,200 ng/ml]) inhibited fusarin C biosynthesis, increased dry weight accumulation, and increased ammonia assimilation. Carbohydrate was found to be the principal component of the increased dry weight in zinc-supplemented cultures. Zinc-deficient cultures synthesized more lipid and lipidlike compounds, such as fusarin C, than did zinc-supplemented cultures. Microscopic examination showed that zinc-deficient hyphae contained numerous lipid globules which were not present in zinc-supplemented cultures. Addition of zinc (3,200 ppb) to 2- and 4-day-old cultures inhibited further fusarin C biosynthesis but did not stimulate additional dry weight accumulation. Iron (10.0 ppm) and cobalt (9.0 ppm) did not affect fusarin C biosynthesis or dry weight accumulation. Manganese (5.1 ppm) did not affect dry weight accumulation but did increase fusarin C biosynthesis in the absence of zinc. Maximum fusarin C levels, 32.3 micrograms/mg (dry weight), were produced when cultures were supplied manganese, whereas minimum fusarin C levels, 0.07 micrograms/mg (dry weight), were produced when zinc, iron, cobalt, and manganese were supplied. These results suggest a multifunctional role for zinc in affecting F. moniliforme metabolism.
Physiological Characterization of Dicarboxylate-Induced Pleomorphic Forms of
Bradyrhizobium japonicum
Authors: Reding H. Keith , Lepo Joe Eugene
Published: January 2020

When
Bradyrhizobium japonicum
I-110 was transferred into medium containing 40 mM succinate or 40 mM fumarate, over 90% of the bacteria acquired a swollen, pleomorphic form similar to that of bacteroids. The induction of pleomorphism was dependent on the carbon substrate and concentration but was independent of the hydrogen ion and sodium ion concentration. Cell extracts of rod-shaped and pleomorphic cells contained enzymes required for sugar catabolism and gluconeogenesis. Variations in these enzyme profiles were correlated with the carbon source used and not with the conversion to the bacteroid-like morphology. Rod-shaped cells cultured on glucose or 10 mM succinate transported glucose and succinate; however, the pleomorphic cells behaved similarly to symbiotic bacteroids in that they lacked the ability to transport glucose and transported succinate at lower rates than did rod-shaped cells.
Kinetics of Nitrate Utilization by Mixed Populations of Denitrifying Bacteria
Authors: Murray Robert E. , Parsons Laura L. , Smith M. Scott
Published: January 2020

Kinetics of nitrate utilization by mixed bacterial populations from two agricultural soils and a pond sediment in Kentucky were measured by using progress curves of nitrous oxide production. Nitrous oxide production from anaerobic soil and sediment slurries containing added nitrate and acetylene exhibited first-order kinetics. Nitrate affinity (

K
m

) for mixed populations of denitrifying bacteria in unfertilized agricultural soils and pond sediments ranged from 1.8 to 13.7 μM. The affinity of bacterial populations for nitrate did not vary with habitat, and the ability to use low concentrations of nitrate was retained by bacterial populations living in environments which received large inputs of nitrate.
Selective medium for isolation of Xanthomonas maltophilia from soil and rhizosphere environments
Authors: Juhnke M E , des Jardin E
Published: January 2020

A selective medium (XMSM) was developed for isolation of Xanthomonas maltophilia from bulk soil and plant rhizosphere environments. The XMSM basal medium contained maltose, tryptone, bromthymol blue, and agar. Antibiotics added to select for X. maltophilia were cycloheximide, nystatin, cephalexin, bacitracin, penicillin G, novobiocin, neomycin sulfate, and tobramycin. A comparison was made between XMSM and 1/10-strength tryptic soy broth agar for recovery of X. maltophilia from sterile and nonsterile soil infested with known X. maltophilia isolates. A recovery rate of 97% or greater for XMSM was demonstrated. XMSM was used to isolate X. maltophilia from a variety of soil and rhizosphere environments.
Medium for Screening
Leuconostoc oenos
Strains Defective in Malolactic Fermentation
Authors: Cavin J. F. , Prevost H. , Lin J. , Schmitt P. , Divies C.
Published: January 2020

A new sensitive medium was developed to screen and isolate mutagenic
Leuconostoc oenos
strains defective in malolactic fermentation. The essential components of the medium included fructose (22 mM),
l
-malic acid (74.6 mM), bromocresol green (as pH indicator), and cellulose powder. The wild-type colonies turned blue, but defective malolactic colonies gave an acid reaction and remained yellow-green.
Nitrogen-Fixing Cyanobacterium with a High Phycoerythrin Content
Authors: Rodriguez Herminia , Rivas Joaquin , Guerrero Miguel G. , Losada Manuel
Published: January 2020

The elemental and molecular composition, pigment content, and productivity of a phycoerythrin-rich nitrogen-fixing cyanobacterium—an
Anabaena
strain isolated from the coastal lagoon Albufera de Valencia, Spain—has been investigated. When compared with other heterocystous species, this strain exhibits similar chlorophyll
a
, carotene, and total phycobiliprotein contents but differs remarkably in the relative proportion of specific phycobiliproteins; the content of C-phycoerythrin amounts to 8.3% (versus about 1% in the other species) of cell dry weight. Absorption and fluorescence spectra of intact phycobilisomes isolated from this
Anabaena
sp. corroborate the marked contribution of phycoerythrin as an antenna pigment, a circumstance that is unusual for cyanobacteria capable of fixing N
2
. The pigment content of cells is affected by variations in irradiance and cell density, these adaptive changes being more patent for C-phycoerythrin than for phycocyanins. The
Anabaena
strain is clumpy and capable of rapid flocculation. It exhibits outdoor productivities higher than 20 g (dry weight) m
−2
day
−1
during summer.
Isolation and identification of Pseudomonas spp. from Schirmacher Oasis, Antarctica
Authors: Shivaji S , Rao N S , Saisree L , Sheth V , Reddy G S , Bhargava P M
Published: January 2020

Ten cultures of Pseudomonas spp. were established from soil samples collected in and around a lake in Antarctica. Based on their morphology, biochemical and physiological characteristics, and moles percent G + C of their DNA, they were identified as P. fluorescens, P. putida, and P. syringae. This is the first report on the identification of Pseudomonas spp. from continental Antarctica.
Complete secretion of activable bovine prochymosin by genetically engineered L forms of Proteus mirabilis
Authors: Klessen C , Schmidt K H , Gumpert J , Grosse H H , Malke H
Published: January 2020

To circumvent problems encountered in the synthesis of active chymosin in a number of bacteria and fungi, a recombinant DNA L-form expression system that directed the complete secretion of fully activable prochymosin into the extracellular culture medium was developed. The expression plasmid constructions involved the in-frame fusion of prochymosin cDNA minus codons 1 to 4 to streptococcal pyrogenic exotoxin type A gene (speA') sequences, including the speA promoter, ribosomal binding site, and signal sequence and five codons of mature SpeA. Secretion of fusion prochymosin enzymatically and immunologically indistinguishable from bovine prochymosin was achieved after transformation of two stable protoplast type L-form strains derived from Proteus mirabilis. The secreted proenzyme was converted by autocatalytic processing to chymosin showing milk-clotting activity. In controlled laboratory fermentation processes, a maximum specific rate of activable prochymosin synthesis of 0.57 x 10(-3)/h was determined from the time courses of biomass dry weight and product formation. Yields as high as 40 +/- 10 micrograms/ml were obtained in the cell-free culture fluid of strain L99 carrying a naturally altered expression plasmid of increased segregational stability. The expression-secretion system described may be generally useful for production of recombinant mammalian proteins synthesized intracellularly as aberrantly folded insoluble aggregates.
Effect of carbonyl cyanide m-chlorophenylhydrazone on Escherichia coli halotolerance
Authors: Ghoul M , Pommepuy M , Moillo-Batt A , Cormier M
Published: January 2020

The growth-inhibitory effect of carbonyl cyanide m-chlorophenylhydrazone (CCCP) was less on members of the family Enterobacteriaceae (halotolerant organisms) than it was on species of Vibrio (moderately halophilic organisms). When sodium chloride concentration increased from 0.5 to 0.85 M, this effect was more pronounced for Escherichia coli; it remained relatively stable for Vibrio spp. The effect of carbonyl cyanide m-chlorophenylhydrazone was antagonized by the addition of glycine betaine or proline or by growth in a rich medium.
Method To Estimate Growth of
Trichoderma reesei
and
Aspergillus wentii
in Mixed Culture on Cellulosic Substrates
Authors: Panda T. , Bisaria V. S. , Ghose T. K.
Published: January 2020

A simple differential method based on measurement of an intracellular pigment of
Aspergillus wentii
was developed for estimation of the individual growths of two fungi,
Trichoderma reesei
and
A. wentii
, in mixed-culture fermentation of an insoluble cellulosic substrate.
Differential regulation of lambda pL and pR promoters by a cI repressor in a broad-host-range thermoregulated plasmid marker system
Authors: Winstanley C , Morgan J A , Pickup R W , Jones J G , Saunders J R
Published: January 2020

Plasmid systems with unique markers were constructed to assess the fate of recombinant DNA and genetically manipulated bacteria in soil and freshwater model environments. On such constructs the marker gene, xylE (for catechol 2,3-dioxygenase), is expressed from the lambda promoter pL or pR, each of which is controlled by the temperature-sensitive lambda repressor c1857. Combinations of these elements were cloned into the broad-host-range plasmid pKT230 to form pLV1010 (pL-xylE), pLV1011 (pL-xylE-c1857), and pLV1013 (pR-xylE-c1857). The recombinant plasmids were introduced into different gram-negative bacteria. The thermoregulated system of pLV1013 functioned well in a range of species, with xylE induction being readily achieved by elevation of the temperature from 28 to 37 degrees C. There was a difference in the induction of catechol 2,3-dioxygenase activity, depending on whether xylE was expressed from pL (pLV1011) or pR (pLV1013). Our observations on testing the different systems in a number of hosts suggest that genes carried by the DNA of genetically engineered microorganisms may not be expressed in a predictable manner following transfer from the release host to other species.
Microcolony epifluorescence microscopy for selective enumeration of injured bacteria in frozen and heat-treated foods
Authors: Rodrigues U M , Kroll R G
Published: January 2020

A rapid (less than 6 h) method for selectively enumerating coliforms, pseudomonads, and staphylococci has been developed which involves counting microcolonies grown on the surface of polycarbonate membranes under selective conditions. The method was not directly applicable to foods containing injured bacteria due to the poor formation of or an inability to form microcolonies under selective conditions. However, the introduction of a 3- to 5-h resuscitation step in tryptone soya broth allowed the method to give reliable estimates of these organisms in a variety of frozen and heat-processed foods. Under nonselective conditions, i.e., for total counts, the microcolony method enabled a rapid count to be made of viable bacteria in heat-treated foods, but these results were also made more consistent by the introduction of a resuscitation step. This method makes results from these foods available far faster than conventional enumeration methods.
Survival and detection of bacteria in an aquatic environment
Authors: Amy P S , Hiatt H D
Published: January 2020

A genetically engineered plasmid, pPSA131, was used as a DNA probe to detect homologous DNA in Escherichia coli HB101(pPSA131) after it was mixed with aquatic microorganisms from Lake Mead, Nevada, water samples. An isolate from the pLAFR1 chromosomal library of Pseudomonas syringae Cit 7 was used to detect parent P. syringae Cit 7 that had been mixed with Lake Mead water. E. coli(pPSA131) was kept in variously treated samples of lake water or buffer, and its survival was measured by viable cell counting on modified Luria-Bertani (LB) agar. Full-strength LB agar proved better than 0.1 x LB agar at recovering E. coli(pPSA131) after survival in low-nutrient environments. Survival of E. coli(pPSA131) remained high in filtered (0.22-micron pore size) lake water and salts buffer on both selective and nonselective agars but was lower in untreated lake water or lake water filtered with a 0.8-micron-pore-size membrane. Total recoverable colonies grown on LB agar were higher when lake water was filter treated (0.8-micron pore size) than when lake water was untreated. Microorganisms recovered from lake water alone grew rapidly on nonselective media, probably because of the "bottle effect." After being mixed with Lake Mead water, E. coli(pPSA131) and P. syringae were detected by colony blotting with non-radioactively labeled DNA probes. E. coli(pPSA131) were recovered at three times during 48 h from variously treated samples of lake water and from a mixture with Lake Mead water organisms. Colonies were supported on either nonselective or selective agar for comparison.(ABSTRACT TRUNCATED AT 250 WORDS)
Transformation of Xenorhabdus nematophilus
Authors: Xu J , Lohrke S , Hurlbert I M , Hurlbert R E
Published: January 2020

The ability of Xenorhabdus nematophilus 19061/1 to be transformed by pHK17 plasmid DNA was studied and optimized. A number of factors, including culture conditions, stage of growth, transformation buffer pH, cation type and concentration required for the production of competency, washing, heat shock conditions, and cell-DNA ratio, were found to affect transformation significantly. On the basis of these observations, a procedure for the routine transformation of X. nematophilus 19061/1 at frequencies of 1 X 10(5) to 10 X 10(5) transformants per microgram of pHK17 plasmid DNA was developed. Maximum transformation was obtained when cells which had reached the mid- to late-logarithmic growth phase (total counts, 2.5 X 10(8) to 5 X 10(8) cells per ml) within 4.5 to 5.5 h were washed once in cold transformation buffer before they were suspended in the same buffer to 0.1 of their original volume. The highest transformation was obtained when dimethyl sulfoxide was added in two steps to the cells immediately before the DNA was added, after which the cell-DNA mixtures were incubated for 30 min on ice before they were given a 3-min heat shock at 37 degrees C. Following these treatments, the transformed cells were incubated in L broth-60 mM CaCl2 for 1 h before they were plated onto selective medium. We also were able to transform X. nematophilus 19061/1 with plasmid pBR325, and we transformed other species of Xenorhabdus with several common plasmids.
Relationship of the Presence and Copy Number of Plasmids to Exopolysaccharide Production and Symbiotic Effectiveness in
Rhizobium fredii
USDA 206
Authors: Barbour W. Mark , Elkan Gerald H.
Published: January 2020

Rhizobium fredii
USDA 206 harbors four large plasmids, one of which carries nodulation and nitrogen fixation genes. Previously isolated groups of plasmid-cured derivatives of strain USDA 206 were compared with each other to determine possible plasmid functions. Mutant strain 206CANS was isolated as a nonmucoid (Muc
−
) derivative of strain 206CA, a mutant that was cured of two plasmids. The Muc
−
phenotype of 206CANS was only expressed when the strain was grown on certain media, particularly those with polyols as carbon sources. Plasmid pRj206b of strain 206CANS was previously shown to have a higher copy number than the same plasmid in strains USDA 206 and 206CA. When this plasmid was transferred to Muc
+
strains, it conferred a nonmucoid phenotype on recipient strains. The symbiotic effectiveness of the wild-type and cured strains was compared. Overall, few differences were shown, but strains 206CA and 206CANS were found to have higher nitrogenase activities than the other strains. Thus, there appeared to be a possible relationship among exopolysaccharide synthesis, plasmid copy number, and symbiotic effectiveness.
Relationships between plasmids and phenotypes of presumptive strains of Vibrio anguillarum isolated from different fish species
Authors: Wiik R , Hoff K A , Andersen K , Daae F L
Published: January 2020

On the basis of plasmid composition as well as serological and biochemical properties, 26 strains identified as Vibrio anguillarum isolated from diseased fish could be assigned to two different groups. Except for three reference strains, these++ strains were isolated from Norwegian fish. The four strains isolated from rainbow trout (Salmo gairdneri), the only strain isolated from char (Salvelinus alpinus), and three of six strains isolated from Atlantic salmon (Salmo salar) harbored a plasmid of 47 megadaltons (MDa). Restriction endonuclease analysis showed that this plasmid and the virulence plasmid pJM1, carried by V. anguillarum strain 775, were very similar but not identical. Strains harboring the 47-MDa plasmid had nearly identical biochemical properties and were serotype O1. Strains isolated from reared coastal cod (Gadus morhua), turbot (Scophthalmus maximus), halibut (Hippoglossus hippoglossus), free-living saithe (Pollachius virens), and partly from reared Atlantic salmon differed from strains harboring the 47-MDa virulence plasmid by not containing this plasmid, by having different biochemical traits, and by being serotype O2. Rainbow trout which were experimentally infected with a strain isolated from cod suffering from vibriosis developed clinical symptoms similar to those in cod but quite different from those usually seen in rainbow trout.
Improvement of
Rhizobium
Inoculants
Authors: Paau Alan S.
Published: January 2020

A practical approach was used to develop a
Rhizobium
(
Bradyrhizobium
)
japonicum
inoculant that increases soybean (
Glycine max
(L.) Merr.) yield in fields with indigenous
Rhizobium
populations, which typically outcompete strains present in existing commercial inoculants and therefore decrease the value of inoculant use. Field tests managed by several universities in the Mississippi delta region averaged a 169-kg/ha (
P
< 0.01) grain yield increase. The inoculant contains a mixture of mutants selected for increased nitrogen fixation ability. These mutants were derived from indigenous wild-type strains that are capable of high-level occupancy of nodules in soybean fields in the Mississippi delta region. To ensure microbiological purity, the inoculant is fermented directly in the point-of-use container with a vermiculite carrier (L. Graham-Weiss, M. L. Bennett, and A. S. Paau, Appl. Environ. Microbiol. 53:2138-2140, 1987). It should be possible to use this approach to produce more effective
Rhizobium
inoculants for any legume in any geographical area.
Kinetics of p-cresol degradation by an immobilized Pseudomonas sp
Authors: O'Reilly K T , Crawford R L
Published: January 2020

A p-cresol (PCR)-degrading Pseudomonas sp. was isolated from creosote-contaminated soil and shown to degrade PCR by conversion to protocatechuate via p-hydroxybenzaldehyde (PBA) and p-hydroxybenzoate (PHB). Cells of the Pseudomonas sp. were immobilized in calcium alginate beads and in polyurethane foam. The relationship between the PCR concentration and the PCR transformation rate was investigated in batch and continuous culture bioreactors. The biodegradation kinetics of PBA and PHB also were investigated. In batch culture reactors, the maximum PCR degradation rate (Vmax) for the alginate-immobilized Pseudomonas sp. cells was 1.5 mg of PCR g of bead-1 h-1 while the saturation constant (Ks) was 0.22 mM. For PHB degradation, the Vmax was 0.62 mg of PHB g of bead-1 h-1 while the Ks was 0.31 mM. For polyurethane-immobilized Pseudomonas sp. cells, the Vmax of PCR degradation was 0.80 mg of PCR g of foam-1 h-1 while the Ks was 0.28 mM. For PHB degradation, the Vmax was 0.21 mg of PHB g of foam-1 h-1 and the Ks was 0.22 mM. In a continuous column alginate bead reactor, the Vmax for PCR transformation was 2.6 mg g of bead-1 h-1 while the Ks was 0.20 mM. The Vmax and Ks for PBA transformation in the presence of PCR were 0.93 mg g of bead-1 h-1 and 0.063 mM, respectively. When PHB alone was added to a reactor, the Vmax was 1.48 mg g of bead-1 h-1 and the Ks was 0.32 mM.(ABSTRACT TRUNCATED AT 250 WORDS)
Bacterial dehalogenation of chlorobenzoates and coculture biodegradation of 4,4'-dichlorobiphenyl
Authors: Adriaens P , Kohler H P , Kohler-Staub D , Focht D D
Published: January 2020

Acinetobacter sp. strain 4CB1 was isolated from a polychlorobiphenyl-contaminated soil sample by using 4-chlorobenzoate as a sole source of carbon and energy. Resting cells of Acinetobacter sp. strain 4CB1 hydrolytically dehalogenated 4-chlorobenzoate under aerobic and anaerobic conditions, but 4-hydroxybenzoate accumulated only under anaerobic conditions. Cell extracts of Acinetobacter sp. strain 4CB1 oxidized 4-hydroxybenzoate by an NADH-dependent monooxygenase to form protocatechuate, which was subsequently oxidized by both ortho- and meta-protocatechuate dioxygenase reactions. When grown on biphenyl, Acinetobacter sp. strain P6 cometabolized 4,4'-dichlorobiphenyl primarily to 4-chlorobenzoate; however, when this strain was grown in a coculture with Acinetobacter sp. strain 4CB1, 4-chlorobenzoate did not accumulate but was converted to inorganic chloride. When resting cells of Acinetobacter sp. strain 4CB1 were incubated anaerobically with 3,4-dichlorobenzoate, they accumulated 4-carboxy-1,2-benzoquinone as a final product. Since 3,4-dichlorobenzoate is a product that is formed from the cometabolism of 3,4-dichloro-substituted tetrachlorobiphenyls by Acinetobacter sp. strain P6, the coculture has a potential application for dehalogenation and mineralization of specific polychlorobiphenyl congeners.
Specific gene probe for detection of biotyped and serotyped Listeria strains
Authors: Notermans S , Chakraborty T , Leimeister-Wächter M , Dufrenne J , Heuvelman K J , Maas H , Jansen W , Wernars K , Guinee P
Published: January 2020

A total of 284 strains of Listeria, including all known serovars and biovars together with Listeria grayi and Listeria murrayi, were biotyped and serotyped. Biotyping and serotyping could be done in 2 days. A gene probe encoding a delayed hypersensitivity factor (DTH) was used in the detection of pathogenic biotypes and serotypes of the tested strains. The gene was found in all 117 tested Listeria monocytogenes strains of serogroups 1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 4c, 4d, 4e, 4ab, and 7. It was also present in Listeria ivanovii. Of 78 L. monocytogenes strains of serogroup 4b, 77 strains contained the gene, whereas it was absent in all 10 tested L. monocytogenes strains of serogroup 4a. Furthermore, the gene was absent in Listeria seeligeri, L. grayi, L. murrayi, and L. innocua of serogroups 3c, 4b, and 6a and in L. welshimeri of serogroups 1/2b, 3b, 6a, and 6b. Since L. monocytogenes and L. ivanovii are the only two biotypes of the genus Listeria considered pathogens, the data obtained indicate that the DNA probe tested may be a useful tool in the detection of virulent Listeria isolates in clinical, environmental, and food samples.
Detection of fecal coliforms in water by using [14C]mannitol
Authors: Reasoner D J , Geldreich E E
Published: January 2020

Interest in rapid bacterial detection methods for sanitary indicator bacteria in water prompted a study of the use of [U-14C]mannitol to detect fecal coliforms (FC). A simple method which used m-FC broth, membrane filtration, and two-temperature incubation (35 degrees C for 2 h followed by 44.5 degrees C for 2.5 h) was developed. [U-14C]mannitol was added to the medium, and the temperature was raised to 44.5 degrees C after 2 h at 35 degrees C. 14CO2 was collected as Ba14CO3 and assayed by liquid scintillation spectroscopy. Correlations were examined between FC cell numbers at the start of incubation (standard 24-h FC test) and Ba14CO3 counts per minute after 4.5 h. Results indicated that FC numbers ranging from 1 x 10(1) to 2.1 x 10(5) cells could be detected in 4.5 h. Within-sample reproducibility at all cell concentrations was good, but sample-to-sample reproducibility was variable. Comparisons between m-FC broth and m-FC broth modified by substituting D-mannitol for lactose indicated that the standard m-FC broth was the better test medium. Results from experiments in which dimethyl sulfoxide was used to increase permeability of FC to [U-14C]mannitol indicated no increase in 14CO2 production due to dimethyl sulfoxide. Detection of FC by this method may be useful for rapid estimation of FC levels in freshwater recreational areas, for estimating the quality of potable source water, and potentially for emergency testing of potable water, suspected of contamination due to distribution line breaks or cross-connections.
Improvement of DNA transfer frequency and transposon mutagenesis of Erwinia carotovora subsp. betavasculorum
Authors: Rella M , Axelrood P E , Weinhold A R , Schroth M N
Published: January 2020

The production of antibiotics and their role in microbial competition under natural conditions can be readily studied by the use of transposon mutants. Several antibiotic-producing strains of Erwinia carotovora subsp. betavasculorum were unable to accept foreign DNA. A plasmid delivery system was developed, using ethyl methanesulfonate mutagenesis, which entailed isolating E. carotovora subsp. betavasculorum mutants able to accept foreign DNA and transfer it to other strains. This enabled transposon mutagenesis of a wild-type antibiotic-producing strain of E. carotovora subsp. betavasculorum. Twelve antibiotic-negative mutants were isolated, and one of these showed a reduction in antibiotic production in vitro. Many of these mutants also showed a reduction in their ability to macerate potato tissue. The mutants were classified into four genetic groups on the basis of their genetic and phenotypic characteristics, indicating that several genes are involved in antibiotic biosynthesis by E. carotovora subsp. betavasculorum.
Population Size and Distribution of
Rhizobium leguminosarum
bv.
trifolii
in Relation to Total Soil Bacteria and Soil Depth
Authors: Bottomley Peter J. , Dughri Muktar H.
Published: January 2020

Bacterial cells small enough to pass through 0.4-μm-pore-size filters made up 5 to 9% of the indigenous bacterial population in 0- to 20-cm-depth samples of Abiqua silty clay loam. Within the same soil samples, cells of a similar dimension were stained with fluorescent antibodies specific to each of four antigenically distinct indigenous serogroups of
Rhizobium leguminosarum
bv.
trifolii
and made up 22 to 34% of the soil population of the four serogroups. Despite the extensive contribution of small cells to these soil populations, no evidence of their being capable of either growth or nodulation was obtained. The density of soil bacteria which could be cultured ranged between 0.5 and 8.5% of the >0.4-μm direct count regardless of media, season of sampling, or soil depth. In the same soil samples, the viable nodulating populations of biovar
trifolii
determined by the plant infection soil dilution technique ranged between 1 and 10% of the >0.4-μm direct-immunofluorescence count of biovar
trifolii.
The <0.4-μm cell populations of both total soil bacteria and biovar
trifolii
changed abruptly between the 10- to 15-cm and 15- to 20-cm soil depth increments, increasing from 5 to 20% and from 20 to 50%, respectively, of their direct-count totals. The increase in density of the small-cell population corresponded to a significant increase in soil bulk density (1.07 to 1.21 g cm
−3
). The percent contribution of the <0.4-μm direct count to individual serogroup totals increased with soil depth by approximately 2-fold (39 to 87%) for serogroups 17 and 21 and by 12-fold (6 to 75%) for serogroups 6 and 36.
Cloning of DNA sequences encoding foreign peptides and their expression in the K88 pili
Authors: Thiry G , Clippe A , Scarcez T , Petre J
Published: January 2020

A genetic system that allows the cloning of a peptide-coding sequence in the Escherichia coli K88ac and K88ad pilin genes and their expression as recombinant pili has been constructed. Two insertion vectors were created by subcloning the pilin genes in a pBR322 plasmid and replacing the coding sequence of two nonconserved clusters by a linker. The K88ac helper genes were subcloned in the compatible pACYC184 plasmid, and expression of pili by bacteria carrying both plasmids occurred by complementation. Two peptide-coding sequences of the influenza hemagglutinin were cloned in both insertion vectors, and recombinant pilins were shown to be assembled in pili. One recombinant pilus was shown to elicit antibodies against the synthetic peptide in immunized rats. The somatostatin-coding sequence was cloned in both vectors and led in one case to detectable pilus production. The fused somatostatin was shown to be recognized by specific monoclonal and polyclonal antibodies.
Levels of bacteria, fungi, and endotoxin in bulk and aerosolized corn silage
Authors: Dutkiewicz J , Olenchock S A , Sorenson W G , Gerencser V F , May J J , Pratt D S , Robinson V A
Published: January 2020

Three samples of silage taken from the surface of a silo and from depths of 20 and 45 cm in the silo were studied for identification of the potential agents causing symptoms of organic dust toxic syndrome. The samples were examined by dilution plating before and after aerosolization in an acoustical dust generator. Aerosol samples were collected by liquid impinger and filter cassettes. The samples were examined for total aerobic bacteria, anaerobic bacteria, gram-negative bacteria, lactobacilli, listeriae, thermophilic actinomycetes, fungi, and endotoxin. Very high levels of total aerobic bacteria and fungi were found in the surface sample (up to 10(9) CFU/g in the bulk sample and up to 10(9) CFU/m3 after aerosolization), whereas the corresponding values from the deepest site were 100 to 50,000 times lower. Aspergillus fumigatus predominated among the fungi, whereas Bacillus and gram-negative organisms (Pseudomonas, Alcaligenes, Citrobacter, and Klebsiella species) prevailed among bacteria. Thermophilic actinomycetes occurred in numbers up to 10(7) CFU/g in the bulk samples, whereas anaerobic bacteria, lactobacilli, and listeriae were only few or absent. The concentration of endotoxin was high in the surface sample (up to 211.4 Endotoxin Units/mg) and about 200-fold lower in the sample from the deepest site. The results show that contact with dust from the surface of silage carries the risk of exposure to high concentrations of microorganisms, of which A. fumigatus and endotoxin-producing bacteria are the most probable disease agents.
Evidence for colonization and destruction of hinge ligaments in cultured juvenile Pacific oysters (Crassostrea gigas) by cytophaga-like bacteria
Authors: Dungan C F , Elston R A , Schiewe M H
Published: January 2020

Several strains of cytophaga-like gliding bacteria (CLB) were isolated as numerically dominant or codominant components of bacterial populations associated with proteinaceous hinge ligaments of cultured juvenile Pacific oysters, Crassostrea gigas. These bacteria were morphologically similar to long, flexible bacilli occurring within degenerative lesions in oyster hinge ligaments. Among bacteria isolated from hinge ligaments, only CLB strains were capable of sustained growth with hinge ligament matrix as the sole source of organic carbon and nitrogen. In vitro incubation of cuboidal portions of ligament resilium with ligament CLB resulted in bacterial proliferation on the surfaces and penetration deep into ligament matrices. Bacterial proliferation was accompanied by loss of resilium structural and mechanical integrity, including complete liquefaction, at incubation temperatures between 10 and 20 degrees C. The morphological, distributional, and degradative characteristics of CLB isolated from oyster hinge ligaments provide compelling, albeit indirect, evidence that CLB are the agents of a degenerative disease affecting juvenile cultured oysters. The motility, metabolic, and hydrolytic characteristics of hinge ligament CLB and the low moles percent G + C values (32.4 to 32.9) determined for three representative strains indicate that they are marine Cytophaga spp.
Viability of Acanthamoeba cysts in ophthalmic solutions
Authors: Brandt F H , Ware D A , Visvesvara G S
Published: January 2020

Acanthamoeba keratitis is a chronic infection of the human cornea. Many people who have this infection wear soft contact lenses. Usually lens wearers clean and maintain their lenses with various ophthalmic solutions including homemade saline. Recently it has been shown that homemade saline solutions play a role in lens contamination and thus in Acanthamoeba keratitis. We therefore evaluated the viability of cysts of three species of Acanthamoeba by exposing them for various time periods to saline, cleaning, and disinfectant solutions generally used to care for these lenses. We found that the viability of the cysts in saline solutions ranged from a minimum of 14 days to 90 days of exposure. In cleaning solutions, the survival times ranged from a minimum of 1 day to 90 days of exposure. Disinfectants, as expected, were the most effective of all tested solutions in killing Acanthamoeba cysts. The survival times ranged from 6 h to 14 days. None of these products were effective in destroying Acanthamoeba cysts in less than 6 h of exposure, which exceeds the suggested time that any given solution should be used for lens care.
Kinetics of ethanolic fermentation of D-xylose by Klebsiella pneumoniae and its mutants
Authors: Banerjee M
Published: January 2020

The microbial production of ethanol from D-xylose by a new soil isolate of Klebsiella pneumoniae and the mutants K. pneumoniae MB-16 and MB-16-1048 was studied. Kinetic and physiological properties of the mutants were compared with those of the original isolate. The volumetric rates of ethanol formation by mutants MB-16-1048 and MB-16 and the original isolate were 1.58, 0.50, and 0.06 g liter-1 h-1, respectively. The cultivation times of mutants MB-16-1048 and MB-16 were 20 and 18 h, respectively, and that of the original isolate was 118 h. Both the mutants exhibited metabolic similarities with the original isolate. Ethanol was the major end product of fermentation in all three strains. Acetic acid and carbon dioxide were the other two important by-products of fermentation. Pyruvic acid was accumulated in significant proportions as an intermediate. The proportion of pyruvate in the original isolate was 54% of the total D-xylose utilized, whereas for MB-16 and MB-16-1048 the values were about 42 and 22%, respectively. The lower fractions of pyruvate in mutants MB-16 and MB-16-1048 showed up as a 41 and 82% improvement, respectively, over the original isolate in terms of the ethanol yield.
C-ring cleavage of flavonoids by human intestinal bacteria
Authors: Winter J , Moore L H , Dowell V R , Bokkenheuser V D
Published: January 2020

Four hitherto undescribed Clostridium strains capable of cleaving the C ring of quercetin, kaempferol, and naringenin at C-3-C-4 were isolated from the fecal flora of humans. None of the strains cleaved catechin. C-ring fission occurred when the substrate was either in solution or in suspension. Mixed cultures of flavonoid-hydrolyzing bacteria, flavonoid-cleaving bacteria, and Escherichia coli, which was used to provide the anaerobic environment, rapidly metabolized rutin to 3,4-dihydroxyphenylacetic acid, indicating that the intestinal half-life of the biologically active aglycone is short. The cleaving strains shared many phenotypic characteristics, including their inability to ferment sugars, but they differed sufficiently to indicate that they represent different species.