bluuebunny/crossref_metadata_2025 huggingface dataset:
Purification and Properties of Catechol 1,2-Dioxygenase from
Rhizobium leguminosarum
biovar
viceae
USDA 2370
Authors: Chen Yung Pin , Lovell Charles R.
Published: January 2020

The catechol 1,2-dioxygenase of
Rhizobium leguminosarum
biovar
viceae
USDA 2370 was purified 296-fold, yielding a homogeneous preparation with a specific activity of 51.1 U mg of protein
-1
. The molecular weight of the native protein was 70,000, with two identical subunits of 34,500 and 1 g-atom of iron per mol. The optimum pH for catalytic activity was 9.0 to 9.5.
Characterization of sodium dodecyl sulfate-resistant proteolytic activity in the hyperthermophilic archaebacterium Pyrococcus furiosus
Authors: Blumentals I I , Robinson A S , Kelly R M
Published: January 2020

Cell extracts from Pyrococcus furiosus were found to contain five proteases, two of which (S66 and S102) are resistant to sodium dodecyl sulfate (SDS) denaturation. Cell extracts incubated at 98 degrees C in the presence of 1% SDS for 24 h exhibited substantial cellular proteolysis such that only four proteins could be visualized by amido black-Coomassie brilliant blue staining of SDS-polyacrylamide gels. The SDS-treated extract retained 19% of the initial proteolytic activity as represented by two proteases, S66 (66 kilodaltons [kDa]) and S102 (102 kDa). Immunoblot analysis with guinea pig sera containing antibodies against protease S66 indicated that S66 is related neither to S102 nor to the other proteases. The results of this analysis also suggest that S66 might be the hydrolysis product of a 200-kDa precursor which does not have proteolytic activity. The 24-h SDS-treated extract showed unusually thermostable proteolytic activity; the measured half-life at 98 degrees C was found to be 33 h. Proteases S66 and S102 were also resistant to denaturation by 8 M urea, 80 mM dithiothreitol, and 5% beta-mercaptoethanol. Purified protease S66 was inhibited by phenylmethylsulfonyl fluoride and diisopropyl fluorophosphate but not by EDTA, ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, or iodoacetic acid. These results indicate that S66 is a serine protease. Amino acid ester hydrolysis studies showed that protease S66 was hydrolytically active towards N-benzoyl-L-arginine ethyl ester.
Isolation of a replication region of a large lactococcal plasmid and use in cloning of a nisin resistance determinant
Authors: von Wright A , Wessels S , Tynkkynen S , Saarela M
Published: January 2020

The replication region of a 28-kilobase-pair (kbp) cryptic plasmid from Lactococcus lactis subsp. lactis biovar diacetylactis SSD207 was cloned in L. lactis subsp. lactis MG1614 by using the chloramphenicol resistance gene from the streptococcal plasmid pGB301 as a selectable marker. The resulting 8.1-kbp plasmid, designated pVS34, was characterized further with respect to host range, potential cloning sites, and location of replication gene(s). In addition to lactococci, pVS34 transformed Lactobacillus plantarum and, at a very low frequency, Staphylococcus aureus but not Escherichia coli or Bacillus subtilis. The 4.1-kbp ClaI fragment representing lactococcal DNA in pVS34 contained unique restriction sites for HindIII, EcoRI, XhoII, and HpaII, of which the last three could be used for molecular cloning. A region necessary for replication was located within a 2.5-kbp fragment flanked by the EcoRI and ClaI restriction sites. A 3.8-kbp EcoRI fragment derived from a nisin resistance plasmid, pSF01, was cloned into the EcoRI site of pVS34 to obtain a nisin-chloramphenicol double-resistance plasmid, pVS39. From this plasmid, the streptococcal chloramphenicol resistance region was subsequently eliminated. The resulting plasmid, pVS40, contains only lactococcal DNA. Potential uses for this type of a nisin resistance plasmid are discussed.
Starvation-specific formation of a peripheral exopolysaccharide by a marine Pseudomonas sp., strain S9
Authors: Wrangstadh M , Szewzyk U , Ostling J , Kjelleberg S
Published: January 2020

The marine bacterium Pseudomonas sp. strain S9 produces exopolysaccharides (EPS) during both growth and total energy source and nutrient starvation. Transmission electron microscopy of immunogold-labeled cells demonstrated that the EPS is closely associated with the cell surface during growth (integral EPS), while both the integral form and a loosely associated extracellular (peripheral) form were observed during starvation. Formation and release of the latter rendered the starvation medium viscous. In addition, after 3 h of starvation in static conditions, less than 5% of the cells were motile, compared with 100% at the onset of starvation and approximately 80% subsequent to release of the peripheral EPS at 27 h of starvation. Inhibition of protein synthesis with chloramphenicol added before 3 h of starvation caused no increase in viscosity. However, addition of chloramphenicol at 3 h did not prevent the subsequent increase in viscosity displayed by S9 cells. The amount of integral EPS increased for both nontreated and chloramphenicol-treated S9 cells during the first hour of starvation, with a subsequent equal decrease. The chloramphenicol-treated cells, as well as cells of a transposon-generated mutant strain deficient in peripheral EPS formation, remained adhesive to a hydrophobic inanimate surface during the initial 5 h of starvation, whereas nontreated wild-type cells had progressively decreased adhesion capacity. During the initial 5 h of starvation, most of the nontreated cells but only a small fraction of the chloramphenicol-treated and mutant cells detached from the hydrophobic substratum.(ABSTRACT TRUNCATED AT 250 WORDS)
Production of poly-(beta-hydroxybutyric-co-beta-hydroxyvaleric) acids
Authors: Ramsay B A , Lomaliza K , Chavarie C , Dubé B , Bataille P , Ramsay J A
Published: January 2020

Alcaligenes latus, Alcaligenes eutrophus, Bacillus cereus, Pseudomonas pseudoflava, Pseudomonas cepacia, and Micrococcus halodenitrificans were found to accumulate poly-(beta-hydroxybutyric-co-beta-hydroxyvaleric) acid [P(HB-co-HV)] copolymer when supplied with glucose (or sucrose in the case of A. latus) and propionic acid under nitrogen-limited conditions. A fed-batch culture of A. eutrophus produced 24 g of poly-beta-hydroxybutyric acid (PHB) liter-1 under ammonium limitation conditions. When the glucose feed was replaced with glucose and propionic acid during the polymer accumulation phase, 17 g of P(HB-co-HV) liter-1 was produced. The P(HB-co-HV) contained 5.0 mol% beta-hydroxyvaleric acid (HV). Varying the carbon-to-nitrogen ratio at a dilution rate of 0.15 h-1 in a chemostat culture of A. eutrophus resulted in a maximum value of 33% (wt/wt) PHB in the biomass. In comparison, A. latus accumulated about 40% (wt/wt) PHB in chemostat culture under nitrogen-limited conditions at the same dilution rate. When propionic acid was added to the first stage of a two-stage chemostat, A. latus produced 43% (wt/wt) P(HB-co-HV) containing 18.5 mol% HV. In the second stage, the P(HB-co-HV) increased to 58% (wt/wt) with an HV content of 11 mol% without further addition of carbon substrate. The HV composition in P(HB-co-HV) was controlled by regulating the concentration of propionic acid in the feed. Poly-beta-hydroxyalkanoates containing a higher percentage of HV were produced when pentanoic acid replaced propionic acid.
Cloning of the citrate permease gene of Lactococcus lactis subsp. lactis biovar diacetylactis and expression in Escherichia coli
Authors: Sesma F , Gardiol D , de Ruiz Holgado A P , de Mendoza D
Published: January 2020

The citrate plasmid (Cit+ plasmid) from Lactococcus lactis subsp. lactis biovar diacetylactis was cloned into the EcoRI site of plasmid pUC18. This recombinant plasmid enabled Escherichia coli K-12 to transport and utilize citrate as a source of energy, indicating expression of the citrate permease from L. lactis biovar diacetylactis. The citrate permease was under the control of the lac promoter of pUC18. Genetic expression of the Cit+ plasmid in maxicells revealed that the plasmid encoded two polypeptides of 47 and 32 kilodaltons, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Maintenance of plasmids pBR322 and pUC8 in nonculturable Escherichia coli in the marine environment
Authors: Byrd J J , Colwell R R
Published: January 2020

Maintenance of plasmids pBR322 and pUC8 in Escherichia coli that was nonculturable after exposure to seawater was studied. E. coli JM83 and JM101, which contained plasmids pBR322 and pUC8, respectively, were placed in sterile artificial seawater for 21 days. Culturability was determined by plating on both nonselective and selective agar, and plasmid maintenance was monitored by direct isolation of plasmid nucleic acid from bacteria collected on Sterivex filters. E. coli JM83 became nonculturable after incubation for 6 days in seawater yet maintained plasmid pBR322 for the entire period of the study, i.e., 21 days. E. coli JM101 was nonculturable after incubation in seawater for 21 days and also maintained plasmid pUC8 throughout the duration of the microcosm experiment. Direct counts of bacterial cells did not change significantly during exposure to seawater, even though plate counts yielded no viable (i.e., platable) cells. We concluded that E. coli cells are capable of maintaining high-copy-number plasmids, even when no longer culturable, after exposure to the estuarine or marine environment.
Acid Tolerance of
Leuconostoc mesenteroides
and
Lactobacillus plantarum
Authors: McDonald L. C. , Fleming H. P. , Hassan H. M.
Published: January 2020

In this study, we determined the internal cellular pH response of
Leuconostoc mesenteroides
and
Lactobacillus plantarum
to the external pH created by the microorganisms themselves or by lactic or acetic acids and their salts added to the growth medium. Growth of
Leuconostoc mesenteroides
stopped when its internal pH reached 5.4 to 5.7, and growth of
L. plantarum
stopped when its internal pH reached 4.6 to 4.8. Variation in growth medium composition or pH did not alter the growth-limiting internal pH reached by these microorganisms.
L. plantarum
maintained its pH gradient in the presence of either 160 mM sodium acetate or sodium lactate down to an external pH of 3.0 with either acid. In contrast, the ΔpH of
Leuconostoc mesenteroides
was zero at pH 4.0 with acetate and 5.0 with lactate. No differences were found between
d
-(−)- and
l
-(+)-lactic acid for the limiting internal pH for growth of either microorganism. The comparatively low growth-limiting internal pH and ability to maintain a pH gradient at high organic acid concentration may contribute to the ability of
L. plantarum
to terminate vegetable fermentations.
Fluoroacetate-metabolizing pseudomonad isolated from Dichapetalum cymosum
Authors: Meyer J J , Grobbelaar N , Steyn P L
Published: January 2020

A pseudomonad was isolated from the fluoroacetate-producing plant Dichapetalum cymosum (Hook) Engl. and identified as Pseudomonas cepacia. We established that this isolate was capable of growing in fluoroacetate-enriched solutions without any reduction in growth rate. Our isolate of P. cepacia was capable of defluorinating 2.69 mg of fluoroacetate per 10(9) cells per h. Fluoroacetate was degraded to CO2 at a rate of 23.53 ng/10(9) cells per h.
Thymidylate synthase gene from Lactococcus lactis as a genetic marker: an alternative to antibiotic resistance genes
Authors: Ross P , O'Gara F , Condon S
Published: January 2020

The potential of the thymidylate synthase thyA gene cloned from Lactococcus lactis subsp. lactis as a possible alternative selectable marker gene to antibiotic resistance markers has been examined. The thyA mutation is a recessive lethal one; thyA mutants cannot survive in environments containing low amounts of thymidine or thymine (such as Luria-Bertani medium) unless complemented by the thyA gene. The cloned thyA gene was strongly expressed in L. lactis subsp. lactis, Escherichia coli, Rhizobium meliloti, and a fluorescent Pseudomonas strain. In addition, when fused to a promoterless enteric lac operon, the thyA gene drove expression of the lac genes in a number of gram-negative bacteria. In transformation experiments with thyA mutants of E. coli and conjugation experiments with thyA mutants of R. meliloti, the lactococcal thyA gene permitted selection of transformants and transconjugants with the same efficiency as did genes for resistance to ampicillin, chloramphenicol, or tetracycline. Starting from the broad-host-range plasmid pGD500, a plasmid, designated pPR602, was constructed which is completely free of antibiotic resistance genes and has the lactococcal thyA gene fused to a promoterless lac operon. This plasmid will permit growth of thyA mutant strains in the absence of thymidine or thymine and has a number of unique restriction sites which can be used for cloning.
Transport and deamination of amino acids by a gram-positive, monensin-sensitive ruminal bacterium
Authors: Chen G , Russell J B
Published: January 2020

Strain F, a recently isolated ruminal bacterium, grew rapidly with glutamate or glutamine as an energy source in the presence but not the absence of Na. Monensin, a Na+/H+ antiporter, completely inhibited bacterial growth and significantly reduced ammonia production (85%), but 3,3',4',5-tetrachlorosalicylanide (a protonophore) and valinomycin had little effect on growth or ammonia production. Dicyclohexylcarbodiimide, a H(+)-ATPase, inhibitor had no effect. The kinetics of glutamate and glutamine transport were biphasic, showing unusually high rates at high substrate concentrations. On the basis of low substrate concentrations (less than 100 microM), the Km values for glutamate and glutamine were 4 and 11 microM, respectively. Strain F had separate carriers for glutamate and glutamine which could be driven by a chemical gradient of Na. An artificial delta psi was unable to drive transport even when Na was present. The glutamate carrier had a single binding site for Na with a Km of 21 mM; the glutamine carrier appeared to have more than one binding site, and the Km was 2.8 mM. Neither carrier could use Li instead of Na. Histidine and serine were also rapidly transported by Na-dependent systems, but serine alone did not allow growth even when Na was present. Because exponentially growing cells at pH 6.9 had little delta psi (-3 mV) and a slightly reversed Z delta pH (+17 mV), it appeared that the membrane bioenergetics of strain F were solely dependent on Na circulation.
Fate of Ice Nucleation-Active
Pseudomonas syringae
Strains in Alpine Soils and Waters and in Synthetic Snow Samples
Authors: Goodnow R. A. , Harrison M. D. , Morris J. D. , Sweeting K. B. , Laduca R. J.
Published: January 2020

The stability of the ice nucleation activity (INA) and viability of INA
Pseudomonas syringae
31a, used as an ice nucleator in the manufacture of synthetic snow, was determined in snow. The viability of
P. syringae
1-2b, a rifampin-resistant mutant selected from strain 31a to improve recovery from test samples, was determined in laboratory tests of three alpine soil and water samples from three different sources. Snow samples were exposed to environmental conditions or held in darkness at −20°C. Samples of soil and water were maintained in darkness at 0, 7.5, or 15°C. Parent strain 31a INA decreased significantly (>99.0%) in snow exposed to sunlight and freeze-thaw, while the INA of the cell population in snow held in darkness at −20°C remained essentially unchanged. No viable strain 31a was detected in snow exposed to the environment after 7 days, while the viability of strain 31a in snow held in darkness at −20°C decreased to <3% of the original inoculation at the test conclusion. Mutant strain 1-2b viability was undetectable or had decreased significantly 19 days postinoculation in soil samples held at 0 or 15°C. In contrast, 1-2b viability remained detectable at low levels for the duration of the test in soils held at 7.5°C. The 1-2b population demonstrated a significantly longer half-life in peatlike soil than in the loam soils tested. The rate of decrease in 1-2b viability was essentially the same in the three alpine water samples tested with respect to water temperature and sample location.
Conditions for growing Mycoplasma canadense and Mycoplasma verecundum in a serum-free medium
Authors: Muñoz G , Sotomayor P
Published: January 2020

Mycoplasma canadense and Mycoplasma verecundum were cultured in a serum-free medium containing bovine serum albumin, cholesterol, oleic acid, and palmitic acid in order to avoid the addition of horse serum. Growth was detected by measurement of A640 and by colony formation. The level of growth attained in this medium was less than that obtained in the horse serum-supplemented media, but colonies retained their distinctive morphology.
Effect of
Methanobrevibacter smithii
on Xylanolytic Activity of Anaerobic Ruminal Fungi
Authors: Joblin K. N. , Naylor G. E. , Williams A. G.
Published: January 2020

Three different ruminal anaerobic fungi,
Neocallimastix frontalis
PNK2,
Sphaeromonas communis
B7, and
Piromonas communis
B19, were grown axenically or in coculture with
Methanobrevibacter smithii
on xylan.
N. frontalis
and
S. communis
in monoculture and coculture accumulated xylobiose, xylose, and arabinose in the growth medium; arabinose was not metabolized, but xylobiose and xylose were subsequently used. The transient accumulation of xylose was much less evident in cocultures. Both the rate and extent of xylan utilization were increased by coculturing, and metabolite profiles became acetogenic as a result of interspecies hydrogen transfer; more acetate and less lactate were formed, while formate and hydrogen did not accumulate. For each of the three fungi, there were marked increases in the specific activities of extracellular xylanase (up to fivefold), α-
l
-arabinofuranosidase (up to fivefold), and β-
d
-xylosidase (up to sevenfold) upon coculturing. The stimulating effect on fungal enzymes from coculturing with
M. smithii
was independent of the growth substrate, and the magnitude of the stimulation varied according to the enzymes and the incubation time. For an
N. frontalis-M. smithii
coculture, the positive stimulation was maintained during an extended (18-day) incubation period, and this affected not only hemicellulolytic enzymes but also polysaccharidase and glycoside hydrolase enzymes that were not involved in xylan breakdown. The specific activity of cell-bound endopeptidase was not increased under the coculture conditions used in this study. The higher enzyme activities in cocultures are discussed in relation to catabolite repression.
Colony formation by sublethally heat-injured Zygosaccharomyces rouxii as affected by solutes in the recovery medium and procedure for sterilizing medium
Authors: Golden D A , Beuchat L R
Published: January 2020

Recovery and colony formation by healthy and sublethally heat-injured cells of Zygosaccharomyces rouxii as influenced by the procedure for sterilizing recovery media (YM agar [YMA], wort agar, cornmeal agar, and oatmeal agar) were investigated. Media were supplemented with various concentrations of glucose, sucrose, glycerol, or sorbitol and sterilized by autoclaving (110 degrees C, 15 min) and by repeated treatment with steam (100 degrees C). An increase in sensitivity was observed when heat-injured cells were plated on glucose-supplemented YMA at an aw of 0.880 compared with aws of 0.933 and 0.998. Colonies which developed from unheated and heated cells on YMA at aws of 0.998 and 0.933 generally exceeded 0.5 mm in diameter within 3.5 to 4 days of incubation at 25 degrees C, whereas colonies formed on YMA at an aw of 0.880 typically did not exceed 0.5 mm in diameter until after 5.5 to 6.5 days of incubation. The number of colonies exceeding 0.5 mm in diameter which were formed by heat-injured cells on YMA at an aw of 0.880 was 2 to 3 logs less than the total number of colonies detected, i.e., on YMA at an aw of 0.933 and using no limits of exclusion based on colony diameter. A substantial portion of cells which survived heat treatment were sublethally injured as evidenced by increased sensitivity to a suboptimum aw (0.880). In no instance was recovery of Z. rouxii significantly affected by medium sterilization procedure when glucose or sorbitol was used as the aw-suppressing solute.(ABSTRACT TRUNCATED AT 250 WORDS)
Conjugal Transfer of Megaplasmid 2 between
Rhizobium meliloti
Strains in Alfalfa Nodules
Authors: Pretorius-Güth Inge-M. , Pühler Alfred , Simon Reinhard
Published: January 2020

A DNA fragment containing the RP4
mob
function, as well as the gentamicin and spectinomycin resistance genes, was inserted by gene replacement onto the megaplasmid 2 (pM2) of
Rhizobium meliloti
0540 (Inf
−
EPS
−
), resulting in PG101 (Inf
−
EPS
−
). The self-transfer of pM2 and the mobilization of pM2 by plasmid RP4-4 were investigated during conjugation between PG101 and
R. meliloti
2526 (Nod
−
). In filter conjugations, pM2 was readily mobilized by RP4-4. In addition to this, the self-transfer of one megaplasmid (pM) was detected at a frequency of 3 × 10
−7
. Bacteria isolated from the nodules of alfalfa and coinoculated with strains PG101 and 2526 showed that pM2 was mobilized at a frequency of approximately 7 × 10
−5
. Bacterial cell numbers were too low in the nodules for detection of the self-transfer of pM2 to occur. No pM2 transfer was detected in the inoculum. A comparison of the transfer frequencies for the various conjugation conditions revealed that pM2 transfer occurred as frequently in the nodules as in filter conjugations. These results indicate that the nodule creates conditions for gene transfer that are comparable to optimal laboratory conditions.
Catabolite Inactivation in the Methylotrophic Yeast
Pichia pastoris
Authors: Murray William D. , Duff Sheldon J. B. , Beveridge Terry J.
Published: January 2020

Inactivation of the alcohol oxidase enzyme system of
Pichia pastoris
, during the whole-cell bioconversion of ethanol to acetaldehyde, was due to catabolite inactivation. Electron microscopy showed that methanol-grown cells contained peroxisomes but were devoid of these microbodies after the bioconversion. Acetaldehyde in the presence of O
2
was the effector of catabolite inactivation. The process was initiated by the appearance of free acetaldehyde, and was characterized by an increase in the level of cyclic AMP, that coincided with a rapid 55% drop in alcohol oxidase activity. Further enzyme inactivation, believed to be due to proteolytic degradation, then proceeded at a constant but slower rate and was complete 21 h after acetaldehyde appearance. The rate of catabolite inactivation was dependent on acetaldehyde concentration up to 0.14 mM. It was temperature dependent and occurred within 24 h at 37°C and by 6 days at 15°C but not at 3°C. Alcohol oxidase activity was psychrotolerant, with only a 17% decrease in initial specific activity over a temperature drop from 37 to 3°C. In contrast, protease activity was inhibited at temperatures below 15°C. When the bioconversion was run at 3°C, catabolite inactivation was prevented. In the presence of 3 M Tris hydrochloride buffer, 123 g of acetaldehyde per liter was produced at 3°C, compared with 58 g/liter at 30°C. By using 0.5 M Tris in a cyclic-batch procedure, 140.6 g of acetaldehyde was produced.
Quantitative Microbiological Analysis of Bacterial Community Shifts in a High-Rate Anaerobic Bioreactor Treating Sulfite Evaporator Condensate
Authors: Ney U. , Macario A. J. L. , de Macario E. Conway , Aivasidis A. , Schoberth S. M. , Sahm H.
Published: January 2020

The bacterial population of a high-rate, anaerobic, fixed-bed loop reactor treating sulfite evaporator condensate from the pulp industry was studied over a 14-month period. This period was divided into seven cycles that included a startup at the beginning of each cycle. Some 82% of the total biomass was immobilized on and between the porous glass rings filling the reactor. The range of the total number of microorganisms in these biofilms was 2 × 10
9
to 7 × 10
9
cells per ml. Enumeration and characterization by microbiological methods and by phase-contrast, epifluorescence, and electron microscopy showed that the samples consisted mainly of the following methanogens: a
Methanobacterium
sp., a
Methanosarcina
sp., a
Methanobrevibacter
sp., and a
Methanothrix
sp., as well as furfural-degrading sulfate-reducing bacteria resembling
Desulfovibrio furfuralis.
Viable counts of hydrogenotrophic methanogens were relatively stable (mostly within the range of 3.2 × 10
8
to 7.5 × 10
8
cells per ml), but
Methanobrevibacter
cells increased from <5 to 30% of the total hydrogenotrophic count after transfer of the fixed bed into a second reactor vessel. Acetotrophic methanogens reached their highest numbers of 1.3 × 10
8
to 2.6 × 10
8
cells per ml in the last fermentation cycles. They showed a morphological shift from sarcinalike packets in early samples to single coccoid forms in later phases of the fermentation. Furfural-degrading sulfate reducers reached counts of 1 × 10
7
to 5.8 × 10
7
cells per ml. The distribution of the chief metabolic groups between free fluid and biofilms was analyzed in the fifth fermentation cycle: 4.5 times more furfural degraders were found in the free fluid than in the biofilms. In contrast, 5.8 times more acetotrophic and 16.6 times more hydrogenotrophic methanogens were found in the biofilms than in the free liquid. The data concerning time shifts of morphotypes among the trophic groups of methanogens corroborated the trends observed by using immunological assays on the same samples.
Transfer of Isolated Nuclei into Protoplasts of
Trichoderma harzianum
Authors: Sivan A. , Harman G. E. , Stasz T. E.
Published: January 2020

Protoplasts released from young hyphae of
Trichoderma harzianum
contained 0 to 10 nuclei per protoplast, and most (about 80%) contained from 4 to 6 nuclei. Most protoplasts were larger than 3 μm in diameter. Nuclei were isolated from protoplasts of an auxotrophic mutant of
T. harzianum
and transferred into protoplasts obtained from another auxotroph of the same strain. This intrastrain nuclear transfer gave rise to numerous progeny which were stable, prototrophic, and heterokaryotic. Interstrain transfers in which nuclei from a wild-type prototroph of one strain were transferred into protoplasts from a lysine-deficient auxotroph of a second strain were also done. Heterokaryotic progeny were recovered from these interstrain transfers when the regenerating protoplasts were provided with a low concentration of lysine 48 h after the initial plating. Heterokaryotic progeny contained 11 to 17% of donor-type nuclei. Progeny homokaryotic for donor-type nuclei were obtained as single-spore isolates. These homokaryotic isolates expressed the isozyme pattern and colony morphology phenotype of the nuclear donor. When regenerating protoplasts were provided with lysine 10 days after the initial plating, only a single progeny was obtained. However, single-spore subprogeny of this nuclear transfer were prototrophic and exhibited a wide range of unstable morphological phenotypes.
Iron-binding compounds and related outer membrane proteins in Vibrio cholerae non-O1 strains from aquatic environments
Authors: Amaro C , Aznar R , Alcaide E , Lemos M L
Published: January 2020

A total of 156 strains of Vibrio cholerae non-O1 from aquatic origins were examined for the presence of iron uptake mechanisms and compared with O1 strains and other Vibrio species. All non-O1 strains were able to grow in iron-limiting conditions, with MICs of ethylenediaminedi (O-hydroxyphenylacetic acid) ranging from 20 microM to 2 mM. The production of siderophores was demonstrated by growth in chrome azurol S agar and cross-feeding assays. All strains produced phenolate-type compounds, as assessed by the chemical tests and by bioassays with Salmonella typhimurium enb-7. Some of the strains also promoted the growth of S. typhimurium enb-1 (which can use only enterobactin as a siderophore) as well as some strains of Vibrio anguillarum deficient in the anguibactin-mediated system. The chromatographic analyses and absorption spectra of siderophores extracted from culture supernatants suggest that vibriobactin may be produced by the strains examined. Interestingly, some strains also produced hydroxamate-type compounds, as determined by chemical tests, and were able to promote the growth of an aerobactin-deficient strain of Escherichia coli. These results were confirmed by the absorption spectra and chromatographic analyses of the culture extracts. The synthesis of iron-regulated outer membrane proteins in representative strains was also examined. The molecular sizes of the main induced proteins ranged from 70 to 78 kilodaltons. These results indicate that several iron uptake mechanisms which could be involved in environmental survival and pathogenicity are present in environmental V. cholerae non-O1 strains.
Tetrachloroethene transformation to trichloroethene and cis-1,2-dichloroethene by sulfate-reducing enrichment cultures
Authors: Bagley D M , Gossett J M
Published: January 2020

Tetrachloroethene, also known as perchloroethylene, was reductively dechlorinated to trichloroethene and cis-1,2-dichloroethene by laboratory sulfate-reducing enrichment cultures. The causative organism or group was not identified. However, tetrachloroethene was dechlorinated to trichloroethene in 50 mM bromoethane-sulfonate-inhibited enrichments and to trichloroethene and cis-1,2-dichloroethene in 3 mM fluoroacetate-inhibited enrichments. Overall transformation varied from 92% tetrachloroethene removal in 13 days to 22% removal in 65 days, depending on conditions of the inoculum, inhibitor used, and auxilliary substrate used. Neither lactate, acetate, methanol, isobutyric acid, valeric acid, isovaleric acid, hexanoic acid, succinic acid, nor hydrogen appeared directly to support tetrachloroethene dechlorination, although lactate-fed inocula demonstrated longer-term dechlorinating capability.
Effectiveness of Different
Frankia
Cell Types as Inocula for the Actinorhizal Plant
Casuarina
Authors: Burleigh Stephen , Torrey John G.
Published: January 2020

The soil bacterium
Frankia
of the
Actinomycetales
, capable of forming N
2
-fixing symbiotic root nodules on a diverse array of actinorhizal plants, has several morphological forms when grown in pure culture. Fresh hydrated preparations of whole cells, hyphae, and spores were all infective on seedlings of
Casuarina
at different dilutions. Desiccated hyphae showed no infection capacity, while desiccated spores remained infective, although at a reduced level. On the basis of most-probable-number statistics, spore suspensions were 3 orders of magnitude more infective than hyphae.
Bacterial Accumulation of Metallic Compounds
Authors: Sakurai I. , Kawamura Y. , Koike H. , Inoue Y. , Kosako Y. , Nakase T. , Kondou Y. , Sakurai S.
Published: January 2020

Xanthomonas maltophilia
accumulated metals or metallic compounds within the cell in the form of one or a few particles, in a peculiar condition similar to that of an electrolyte solution in an electric cell. The particles consisted of many little granules, most of which were assumed to be composed mainly of a compound of zinc and phosphorus in an amorphous state.
Occurrence of an Affinity Site apart from the Active Site on the Raw-Starch-Digesting but Non-Raw-Starch-Adsorbable
Bacillus subtilis
65 α-Amylase
Authors: Hayashida Shinsaku , Teramoto Yuji , Inoue Takehiro , Mitsuiki Shinji
Published: January 2020

α-Cyclodextrin specifically inhibited raw starch digestion by
Bacillus subtilis
65 α-amylase. The raw starch digestibility and α-cyclodextrin-Sepharose 6B adsorbability of this α-amylase were simultaneously lost when the specific domain corresponding to the affinity site essential for raw starch digestion was deleted by proteolysis. Occurrence of the affinity site on raw-starch-digesting enzymes was proven also with bacterial amylase.
Inactivation of human immunodeficiency virus type 1 in blood samples stored as high-salt lysates
Authors: Zolg J W , Lanciotti R S , Wendlinger M , Meyer W A
Published: January 2020

Blood samples to be tested for the presence of parasite DNA by using specific DNA probes are routinely stored in our laboratory as high-salt lysates (HSL). To safeguard against the risk of accidental infection with etiological agents such as the human immunodeficiency virus type 1 (HIV-1) while manipulating large numbers of blood samples in preparation for DNA probing, we determined the residual infectivity of HIV-1 after exposure to HSL components. Both high-titer virus stocks or provirus-carrying cells, suspended either in tissue culture medium or freshly drawn blood, were completely inactivated upon contact with the HSL components. This was verified by the absence of any detectable HIV-1-specific antigen in the supernatants of long-term cultures and the absence of virus-specific DNA fragments after amplification by polymerase chain reaction with DNA from such cultures as target DNA. These results support the conclusion that the virus is in fact completely inactivated by contact with the HSL components, rendering blood specimens stored as HSL noninfectious in regard to HIV-1.
Inducible overexpression of the FUM1 gene in Saccharomyces cerevisiae: localization of fumarase and efficient fumaric acid bioconversion to L-malic acid
Authors: Peleg Y , Rokem J S , Goldberg I , Pines O
Published: January 2020

Cloning of the Saccharomyces cerevisiae FUM1 gene downstream of the strong GAL10 promoter resulted in inducible overexpression of fumarase in the yeast. The overproducing strain exhibited efficient bioconversion of fumaric acid to L-malic acid with an apparent conversion value of 88% and a conversion rate of 80.4 mmol of fumaric acid/h per g of cell wet weight, both of which are much higher than parameters known for industrial bacterial strains. The only product of the conversion reaction was L-malic acid, which was essentially free of the unwanted by-product succinic acid. The GAL10 promoter situated upstream of a promoterless FUM1 gene led to production and correct distribution of the two fumarase isoenzyme activities between cytosolic and mitochondrial subcellular fractions. The amino-terminal sequence of fumarase contains the mitochondrial signal sequence since (i) 92 of 463 amino acid residues from the amino terminus of fumarase are sufficient to localize fumarase-lacZ fusions to mitochondria and (ii) fumarase and fumarase-lacZ fusions lacking the amino-terminal sequence are localized exclusively in the cytosol. The possibility that both mitochondrial and cytosolic fumarases are derived from the same initial translation product is discussed.
Genetic relatedness of Legionella longbeachae isolates from human and environmental sources in Australia
Authors: Lanser J A , Adams M , Doyle R , Sangster N , Steele T W
Published: January 2020

The genetic relatedness of Legionella longbeachae isolated in Australia since 1987 was investigated by restriction fragment length polymorphism (RFLP) analysis and allozyme electrophoresis. Three radiolabeled probes were used in Southern hybridizations for the RFLP studies. They were Escherichia coli 16S and 23S rRNA and cloned fragments of L. longbeachae selected empirically from genomal banks in lambda and a cosmid. The legionellae included in the study comprised 11 Legionella longbeachae serogroup 1 organisms isolated from humans, 28 L. longbeachae serogroup 1 isolates from environmental sources, and 3 L. longbeachae serogroup 2 environmental isolates. These were compared with the American Type Culture Collection reference strains of both serogroups and some other related Legionella species. Results of allozyme and RFLP analysis showed that all the isolates from humans and all but three of the environmental L. longbeachae serogroup 1 isolates were closely related. They were also closely related to L. longbeachae serogroup 1 ATCC 33462. There was wider variation among the three L. longbeachae serogroup 2 environmental isolates. One of these was closely related to L. longbeachae serogroup 2 ATCC 33484. RFLP studies with the rRNA probe provided the most discrimination among isolates but did not distinguish between the two serogroups.
Growth and Maintenance of
Thiobacillus ferrooxidans
Cells
Authors: Barron Jean LaCombe , Lueking Donald R.
Published: January 2020

A rapid and sensitive spectrophotometric procedure was developed for monitoring the growth of
Thiobacillus ferrooxidans
in liquid culture. Values determined for the optical densities at 500 nm of washed
T. ferrooxidans
cell suspensions were directly proportional to both total cell number and total cell protein concentration and provided an accurate measurement of culture growth rate. The utility of this procedure was demonstrated by conducting physiological studies on the influence of CO
2
and FeSO
4
availability on the growth of
T. ferrooxidans
. In addition, we describe a procedure for the long-term maintenance of cells
T. ferrooxidans
that ensures culture purity and genetic stability.
Transformation of
Rhodococcus fascians
by High-Voltage Electroporation and Development of
R. fascians
Cloning Vectors
Authors: Desomer Jan , Dhaese Patrick , Montagu Marc Van
Published: January 2020

The analysis of the virulence determinants of phytopathogenic
Rhodococcus fascians
has been hampered by the lack of a system for introducing exogenous DNA. We investigated the possibility of genetic transformation of
R. fascians
by high-voltage electroporation of intact bacterial cells in the presence of plasmid DNA. Electrotransformation in
R. fascians
D188 resulted in transformation frequencies ranging from 10
5
/μg of DNA to 10
7
/μg of DNA, depending on the DNA concentration. The effects of different electrical parameters and composition of electroporation medium on transformation efficiency are presented. By this transformation method, a cloning vector (pRF28) for
R. fascians
based on an indigenous 160-kilobase (chloramphenicol and cadmium resistance-encoding) plasmid pRF2 from strain NCPPB 1675 was developed. The origin of replication and the chloramphenicol resistance gene on pRF28 were used to construct cloning vectors that are capable of replication in
R. fascians
and
Escherichia coli.
The electroporation method presented was efficient enough to allow detection of the rare integration of replication-deficient pRF28 derivatives in the
R. fascians
D188 genome via either homologous or illegitimate recombination.
Potential of
Agrobacterium tumefaciens
and Octopine-Utilizing Fluorescent
Pseudomonas
Strains To Attach to Susceptible Potato Tissues
Authors: Chan James W. Y. F. , Ramey William D. , Moore Larry W. , Bell Colin R.
Published: January 2020

The binding characteristics of two octopine-catabolizing pseudomonads,
Pseudomonas fluorescens
B99A and E175D, which were isolated from crown galls, have been examined. The binding of strain B99A to potato disks was very weak, followed a Freundlich isotherm, and was temperature and pH independent. Strain E175D displayed strong attachment and followed a Langmuir isotherm. Despite these fundamental differences in binding characteristics, when each strain was placed in competitive binding assays with either
Agrobacterium tumefaciens
B6 or
A. tumefaciens
ATCC 15955, the number of bound pseudomonad cells decreased compared with those obtained in independent trials. Furthermore, the binding of
A. tumefaciens
cells was increased. In prebinding experiments, in which the potato disks were bound with the pseudomonads before exposure to the agrobacteria, the number of bound pseudomonad cells again decreased. This implies that increased desorption was occurring. In these prebinding studies, the numbers of bound
A. tumefaciens
ATCC 15955 increased, but the number of bound
A. tumefaciens
B6 remained the same. The mechanism for this observed synergism on the binding of agrobacterial cells and the depression in bound pseudomonad cells is believed to be alterations in the electrostatic or ionic charges on the plant and bacterial cell surfaces. The synergistic effect on
A. tumefaciens
undermines the use of these pseudomonads as potential biocontrol agents for crown gall.
Identification of lactococci and enterococci by colony hybridization with 23S rRNA-targeted oligonucleotide probes
Authors: Betzl D , Ludwig W , Schleifer K H
Published: January 2020

Specific sequences of 23S rRNA of Lactococcus lactis, Enterococcus faecalis, Enteroccus faecium, and Enterococcus malodoratus/Enterococcus avium were identified, and complementary oligonucleotide probes were synthesized. The specificity of the probes was evaluated by dot blot and colony hybridizations. The probes can be used for the specific detection and identification of colonies of the corresponding species in mixed cultures.
Selection of Astaxanthin-Overproducing Mutants of
Phaffia rhodozyma
with β-Ionone
Authors: Lewis M. J. , Ragot N. , Berlant M. C. , Miranda M.
Published: January 2020

β-Ionone, an end ring analog of β-carotene, inhibits astaxanthin production in the red yeast
Phaffia rhodozyma
. Astaxanthin-overproducing mutants of this yeast are easily spotted on β-ionone-containing yeast malt agar plates. β-Ionone appears to block astaxanthin synthesis at the β-carotene level.
Co-utilization of polymerized carbon sources by Bacteroides ovatus grown in a two-stage continuous culture system
Authors: MacFarlane G T , Gibson G R
Published: January 2020

Bacteroides ovatus NCTC 11153 was grown in a two-stage continuous culture system at various growth rates (vessel 1, D = 0.06 to 0.19 h-1; vessel 2, D = 0.03 to 0.09 h-1) on media containing mixtures of starch and arabinogalactan as carbon sources. The cell-associated enzyme activities needed to hydrolyze both substrates (amylase, arabinogalactanase, alpha-glucosidase, beta-galactosidase, and alpha-arabinofuranosidase) were variously influenced by growth rate and polysaccharide availability but were detected under all growth conditions tested. Measurements of residual carbohydrate in spent culture media showed that both polysaccharides were co-utilized during growth under putative C-limited conditions. The arabinogalactan was partly depolymerized in N-limited chemostats, and significant amounts of arabinose- and galactose-containing oligosaccharides accumulated in the cultures, indicating that starch was being preferentially utilized. Acetate, propionate, and succinate were the major fermentation products formed by C-limited bacteria, but under N limitation, lactate was also produced. Molar ratios of succinate increased concomitantly with the dilution rate in C-limited chemostats, whereas molar ratios of propionate decreased. During N-limited growth, however, decarboxylation of succinate to propionate was relatively independent of growth rate. Cell viability was higher in C-limited cultures compared with those grown under N limitation and was greatest at high dilution rates, irrespective of nutrient limitation.
Hydraulic Permeability of Immobilized Bacterial Cell Aggregates
Authors: Fowler Jeffrey D. , Robertson Channing R.
Published: January 2020

A dense aggregate of cells was retained in a reactor by a supported porous membrane. A continuous flow of nutrient medium was maintained through the cell aggregate and membrane. The hydraulic resistance of the cell aggregate was monitored throughout experiments with either growing or chemically cross-linked cells, under conditions of varying flow rates. Digital image analysis was used to characterize the sizes, separations, and orientations of several thousand individual cells in electron micrographs of chemically cross-linked cell aggregates. Two nonlinear phenomena were observed. First, the hydraulic resistance varied in direct relation to and reversibly with flow rate. Second, in constant flow-rate experiments the hydraulic resistance increased with time at a faster rate than could be attributed to cell growth. Both of these phenomena were dependent upon and could be explained by the ability of cells to move with respect to one another, under the influences of Brownian motion and of convection. Such relative motion could allow changes in net alignment of cells in the direction of flow and in the volume fraction of cells in the aggregate. This explanation is consistent with image analysis data. The observed sensitivity of hydraulic resistance to flow rate was inconsistent with a model that assumed elastic deformation of individual cells, and no evidence of cell deformation was found in electron micrographs.
Temperature Effects on Bacterial Leaching of Sulfide Minerals in Shake Flask Experiments
Authors: Ahonen Lasse , Tuovinen Olli H.
Published: January 2020

The microbiological leaching of a sulfide ore sample was investigated in shake flask experiments. The ore sample contained pyrite, pyrrhotite, pentlandite, sphalerite, and chalcopyrite as the main sulfide minerals. The tests were performed at eight different temperatures in the range of 4 to 37°C. The primary data were used for rate constant calculations, based on kinetic equations underlying two simplified models of leaching, i.e., a shrinking particle model and a shrinking core model. The rate constants thus derived were further used for the calculation of activation energy values for some of the sulfide minerals present in the ore sample. The chalcopyrite leaching rates were strongly influenced by the interaction of temperature, pH, and redox potential. Sphalerite leaching could be explained with the shrinking particle model. The data on pyrrhotite leaching displayed good fit with the shrinking core model. Pyrite leaching was found to agree with the shrinking particle model. Activation energies calculated from the rate of constants suggested that the rate-limiting steps were different for the sulfide minerals examined; they could be attributed to a chemical or biochemical reaction rather than to diffusion control.
Isolation of amoebae and Pseudomonas and Legionella spp. from eyewash stations
Authors: Paszko-Kolva C , Yamamoto H , Shahamat M , Sawyer T K , Morris G , Colwell R R
Published: January 2020

Forty eyewash units were sampled for protozoa, bacteria, and fungi. Total heterotrophic bacterial counts on nutrient agar and R2A agar (Difco Laboratories, Detroit, Mich.) ranged from 0 to 10(5) CFU/ml, with Pseudomonas spp. being the most frequently isolated. Total counts of 10(4) and 10(8) cells per ml were obtained with the acridine orange staining procedure. All samples were examined for Legionella spp. by direct fluorescent-antibody staining and by culturing on buffered charcoal-yeast extract agar containing alpha-ketoglutarate and glycine and supplemented with cycloheximide, vancomycin, and polymyxin B. DNA-DNA hybridization was used to confirm identification of the Legionella isolates. Legionellae were detected in 35 of 40 (87.5%) samples by direct fluorescent-antibody staining, with 3 samples yielding both Legionella spp. and amoebae. Amoebae identified as Hartmannella, Vahlkampfia, Acanthamoeba, and Cochliopodium spp. were detected in 19 of 40 (47:5%) samples. Sabouraud dextrose agar was used to obtain a crude estimate of viable fungal populations, pH, hardness, and ammonia, alkalinity, chlorine, copper, and iron contents were recorded for all water samples collected from eyewash stations; 33% of the samples had greater than or equal to 10 mg of CO2 per liter. It is concluded that eyewash stations not regularly flushed and/or cleaned and used to flush traumatized eye tissue may be a source of infection and can contaminate laboratory environments via aerosol transmission.