bluuebunny/crossref_metadata_2025 huggingface dataset:
Degradation of mono-, di-, and trihalogenated benzoic acids by Pseudomonas aeruginosa JB2
Authors: Hickey W J , Focht D D
Published: January 2020

Pseudomonas aeruginosa JB2 was isolated from a polychlorinated biphenyl-contaminated soil by enrichment culture containing 2-chlorobenzoate as the sole carbon source. Strain JB2 was subsequently found also to grow on 3-chlorobenzoate, 2,3- and 2,5-dichlorobenzoates, 2,3,5-trichlorobenzoate, and a wide range of other mono- and dihalogenated benzoic acids. Cometabolism of 2,4-dichlorobenzoate was also observed. Chlorocatechols were the central intermediates of all chlorobenzoate catabolic pathways. Degradation of 2-chlorobenzoate was routed through 3-chlorocatechol, whereas 4-chlorocatechol was identified from the metabolism of both 2,3- and 2,5-dichlorobenzoate. The initial attack on chlorobenzoates was oxygen dependent and most likely mediated by dioxygenases. Although plasmids were not detected in strain JB2, spontaneous mutants were detected in 70% of glycerol-grown colonies. The mutants were all of the following phenotype: benzoate+, 3-chlorobenzoate+, 2-chlorobenzoate-, 2,3-dichlorobenzoate-, 2,5-dichlorobenzoate-. While chlorocatechols were oxidized by the mutants at wild-type levels, oxidation of 2-chloro- and 2,3- and 2,5-dichlorobenzoates was substantially diminished. These findings suggested that strain JB2 possessed, in addition to the benzoate dioxygenase, a halobenzoate dioxygenase that was necessary for the degradation of chlorobenzoates substituted in the ortho position.
Characterization of Naturally Occurring Atrazine-Resistant Isolates of the Purple Non-Sulfur Bacteria
Authors: Brown Alfred E. , Luttrell Robin , Highfill Claudia T. , Rushing Ann E.
Published: January 2020

Six isolates of the purple non-sulfur bacteria, which upon primary isolation were naturally resistant to the herbicide atrazine, were characterized with respect to their taxonomic identity and the mechanism of their resistance. On the basis of electron microscopy, photopigment analysis, and other criteria, they were identified as strains of
Rhodopseudomonas acidophila, Rhodopseudomonas palustris
, or
Rhodocyclus gelatinosus
. These isolates exhibited degrees of atrazine resistance which ranged from 1.5 to about 4 times greater than that of cognate reference strains (American Type Culture Collection) tested. Furthermore, all of the reference strains tested were more intrinsically resistant to atrazine than was
Rhodobacter sphaeroides
. No unique plasmids which might encode for herbicide degradation or inactivation were found in these isolates. Resistance to the herbicide in these isolates was not the result of diminished binding of the herbicide to the L subunit of the bacterial reaction center. Differences in herbicide resistance among the various species of this group may be the result of compositional and chemical differences in the individual reaction centers. However, the increase in atrazine resistance for the isolates characterized in this study probably occurs by undefined mechanisms and not necessarily by changes in the binding of the herbicide to the L subunit of the photosynthetic reaction center.
Fuel alcohol production: effects of free amino nitrogen on fermentation of very-high-gravity wheat mashes
Authors: Thomas K C , Ingledew W M
Published: January 2020

Although wheat mashes contain only growth-limiting amounts of free amino nitrogen, fermentations by active dry yeast (Saccharomyces cerevisiae) were completed (all fermentable sugars consumed) in 8 days at 20 degrees C even when the mash contained 35 g of dissolved solids per 100 ml. Supplementing wheat mashes with yeast extract, Casamino Acids, or a single amino acid such as glutamic acid stimulated growth of the yeast and reduced the fermentation time. With 0.9% yeast extract as the supplement, the fermentation time was reduced from 8 to 3 days, and a final ethanol yield of 17.1% (vol/vol) was achieved. Free amino nitrogen derived in situ through the hydrolysis of wheat proteins by a protease could substitute for the exogenous nitrogen source. Studies indicated, however, that exogenously added glycine (although readily taken up by the yeast) reduced the cell yield and prolonged the fermentation time. The results suggested that there are qualitative differences among amino acids with regard to their suitability to serve as nitrogen sources for the growth of yeast. The complete utilization of carbohydrates in wheat mashes containing very little free amino nitrogen presumably resulted because they had the "right" kind of amino acids.
Characterization of the Opine-Utilizing Microflora Associated with Samples of Soil and Plants
Authors: Nautiyal C. S. , Dion P.
Published: January 2020

Microorganisms utilizing an opine as the sole carbon source were recovered from crown gall tumors, soil, and surface-disinfected potato tubers. The effect of the opines octopine, nopaline, succinamopine, and mannopine as selective substrates was compared with that of the auxin indoleacetic acid. Selection on octopine and indoleacetic acid favored the fluorescent pseudomonads, whereas mannopine allowed the frequent recovery of agrobacteria. Coryneforms which utilized succinamopine or mannopine were detected in soil, but not in tumors. Fungi growing on succinamopine or mannopine and a mannopine-utilizing
Pseudomonas putida
were isolated from tumor and soil, respectively.
Cloning and expression of the Clostridium thermosulfurogenes glucose isomerase gene in Escherichia coli and Bacillus subtilis
Authors: Lee C Y , Bhatnagar L , Saha B C , Lee Y E , Takagi M , Imanaka T , Bagdasarian M , Zeikus J G
Published: January 2020

The gene that encodes thermostable glucose isomerase in Clostridium thermosulfurogenes was cloned by complementation of glucose isomerase activity in a xylA mutant of Escherichia coli. A new assay method for thermostable glucose isomerase activity on agar plates, using a top agar mixture containing fructose, glucose oxidase, peroxidase, and benzidine, was developed. One positive clone, carrying plasmid pCGI38, was isolated from a cosmid library of C. thermosulfurogenes DNA. The plasmid was further subcloned into a Bacillus cloning vector, pTB523, to generate shuttle plasmid pMLG1, which is able to replicate in both E. coli and Bacillus subtilis. Expression of the thermostable glucose isomerase gene in both species was constitutive, whereas synthesis of the enzyme in C. thermosulfurogenes was inducible by D-xylose. B. subtilis and E. coli produced higher levels of thermostable glucose isomerase (1.54 and 0.46 U/mg of protein, respectively) than did C. thermosulfurogenes (0.29 U/mg of protein). The glucose isomerases synthesized in E. coli and B. subtilis were purified to homogeneity and displayed properties (subunit Mr, 50,000; tetrameric molecular structure; thermostability; metal ion requirement; and apparent temperature and pH optima) identical to those of the native enzyme purified from C. thermosulfurogenes. Simple heat treatment of crude extracts from E. coli and B. subtilis cells carrying the recombinant plasmid at 85 degrees C for 15 min generated 80% pure glucose isomerase. The maximum conversion yield of glucose (35%, wt/wt) to fructose with the thermostable glucose isomerase (10.8 U/g of dry substrate) was 52% at pH 7.0 and 70 degrees C.
Coexisting Bacterial Populations Responsible for Multiphasic Mineralization Kinetics in Soil
Authors: Schmidt S. K. , Gier M. J.
Published: January 2020

Experiments were conducted to study populations of indigenous microorganisms capable of mineralizing 2,4-dinitrophenol (DNP) in two soils. Previous kinetic analyses indicated the presence of two coexisting populations of DNP-mineralizing microorganisms in a forest soil (soil 1). Studies in which eucaryotic and procaryotic inhibitors were added to this soil indicated that both populations were bacterial. Most-probable-number counts with media containing different concentrations of DNP indicated that more bacteria could mineralize low concentrations of DNP than could metabolize high concentrations of it. Enrichments with varying concentrations of DNP and various combinations of inhibitors consistently resulted in the isolation of the same two species of bacteria from soil 1. This soil contained a large number and variety of fungi, but no fungi capable of mineralizing DNP were isolated. The two bacterial isolates were identified as a
Janthinobacterium
sp. and a
Rhodococcus
sp. The
Janthinobacterium
sp. had a low μ
max
and a low
K

m

for DNP mineralization, whereas the
Rhodococcus
sp. had much higher values for both parameters. These differences between the two species of bacteria were similar to differences seen when soil was incubated with different concentrations of DNP. Values for μ
max
from soil incubations were similar to μ
max
values obtained in pure culture studies. In contrast,
K

s

and
K

m

values showed greater variation between soil and pure culture studies. The results of this study help to confirm predictions that two physiologically distinct bacterial populations are responsible for the multiphasic mineralization kinetics observed in the soil studied.
Polymerase chain reaction identification of Vibrio vulnificus in artificially contaminated oysters
Authors: Hill W E , Keasler S P , Trucksess M W , Feng P , Kaysner C A , Lampel K A
Published: January 2020

DNAs extracted from Vibrio vulnificus seeded into oyster homogenates were evaluated as templates for the polymerase chain reaction. Several extraction procedures were examined, and it was determined that DNA recovered from cells lysed by guanidine isothiocyanate, extracted with chloroform, and precipitated with ethanol was most suitable for use as a polymerase chain reaction template. The region targeted was a 519-bp portion of the cytotoxin-hemolysin gene of V. vulnificus. This region was amplified only when DNA from this species was present in the homogenate. V. vulnificus seeded into oyster homogenates at an initial level of 10(2) CFU/g of oyster meat was consistently observed after 24 h of incubation in alkaline peptone water.
Control of Salmonella enteritidis infections in poultry by polymyxin B and trimethoprim
Authors: Goodnough M C , Johnson E A
Published: January 2020

Antimicrobial compounds were screened in vitro in Trypticase soy broth for antimicrobial activity against a virulent strain of Salmonella enteritidis. Of the several compounds tested, polymyxin B showed the strongest inhibition in vitro, preventing growth at a concentration of less than or equal to 10 micrograms/ml. Polymyxin B administered in the drinking water was effective in vivo for preventing infections in 1-day-old chickens but did not remove established infections in 1-week-old chickens. It was found that trimethoprim, which was not active in vitro, prevented colonization and removed existing infections in 1-day-old chickens when it was administered together with polymyxin B sulfate. Enrichment cultures in which selenite-cystine and tetrathionate broth media were used showed that chickens given a combination of 100 micrograms of polymyxin B sulfate per ml and 250 micrograms of trimethoprim per ml 24 h prior to oral inoculation with 10(8) to 10(9) CFU were negative for S. enteritidis after 7 days. Established infections (10(5) to 10(6) CFU/g of feces) in 1-week-old chickens were eliminated by treatment with the polymyxin-trimethoprim system. This antimicrobial agent treatment may be useful for preventing colonization in poultry and for eliminating S. enteritidis from infected flocks.
Characterization of the Cell Wall-Bound Proteinase of
Lactobacillus casei
HN14
Authors: Kojic M. , Fira D. , Banina A. , Topisirovic L.
Published: January 2020

Lactobacillus casei
HN14, which was isolated from homemade cheese, produces an extracellular, cell wall-bound proteinase. The HN14 proteinase can be removed from the cell envelope by washing the cells in a Ca
2+
-free buffer. The activity of the crude proteinase extract is inhibited by phenylmethylsulfonyl fluoride, showing that the enzyme is a serine-type proteinase. Considering the substrate specificity, the HN14 proteinase is similar to the lactococcal PI-type enzyme, since it hydrolyzes β-casein only.
Lactobacillus casei
HN14 appeared to be plasmid free, which suggests that the proteinase gene is chromosomally located. Chromosomal DNA of this strain hybridizes with DNA probes Q1 (which contains a fragment of the
prtM
gene) and Q6 and Q92 (which contain fragments of the
prtP
gene); all three probes originated from the proteinase gene region of
Lactococcus lactis
subsp.
cremoris
Wg2. A restriction enzyme map of the proteinase region of
Lactobacillus casei
HN14 was constructed on the basis of hybridization experiments. Comparison of the restriction enzyme maps of the
Lactobacillus casei
HN14 proteinase gene region and those of lactococcal proteinase gene regions studied so far indicates that they are highly similar.
Molecular structure of the replication origin of a Bacillus subtilis (natto) plasmid, pUH1
Authors: Hara T , Nagatomo S , Ogata S , Ueda S
Published: January 2020

The structure of a 2.0-kb BstEII DNA sequence necessary and sufficient for the replication of a 5.7-kb Natto plasmid, pUH1, which is responsible for gamma-polyglutamate production by Bacillus subtilis (natto), has been characterized by using a trimethoprim resistance gene derived from B. subtilis chromosomal DNA as a selective marker. The 2.0-kb DNA sequence contains an open reading frame, rep, stretching for 999 bp; a promoter region for rep expression; and a possible replication origin for the plasmid upstream of the promotor. The predicted Rep protein has highly homologous amino acid sequences with rep14 of pFTB14 in B. amyloliquefaciens, RepB of pUB110, and protein A, which is necessary for pC194 replication in staphylococci throughout the protein molecule, but is not homologous with RepC of staphylococcal plasmid pT181.
Development and application of a multiple typing system for Clostridium difficile
Authors: Mahony D E , Clow J , Atkinson L , Vakharia N , Schlech W F
Published: January 2020

A combination of bacteriocin, bacteriophage, and plasmid typing techniques was used to differentiate strains of Clostridium difficile. A typing set of 20 bacteriocin-producing strains was established after 400 isolates of C. difficile were screened for the ability to produce bacteriocin. These strains were used to type a collection of 114 isolates of C. difficile. Forty-six (40%) of the 114 isolates were typeable, and 31 typing patterns were distinguishable. Plasmid typing of the same 114 isolates of C. difficile showed that 67 (59%) of the isolates carried up to four plasmids ranging from 7 to 60 kb in size, although most strains contained only one or two plasmids. Twenty different plasmid typing patterns were observed among the isolates. A combination of bacteriocin and plasmid typing provided 77% typeability. Fifteen (13%) of the 114 strains were typeable with five bacteriophages isolated in our laboratory, but the increase in typeability of strains over that obtainable by plasmid and bacteriocin typing was only 1.8%. Isolates that were nontypeable by bacteriocins, plasmids, or phages could be divided into two groups on the basis of positive or negative cytotoxin production. This further division of strains would increase the typeability potential by 7%; i.e., the ability to differentiate strains would rise from 77 to 84%, or perhaps 86%, if phage typing were included. We conclude that more than one of the techniques reported in this paper must be used to achieve an acceptable level of typeability of this species.
Effect of C6 to C9 alkenals on aflatoxin production in corn, cottonseed, and peanuts
Authors: Zeringue H J
Published: January 2020

The effect on aflatoxin production in Aspergillus flavus-inoculated corn, cottonseed, and peanuts in static culture in the presence of gaseous phase C6 to C9 alkenals was investigated. Aflatoxin B1 production was stimulated in corn at the lowest alkenal concentration (1-microliters level) tested. Aflatoxin B1 was completely eliminated at the highest alkenal concentrations (20-microliters level) tested in both treated corn and cottonseed cultures.
Chromosomal integration of plasmid DNA by homologous recombination in Enterococcus faecalis and Lactococcus lactis subsp. lactis hosts harboring Tn919
Authors: Casey J , Daly C , Fitzgerald G F
Published: January 2020

Integration of pCI192, a pBR322-derived vector plasmid containing homology to the chromosomally located conjugative transposon Tn919 was observed in two strains that harbor Tn919, namely, Enterococcus faecalis GF590 and Lactococcus lactis subsp. lactis CH919. Hybridization analysis indicated that single-copy integration of the plasmid had occurred at low frequency. The Tn919::plasmid structure was conjugated from an E. faecalis donor to a L. lactis recipient, although at lower frequencies than was Tn919. Segregation of the tetracycline and chloramphenicol resistance markers during conjugation was observed. The integration strategy described allows for DNA manipulations to be performed in an easily manipulated model host strain with the subsequent transfer of integrated structures by conjugation to any strain capable of receiving Tn919. The results indicate that homologous recombination events may be used to introduce plasmid-encoded genes to the lactococcal chromosome.
Use of Bioluminescence for Detection of Genetically Engineered Microorganisms Released into the Environment
Authors: Shaw Joe J. , Dane Fenny , Geiger Dorothy , Kloepper Joseph W.
Published: January 2020

The persistence and movement of strain JS414 of
Xanthomonas campestris
pv. campestris, which was genetically engineered to bioluminesce, were monitored during a limited field introduction. Bioluminescence and traditional dilution plate counts were determined. Strain JS414 was applied to cabbage plants and surrounding soil by mist inoculation, by wound inoculation, by scattering infested debris among plants, and by incorporating bacteria into the soil. Bioluminescent
X. campestris
pv. campestris was detected in plant samples and in the rhizosphere up to 6 weeks after inoculation. Movement to uninoculated plants was detected on one occasion, but movement from the immediate release area was not detected. Strain JS414 was detected in soil samples beneath mist- and wound-inoculated plants only at intentionally infested locations and in aerial samples only on the day of inoculation. Our bioluminescence methods proved to be as sensitive as plating methods for detecting the genetically engineered microorganisms in environmental samples. Our results demonstrate that transgenic incorporation of the
luxCDABE
operon provides a non-labor-intensive, sensitive detection method for monitoring genetically engineered microorganisms in nature.
Electrical Energy Changes Conductivity and Determines Optimal Electrotransformation Frequency in Gram-Negative Bacteria
Authors: Bowen Benjamin A. , Kosslak Renee M.
Published: January 2020

In many bacterial electrotransformation protocols, pulse time is related to the time constant for a capacitor discharging across a sample of fixed resistance. Using an electroporator which controls pulse time independently of the capacitor time constant, we found that the resistance of bacterial suspensions fluctuates widely during capacitor discharge. With three gram-negative species of bacteria, electrotransformation frequency and survival could be more simply related to the electrical energy delivered in each pulse than to component parameters, such as initial field strength, capacitance, and pulse time. In each case, the number of transformants per survivor increased exponentially and leveled off when more than 0.5 to 1.0 J of electrical energy was delivered. An inverse log-linear relationship between survival and energy delivered was also observed for all three species.
Riboflavin Production during Growth of
Micrococcus luteus
on Pyridine
Authors: Sims Gerald K. , O'Loughlin Edward J.
Published: January 2020

Micrococcus luteus
produced 29 μM riboflavin during growth on 6.5 mM pyridine but not during growth on other substrates. On the basis of the results of radiolabelling studies, riboflavin was not directly synthesized from pyridine. Pyridine may interfere with riboflavin biosynthesis or elicit a general stress response in
M. luteus.
Cloning of a creatinase gene from Pseudomonas putida in Escherichia coli by using an indicator plate
Authors: Chang M C , Chang C C , Chang J C
Published: January 2020

A genomic library of Pseudomonas putida DNA was constructed by using plasmid pBR322. Transformants of Escherichia coli in combination with Proteus mirabilis cells grown on creatinase test plates were screened for creatinase activity; transformants were considered positive for creatinase activity if a red-pink zone appeared around the colonies. One creatinase-positive clone was further analyzed, and the gene was reduced to a 2.7-kb DNA fragment. A unique protein band (with a molecular weight of approximately 50,000) was observed in recombinant E. coli by minicell analysis.
Enterotoxigenicity of Staphylococcus strains isolated from Spanish dry-cured hams
Authors: Marín M E , de la Rosa M C , Cornejo I
Published: January 2020

The ability of 135 Staphylococcus strains isolated from Spanish dry-cured hams to produce enterotoxins in culture was investigated by the reversed passive latex agglutination method. A high percentage of enterotoxigenic Staphylococcus aureus strains (85.9%) was recorded, and 54.3% of these produced enterotoxin A. One of the two Staphylococcus epidermidis strains produced enterotoxin C. The reversed passive latex agglutination method yielded satisfactory results.
Toxin Production by
Microcystis aeruginosa
as a Function of Light in Continuous Cultures and Its Ecological Significance
Authors: Utkilen Hans , Gjølme Nina
Published: January 2020

The effects of light intensity and light quality on toxin production by
Microcystis aeruginosa
were examined in continuous cultures. Light intensity had a pronounced effect on toxicity and the toxin production rate. Toxicity and the toxin production rate increased with light intensity up to an intensity of about 40 microeinsteins m
-2
s
-1
and decreased at higher light intensities, while the ratio of toxin to protein was constant at intensities of more than 40 microeinsteins m
-2
s
-1
. Light quality had only slight effects on toxicity. The results of our laboratory experiments were supported by the results of field work in which we examined toxin production at different depths in a lake. Our observations explain the mixed pattern of high and low toxicity found in a surface bloom of
M. aeruginosa.
Our findings also indicate that production of the peptide toxin can be uncoupled from general protein synthesis.
Enzymic Dehalogenation of 4-Chlorobenzoyl Coenzyme A in
Acinetobacter
sp. Strain 4-CB1
Authors: Copley Shelley D. , Crooks Gwen P.
Published: January 2020

4-Chlorobenzoate degradation in cell extracts of
Acinetobacter
sp. strain 4-CB1 occurs by initial synthesis of 4-chlorobenzoyl coenzyme A (4-chlorobenzoyl CoA) from 4-chlorobenzoate, CoA, and ATP. 4-Chlorobenzoyl CoA is dehalogenated to 4-hydroxybenzoyl CoA. Following the dehalogenation reaction, 4-hydroxybenzoyl CoA is hydrolyzed to 4-hydroxybenzoate and CoA. Possible roles for the CoA moiety in the dehalogenation reaction are discussed.
Development of a gene transfer system for curing of plasmids in the marine fish pathogen Vibrio salmonicida
Authors: Valla S , Frydenlund K , Coucheron D H , Haugan K , Johansen B , Jørgensen T , Knudsen G , Strøm A
Published: January 2020

All reported natural isolates of the marine fish pathogen Vibrio salmonicida contain plasmids, and in another marine fish pathogen, Vibrio anguillarum, it has been shown that a plasmid is important for expression of virulence by the organism. To study the function of the plasmids in V. salmonicida, we developed a gene transfer system based on the plasmid RSF1010 replicon. The gene transfer system was used to construct a plasmid-free strain, and this strain was found to behave similarly to the wild type in a fish pathogenicity test based on intraperitoneal injection of the bacteria. We were unable to detect any other phenotypic differences between the two strains. It could therefore be concluded that at least in the V. salmonicida strain tested, extrachromosomal DNA is not required for expression of virulence.
Application of DNA probes to analysis of bacteriophage distribution patterns in the environment
Authors: Ogunseitan O A , Sayler G S , Miller R V
Published: January 2020

Radiolabeled bacteriophage DNA probes have been used in this study to determine the distribution of Pseudomonas aeruginosa-infecting bacteriophages in natural samples of lake water, sediment, soil, and sewage. The sensitivity of detection of bacteriophage with the DNA probes was between 10(3) and 10(4) PFU and 10(6) to 10(7) CFU of lysogenized bacteria detectable with a homologous phage DNA probe. Analyses of environmental samples suggest that up to 40% of P. aeruginosa in natural ecosystems contain DNA sequences homologous to phage genomes. By using different bacteriophage DNA probes, the diversity of the bacteriophage population in sewage was estimated to be higher than that in other natural samples. The indication that transducing phages and prophages are widely distributed in the Pseudomonas populations investigated has considerable implications for the frequency of natural gene transfer by transduction and of lysogenic conversion of host bacteria in natural ecosystems.
Lipoxygenase inhibitors shift the yeast/mycelium dimorphism in Ceratocystis ulmi
Authors: Jensen E C , Ogg C , Nickerson K W
Published: January 2020

The yeast-mycelium dimorphism in Ceratocystis ulmi, the causative agent of Dutch elm disease, was switched by gossypol, nordihydroguaiaretic acid, and propylgallate. In each case the mycelial form was converted to the yeast form. These compounds are recognized lipoxygenase inhibitors. Inhibitors of cyclooxygenase and thromboxane synthetase did not cause mycelia to shift to the yeast form. We suggest the following two-part hypothesis: (i) that lipoxygenase is a target for antifungal antibiotics and (ii) that many phytoalexins (antimicrobial compounds of plant origin) are targeted toward fungal lipoxygenases. In addition, in a study to determine potential lipoxygenase substrates, a fatty acid analysis indicated that C. ulmi conidiospores contained high levels of oleic, linoleic, and linolenic acids but no arachidonic acid.
Cloning and nucleotide sequence of the gene coding for aspartokinase II from a thermophilic methylotrophic Bacillus sp
Authors: Schendel F J , Flickinger M C
Published: January 2020

The structural gene coding for the lysine-sensitive aspartokinase II of the methylotrophic thermotolerant Bacillus sp. strain MGA3 was cloned from a genomic library by complementation of an Escherichia coli auxotrophic mutant lacking all three aspartokinase isozymes. The nucleotide sequence of the entire 2.2-kb PstI fragment was determined, and a single open reading frame coding for the aspartokinase II enzyme was found. Aspartokinase II was shown to be an alpha 2 beta 2 tetramer (M(r) 122,000) with the beta subunit (M(r) 18,000) encoded within the alpha subunit (M(r) 45,000) in the samea reading frame. The enzyme was purified, and the N-terminal sequences of the alpha and beta subunits were identical with those predicted from the gene sequences. The predicted amino acid sequence was 76% identical with the sequence of the Bacillus subtilis aspartokinase II. The transcription initiation site was located approximately 350 bp upstream of the translation start site, and putative promoter regions at -10 (TATGCT) and -35 (ATGACA) were identified. A 300-nucleotide intervening sequence between the transcription initiation and translational start sites suggests a possible attenuation mechanism for the regulation of transcription of this enzyme in the presence of lysine.
Dispersion of Small Ceramic Particles (Al
2
O
3
) with
Azotobacter vinelandii
Authors: Ren Tao , Pellerin Nancy B. , Graff Gordon L. , Aksay Ilhan A. , Staley James T.
Published: January 2020

The high surface charge of small ceramic particles such as alumina particles prevents them from dispersing evenly in aqueous suspensions and forming high-density compacts. However, suspensions of 400-nm-diameter alumina particles treated with alginate from the bacterium
Azotobacter vinelandii
were well dispersed. The alginate bound firmly to the particle surface and could not be removed by repeated washing with distilled water (2.82 mg of the bacterial alginate adsorbed to 1 g of the alumina particles). Furthermore,
A. vinelandii
grew and produced alginate in the presence of up to 15% (vol/vol) alumina particles. These results suggest that an in situ process using this bacterium to coat ceramic particles with alginate might be developed. In in situ processing experiments, the particle-packing densities were significantly increased and the viscosities of 5 and 10% (vol/vol) suspensions were reduced 4- and 60-fold, respectively, over those of controls. The bacteria were readily removed from the alumina particles by washing.
Characterization of RFRS9, a second member of the Rhizobium fredii repetitive sequence family from the nitrogen-fixing symbiont R. fredii USDA257
Authors: Krishnan H B , Pueppke S G
Published: January 2020

The genome of the nitrogen-fixing symbiont, Rhizobium fredii USDA257, contains nine copies of repetitive sequences known as the R. fredii repetitive sequence (RFRS) family. We previously sequenced RFRS3, which is linked to symbiosis plasmid-borne nodulation genes of this organism and has substantial homology to the T-DNA of Agrobacterium rhizogenes and lesser homology to reiterated sequences of Bradyrhizobium japonicum. Here we characterize a second family member, RFRS9. The EcoRI fragment containing RFRS9 is 1,248 bp in length and contains a single 666-bp open reading frame that is flanked by perfect 8-bp inverted repeats. Nucleic and amino acid sequences corresponding to the C terminus of the putative RFRS9 protein are nearly identical to those of RFRS3, and they retain homology to DNA from A. rhizogenes. The central portion of the RFRS9 protein also appears to be related to the S locus-specific glycoprotein family of pollen stigma incompatibility glycoproteins from Brassica oleracea, which are involved in signal perception. Sequences that define the RFRS family are restricted to the open reading frame of RFRS9 and associated upstream sequences. These regions also contain a second group of repetitive sequences, which is present in four copies within the genome of USDA257. Both families of repetitive sequences are ubiquitous in R. fredii, and they are preferentially localized on symbiosis plasmids. Southern hybridization confirms that sequences homologous to RFRS9 are present in broad-host-range Rhizobium sp. strain NGR234, in A. rhizogenes, and in two biotype 3 strains of Agrobacterium tumefaciens.
Invasion and Exclusion among Coexisting
Pseudomonas syringae
Strains on Leaves
Authors: Kinkel Linda L. , Lindow Steven E.
Published: January 2020

The invasion and exclusion abilities of coexisting
Pseudomonas syringae
strains were quantified on leaves. Twenty-nine
P. syringae
strains were inoculated onto plants in 107 pairwise combinations. All pairs were duplicated so that each strain was inoculated both first as an antagonist strain (day 0) and second as a challenge strain (day 3). The population size of each strain in a mixture was quantified on day 6 following incubation under moist conditions. For
P. syringae
strains, the presence of an established population often significantly reduced the growth of subsequently arriving challenge strains on the leaf surface. Invasion and exclusion abilities, quantified by contrasting population sizes of challenge strains in the presence and in the absence of another strain, varied significantly among
P. syringae
strains and were partly a function of the particular strain pair. The population size of a strain when present alone on a leaf was not predictive of invasion or exclusion ability. Successful invaders were significantly less likely to exclude challenge populations than were nonsuccessful invaders. Population sizes of successful excluders were negatively correlated with population sizes of coexisting challenge strains, while population sizes of successful invaders were positively correlated with those of coexisting antagonist strains. The patterns of interaction among coexisting strains suggest mechanisms for successful invasion and exclusion among
P. syringae
strains on leaves.
A model system for the investigation of heterologous protein secretion pathways in Lactococcus lactis
Authors: Wells J M , Wilson P W , Norton P M , Le Page R W
Published: January 2020

The capacity of recombinant strains of Lactococcus lactis to secrete a heterologous protein was investigated by constructing two expression-secretion vectors (pLET2 and pLET3) for use with a lactococcal gene expression system driven by the highly active T7 RNA polymerase. The vectors incorporated different lactococcal secretion leaders and translation initiation sequences. When tetanus toxin fragment C (TTFC) was used as a test protein, the quantities of TTFC produced by the pLET2-TTFC strain exceeded the rate of secretion of TTFC into the growth medium. However, nearly all of the soluble TTFC associated with the cell (3.4%) was translocated through the cell membrane. The pLET3-TTFC strain did not accumulate TTFC intracellularly and exhibited growth characteristics and viability identical to the growth characteristics and viability of the control strain. This strain secreted approximately 2.9 mg of TTFC per liter into the growth medium after 6 h of growth under test tube conditions. Our results indicate that L. lactis is capable of secreting substantial amounts of heterologous protein and also confirm the findings of other workers that the cell wall may serve as a functional barrier to the diffusion of some secreted proteins into the growth medium.
Protozoa in Subsurface Sediments from Sites Contaminated with Aviation Gasoline or Jet Fuel
Authors: Sinclair James L. , Kampbell Don H. , Cook Mike L. , Wilson John T.
Published: January 2020

Numbers of protozoa in the subsurface of aviation gasoline and jet fuel spill areas at a Coast Guard base at Traverse City, Mich., were determined. Boreholes were drilled in an uncontaminated location, in contaminated but untreated parts of the fuel plumes, and in the aviation gasoline source area undergoing H
2
O
2
biotreatment. Samples were taken from the unsaturated zone to depths slightly below the floating free product in the saturated zone. Protozoa were found to occur in elevated numbers in the unsaturated zone, where fuel vapors mixed with atmospheric oxygen, and below the layer of floating fuel, where uncontaminated groundwater came into contact with fuel. The same trends were noted in the biotreatment area, except that numbers of protozoa were higher. Numbers of protozoa in some contaminated areas equalled or exceeded those found in surface soil. The abundance of protozoa in the biotreatment area was high enough that it would be expected to significantly reduce the bacterial community that was degrading the fuel. Little reduction in hydraulic conductivity was observed, and no bacterial fouling of the aquifer was observed during biotreatment.
Inhibition of Butyrate Oxidation by Formate during Methanogenesis
Authors: Bae Jaeho , McCarty Perry L.
Published: January 2020

A mixed methanogenic culture fed with glucose was perturbed with butyrate and formate to investigate the role of formate in the acetogenesis of butyrate. A free energy analysis suggests that formate rather than H
2
was the interspecies electron carrier for butyrate conversion into acetate for the culture studied.
Detection of enteric viruses in oysters by using the polymerase chain reaction
Authors: Atmar R L , Metcalf T G , Neill F H , Estes M K
Published: January 2020

A procedure for the detection of enteric viral nucleic acid in oysters by the polymerase chain reaction was developed. Known quantities of poliovirus type 1 were seeded into oysters. Virus was extracted and concentrated by using organic flocculation and polyethylene glycol precipitation. Inhibitors of reverse transcription-polymerase chain reaction were present in the oyster extracts, preventing amplification of target viral nucleic acid. The use of cetyltrimethylammonium bromide precipitation sufficiently removed inhibitors to allow detection of as few as 10 PFU of poliovirus. Norwalk virus also could be detected after being seeded into oysters. This methodology may be useful for the detection of these and other shellfish-borne viral pathogens.
Sorption of Heavy Metals to the Filamentous Bacterium
Thiothrix
Strain A1
Authors: Shuttleworth Kay L. , Unz Richard F.
Published: January 2020

A study was undertaken to determine the ability of the filamentous bacterium
Thiothrix
strain A1 to sorb heavy metals from solution. Cells of
Thiothrix
strain A1 were harvested, washed, and suspended in solutions of metals. After an equilibration period, biomass was separated from solution and the metal content in acid-digested cells and/or filtrates was determined by atomic absorption spectrophotometry. Sorption of nickel and zinc was very rapid; most of the sorbed metal was bound in less than 10 min. The sorption data for copper fit the Freundlich isotherm, and nickel and zinc data fit biphasic Freundlich isotherms. Sorption of both nickel and zinc was dependent on cell age. Cells harvested 24 h after inoculation sorbed approximately one-half of the amount of metal per gram cell protein than did cells harvested after 48, 72, or 96 h. Calcium and magnesium effectively competed with zinc for binding sites, whereas potassium had only a slight effect on the capacity of cells to sorb zinc. The primary mechanism of metal sorption apparently was ion exchange, because 66 to 75% of nickel or zinc could be desorbed by placing metal-laden cells in a solution of 5 mM CaCl
2
. A competition experiment with nickel and zinc indicated that both metals occupied the same sorption sites. The strong chelating agents EDTA and NTA effectively prevented metal uptake, but lactate enhanced the uptake of nickel.
Thiothrix
strain A1 grown in nickel-containing medium had a relatively low uptake of nickel compared with uptake by resting cells suspended in a simple buffer solution.
Modelling the growth kinetics of Phanerochaete chrysosporium in submerged static culture
Authors: Barclay C D , Legge R L , Farquhar G F
Published: January 2020

The potential commercial application of Phanerochaete chrysosporium requires methods for quantitatively predicting growth and substrate utilization. The growth kinetics of P. chrysosporium INA-12 (CNCM I-398) were investigated and modelled under nonlimiting nitrogen and carbon conditions in submerged static culture. This strain, unlike other strains, does not require nutrient limitation for induction of lignin peroxidase. Maximum levels of lignin peroxidase activity were reached 7 days after culture initiation, when almost 80% of the initial glycerol and 70% of the initial nitrogen were still present. Lignin peroxidase levels then decreased, while biomass levels increased until about day 14. The ratio of cell dry weight to wet weight was constant until the maximum biomass concentration was achieved, after which there was a decrease in the water content. The change in this ratio reflects cell lysis as it correlated with increased concentrations of nitrogen in the media, arising from cell leakage. The suitability of four growth models to predict growth, and in some cases glycerol consumption, was evaluated. A simple linear model and the Emerson model performed poorly for the early stages of growth, while a modified Williams model and the Monod model predicted substrate and biomass concentrations equally well. All models will predict biomass concentrations during the active growth phase, but they should not be used to predict biomass concentrations after the stationary growth phase, when cell lysis becomes significant.
Cloning of the gene encoding Streptococcin A-FF22, a novel lantibiotic produced by Streptococcus pyogenes, and determination of its nucleotide sequence
Authors: Hynes W L , Ferretti J J , Tagg J R
Published: January 2020

Streptococcin A-FF22 (SA-FF22) is a lantibiotic produced by Streptococcus pyogenes FF22. The nucleotide sequence of the SA-FF22 structural gene (scnA) was determined and shown to encode a 51-amino-acid prepeptide. The proteolytic processing site of the SA-FF22 prepeptide differs from that which characterizes other type A lantibiotics.
Polyvinylpyrrolidone-Agarose Gel Electrophoresis Purification of Polymerase Chain Reaction-Amplifiable DNA from Soils
Authors: Young Charles C. , Burghoff Robert L. , Keim Lois G. , Minak-Bernero Vera , Lute James R. , Hinton Stephen M.
Published: January 2020

This communication describes a modification of agarose gel electrophoresis to provide a rapid and simple method for the purification of polymerase chain reaction-amplifiable DNA from soil. This modification is to add polyvinylpyrrolidone to the agarose gel. The polyvinylpyrrolidone addition retards the electrophoretic mobility of denaturing phenolic compounds so that they do not comigrate with nucleic acids.
Effect of X-Prolyl Dipeptidyl Aminopeptidase Deficiency on
Lactococcus lactis
Authors: Mayo Baltasar , Kok Jan , Bockelmann Wilhelm , Haandrikman Alfred , Leenhouts Kees J. , Venema Gerard
Published: January 2020

The genetic determinant (
pepXP
) of an X-prolyl dipeptidyl aminopeptidase (PepXP) has recently been cloned and sequenced from both
Lactococcus lactis
subsp.
cremoris
(B. Mayo, J. Kok, K. Venema, W. Bockelmann, M. Teuber, H. Reinke, and G. Venema, Appl. Environ. Microbiol. 57:38-44, 1991) and
L. lactis
subsp.
lactis
(M. Nardi, M.-C. Chopin, A. Chopin, M.-M. Cals, and J.-C. Gripon, Appl. Environ. Microbiol. 57:45-50, 1991). To examine the possible role of the enzyme in the breakdown of caseins required for lactococci to grow in milk, integration vectors have been constructed and used to specifically inactivate the
pepXP
gene. After inactivation of the gene in
L. lactis
subsp.
lactis
MG1363, which is Lac
-
and Prt
-
, the Lac
+
Prt
+
determinants were transferred by conjugation by using
L. lactis
subsp.
lactis
712 as the donor. Since growth of the transconjugants relative to the PepXP
+
strains was not retarded in milk, it was concluded that PepXP is not essential for growth in that medium. It was also demonstrated that the open reading frame ORF1, upstream of
pepXP
, was not required for PepXP activity in
L. lactis.
A marked difference between metenkephalin degradation patterns was observed after incubation of this pentapeptide with cell extracts obtained from wild-type lactococci and
pepXP
mutants. Therefore, altered expression of the
pepXP
-encoded general dipeptidyl aminopeptidase activity may change the peptide composition of fermented milk products.
Mobilization of a Recombinant IncQ Plasmid between Bacteria on Agar and in Soil via Cotransfer or Retrotransfer
Authors: Smit Eric , Venne Dorine , van Elsas Jan D.
Published: January 2020

Mobilization of a genetically engineered IncQ plasmid, pSKTG, was studied in vitro and in sterile and nonsterile soils. In biparental and triparental filter matings, the mobilization frequencies of pSKTG were identical, and the plasmid was mobilized only in the presence of self-transmissible plasmid RP4p. In sterile soil, mobilization was probably limited by reduced cell-to-cell contact, since the frequencies of mobilization were approximately 100-fold lower than the frequencies in the filter matings. The transfer frequency of pSKTG in sterile soil when RP4p was present in the same strain was about 100-fold higher than the transfer frequency when RP4p was present in a separate strain. In studies in natural soil, pSKTG was also found to be transferred to indigenous bacteria. However, natural mobilization by genetic elements present in the indigenous soil microflora could not be detected. In vitro studies of natural transfer suggested that such genetic elements occur in soil bacteria.
Methods to investigate the expression of lignin peroxidase genes by the white rot fungus Phanerochaete chrysosporium
Authors: Reiser J , Walther I S , Fraefel C , Fiechter A
Published: January 2020

Two methods allowing the analysis of expression of specific lignin peroxidase (LPO) genes from white rot fungi are presented. In the first method, degenerate oligonucleotide primers derived from amino acid sequence motifs held in common among all members of the LPO gene family are used to prime the polymerase chain reaction (PCR) amplification of LPO-related nucleotide sequences from cDNA prepared by using RNA from ligninolytic cultures. The PCR products are cloned and analyzed by restriction cleavage and DNA sequencing. This method was applied to the analysis of transcripts from carbon-limited cultures of Phanerochaete chrysosporium BKM-F-1767, revealing two major classes of PCR products. One class showed DNA sequences with a high degree of similarity to the previously described CLG4 cDNA sequence (H. A. De Boer, Y. Zhang, C. Collins, and C. A. Reddy, Gene 60:93-102, 1987), whereas the other harbored DNA sequences with similarities to the L18 cDNA sequence previously described for P. chrysosporium OGC101 (T. G. Ritch, Jr., V. J. Nipper, L. Akileswaran, A. J. Smith, D. G. Pribnow, and M. H. Gold, Gene 107:119-126, 1991). The second method is based on nuclease protection assays involving isoenzyme-specific RNA probes. By using this method, the L18-related gene of P. chrysosporium BKM-F-1767 was found to be expressed under conditions of carbon and of nitrogen limitation, although the transcript levels were found to be higher in carbon-limited cultures. Furthermore, it was found that omission of veratryl alcohol addition to the culture did not affect the levels of the L18-related transcripts in carbon-limited cultures.
Effect of water and nitrogen additions on free-living nitrogen fixer populations in desert grass root zones
Authors: Herman R P , Provencio K R , Torrez R J , Seager G M
Published: January 2020

In this study we measured changes in population levels of free-living N2-fixing bacteria in the root zones of potted Bouteloua eriopoda and Sporobolus flexuosus plants as well as the photosynthetic indices of the plants in response to added nitrogen, added water, and added water plus nitrogen treatments. In addition, N2 fixer population changes in response to added carbon source and nitrogen were measured in plant-free soil columns. There were significant increases in the numbers of N2 fixers associated with both plant species in the water and the water plus nitrogen treatments. Both treatments increased the photosynthetic index, suggesting that plant exudates were driving N2 fixer population changes. Population increases were greatest in the water plus nitrogen treatments, indicating that added nitrogen was synergistic with added water and suggesting that nitrogen addition spared bacteria the metabolic cost of N2 fixation, allowing greater reproduction. Plant-free column studies demonstrated a synergistic carbon-nitrogen effect when carbon levels were limiting (low malate addition) but not when carbon was abundant (high malate), further supporting this hypothesis. The results of this study indicate the presence of N2 fixer populations which interact with plants and which may play a role in the nitrogen balance of desert grasslands.
A Gas Lift Bioreactor for Removal of Contaminants from the Vapor Phase
Authors: Ensley B. D. , Kurisko P. R.
Published: January 2020

The cometabolic degradation of trichloroethylene (TCE) as a vapor by two aromatic-metabolizing pseudomonads was evaluated in an airlift reactor. These microorganisms were able to degrade 90 to 95% of TCE in air at concentrations at the reactor inlet of 300 to 4,000 μg/liter. Although exposure of the cells to high inlet concentrations of TCE (4 mg/liter) caused a decline in enzyme-specific activity and TCE removal efficiency, this loss in activity could be prevented or delayed by increasing the rate of cosubstrate addition. Under the appropriate operating conditions, the microorganisms were able to degrade even high concentrations of TCE and activity of the cells in the reactor could be maintained for periods of at least 2 weeks.