bluuebunny/crossref_metadata_2025 huggingface dataset:
Purification and Comparison of the Periplasmic and Extracellular Forms of the Cellodextrinase from
Bacteroides succinogenes
Authors: Huang Li , Forsberg Cecil W.
Published: January 2020

Both the periplasmic and the extracellular cellodextrinases from
Bacteroides succinogenes
S85 grown on Avicel microcrystalline cellulose were purified to homogeneity by column chromatography and characterized. Over 70% of the total cellobiosidase activity displayed by cells was accounted for by these enzymes. The periplasmic and extracellular cellodextrinases had identical molecular weights (50,000), as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and identical isoelectric points (4.9). In addition, the two enzymes were similar in catalytic properties, with

K
m

and
V
max
values of approximately 0.24 mM and 21 μmol/min per mg of protein, respectively. Examination of the two enzymes by using peptide mapping and immunoblotting techniques provided additional evidence indicating their identical nature. Immunoblotting of the extracellular culture fluid with affinity-purified antibody to the periplasmic cellodextrinase revealed one band with a molecular weight corresponding to that of the periplasmic cellodextrinase. The stability of the purified periplasmic cellodextrinase in aqueous solution was markedly enhanced by increased protein content. This enzyme showed a low affinity for crystalline cellulose.
Cloning of the cellulase gene from Ruminococcus albus and its expression in Escherichia coli
Authors: Ohmiya K , Nagashima K , Kajino T , Goto E , Tsukada A , Shimizu S
Published: January 2020

The gene for cellulase from Ruminococcus albus F-40 was cloned in Escherichia coli HB101 with pBR322. A 3.4-kilobase-pair HindIII fragment encoding cellulase hybridized with the chromosomal DNA of R. albus. The Ouchterlony double-fusion test gave a single precipitation line between the cloned enzyme and the cellulase from R. albus. The size of the cloned fragment was reduced by using HindIII and EcoRI. The resulting active fragment had a size of 1.9 kilobase pairs; and the restriction sites EcoRI, BamHI, PvuII, EcoRI, PvuII, and HindIII, in that order, were ligated into pUC19 at the EcoRI and HindIII sites (pURA1). Cellulase production by E. coli JM103(pURA1) in Luria-Bertani broth was remarkably enhanced, up to approximately 80 times, by controlling the pH at 6.5 and by reducing the concentration of NaCl in the broth to 80 mM.
l
-Methionine, a Precursor of Trace Methane in Some Proteolytic Clostridia
Authors: Rimbault Alain , Niel Philippe , Virelizier Henri , Darbord Jacques Christian , Leluan Georges
Published: January 2020

The in vivo formation of methane and of several
S
-methyl volatile compounds from the terminal
S
-methyl group of
l
-methionine is reported for growing cultures of four
Clostridium
strains (
C. hastiforme, C. histolyticum, C. subterminale
, and
Clostridium
sp. strain DSM 1786). After growth in 5 ml of unamended medium,
C. hastiforme
formed the highest amount of methane (408 nmol per tube in the headspace). When the culture medium was amended with 100 mM
l
-[
S-methyl
-
2
H
3
]methionine, the four strains formed [
2
H
3
]methane (proportion in the methane peak, >85%) as well as methanethiol, dimethyl disulfide, dimethyl trisulfide, and
S
-methyl thioacetate labeled on the methyl moiety. Methanethiol is also a precursor of methane for
Clostridium
sp. strain DSM 1786. The trace methane formation observed for these four proteolytic, nonglucidolytic
Clostridium
strains can be of ecological interest, particularly in aquatic sediments and in the gastrointestinal tract of humans and animals. It can explain in part the trace methane formation which cannot be ascribed to methanogens sensu stricto.
Conversion of Glucose to 2-Keto-
l
-Gulonate, an Intermediate in
l
-Ascorbate Synthesis, by a Recombinant Strain of
Erwinia citreus
Authors: Grindley J. F. , Payton M. A. , van de Pol H. , Hardy K. G.
Published: January 2020

A gene for 2,5-diketo-
d
-gluconate (25DKG) reductase, which encodes an enzyme composed of 277 amino acid residues catalyzing the reduction of 25DKG to 2-keto-
l
-gulonate (2KLG), was cloned from
Corynebacterium
sp. strain SHS752001 and expressed in
Erwinia citreus
SHS2003, a strain which oxidizes glucose to 25DKG. The recombinant microorganism converted glucose to 2KLG, a compound which can be readily converted to
l
-ascorbate (vitamin C). Improvements in the yield of 2KLG were obtained by changing fermentation conditions, using the
p

l

promoter of bacteriophage lambda to express the reductase, and selecting a mutant of
E. citreus
which could use neither 25DKG nor 2KLG as a sole carbon source for growth. When a culture of the recombinant strain was fed with glucose to a total of 40 g/liter, 49.4% of the glucose was converted to 2KLG during a 72-h fermentation.
Mechanism for nitrosation of 2,3-diaminonaphthalene by Escherichia coli: enzymatic production of NO followed by O2-dependent chemical nitrosation
Authors: Ji X B , Hollocher T C
Published: January 2020

The mechanism by which Escherichia coli can catalyze the nitrite-dependent nitrosation of 2,3-diaminonaphthalene (DAN), with formation of the corresponding fluorescent triazole, was studied. The reaction was dependent on production of a gaseous compound which can nitrosylate DAN upon contact with air. This compound was identified as nitric oxide (NO), and the kinetics of NO and triazole production are reported. NO and triazole were produced proportionally in a stoichiometric ratio, NO/triazole, of 1.4 to 1.7. Given the requirement for air, nitrosation of DAN probably proceeds via formation of the well-known strong nitrosylating agents N2O3 and N2O4 from NO. The parallel inhibition of NO and triazole production by azide and nitrate served to reinforce the link between nitrosation and nitrate reductase that had been established previously by others on genetic grounds.
Effect of Temperature and Prey Availability on Growth of
Paramoeba invadens
in Monoxenic Culture
Authors: Jellett Joanne F. , Scheibling Robert E.
Published: January 2020

Paramoeba invadens
Jones 1985 is a pathogenic marine amoeba responsible for mass mortalities of sea urchins (
Strongylocentrotus droebachiensis
) of Nova Scotia between 1980 and 1983. A direct relationship between temperature and sea urchin paramoebiasis has been shown in previous laboratory and field studies. This study examined the effect of prey availability and temperature on the growth of
P. invadens
in monoxenic culture (with the marine bacterium
Pseudomonas nautica
). At 15°C, the specific growth rate of
P. invadens
increased with bacterial prey concentration and was highest at 10
8
bacterial cells ml
−1
. Growth rate of
P. invadens
was maximal at 15 to 20°C (which corresponds to annual sea temperature maxima in the natural environment) and the minimum generation time was 19.41 h at 20°C. At 10 and 12°C, generation times were 91.18 and 73.39 h, respectively; at 2 and 5°C, there was no growth.
P. invadens
did not survive in monoxenic culture at 27°C. Growth rates of
P. invadens
in vitro were positively correlated with time to morbidity of infected
S. droebachiensis
.
Pseudomonas oleovorans
as a Source of Poly(β-Hydroxyalkanoates) for Potential Applications as Biodegradable Polyesters
Authors: Brandl Helmut , Gross Richard A. , Lenz Robert W. , Fuller R. Clinton
Published: January 2020

Pseudomonas oleovorans
was grown in homogeneous media containing
n
-alkanoic acids, from formate to decanoate, as the sole carbon sources. Formation of intracellular poly(β-hydroxyalkanoates) was observed only for hexanoate and the higher
n
-alkanoic acids. The maximum isolated polymer yields were approximately 30% of the cellular dry weight with growth on either octanoate or nonanoate. In most cases, the major repeating unit in the polymer had the same chain length as the
n
-alkanoic acid used for growth, but units with two carbon atoms less or more than the acid used as a carbon source were also generally present in the polyesters formed. Indeed, copolymers containing as many as six different types of β-hydroxyalkanoate units were formed. The weight average molecular weights of the poly(β-hydroxyalkanoate) copolymers produced by
P. oleovorans
ranged from 90,000 to 370,000. In spite of the higher cell yields obtained with octanoate and nonanoate, the use of hexanoate and heptanoate yielded higher-molecular-weight polymers. These copolyesters represent an entirely new class of biodegradable thermoplastics.
Sodium-Dependent
Azotobacter chroococcum
Strains Are Aeroadaptive, Microaerophilic, Nitrogen-Fixing Bacteria
Authors: Page William J. , Jackson Linda , Shivprasad Shailaja
Published: January 2020

Na
+
-dependent strains of
Azotobacter chroococcum
were observed to have very low reactivities with the H
2
O
2
spot test for catalase. The cell extract of the representative Na
+
-dependent strain 184 contained a catalase specific activity that was 10-to 600-fold lower than those found in Na
+
-independent strains of
A. chroococcum.
Peroxidase and superoxide dismutase activities existed in all strains, although only certain Na
+
-dependent strains contained a peroxidase reactive with
p
-phenylenediamine. The activities of catalase and peroxidase in the Na
+
-dependent strain 184 were dependent on iron availability, which helped to explain the iron-dependent growth characteristic of this strain. The activities of these enzymes were not increased by subjecting the cells to increased aeration, nitrogen-fixing conditions, or paraquat. Strain 184 was found to be very sensitive to H
2
O
2
or paraquat, even under iron-sufficient conditions, and was difficult to recover quantitatively on solid plating media. Strain 184 was more susceptible to H
2
O
2
when grown under low-aeration, nitrogen-fixing conditions than when it was grown in the presence of NH
4
+
. Low population densities of strain 184 grew in nitrogen-free medium under microaerophilic conditions, while more dense populations were able to fix nitrogen under aerobic conditions. Therefore, these bacteria appeared to be aeroadaptive, microaerophilic, nitrogen-fixing bacteria.
Calculation of Cell Production from [
3
H]Thymidine Incorporation with Freshwater Bacteria
Authors: Smits Jan D. , Riemann Bo
Published: January 2020

The conversion factor for the calculation of bacterial production from rates of [
3
H]thymidine incorporation was examined with diluted batch cultures of freshwater bacteria. Natural bacterial assemblages were grown in aged, normal, and enriched media at 10 to 20°C. The generation time during 101 growth cycles covered a range from 4 to >200 h. The average conversion factor was 2.15 × 10
18
cells mol
-1
of thymidine incorporated into the trichloroacetic acid (TCA) precipitate (standard error = 0.29 × 10
18
;
n
= 54), when the generation time exceeded 20 h. At generation times of <20 h, the average conversion factor was 11.8 × 10
18
cells mol
-1
of thymidine incorporated into TCA precipitate (standard error = 1.72 × 10
18
;
n
= 47). The amount of radioactivity in purified DNA increased with decreasing generation time and increasing conversion factor (calculated from the TCA precipitate), corresponding to a decrease in the percentage in protein. The conversion factors calculated from purified DNA or from the TCA precipitate gave the same variability. Conversion factors did not change significantly with the medium, but were significantly higher at 20°C than at 15 and 10°C. A detailed examination of the [
3
H]thymidine concentrations that were needed to achieve maximum labeling in DNA was carried out 6 times during a complete growth cycle. During periods with low generation times and high conversion factors, 15 nM [
3
H]thymidine was enough for the maximum labeling of the TCA precipitate. This suggests that incorporation of [
3
H]thymidine into DNA is probably limited by uptake during periods with generation times of <20 h and that freshwater bacterioplankton cell production sometimes is underestimated when a conversion factor of 2.15 × 10
18
cells mol
-1
of thymidine incorporated is used.
Combined Oxygen and Nitrous Oxide Microsensor for Denitrification Studies
Authors: Revsbech Niels Peter , Nielsen Lars Peter , Christensen Peter Bondo , Sørensen Jan
Published: January 2020

The construction of a microsensor which can be used to measure O
2
and N
2
O simultaneously is described. The microsensor exhibited a linear response to both O
2
and N
2
O, and the response to N
2
O was independent of the O
2
concentration and vice versa. The N
2
O detection limit of a microsensor with a tip diameter of 20 μm was around 1 μmol liter
−1
. The signals for O
2
and N
2
O were affected by hydrogen sulfide, but other interfering agents were not observed in the biofilms and sediments analyzed. Microprofiles of O
2
and N
2
O were measured in a biofilm which was exposed to acetylene to block the N
2
O reductase activity of denitrifying bacteria. O
2
penetrated about 0.5 mm into the biofilm and was not affected by acetylene, but the N
2
O concentration at 1.4 mm depth increased from 32 to 411 μmol liter
−1
after the addition of the inhibitor. The shape of the N
2
O profile after the addition of acetylene showed that denitrification (denitrifying activity) was detectable in all anoxic layers of the biofilm.
Toxicity and partial structure of a hepatotoxic peptide produced by the cyanobacterium Nodularia spumigena Mertens emend. L575 from New Zealand
Authors: Carmichael W W , Eschedor J T , Patterson G M , Moore R E
Published: January 2020

A clonal isolate, termed L575, of the filamentous brackish-water cyanobacterium Nodularia spumigena Mertens emend. was found to produce a potent hepatotoxic peptide (50% lethal intraperitoneal dose for the mouse, 60 micrograms/kg) with chemical and toxicological properties similar to those of the hepatotoxic heptapeptides produced by other freshwater planktonic cyanobacteria. The isolate was made from a water sample collected in Lake Ellesmere, New Zealand, in 1980. The toxin, isolated and purified by high-performance liquid chromatography (HPLC) and analyzed by HPLC amino acid analysis, contained glutamic acid, beta-methyla-spartic acid, and arginine units in equivalent amounts. The fast-atom-bombardment mass spectrum of the toxin indicated the molecular weight to be 824. Batch cultures of strain L575 showed that the toxin content varied between 1.96 and 2.99 mg/g of cells and that a positive correlation between toxin content and chlorophyll a, but not biomass, was present.
Effect of Plant Species and Environmental Conditions on Ice Nucleation Activity of
Pseudomonas syringae
on Leaves
Authors: O'Brien R. Douglas , Lindow Steven E.
Published: January 2020

Selected plant species and environmental conditions were investigated for their influences on expression of ice nucleation activity by 15
Pseudomonas syringae
strains grown on plants in constant-temperature growth chamber studies. Ice nucleation frequencies (INFs), the fraction of cells that expressed ice nucleation at −5 or −9°C, of individual strains varied greatly, both on plants and in culture. This suggests that the probability of frost injury, which is proportional to the number of ice nuclei on leaf surfaces, is strongly determined by the particular bacterial strains that are present on a leaf surface. The INFs of strains were generally higher when they were grown on plants than when they were grown in culture. In addition, INFs in culture did not correlate closely with INFs on plants, suggesting that frost injury prediction should be based on INF measurements of cells grown on plants rather than in culture. The relative INFs of individual strains varied with plant host and environment. However, none of seven plant species tested optimized the INFs of all 15 strains. Similarly, incubation for 48 h at near 100% relative humidity with short photoperiods did not always decrease the INF when compared with a 72 h, 40% relative humidity, long-photoperiod incubation. Pathogenic strains on susceptible hosts were not associated with higher or lower INFs relative to their INFs on nonsusceptible plant species. The ice nucleation activity of individual bacterial strains on plants therefore appears to be controlled by complex and interacting factors such as strain genotype, environment, and host plant species.
Molecular cloning and expression in Escherichia coli of a cellodextrinase gene from Bacteroides succinogenes S85
Authors: Gong J H , Lo R Y , Forsberg C W
Published: January 2020

A DNA fragment coding for a cellodextrinase of Bacteroides succinogenes S85 was isolated by screening of a pBR322 gene library in Escherichia coli HB101. Of 100,000 colonies screened on a complex medium with methylumbelliferyl-beta-D-cellobioside as the indicator substrate, two cellodextrinase-positive clones (CB1 and CB2) were isolated. The DNA inserts from the two recombinant plasmids were 7.7 kilobase pairs in size and had similar restriction maps. After subcloning from pCB2, a 2.5-kilobase-pair insert which coded for cellodextrinase activity was isolated. The enzyme was located in the cytoplasm of the E. coli host. It exhibited no activity on carboxymethyl cellulose, Avicel microcrystalline cellulose, acid-swollen cellulose, or cellobiose but hydrolyzed p-nitrophenyl-beta-D-cellobioside and p-nitrophenyl-beta-D-lactoside. The Km (0.1 mM) for the hydrolysis of p-nitrophenyl-cellobioside by the enzyme expressed in E. coli was similar to that reported for the purified enzyme from B. succinogenes. Expression of the cellodextrinase gene was subjected to catabolite repression by glucose and was not induced by cellobiose. The origin of the DNA insert from B. succinogenes was confirmed by Southern blot analysis. Western blotting (immunoblotting) using antibodies raised against the purified B. succinogenes cellodextrinase revealed a protein with a molecular weight of approximately 50,000 in E. coli clones which comigrated with the native enzyme isolated from B. succinogenes. These data indicate that the cellodextrinase gene expressed in E. coli is fully functional and codes for an enzyme with properties similar to those of the native enzyme.
Associated bacterial flora, growth, and toxicity of cultured benthic dinoflagellates Ostreopsis lenticularis and Gambierdiscus toxicus
Authors: Tosteson T R , Ballantine D L , Tosteson C G , Hensley V , Bardales A T
Published: January 2020

The growth, toxicity, and associated bacterial flora of 10 clonal cultures of the toxic benthic dinoflagellates Ostreopsis lenticularis and Gambierdiscus toxicus isolated from the coastal waters of southwest Puerto Rico have been examined. Clonal cultures of O. lenticularis grew more rapidly and at broader temperature ranges than those of G. toxicus. All five Ostreopsis clones were toxic, while only one of the five Gambierdiscus clones was poisonous. The degree of toxicity among poisonous clones was highly variable. The number of associated bacterial genera and their frequency of occurrence were quite variable among clones of both dinoflagellate genera. Bacterial isolates represented six genera (Nocardia, Pseudomonas, Vibrio, Aeromonas, Flavobacterium, and Moraxella) in addition to coryneform bacteria. Extracts of dinoflagellate-associated bacteria grown in pure culture were not toxic. Gambierdiscus clones were characterized by the frequent presence of Pseudomonas spp. (four of five clones) and the absence of coryneforms. In O. lenticularis, only one of five clones showed the presence of Pseudomonas spp., and Moraxella sp. was absent altogether. Detailed analyses of toxicity and associated microflora in a selected Ostreopsis clone, repeatedly cultivated (four times) over a period of 160 days, showed that peak cell toxicities developed in the late static and early negative culture growth phases. Peak Ostreopsis cell toxicities in the stationary phase of culture growth were correlated with significant increases in the percent total bacteria directly associated with these cells. Changes in the quantity of bacteria directly associated with microalgal cell surfaces and extracellular matrices during culture growth may be related to variability and degree of toxicity in these laboratory-cultured benthic dinoflagellates.
Intact Soil-Core Microcosms for Evaluating the Fate and Ecological Impact of the Release of Genetically Engineered Microorganisms
Authors: Bentjen S. A. , Fredrickson J. K. , Van Voris P. , Li S. W.
Published: January 2020

Intact soil-core microcosms were studied to determine their applicability for evaluating the transport, survival, and potential ecosystem effects of genetically engineered microorganisms before they are released into the environment. Soil-core microcosms were planted with wheat and maize seeds and inoculated with
Azospirillum lipoferum
SpBr17 and SpRG20a Tn
5
mutants, respectively. Microcosm leachate, rhizosphere soil, plant endorhizosphere, insects, and xylem exudate were sampled for
A. lipoferum
Tn
5
mutant populations.
A. lipoferum
Tn
5
populations, determined by most-probable-number technique-DNA hybridization, varied from below detection to 10
6
g of dry root
−1
in the rhizosphere, with smaller populations detected in the endorhizosphere. Intact soil-core microcosms were found to maintain some of the complexities of the natural ecosystem and should be particularly useful for initial evaluations of the fate of plant-associated genetically engineered bacteria.
In vitro metabolism of 2,2'-diaminopimelic acid from gram-positive and gram-negative bacterial cells by ruminal protozoa and bacteria
Authors: Denholm A M , Ling J R
Published: January 2020

Bacillus megaterium GW1 and Escherichia coli W7-M5 were specifically radiolabeled with 2,2'-diamino[G-3H]pimelic acid [( 3H]DAP) as models of gram-positive and gram-negative bacteria, respectively. These radiolabeled bacterial mutants were incubated alone (control) and with mixed ruminal bacteria or protozoa, and the metabolic processes, rates, and patterns of radiolabeled products released from them were studied. Control incubations revealed an inherent difference between the two substrates; gram-positive supernatants consistently contained 5% radioactivity, whereas even at 0 h, those from the gram-negative mutant released 22%. Incubations with ruminal microorganisms showed that the two mutants were metabolized differently and that protozoa were the major effectors of their metabolism. Protozoa exhibited differential rates of engulfment (150 B. megaterium GW1 and 4,290 E. coli W7-M5 organisms per protozoan per h), and they extensively degraded [3H]DAP-labeled B. megaterium GW1 at rates up to nine times greater than those of ruminal bacteria. By contrast, [3H]DAP-labeled E. coli W7-M5 degradation by either ruminal bacteria or ruminal protozoa was more limited. These fundamental differences in the metabolism of the two mutants, especially by ruminal protozoa, were reflected in the patterns and rates of radiolabeled metabolites produced; many were rapidly released from [3H]DAP-labeled B. megaterium GW1, whereas few were slowly released from [3H]DAP-labeled E. coli W7-M5. Most radiolabeled products derived from [3H]DAP-labeled B. megaterium GW1 were peptides of bacterial peptidoglycan origin. The ruminal metabolism of DAP-containing gram-positive and gram-negative bacteria, even with the same peptidoglycan chemotype, is thus likely to be profoundly different.(ABSTRACT TRUNCATED AT 250 WORDS)
Effect of Emulsan on Biodegradation of Crude Oil by Pure and Mixed Bacterial Cultures
Authors: Foght Julia M. , Gutnick David L. , Westlake Donald W. S.
Published: January 2020

Crude oil was treated with purified emulsan, the heteropolysaccharide bioemulsifier produced by
Acinetobacter calcoaceticus
RAG-1. A mixed bacterial population as well as nine different pure cultures isolated from various sources was tested for biodegradation of emulsan-treated and untreated crude oil. Biodegradation was measured both quantitatively and qualitatively. Recovery of
14
CO
2
from mineralized
14
C-labeled substrates yielded quantitative data on degradation of specific compounds, and capillary gas chromatography of residual unlabeled oil yielded qualitative data on a broad spectrum of crude oil components. Biodegradation of linear alkanes and other saturated hydrocarbons, both by pure cultures and by the mixed population, was reduced some 50 to 90% after emulsan pretreatment. In addition, degradation of aromatic compounds by the mixed population was reduced some 90% in emulsan-treated oil. In sharp contrast, aromatic biodegradation by pure cultures was either unaffected or slightly stimulated by emulsification of the oil.
In vitro total-gas, CH4, H2, volatile fatty acid, and lactate kinetics studies on luminal contents from the small intestine, cecum, and colon of the pig
Authors: Robinson J A , Smolenski W J , Ogilvie M L , Peters J P
Published: January 2020

Two experiments were conducted to assess differences in fermentative activities of digesta obtained from various regions of the pig gastrointestinal tract. In experiment 1, the contents of small intestines, ceca, and colons of 110-kg pigs were collected, diluted twofold, and incubated for 2 h at 37 degrees C. In experiment 2, colonic samples from 16,100-kg pigs were similarly treated, except that the incubation period was 5 h. Total gas (gas pressure), CH4, H2, lactate, formate, acetate, propionate, butyrate, valerate, and isovalerate were measured in experiment 1. Only the gas variables were measured in experiment 2. Statistically significant differences (P greater than 0.05) were not observed among the gas production rate estimates across the small-intestinal, cecal, and colonic regions in experiment 1. Furthermore, all the small-intestinal samples and half the cecal samples assayed in experiment 1 were nonmethanogenic. The mean methanogenic and total-gas production rate estimates for the colonic samples in experiment 1 were 0.052 ml g of wet contents-1 h-1 and 1.7 ml of total gas g of wet contents-1 h-1, respectively. No differences in the methanogenic rate estimates were detected between the proximal, middle, and distal thirds of the pig colons (P greater than 0.05). The volatile fatty acid and lactate molar percentages measured in experiment 1 were consistent with previously published observations. Hydrogen accumulated to the greatest extent (7 microM on average) in the in vitro incubations of small-intestinal contents, whereas the H2 concentrations ranged from 0.5 to 1 microM for the incubated cecal and colonic samples in experiment 1.(ABSTRACT TRUNCATED AT 250 WORDS)
Improved electroporation efficiency of intact Lactococcus lactis subsp. lactis cells grown in defined media
Authors: McIntyre D A , Harlander S K
Published: January 2020

The impact of growth conditions on electroporation of Lactococcus lactis subsp. lactis LM0230 (previously designated Streptococcus lactis LM0230) was evaluated. Cells grown in M17 broth supplemented with 0.5% glucose (M17-Glu) and two chemically defined synthetic media, FMC and RPMI 1640, all supplemented with 0.24% DL-threonine or 0.5% glycine, were harvested, washed with double-distilled water, diluted, and porated in the presence of 1 microgram of pGB301 DNA with a Transfector 100 (BTX, Inc., San Diego, Calif.) or a Gene Pulser (Bio-Rad Laboratories, Richmond, Calif.). Transformants were recovered at consistently higher efficiencies for cells grown in FMC or RPMI 1640 (10(3) to 10(4) transformants per micrograms of DNA) than for cells grown in M17-Glu (10(1) to 10(2) transformants per micrograms of DNA). Other parameters influencing electroporation of L. lactis cells grown in chemically defined media were growth phase and final concentration of cells, concentration of plasmid DNA, voltage achieved during poration, and expression conditions. A high degree of variability in transformation efficiencies was evident for replicate samples of cells pulsed with either electroporation machine. A trend toward decreased variability was observed for duplicate samples of cells prepared on the same day. In addition, storage studies done with a large batch of cells prepared on the same day indicated that freezing dry cell pellets at -60 degrees C had no deleterious effect on transformation efficiencies over a 30-day period when a new 0.2-cm cuvette was used for porating each sample.
Effect of Growth Rate and Starvation-Survival on Cellular DNA, RNA, and Protein of a Psychrophilic Marine Bacterium
Authors: Moyer Craig L. , Morita Richard Y.
Published: January 2020

DNA, RNA, and protein concentrations from starved ANT-300 cell populations grown at different growth rates fluctuated corresponding to the three stages of starvation-survival on total and viable cell bases. During stage 1 of starvation-survival, two to three peaks in the concentration levels for all three macromolecules were characteristic. During stage 2, DNA per total cell dropped to between 4.2 and 8.3% of the original amount for all of the cell populations examined, and it stabilized throughout stage 3. The decrease in DNA per cell was also observed in electron micrographs of cellular DNA in unstarved compared with starved cells. The fluctuations of RNA and protein per total cell concentrations observed during stage 2 coincided in all cases, except for the cells from dilution rate (
D
) = 0.015 h
−1
. This ANT-300 cell population showed a decrease in RNA per total cell to only 29.2% and an increase in protein to 129.7% of the original amount after 98 days of starvation. During stage 3, DNA, RNA, and protein concentrations per total cell also stabilized to continuous levels. Cells from the faster-growth-rate cell populations of
D
= 0.170 h
−1
and batch culture had elevated protein per total cell concentrations, which remained primarily residual during the starvation period. Starved cells from
D
= 0.015 h
−1
had estimated nucleoid and cell volumes of 0.018 and 0.05 μm
3
, respectively, yielding a nucleoid volume/cell volume ratio of 0.40. We consider these data to indicate that slow-growth-rate cells are better adapted for starvation-survival than their faster-growth-rate counterparts.
Inducible Secretion of a Cellulase from
Clostridium thermocellum
in
Bacillus subtilis
Authors: Joliff Gwennael , Edelman Alex , Klier Andre , Rapoport Georges
Published: January 2020

A host-vector system for inducible secretion during the logarithmic growth phase in
Bacillus subtilis
has been developed. The
B. subtilis
levansucrase gene promoter and the region encoding its signal sequence have been used. The endoglucanase A of
Clostridium thermocellum
was used as a model protein to test the efficiency of the system. Effective inducible secretion of the endoglucanase A was observed when either the levansucrase signal sequence or its own signal sequence was used. Expression of the endoglucanase A in different genetic backgrounds of
B. subtilis
showed that its regulation was similar to that of levansucrase, and high enzyme activity was recovered from the culture supernatant of a hyperproducing
B. subtilis sacU
(Hy) strain. The molecular weight of 46,000 estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the secreted endoglucanase A is compatible with the calculated molecular weight of the mature polypeptide.
Specificity of Cellular DNA-Binding Sites of Microbial Populations in a Florida Reservoir
Authors: Paul John H. , Pichard Scott L.
Published: January 2020

The substrate specificity of the DNA-binding mechanism(s) of bacteria in a Florida reservoir was investigated in short- and long-term uptake studies with radiolabeled DNA and unlabeled competitors. Thymine oligonucleotides ranging in size from 2 base pairs to 19 to 24 base pairs inhibited DNA binding in 20-min incubations by 43 to 77%. Deoxynucleoside monophosphates, thymidine, and thymine had little effect on short-term DNA binding, although several of these compounds inhibited the uptake of the radiolabel from DNA in 4-h incubations. Inorganic phosphate and glucose-1-phosphate inhibited neither short- nor long-term binding of [
3
H]- or [
32
P]DNA, indicating that DNA was not utilized as a phosphorous source in this reservoir. RNA inhibited both short- and long-term radiolabeled DNA uptake as effectively as unlabeled DNA. Collectively these results indicate that aquatic bacteria possess a generalized nucleic acid uptake/binding mechanism specific for compounds containing phosphodiester bonds and capable of recognizing oligonucleotides as short as dinucleotides. This binding site is distinct from nucleoside-, nucleotide-, phosphomonoester-, and inorganic phosphate-binding sites. Such a nucleic acid-binding mechanism may have evolved for the utilization of extracellular DNA (and perhaps RNA), which is abundant in many marine and freshwater environments.
Occurrence and characteristics of agglutination of Vibrio cholerae by serum from the eastern oyster, Crassostrea virginica
Authors: Tamplin M L , Fisher W S
Published: January 2020

Cell-free hemolymph (serum) of the eastern oyster, Crassostrea virginica, agglutinated Vibrio cholerae, including all O1 serovars and biovars. Seventy-nine other strains of bacteria, including 14 genera and 26 species, were not agglutinated. The A, B, and C factors of O1 antigen were not involved in agglutination. Bacterial agglutinating (BA) activity was demonstrated for oysters inhabiting different environments of the U.S. Atlantic and Gulf coasts. Oyster serum BA titers showed high individual variation. The serum component(s) involved in BA was inhibited by 80 degrees C heat, pronase, EDTA, mucin, and fetuin treatments. N-Acetylneuraminic acid (10 mg/ml) weakly inhibited BA activity. Ligands of V. cholerae were sensitive to neuraminidase and resistant to 80 degrees C and pronase. High salinities (24 and 30%) enhanced BA. Cross-adsorption tests with V. cholerae and human O+ erythrocytes indicated that BA and hemagglutinating activities may involve different serum components. These results imply that the ecology of V. cholerae in C. virginica is influenced by agglutinating activity of oyster serum.
Influence of modified-atmosphere storage on the growth of uninjured and heat-injured Aeromonas hydrophila
Authors: Golden D A , Eyles M J , Beuchat L R
Published: January 2020

The growth of uninjured and heat-injured Aeromonas hydrophila incubated at 5 degrees C (22 days) and 30 degrees C (31 h) under air, N2, and CO2 was investigated. At 30 degrees C, the growth patterns of cells on brain heart infusion agar incubated under air and N2 were similar, although slight differences in the lengths of the lag phases and the final populations were detected. The lag phases of cells incubated under air and N2 were substantially longer at 5 degrees C than at 30 degrees C. The population of uninjured A. hydrophila incubated at 5 degrees C under air and N2 remained constant, whereas the number of injured cells declined before the exponential growth phase. Growth at 5 degrees C was enhanced when uninjured and heat-injured A. hydrophila were incubated under N2. At 30 degrees C, cells incubated under CO2 exhibited noticeably longer lag phases and lower growth rates than did cells incubated under air and N2. The viable populations of uninjured and heat-injured cells incubated at 5 degrees C under CO2 declined steadily throughout incubation.
Growth of genetically engineered Pseudomonas aeruginosa and Pseudomonas putida in soil and rhizosphere
Authors: Yeung K H , Schell M A , Hartel P G
Published: January 2020

The effect of the addition of a recombinant plasmid containing the pglA gene encoding an alpha-1,4-endopolygalacturonase from Pseudomonas solanacearum on the growth of Pseudomonas aeruginosa and Pseudomonas putida in soil and rhizosphere was determined. Despite a high level of polygalacturonase production by genetically engineered P. putida and P. aeruginosa, the results suggest that polygalacturonase production had little effect on the growth of these strains in soil or rhizosphere.
Microbiological Oxidation of Ferrous Iron at Low Temperatures
Authors: Ahonen Lasse , Tuovinen Olli H.
Published: January 2020

Acidophilic iron-oxidizing bacteria were enriched from mine water samples with ferrous sulfate as the substrate at incubation temperatures in the range of 4 to 46°C. After several subcultures at each test temperature except 46°C, which was prohibitive to growth, the rates of iron oxidation were determined in batch cultures. The results yielded linear rates in a semilogarithmic scale. The rate constants of iron oxidation by growing cultures were fitted into the Arrhenius equation, which displayed linearity in the 4 to 28°C range and yielded an activation energy value of 83 ± 3 kJ/mol.
Survey of the Anaerobic Biodegradation Potential of Organic Chemicals in Digesting Sludge
Authors: Battersby Nigel S. , Wilson Valerie
Published: January 2020

The degradation potential of 77 organic chemicals under methanogenic conditions was examined with an anaerobic digesting sludge from the United Kingdom. Degradation was assessed in terms of net total gas (CH
4
plus CO
2
) produced, expressed as a percentage of the theoretical production (ThGP). The compounds tested were selected from various chemical groups and included substituted phenols and benzoates, pesticides, phthalic acid esters, homocyclic and heterocyclic ring compounds, glycols, and monosubstituted benzenes. The results obtained were in good agreement with published surveys of biodegradability in U.S. digesting sludges and other methanogenic environments. In general, the presence of chloro or nitro groups inhibited anaerobic gas production, while carboxyl and hydroxyl groups facilitated biodegradation. The relationship between substituent position and susceptibility to methanogenic degradation was compound dependent. The following chemicals were completely degraded (≥80% ThGP) at a concentration of 50 mg of carbon per liter: phenol, 2-aminophenol, 4-cresol, catechol, sodium benzoate, 4-aminobenzoic acid, 3-chlorobenzoic acid, phthalic acid, ethylene glycol, diethylene glycol, triethylene glycol, sodium stearate, and quinoline. 3-Cresol, 4-chlorobenzoic acid, dimethyl phthalate, and pyridine were partially degraded. Although the remaining chemicals tested were either persistent or toxic, their behavior may differ at more environmentally realistic chemical-to-biomass ratios. Our findings suggest that biodegradability assessments made with sludge from one source can be extrapolated to sludge from another source with a reasonable degree of confidence and should help in predicting the fate of an organic chemical during the anaerobic digestion of sewage sludge.
Pseudomonas pellicle in disinfectant testing: electron microscopy, pellicle removal, and effect on test results
Authors: Cole E C , Rutala W A , Carson J L , Alfano E M
Published: January 2020

Pseudomonas aeruginosa ATCC 15442 is a required organism in the Association of Official Analytical Chemists use-dilution method for disinfectant efficacy testing. When grown in a liquid medium, P. aeruginosa produces a dense mat or pellicle at the broth/air interface. The purpose of this investigation was to examine the pellicle by scanning electron microscopy, to evaluate three pellicle removal methods, and to determine the effect of pellicle fragments on disinfectant efficacy test results. The efficacies of three methods of pellicle removal (decanting, vacuum suction, and filtration) were assessed by quantifying cell numbers on penicylinders. The Association of Official Analytical Chemists use-dilution method was used to determine whether pellicle fragments in the tubes used to inoculate penicylinders affected test results. Scanning electron micrographs showed the pellicle to be a dense mass of intact, interlacing cells at least 10 microns thick. No significant differences in pellicle removal methods were observed, and the presence of pellicle fragments usually increased the number of positive tubes in the use-dilution method significantly.
Significance of microflora in proteolysis in the colon
Authors: Gibson S A , McFarlan C , Hay S , MacFarlane G T
Published: January 2020

Protease activities in human ileal effluent and feces were compared by using a variety of native and diazotized protein substrates. In many cases the diazotized proteins had altered susceptibilities to hydrolysis compared with the native proteins. Proteolytic activity was significantly greater than (P less than 0.001) in small intestinal effluent than in feces (319 +/- 45 and 11 +/- 6 mg of azocasein hydrolyzed per h per g, respectively). Moreover, fecal proteolysis was qualitatively different in that ileal effluent did not hydrolyze the highly globular protein bovine serum albumin, whereas all fecal samples tested degraded this substrate. Inhibition experiments provided further evidence that fecal protease activity differed from that in the small intestine. Physical disruption of fecal bacteria released large quantities of proteases, indicating that the lysis of bacteria in the colon may contribute to the extracellular proteolytic activity in feces. Protease inhibition studies with washed fecal bacteria showed that they produced serine, cystine, and metalloproteases, and experiments with synthetic p-nitroanilide substrates indicated that low levels of trypsin- and chymotrypsin-like activities were associated with whole cells. An elastase-like enzyme was bound to the outer membranes of some fecal bacteria.
Microelectrode Measurements of Nitrate Gradients in the Littoral and Profundal Sediments of a Meso-Eutrophic Lake (Lake Vechten, The Netherlands)
Authors: Sweerts Jean-Pierre R. A. , de Beer Dirk
Published: January 2020

NO
3
−
concentration profiles were measured in the sediments of a meso-eutrophic lake with a newly developed microelectrode. The depth of penetration of NO
3
−
varied from only 1.3 mm in organic-rich profundal silty sediments to 5 mm in organic-poor littoral sandy sediments. The thickness of the zone of denitrification in the organic-rich sediments was <500 μm. Oxygen profiles measured simultaneously revealed that the zone of denitrification was directly adjacent to the aerobic zone. The results demonstrate high denitrification rates (0.26 to 1.31 mmol m
−2
day
−1
) at in situ nitrate concentrations in the overlying water (0.030 mmol liter
−1
) and limitation of denitrification by nitrate availability.
Isolation and characterization of quinoline-degrading bacteria from subsurface sediments
Authors: Brockman F J , Denovan B A , Hicks R J , Fredrickson J K
Published: January 2020

Two gram-negative, motile bacteria isolated from deep subsurface sediments mineralized the nitrogen-containing polyaromatic hydrocarbon quinoline under aerobic conditions and transformed quinoline to soluble intermediates under anaerobic conditions. Many aromatic compounds were also able to serve as the sole source of carbon and energy under aerobic conditions. Rapid aerobic mineralization of quinoline at concentrations as low as 0.002 microgram ml-1 indicates that these organisms possess a high-affinity uptake and utilization system, which may reflect the oligotrophic nature of deep subsurface environments. Both bacteria harbored four plasmids of identical size, ranging from 50 to 440 kilobases.
Kinetics of biotransformation of 1,1,1-trichloroethane by Clostridium sp. strain TCAIIB
Authors: Gälli R , McCarty P L
Published: January 2020

Batch experiments were conducted to examine the effects of high concentrations of 1,1,1-trichloroethane (TCA) on the biotransformation of TCA by Clostridium sp. strain TCAIIB. The biotic dehalogenation of TCA to 1,1-dichloroethane by nongrowing cells was measured at 35 degrees C, and the data were used to obtain the kinetic parameters of the Monod relationship half-velocity coefficient Ks (31 microM) and the coefficient of maximum rate of TCA biotransformation (kTCA; 0.28 mumol per mg per day). The yield of biomass decreased with an increase in the TCA concentration, although TCA concentrations up to 750 microM did not completely inhibit bacterial growth. Also, kTCA was higher in the presence of high concentrations of TCA. A mathematical model based on a modified Monod equation was used to describe the biotransformation of TCA. The abiotic transformation of TCA to 1,1-dichloroethene was measured at 35 degrees C, and the first-order formation rate coefficient for 1,1-dichloroethene (ke) was determined to be 0.86 per year.
Nonphotosynthetic pigmented bacteria in a potable water treatment and distribution system
Authors: Reasoner D J , Blannon J C , Geldreich E E , Barnick J
Published: January 2020

The occurrence of pigmented bacteria in potable water, from raw source water through treatment to distribution water, including dead-end locations, was compared at sample sites in a large municipal water system. Media used to enumerate heterotrophic bacteria and differentiate pigmented colonies were standard method plate count (SPC), m-SPC, and R2A agars, incubated up to 7 days at 35 degrees C. The predominant pigmented bacteria at most sample locations were yellow and orange, with a small incidence of pink organisms at the flowing distribution site. Seasonal variations were seen, with the yellow and orange organisms shifting in dominance. SPC agar was the least productive medium for both heterotroph counts and pigmented bacteria differentiation. At the flowing distribution site, percentages of pigmented bacteria on SPC medium ranged from 2.3 to 9.67 times less than on m-SPC and from 2.3 to 9.86 times less than on R2A. At the same site, seasonal trends in the percentage of pigmented bacteria were the same for m-SPC and R2A media, and the highest and lowest percentages occurred in the fall and winter, respectively. At site 6, there appeared to be an inverse relationship between the yellow and orange pigmented groups, but upon analysis, this did not hold and all correlations between yellow and orange pigmented bacteria were positive. The study results indicate that pigmented bacteria could readily be detected by using plate counting media developed for heterotroph enumeration in potable waters with incubation periods of 7 days. Pigmented bacteria can be used as an additional marker for monitoring changes in water quality. High numbers of heterotrophs, including pigmented forms, were found at dead-end locations, usually in the absence of a free chlorine residual and when the water temperature was greater than 16 degrees C. The association of some pigmented bacteria with nosocomial and other infections raises concern that the organisms may have originated from the potable water supply. High levels of pigmented bacteria could pose an increased health risk to immunologically compromised individuals. Therefore, the bacterial quality of the distribution water should be controlled to prevent the development of high concentrations of heterotrophic plate count bacteria, including the pigmented forms.
Lignin Degradation and Humus Formation in Alluvial Soils and Sediments
Authors: Fustec Eliane , Chauvet Eric , Gas Gilbert
Published: January 2020

The contribution of lignin to the formation of humic compounds was examined in different environments of the terrestrial-aquatic interface in the Garonne River valley in southwestern France. Alluvial soils and submerged or nonsubmerged river and pond sediments containing alder, poplar, or willow [
14
C-lignin]ligno-celluloses were incubated. After a 49-day incubation period, 10 to 15% of labeled lignins in alluvial soils was recovered as evolved
14
CO
2
. In nonsubmerged sediments, 10% of the applied activity was released as
14
CO
2
, and in submerged sediments, only 5% was released after 60 days of incubation. In the different alluvial soils and sediments, the bulk of residual activity (70 to 85%) remained in the two coarsest-grain fractions (2,000 to 100 and 100 to 50 μm). Only 2 to 6% of the residual activity of these two coarse fractions was recovered as humic and fulvic acids, except in the case of alder [
14
C-lignin]lignocellulose, which had decomposed in a soil collected beneath alders. In this one 55% of the residual activity was extracted as humic substances from the 2,000- to 100-μm fraction. Humic and fulvic acids represented from 6 to 50% of the residual activity in the finest-grain fractions (50 to 20 and 20 to 0 μm). The highest percentages were obtained in soil collected beneath alders and in submerged pond sediment. The contribution of different groups of microorganisms, as well as nutrients and clay content, may influence humic-substance formation in such environments. Physical stability also may be an important factor for complex microbial activity involved in this process.