bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparison of putative virulence factors in Aeromonas hydrophila strains isolated from the marine environment and human diarrheal cases in southern Italy
Authors: Krovacek K , Pasquale V , Baloda S B , Soprano V , Conte M , Dumontet S
Published: January 2020

Aeromonas hydrophila strains isolated from the same geographical region (southern Italy) but from different sources (sea sediments and human diarrhea cases) were characterized for the production of potential virulence determinants, such as production of cytotoxins, cytotonic toxins, hemolysin, and dermonecrotic factors and their capacity to adhere to human intestinal 407 cells in vitro. The results showed that isolates from both the sources produced all or some of the virulence factors which may be involved in the pathogenesis of Aeromonas-associated infections. Our study indicates that further epidemiological studies are necessary to elucidate the public health significance of infections caused by Aeromonas spp.
A Simple Method That Uses Differential Staining and Light Microscopy To Assess the Selectivity of Wood Delignification by White Rot Fungi
Authors: Srebotnik Ewald , Messner Kurt
Published: January 2020

Cryostat microtome sections of birch wood degraded by white rot fungi were examined by light microscopy after treatment with two stains: astra-blue, which stains cellulose blue only in the absence of lignin, and safranin, which stains lignin regardless of whether cellulose is present. The method provided a simple and reliable screening procedure that distinguishes between fungi that cause decay by selectively removing lignin and those that degrade both cellulose and lignin simultaneously. Moreover, morphological characteristics specific to selective delignification were revealed.
Effect of Residual Lignin Type and Amount on Bleaching of Kraft Pulp by
Trametes versicolor
Authors: Reid Ian D. , Paice Michael G.
Published: January 2020

The white rot fungus
Trametes (Coriolus) versicolor
can delignify and brighten unbleached hardwood kraft pulp within a few days, but softwood kraft pulps require longer treatment. To determine the contributions of higher residual lignin contents (kappa numbers) and structural differences in lignins to the recalcitrance of softwood kraft pulps to biobleaching, we tested softwood and hardwood pulps cooked to the same kappa numbers, 26 and 12. A low-lignin-content (overcooked) softwood pulp resisted delignification by
T. versicolor
, but a high-lignin-content (lightly cooked) hardwood pulp was delignified at the same rate as a normal softwood pulp. Thus, the longer time taken by
T. versicolor
to brighten softwood kraft pulp than hardwood pulp results from the higher residual lignin content of the softwood pulp; possible differences in the structures of the residual lignins are important only when the lignin becomes highly condensed. Under the conditions used in this study, when an improved fungal inoculum was used, six different softwood pulps were all substantially brightened by
T. versicolor.
Softwood pulps whose lignin contents were decreased by extended modified continuous cooking or oxygen delignification to kappa numbers as low as 15 were delignified by
T. versicolor
at the same rate as normal softwood pulp. More intensive O
2
delignification, like overcooking, decreased the susceptibility of the residual lignin in the pulps to degradation by
T. versicolor.
Effect of Motility on Surface Colonization and Reproductive Success of
Pseudomonas fluorescens
in Dual-Dilution Continuous Culture and Batch Culture Systems
Authors: Korber Darren R. , Lawrence John R. , Caldwell Douglas E.
Published: January 2020

The colonization of glass surfaces by motile and nonmotile strains of
Pseudomonas fluorescens
was evaluated by using dual-dilution continuous culture (DDCC), competitive and noncompetitive attachment assays, and continuous-flow slide culture. Both strains possessed identical growth rates whether in the attached or planktonic state. Results of attachment assays using radiolabeled bacteria indicated that both strains obeyed first-order (monolayer) adsorption kinetics in pure culture. However, the motile strain attached about four times more rapidly and achieved higher final cell densities on surfaces than did the nonmotile strain (2.03 × 10
8
versus 5.57 × 10
7
cells vial
-1
) whether evaluated alone or in cocultures containing motile and nonmotile
P. fluorescens.
These kinetics were attributed to the increased transport of motile cells from the bulk aqueous phase to the hydrodynamic boundary layer where bacterial attachment, growth, and recolonization could occur. First-order attachment kinetics were also observed for both strains by using continuous-flow slide culture assays analyzed by image analysis. The DDCC system contained both aqueous and particulate phases which could be diluted independently. DDCC results indicated that when cocultures containing motile and nonmotile
P. fluorescens
colonized solid particles, the motile strain replaced the nonmotile strain in the system over time. Increasing the aqueous-phase rates of dilution decreased the time required for extinction of the nonmotile strain while concurrently decreasing the overall carrying capacity of the DDCC system for both strains. These results confirmed that bacterial motility conveyed a selective advantage during surface colonization even in aqueous-phase systems not dominated by laminar flow.
Effect of Grain Size on Bacterial Penetration, Reproduction, and Metabolic Activity in Porous Glass Bead Chambers
Authors: Sharma Pramod K. , McInerney Michael J.
Published: January 2020

We determined the effects of grain size and nutritional conditions on the penetration rate and metabolic activity of
Escherichia coli
strains in anaerobic, nutrient-saturated chambers packed with different sizes of glass beads (diameters, 116 to 767 μm) under static conditions. The chambers had nearly equal porosities (38%) but different calculated pore sizes (range, 10 to 65 μm). Motile strains always penetrated faster than nonmotile strains, and nutrient conditions that resulted in faster growth rates (fermentative conditions versus nitrate-respiring conditions) resulted in faster penetration rates for both motile and nonmotile strains for all of the bead sizes tested. The penetration rate of nonmotile strains increased linearly when bead size was increased, while the penetration rate of motile strains became independent of the bead size when beads having diameters of 398 μm or greater were used. The rate of H
2
production and the final amount of H
2
produced decreased when bead size was decreased. However, the final protein concentrations were similar in chambers packed with 116-, 192-, and 281-μm beads and were only slightly higher in chambers packed with 398- and 767-μm beads. Our data indicated that conditions that favored faster growth rates also resulted in faster penetration times and that the lower penetration rates observed in chambers packed with small beads were due to restriction of bacterial activity in the small pores. The large increases in the final amount of hydrogen produced without corresponding increases in the final amount of protein made indicated that metabolism became uncoupled from cell mass biosynthesis as bead size increased, suggesting that pore size influenced the efficiency of substrate utilization.
Diel Migrations of Microorganisms within a Benthic, Hypersaline Mat Community
Authors: Garcia-Pichel Ferran , Mechling Margaret , Castenholz Richard W.
Published: January 2020

We studied the diel migrations of several species of microorganisms in a hypersaline, layered microbial mat. The migrations were quantified by repeated coring of the mat with glass capillary tubes. The resulting minicores were microscopically analyzed by using bright-field and epifluorescence (visible and infrared) microscopy to determine depths of coherent layers and were later dissected to determine direct microscopic counts of microorganisms. Microelectrode measurements of oxygen concentration, fiber optic microprobe measurements of light penetration within the mat, and incident irradiance measurements accompanied the minicore sampling. In addition, pigment content, photosynthesis and irradiance responses, the capacity for anoxygenic photosynthesis, and gliding speeds were determined for the migrating cyanobacteria. Heavily pigmented
Oscillatoria
sp. and
Spirulina
cf.
subsalsa
migrated downward into the mat during the early morning and remained deep until dusk, when upward migration occurred. The mean depth of the migration (not more than 0.4 to 0.5 mm) was directly correlated with the incident irradiance over the mat surface. We estimated that light intensity at the upper boundary of the migrating cyanobacteria was attenuated to such an extent that photoinhibition was effectively avoided but that intensities which saturated photosynthesis were maintained through most of the daylight hours. Light was a cue of paramount importance in triggering and modulating the migration of the cyanobacteria, even though the migrating phenomenon could not be explained solely in terms of a light response. We failed to detect diel migration patterns for other cyanobacterial species and filamentous anoxyphotobacteria. The sulfide-oxidizing bacterium
Beggiatoa
sp. migrated as a band that followed low oxygen concentrations within the mat during daylight hours. During the nighttime, part of this population migrated toward the mat surface, but a significant proportion remained deep.
Compositional Changes in Compost during Composting and Growth of
Agaricus bisporus
Authors: Iiyama Kenji , Stone Bruce A. , Macauley Barry J.
Published: January 2020

Samples from conventional compost taken at various stages of composting and mushroom (
Agaricus bisporus
) growth were analyzed for changes in 80% ethanol and water extracts, monosaccharides in acid hydrolysates of polysaccharides, lignin concentration, and lignin structural features. Variable amounts of extraneous inorganic solids in the form of fine sandy particles were removed by sedimentation of the samples in a carbon tetrachloride-dibromomethane mixture. During composting, about two-thirds of the initial wall polysaccharides were consumed by compost microorganisms, and only 17% of the total polysaccharides were used during mushroom production. The relative lignin content of composts as measured by the acetyl bromide procedure increased, both during composting and mushroom growth, and the chemical structure of lignin was altered by condensation and oxidation reactions.
Quantitative cell lysis of indigenous microorganisms and rapid extraction of microbial DNA from sediment
Authors: Moré M I , Herrick J B , Silva M C , Ghiorse W C , Madsen E L
Published: January 2020

This study reports improvements in two of the key steps, lysis of indigenous cells and DNA purification, required for achieving a rapid nonselective protocol for extracting nucleic acids directly from sodium dodecyl sulfate (SDS)-treated sediment rich in organic matter. Incorporation of bead-mill homogenization into the DNA extraction procedure doubled the densitometrically determined DNA yield (11.8 micrograms of DNA.g [dry weight] of sediment-1) relative to incorporation of three cycles of freezing and thawing (5.2 micrograms of DNA.g [dry weight] of sediment-1). The improved DNA extraction efficiency was attributed to increased cell lysis, measured by viable counts of sediment microorganisms which showed that 2 and 8%, respectively, survived the bead-mill homogenization and freeze-thaw procedures. Corresponding measurements of suspensions of viable Bacillus endospores demonstrated that 2 and 94% of the initial number survived. Conventional, laser scanning epifluorescence phase-contrast, and differential interference-contrast microscopy revealed that small coccoid bacterial cells (1.2 to 0.3 micron long) were left intact after combined SDS and bead-mill homogenization of sediment samples. Estimates of the residual fraction of the fluorescently stained cell numbers indicated that 6% (2.2 x 10(8) cells.g [dry weight] of sediment-1) of the original population (3.8 x 10(9) cells.g [dry weight] of sediment-1) remained after treatment with SDS and bead-mill homogenization. Thus, lysis of total cells was less efficient than that of cells which could be cultured. The extracted DNA was used to successfully amplify nahR, the regulatory gene for naphthalene catabolism in Pseudomonas putida G7, by PCR.(ABSTRACT TRUNCATED AT 250 WORDS)
Identification of Chloroacetaldehyde Dehydrogenase Involved in 1,2-Dichloroethane Degradation
Authors: van der Ploeg Jan , Smidt Marten P. , Landa Andrew S. , Janssen Dick B.
Published: January 2020

The degradation of 1,2-dichloroethane and 2-chloroethanol by
Xanthobacter autotrophicus
GJ10 proceeds via chloroacetaldehyde, a reactive and potentially toxic intermediate. The organism produced at least three different aldehyde dehydrogenases, of which one is plasmid encoded. Two mutants of strain GJ10, designated GJ10M30 and GJ10M41, could no longer grow on 2-chloroethanol and were found to lack the NAD-dependent aldehyde dehydrogenase that is the predominant protein in wild-type cells growing on 2-chloroethanol. Mutant GJ10M30, selected on the basis of its resistance to 1,2-dibromoethane, also had lost haloalkane dehalogenase activity and Hg
2+
resistance, indicating plasmid loss. From a gene bank of strain GJ10, different clones that complemented one of these mutants were isolated. In both transconjugants, the aldehyde dehydrogenase that was absent in the mutants was overexpressed. The enzyme was purified and was a tetrameric protein of 55-kDa subunits. The substrate range was rather broad, with the highest activity measured for acetaldehyde. The

K
m

value for chloroacetaldehyde was 160 μM, higher than those for other aldehydes tested. It is concluded that the ability of GJ10 to grow with 2-chloroethanol is due to the high expression level of an aldehyde dehydrogenase with a rather low activity for chloroacetaldehyde.
Fumonisin occurrence in corn from high- and low-risk areas for human esophageal cancer in China
Authors: Yoshizawa T , Yamashita A , Luo Y
Published: January 2020

Forty-seven corn samples were collected in 1989 from Linxian and Shangqiu Counties in Henan Province, the high- and low-risk areas, respectively, for human esophageal cancer in the People's Republic of China. The samples were analyzed for fumonisin (fumonisin B1 [FB1] and FB2) contamination. Of the fumonisin-positive samples, the mean levels in Linxian corn were found to be 872 ng/g for FB1 and 448 ng/g for FB2, while the Shangqiu corns had 890 ng of FB1 and 330 ng of FB2 per g. The incidence of fumonisin contamination of Linxian corn (48%) was about two times higher than that of Shangqiu corn (25%), and the former corn samples were frequently cocontaminated with trichothecenes. Fusarium species isolated from corn from Linxian County produced FB1 at levels ranging from 1,280 to 11,300 micrograms/g.
Sodium chloride induces an NhaA/NhaR-independent acid sensitivity at neutral external pH in Escherichia coli
Authors: Rowbury R J , Goodson M , Humphrey T J
Published: January 2020

Escherichia coli previously grown in low-salt broth, pH 7.0, produced organisms which were markedly more acid sensitive when subsequently cultured in the same broth with 200 mM or more salt (NaCl) added. Induction of acid sensitivity occurred rapidly at both 37 and 30 degrees C, with a substantial effect within 15 min. Sensitization was partially inhibited by chloramphenicol and tetracycline and may depend on both protein synthesis-dependent and -independent physiological changes in the NaCl-induced organisms; sensitization did not result from osmotic shocking on transfer to challenge medium. Induction of acid sensitivity was affected by neither the sodium ion pore inhibitor amiloride nor the DNA synthesis inhibitor nalidixic acid; rifampin had a small effect, similar to that of chloramphenicol. Chlorides of other monovalent cations, especially Li+ and NH4+, also produced sensitization to acid, although CsCl was ineffective but did not interfere with sensitization by NaCl. Other sodium salts were also active as sensitizers, as were chlorides of divalent cations, but although sucrose (but not glycerol) was a good inducer, the results were not fully in accord with triggering of induction solely by the NaCl-associated increase in osmotic pressure. Sensitization was not prevented by deletion of the nhaA, nhaR, or nhaB gene. Acid sensitivity of NaCl-induced cells was slightly reduced after 90 min of growth at 37 degrees C in low-salt broth but was completely lost after 240 min. For NaCl-induced cells, acid killing in challenge media was not inhibited by amiloride. The NaCl-induced sensitization is distinct from the phenomenon of acid sensitivity induction in E. coli at alkaline external pH.
Evaluation of Fleroxacin Activity against Established
Pseudomonas fluorescens
Biofilms
Authors: Korber D. R. , James G. A. , Costerton J. W.
Published: January 2020

Scanning confocal laser microscopy (SCLM) and fluorescent molecular probes were used to evaluate the effect of the fluoroquinolone fleroxacin on the architecture of established
Pseudomonas fluorescens
biofilms. Control
P. fluorescens
biofilms were heterogeneous, consisting of cell aggregates extending from the attachment surface to maximum measured depths of ∼90 μm (mean biofilm depth at 72 h, 42 ± 28 μm) and penetrated by an array of channels. In contrast, fleroxacin-treated biofilms were less deep (mean biofilm depth at 72 h, 29 ± 8 μm), varied little in depth over large areas, and consisted of a homogeneous distribution of cells. Fleroxacin also caused cells to elongate, with cells located near the biofilm-liquid interface lengthening significantly more than cells located at the attachment surface. By using SCLM, acridine orange, and image analysis it was found that ∼59% of cells within fleroxacin-treated biofilms emitted red fluorescence whereas >99% of cells from control biofilms emitted green fluorescence. The fleroxacin-treated cells which emitted red fluorescence were observed to be the population of cells which elongated.
Tellurium and Selenium Resistance in Rhizobia and Its Potential Use for Direct Isolation of
Rhizobium meliloti
from Soil
Authors: Kinkle B. K. , Sadowsky M. J. , Johnstone K. , Koskinen W. C.
Published: January 2020

Forty-eight
Rhizobium
and
Bradyrhizobium
strains were screened for resistance to tellurite, selenite, and selenate. High levels of resistance to the metals were observed only in
Rhizobium meliloti
and
Rhizobium fredii
strains; the MICs were 2 to 8 mM for Te(IV), >200 mM for Se(VI), and 50 to 100 mM for Se(IV). Incorporation of Se and Te into growth media permitted us to directly isolate
R. meliloti
strains from soil. Mutant strains of rhizobia having decreased levels of Se and Te resistance were constructed by Tn
5
mutagenesis and were found to have transposon insertions in DNA fragments of different sizes. Genomic DNAs from Te
r
rhizobium strains failed to hybridize with Te
r
determinants from plasmids RP4, pHH1508a, and pMER610.
Isolation of Vibrio cholerae O139 synonym Bengal from the aquatic environment in Bangladesh: implications for disease transmission
Authors: Islam M S , Hasan M K , Miah M A , Yunus M , Zaman K , Albert M J
Published: January 2020

Currently, Bangladesh is experiencing an epidemic of acute watery diarrhea caused by Vibrio cholerae O139. Surface waters were collected and cultured for vibrious following enrichment. Twelve percent (11 of 92) of samples yielded V. cholerae O139, and all of them were positive for cholera toxin. The data suggest that V. cholerae O139 is easily culturable from surface water samples.
Distribution and evolution of nisin-sucrose elements in Lactococcus lactis
Authors: Rauch P J , Beerthuyzen M M , de Vos W M
Published: January 2020

The distribution, architecture, and conjugal capacity of nisin-sucrose elements in wild-type Lactococcus lactis strains were studied. Element architecture was analyzed with the aid of hybridizations to different probes derived from the nisin-sucrose transposon Tn5276 of L. lactis NIZO R5, including its left and right ends, the nisA gene, and IS1068 (previously designated iso-IS904), located between the left end and the nisA gene. Three classes of nisin-sucrose elements could be distinguished in the 13 strains investigated. Classes I and II consist of conjugative transposons containing a nisA gene and a nisZ gene, respectively. Representative conjugative transposons of these classes include Tn5276 (class I) from L. lactis NIZO R5 and Tn5278 (class II) from L. lactis ILC11. The class II transposon found in L. lactis NCK400 and probably all class II elements are devoid of IS1068-like elements, which eliminates the involvement of an iso-IS1068 element in conjugative transposition. Members of class III contain a nisZ gene, are nonconjugative, and do not contain sequences similar to the left end of Tn5276 at the appropriate position. The class III element from L. lactis NIZO 22186 was found to contain an iso-IS1068 element, termed IS1069, at a position corresponding to that of IS1068 in Tn5276 but in the inverted orientation. The results suggest that an iso-IS1068-mediated rearrangement is responsible for the dislocation of the transposon's left end in this strain. A model for the evolution of nisin-sucrose elements is proposed, and the practical implications for transferring nisin A or nisin Z production and immunity are discussed.
Accumulation of New Polypeptides in Ri T-DNA-Transformed Roots of Tomato (
Lycopersicon esculentum
) during the Development of Vesicular-Arbuscular Mycorrhizae
Authors: Simoneau Philippe , Louisy-Louis Nathalie , Plenchette Christian , Strullu Désiré Georges
Published: January 2020

Root-inducing transferred-DNA (Ri T-DNA)-transformed roots of tomato (
Lycopersicon esculentum
) were in vitro inoculated with surface-sterilized vesicular-arbuscular mycorrhizal leek root pieces. About 1 week after inoculation, the infection of the transformed root culture by the fungal endophyte was confirmed by photonic microscopy. Total proteins were extracted from the mycorrhizal roots and analyzed by two-dimensional polyacrylamide gel electrophoresis. Control gels were run with proteins extracted from noninoculated roots mixed with purified intraradical vesicles and extraradical hyphae. Comparison of the resulting patterns revealed the presence of two polypeptides with estimated apparent masses of 24 and 39 kDa that were detected only in infected roots. Polypeptides with similar migration parameters were not detected in roots challenged with spore extracts, suggesting that the accumulation of the polypeptides was directly linked to root colonization by the fungus rather than to induction by fungus-derived elicitors.
Influence of Plumbing Materials on Biofilm Formation and Growth of
Legionella pneumophila
in Potable Water Systems
Authors: Rogers Julie , Dowsett A. B. , Dennis P. J. , Lee J. V. , Keevil C. W.
Published: January 2020

A two-stage chemostat model of a plumbing system was developed, with tap water as the sole nutrient source. The model system was populated with a naturally occurring inoculum derived from an outbreak of Legionnaires' disease and containing
Legionella pneumophila
along with associated bacteria and protozoa. The model system was used to develop biofilms on the surfaces of a range of eight plumbing materials under controlled, reproducible conditions. The materials varied in their abilities to support biofilm development and the growth of
L. pneumophila.
Elastomeric surfaces had the most abundant biofilms supporting the highest numbers of
L. pneumophila
CFU; this was attributed to the leaching of nutrients for bacterial growth from the materials. No direct relationship existed between total biofouling and the numbers of
L. pneumophila
CFU.
Nitrogen Fixation and Hydrogen Metabolism in Relation to the Dissolved Oxygen Tension in Chemostat Cultures of the Wild Type and a Hydrogenase-Negative Mutant of
Azorhizobium caulinodans
Authors: Boogerd Fred C. , Ferdinandy-van Vlerken Marijke M. A. , Mawadza Crispen , Pronk Annemieke F. , Stouthamer Adriaan H. , van Verseveld Henk W.
Published: January 2020

Both the wild type and an isogenic hydrogenase-negative mutant of
Azorhizobium caulinodans
growing ex planta on N
2
as the N source were studied in succinate-limited steady-state chemostat cultures under 0.2 to 3.0% dissolved O
2
tension. Production or consumption of O
2
, H
2
, and CO
2
was measured with an on-line-connected mass spectrometer. In the range of 0.2 to 3.0%, growth of both the wild type and the mutant was equally dependent on the dissolved O
2
tension: the growth yield decreased, and the specific O
2
consumption and CO
2
production increased. A similar dependency on the dissolved O
2
tension was found for the mutant with 2.5% H
2
in the influent gas. The H
2
/N
2
ratio (moles of H
2
evolved per mole of N
2
consumed via nitrogenase) of the mutant, growing with or without 2.5% H
2
, increased with increasing dissolved O
2
tensions. This increase in the H
2
/N
2
ratio was small but significant. The dependencies of the ATP/N
2
ratio (moles of ATP consumed per mole of N
2
fixed) and the ATP/2e
-
ratio [moles of ATP consumed per mole of electron pairs transferred from NAD(P)H to nitrogenase] on the dissolved O
2
tension were estimated. These dependencies were interpreted in terms of the physiological concepts of respiratory protection and autoprotection.
Autotrophic, Hydrogen-Oxidizing, Denitrifying Bacteria in Groundwater, Potential Agents for Bioremediation of Nitrate Contamination
Authors: Smith Richard L. , Ceazan Marnie L. , Brooks Myron H.
Published: January 2020

Addition of hydrogen or formate significantly enhanced the rate of consumption of nitrate in slurried core samples obtained from an active zone of denitrification in a nitrate-contaminated sand and gravel aquifer (Cape Cod, Mass.). Hydrogen uptake by the core material was immediate and rapid, with an apparent

K
m

of 0.45 to 0.60 μM and a
V
max
of 18.7 nmol cm
-3
h
-1
at 30°C. Nine strains of hydrogen-oxidizing denitrifying bacteria were subsequently isolated from the aquifer. Eight of the strains grew autotrophically on hydrogen with either oxygen or nitrate as the electron acceptor. One strain grew mixotrophically. All of the isolates were capable of heterotrophic growth, but none were similar to
Paracoccus denitrificans
, a well-characterized hydrogen-oxidizing denitrifier. The kinetics for hydrogen uptake during denitrification were determined for each isolate with substrate depletion progress curves; the

K
m

s ranged from 0.30 to 3.32 μM, with
V
max
s of 1.85 to 13.29 fmol cell
-1
h
-1
. Because these organisms appear to be common constituents of the in situ population of the aquifer, produce innocuous end products, and could be manipulated to sequentially consume oxygen and then nitrate when both were present, these results suggest that these organisms may have significant potential for in situ bioremediation of nitrate contamination in groundwater.
Enzyme Immunoassay Detection of
Nitrosomonas europaea
Authors: Saraswat N. , Alleman J. E. , Smith T. J.
Published: January 2020

An exploratory effort to selectively detect the presence of a nitrifying bacterium,
Nitrosomonas europaea
, successfully demonstrated the fundamental utility of an enzyme-based immunoassay protocol. The applied polyclonal antibody test seemingly offered a marked improvement over the available analytical options, including plating, activity, and fluorescence immunoassay techniques. Following an initial purification step to enhance overall specificity, this procedure had an apparent lower limit of detection of ∼5 × 10
6
cells per ml. Tests conducted with activated sludge samples exhibited a distinct difference between nitrifying and nonnitrifying mixed liquors, although the highest
Nitrosomonas
levels observed (i.e., at 1 to 2% of the overall viable cell density) were relatively close to the latter detection boundary.
4-Ethylphenol metabolism by Aspergillus fumigatus
Authors: Jones K H , Trudgill P W , Hopper D J
Published: January 2020

Aspergillus fumigatus ATCC 28282 was found to be capable of growth on 4-ethylphenol as its sole carbon and energy source. A pathway for the metabolism of this compound has been proposed. The initial step involves hydroxylation of the methylene group of 4-ethylphenol to form 1-(4'-hydroxyphenyl)ethanol, followed by oxidation to 4-hydroxyacetophenone. The hydroxylase was NADPH and oxygen dependent, which is a characteristic of a monooxygenase type of enzyme. The 1-(4'-hydroxyphenyl)ethanol isolated from growth medium was a racemic mixture of R-(+) and S-(-) enantiomers. 4-Hydroxyacetophenone undergoes an NADPH-dependent Baeyer-Villiger type of oxygenation to give 4-hydroxyphenyl acetate, which is hydrolyzed to form hydroquinone (1,4-dihydroxybenzene). Hydroxylation of hydroquinone by an NADPH-dependent enzyme produces 1,2,4-trihydroxybenzene, the ring fission substrate, which is cleaved by ortho fission to form maleylacetate. The pathway was elucidated by various kinds of investigations. Analysis of culture medium sampled during growth on 4-ethylphenol revealed the transient appearance of 1-(4'-hydroxyphenyl)ethanol, 4-hydroxyacetophenone, and hydroquinone. Cells grown on 4-ethylphenol were able to oxidize all of these compounds immediately, whereas oxidation by succinate-grown cells showed a lag period. Extracts prepared from cells grown on 4-ethylphenol contained enzyme activities for all of the proposed steps. Apart from a low level of esterase activity towards 4-hydroxyphenyl acetate, extracts prepared from cells grown on succinate did not contain any of these enzyme activities.
Characterization of a restriction endonuclease, PhaI, from Pasteurella haemolytica serotype A1 and protection of heterologous DNA by a cloned PhaI methyltransferase gene
Authors: Briggs R E , Tatum F M , Casey T A , Frank G H
Published: January 2020

Pasteurella haemolytica is the leading cause of economic loss to the beef cattle industry in the United States and an important etiologic agent worldwide. Study of P. haemolytica is hindered by researchers' inability to genetically manipulate the organism. A new restriction endonuclease, PhaI, an isoschizomer of SfaNI (R. J. Roberts, Methods Enzymol. 65:19-36, 1980), was isolated from P. haemolytica serotype 1, strain NADC-D60, obtained from pneumonic bovine lung. PhaI recognizes the 5-base nonpalindromic sequences 5'-GCATC-3' and 5'-GATGC-3'. Cleavage occurs 5 bases 3' from the former recognition site and 9 bases 5' from the latter recognition site. A gene encoding a methyltransferase which protects against PhaI cleavage was cloned from P. haemolytica NADC-D60 into Escherichia coli. Whereas unmethylated plasmid DNA containing a P. haemolytica origin of replication was unable to transform P. haemolytica when introduced by electroporation, the same plasmid DNA obtained from E. coli which contained a cloned PhaI methyltransferase gene could do so. The data indicate that PhaI is an effective barrier to the introduction and establishment of exogenous DNA in P. haemolytica.
Sunlight Inactivation of Enterococci and Fecal Coliforms in Sewage Effluent Diluted in Seawater
Authors: Davies-Colley Robert J. , Bell Robert G. , Donnison Andrea M.
Published: January 2020

Inactivation (loss of culturability) by sunlight of enterococci and fecal coliforms within sewage effluent diluted in seawater was investigated in field experiments. In most experiments, 500-ml flasks of pure silica were used to confine activated sludge effluent diluted to 2% (vol/vol) in seawater. Inactivation of bacteria in these flasks (diameter, 0.1 m) was faster than in either open chambers (depth, 0.25 m) or patches of dyed effluent (depth of order, 1 m), probably because of the longer light paths in the latter two types of experiment, which caused greater attenuation of sunlight. Inactivation of 90% of enterococci generally required 2.3 times the insolation required for 90% inactivation of fecal coliforms, because of both the presence of larger initial shoulders on survival curves and a lower final inactivation rate. Two parameters are required to model inactivation of enterococci, a shoulder constant as well as a rate coefficient. The depth dependence of inactivation rate for both fecal indicators matched the attenuation profile of UV-A radiation at about 360 nm. Inactivation by UV-B radiation (290 to 320 nm), which penetrates much less into seawater, is of minor importance compared with the UV-A and visible radiation in sunlight, contrary to expectations in consideration of published action spectra for bacterial inactivation.
Inactivation of Escherichia coli O157:H7, salmonellae, and Campylobacter jejuni in raw ground beef by gamma irradiation
Authors: Clavero M R , Monk J D , Beuchat L R , Doyle M P , Brackett R E
Published: January 2020

Raw ground beef patties inoculated with stationary-phase cells of Escherichia coli O157:H7, salmonellae, or Campylobacter jejuni were subjected to gamma irradiation (60Co) treatment, with doses ranging from 0 to 2.52 kGy. The influence of two levels of fat (8 to 14% [low fat] and 27 to 28% [high fat]) and temperature (frozen [-17 to -15 degrees C] and refrigerated [3 to 5 degrees C]) on the inactivation of each pathogen by irradiation was investigated. In ascending order of irradiation resistance, the D10 values ranged from 0.175 to 0.235 kGy (C. jejuni), from 0.241 to 0.307 kGy (E. coli O157:H7), and from 0.618 to 0.800 kGy (salmonellae). Statistical analysis revealed that E. coli O157:H7 had a significantly (P < 0.05) higher D10 value when irradiated at -17 to -15 degrees C than when irradiated at 3 to 5 degrees C. Regardless of the temperature during irradiation, the level of fat did not have a significant effect on the D10 value. Salmonellae behaved like E. coli O157:H7 in low-fat beef, but temperature did not have a significant effect when the pathogen was irradiated in high-fat ground beef. Significantly higher D10 values were calculated for C. jejuni irradiated in frozen than in refrigerated low-fat beef. C. jejuni was more resistant to irradiation in low-fat beef than in high-fat beef when treatment was at -17 to -15 degrees C. Regardless of the fat level and temperature during inactivation, these pathogens were highly sensitive to gamma irradiation.(ABSTRACT TRUNCATED AT 250 WORDS)
Estimation of Nitrification and Denitrification from Microprofiles of Oxygen and Nitrate in Model Sediment Systems
Authors: Jensen Kim , Sloth Niels P. , Risgaard-Petersen Nils , Rysgaard Søren , Revsbech Niels Peter
Published: January 2020

The coupling between nitrification and denitrification and the regulation of these processes by oxygen were studied in freshwater sediment microcosms with O
2
and NO
3
-
microsensors. Depth profiles of nitrification (indicated as NO
3
-
production), denitrification (indicated as NO
3
-
consumption), and O
2
consumption activities within the sediment were calculated from the measured concentration profiles. From the concentration profiles, it was furthermore possible to distinguish between the rate of denitrification based on the diffusional supply of NO
3
-
from the overlying water and the rate based on NO
3
-
supplied by benthic nitrification (

D
w

and

D
n

, respectively). An increase in O
2
concentration caused a deeper O
2
penetration while a decrease in

D
w

and an increase in

D
n

were observed. The relative importance for total denitrification of NO
3
-
produced by nitrification thus increased compared with NO
3
-
supplied from the water phase. The decrease in

D
w

at high oxygen was due to an increase in diffusion path for NO
3
-
from the overlying water to the denitrifying layers in the anoxic sediment. At high O
2
concentrations, nitrifying activity was restricted to the lower part of the oxic zone where there was a continuous diffusional supply of NH
4
+
from deeper mineralization processes, and the long diffusion path from the nitrification zone to the overlying water compared with the path to the denitrifying layers led to a stimulation in

D
n

.
Siderophore-Mediated Iron Sequestering by
Shewanella putrefaciens
Authors: Gram Lone
Published: January 2020

The iron-sequestering abilities of 51 strains of
Shewanella putrefaciens
isolated from different sources (fish, water, and warm-blooded animals) were assessed. Thirty strains (60%) produced siderophores in heat-sterilized fish juice as determined by the chrome-azurol-S assay. All cultures were negative for the catechol-type siderophore, whereas 24 of the 30 siderophore-producing strains tested positive in the Csáky test, indicating the production of siderophores of the hydroxamate type. Siderophore-producing
S. putrefaciens
could to some degree cross-feed on the siderophores of other
S. putrefaciens
strains and on compounds produced by an
Aeromonas salmonicida
strain under iron-limited conditions. The siderophores of
S. putrefaciens
were not sufficiently strong to inhibit growth of other bacteria under iron-restricted conditions. However, siderophore-producing
Pseudomonas
bacteria were always inhibitory to
S. putrefaciens
under iron-limited conditions. Growth of siderophore-producing strains under iron-limited conditions induced the formation of one major new outer membrane protein of approximately 72 kDa. Two outer membrane proteins of approximately 53 and 23 kDa were not seen when iron was restricted.
Efficacy of Selected Respiratory Protective Equipment Challenged with
Bacillus subtilis
subsp.
niger
Authors: Johnson B. , Martin D. D. , Resnick I. G.
Published: January 2020

The efficacy of powered air-purifying respirators, surgical masks, dust/mist respirators, and high-efficiency respirators were tested with a biological aerosol under simulated breathing conditions. Protective ability ranged from 67 to 99.95%. The majority of penetration in negative-pressure respirators occurred at the face-mask interface rather than through the filter material.
Rates of Benthic Protozoan Grazing on Free and Attached Sediment Bacteria Measured with Fluorescently Stained Sediment
Authors: Starink Mathieu , Krylova Irina N. , Bär-Gilissen Marie-José , Bak Rolf P. M. , Cappenberg Thomas E.
Published: January 2020

In order to determine the importance of benthic protozoa as consumers of bacteria, grazing rates have been measured by using monodispersed fluorescently labeled bacteria (FLB). However, high percentages of nongrazing benthic protists are reported in the literature. These are related to serious problems of the monodispersed FLB method. We describe a new method using 5-(4,6-dichlorotriazin-2-yl)-aminofluorescein (DTAF)-stained sediment to measure in situ bacterivory by benthic protists. This method is compared with the monodispersed FLB technique. Our estimates of benthic bacterivory range from 61 to 73 bacteria protist
-1
h
-1
and are about twofold higher than the results of the monodispersed FLB method. The number of nongrazing protists after incubation for 15 min with DTAF-stained sediment is in agreement with theoretical expectation. We also tested the relative affinity for FLB of protists and discuss the results with respect to a grazing model.
Uptake of Zinc in
Pseudomonas
sp. Strain UDG26
Authors: Mago Rohit , Srivastava Sheela
Published: January 2020

Zinc resistance in
Pseudomonas
sp. strain UDG26 was inducible. Induction led to enhanced uptake of the metal. A zinc-sensitive variant (UDG86) took up significantly less metal ion than the resistant one did. The affinity of uninduced and sensitive cells to zinc was less than that of resistant, induced cells. Metal accumulation by induced cells was not inhibited by azide, while 2,4-dinitrophenol and
N-N′
-dicyclohexylcarbodiimide enhanced zinc uptake because of inhibition of efflux. Transcription and translation inhibitors drastically reduced zinc accumulation, bringing it to the level found in the sensitive strain. These results suggest the involvement of protein(s) in zinc resistance.
Evidence for a novel pathway in the degradation of fluorene by Pseudomonas sp. strain F274
Authors: Grifoll M , Selifonov S A , Chapman P J
Published: January 2020

A fluorene-utilizing microorganism, identified as a species of Pseudomonas, was isolated from soil severely contaminated from creosote use and was shown to accumulate six major metabolites from fluorene in washed-cell incubations. Five of these products were identified as 9-fluorenol, 9-fluorenone, (+)-1,1a-dihydroxy-1-hydro-9-fluorenone, 8-hydroxy-3,4-benzocoumarin, and phthalic acid. This last compound was also identified in growing cultures supported by fluorene. Fluorene assimilation into cell biomass was estimated to be approximately 50%. The structures of accumulated products indicate that a previously undescribed pathway of fluorene catabolism is employed by Pseudomonas sp. strain F274. This pathway involves oxygenation of fluorene at C-9 to give 9-fluorenol, which is then dehydrogenated to the corresponding ketone, 9-fluorenone. Dioxygenase attack on 9-fluorenone adjacent to the carbonyl group gives an angular diol, 1,1a-dihydroxy-1-hydro-9-fluorenone. Identification of 8-hydroxy-3,4-benzocoumarin and phthalic acid suggests that the five-membered ring of the angular diol is opened first and that the resulting 2'-carboxy derivative of 2,3-dihydroxy-biphenyl is catabolized by reactions analogous to those of biphenyl degradation, leading to the formation of phthalic acid. Cell extracts of fluorene-grown cells possessed high levels of an enzyme characteristic of phthalate catabolism, 4,5-dihydroxyphthalate decarboxylase, together with protocatechuate 4,5-dioxygenase. On the basis of these findings, a pathway of fluorene degradation is proposed to account for its conversion to intermediary metabolites. A range of compounds with structures similar to that of fluorene was acted on by fluorene-grown cells to give products consistent with the initial reactions proposed.
Anaerobic degradation of methylmercaptan and dimethyl sulfide by newly isolated thermophilic sulfate-reducing bacteria
Authors: Tanimoto Y , Bak F
Published: January 2020

The complete oxidation of methylmercaptan (MSH) and dimethyl sulfide (DMS) with sulfate or nitrate as electron acceptors was observed in enrichment cultures and dilution series using thermophilic fermentor sludge as the inoculum. Three new strains of thermophilic sulfate reducers were isolated in pure culture (strains MTS5, TDS2, and SDN4). Strain MTS5 grew on MSH and strain TDS2 grew on DMS whereas strain SDN4 grew on either MSH or DMS. The cellular growth yields were 2.57 g (dry weight)/mol of MSH for strain MTS5 and 6.02 g (dry weight)/mol of DMS for strain TDS2. All strains used sulfate, sulfite, or thiosulfate as electron acceptors, but only strain SDN4 used nitrate. DMS and MSH were oxidized to CO2 and sulfide with either sulfate or nitrate as the electron acceptor. Sulfate was stoichiometrically reduced to sulfide while nitrate was reduced to ammonium. All strains were motile rods, required biotin for growth, lacked desulfoviridin, had DNA with G+C contents of 48 to 57 mol% and probably belonged to the genus Desulfotomaculum. This is the first report of the oxidation of MSH and DMS by pure cultures of sulfate-reducing bacteria.
Sequence analysis of 16S rRNA from mycoplasmas by direct solid-phase DNA sequencing
Authors: Pettersson B , Johansson K E , Uhlén M
Published: January 2020

Automated solid-phase DNA sequencing was used for determination of partial 16S ribosomal DNA sequences of mycoplasmas. The sequence information was used to establish phylogenetic relationships of 11 different mycoplasmas whose 16S rRNA sequences had not been determined earlier. A biotinylated fragment corresponding to positions 344 to 939 in the Escherichia coli sequence was generated by PCR. The PCR product was immobilized onto streptavidin-coated paramagnetic beads, and direct sequencing was performed in both directions. One previously unclassified avian mycoplasma was found to belong to the Mycoplasma lipophilum cluster of the hominis group. Microheterogeneities were discovered in the rRNA operons of Mycoplasma mycoides subsp. mycoides (SC type), confirming the existence of two different rRNA operons. The 16S rRNA sequence of M. mycoides subsp. capri was identical to that of M. mycoides subsp. mycoides (type SC), except that no microheterogeneities were revealed. Furthermore, automated solid-phase DNA sequencing was used to identify a mycoplasmal contamination of a cell culture as Mycoplasma hyorhinis, which proved to be very difficult by conventional methods. The results suggest that the direct solid-phase DNA sequencing procedure is a powerful tool for identification of mycoplasmas and is also useful in taxonomic studies.
Structural and functional analysis of pyruvate kinase from Corynebacterium glutamicum
Authors: Jetten M S , Gubler M E , Lee S H , Sinskey A J
Published: January 2020

Pyruvate kinase activity is an important element in the flux control of the intermediate metabolism. The purified enzyme from Corynebacterium glutamicum demonstrated a marked sigmoidal dependence of the initial rate on the phosphoenolpyruvate concentration. In the presence of the negative allosteric effector ATP, the phosphoenolpyruvate concentration at the half-maximum rate (S0.5) increased from 1.2 to 2.8 mM, and cooperation, as expressed by the Hill coefficient, increased from 2.0 to 3.2. AMP promoted opposite effects: the S0.5 was decreased to 0.4 mM, and the enzyme exhibited almost no cooperation. The maximum reaction rate was 702 U/mg, which corresponded to an apparent kcat of 2,540 s-1. The enzyme was not influenced by fructose-1,6-diphosphate and used Mn2+ or Co2+ as cations. Sequence determination of the C. glutamicum pyk gene revealed an open reading frame coding for a polypeptide of 475 amino acids. From this information and the molecular mass of the native protein, it follows that the pyruvate kinase is a tetramer of 236 kDa. Comparison of the deduced polypeptide sequence with the sequences of other bacterial pyruvate kinases showed 39 to 44% homology, with some regions being very strongly conserved.
Dual Mechanisms of Tricarboxylate Transport and Catabolism by
Acidaminococcus fermentans
Authors: Cook Gregory M. , Russell James B.
Published: January 2020

Acidaminococcus fermentans
utilized citrate or the citrate analog aconitate as an energy source for growth, and these tricarboxylates were used simultaneously. Citrate utilization and uptake showed biphasic kinetics. High-affinity citrate uptake had a

K
t

of 40 μM, but the V
max
was only 25 nmol/mg of protein per min. Low-affinity citrate utilization had a 10-fold higher V
max
, but the

K
s

was greater than 1.0 mM. Aconitate was a competitive inhibitor (

K
i

= 34μM) of high-affinity citrate uptake, but low-affinity aconitate utilization had a 10-fold-lower requirement for sodium than did low-affinity citrate utilization. On the basis of this large difference in sodium requirements, it appeared that
A. fermentans
probably has two systems of tricarboxylate uptake: (i) a citrate/aconitate carrier with a low affinity for sodium and (ii) an aconitate carrier with a high affinity for sodium. Citrate was catabolized by a pathway involving a biotin-requiring, avidin-sensitive, sodium-dependent, membrane-bound oxaloacetate decarboxylase. The cells also had aconitase, but this enzyme was unable to convert citrate to isocitrate. Since cell-free extracts converted either aconitate or glutamate to 2-oxoglutarate, it appeared that aconitate was being catabolized by the glutaconyl-CoA decarboxylase pathway. Exponentially growing cultures on citrate or citrate plus aconitate were inhibited by the sodium/proton antiporter, monensin. Because monensin had no effect on cultures growing with aconitate alone, it appeared that citrate metabolism was acting as an inducer of monensin sensitivity.
A. fermentans
cells always had a low proton motive force (<50 mV), and cells treated with the protonophore TCS (3,3′,4′,5-tetrachlorosalicylanide) grew even though the proton motive force was less than 20 mV. On the basis of these results, it appeared that
A. fermentans
was depending almost exclusively on a sodium motive force for its membrane energetics.