bluuebunny/crossref_metadata_2025_split huggingface dataset:
Combined Molecular and Conventional Analyses of Nitrifying Bacterium Diversity in Activated Sludge:
Nitrosococcus mobilis
and
Nitrospira
-Like Bacteria as Dominant Populations
Authors: Juretschko Stefan , Timmermann Gabriele , Schmid Markus , Schleifer Karl-Heinz , Pommerening-Röser Andreas , Koops Hans-Peter , Wagner Michael 
Published: December 2019

ABSTRACT

The ammonia-oxidizing and nitrite-oxidizing bacterial populations occurring in the nitrifying activated sludge of an industrial wastewater treatment plant receiving sewage with high ammonia concentrations were studied by use of a polyphasic approach. In situ hybridization with a set of hierarchical 16S rRNA-targeted probes for ammonia-oxidizing bacteria revealed the dominance of
Nitrosococcus mobilis
-like bacteria. The phylogenetic affiliation suggested by fluorescent in situ hybridization (FISH) was confirmed by isolation of
N. mobilis
as the numerically dominant ammonia oxidizer and subsequent comparative 16S rRNA gene (rDNA) sequence and DNA-DNA hybridization analyses. For molecular fine-scale analysis of the ammonia-oxidizing population, a partial stretch of the gene encoding the active-site polypeptide of ammonia monooxygenase (
amoA
) was amplified from total DNA extracted from ammonia oxidizer isolates and from activated sludge. However, comparative sequence analysis of 13
amoA
clone sequences from activated sludge demonstrated that these sequences were highly similar to each other and to the corresponding
amoA
gene fragments of
Nitrosomonas europaea
Nm50 and the
N. mobilis
isolate. The unexpected high sequence similarity between the
amoA
gene fragments of the
N. mobilis
isolate and
N. europaea
indicates a possible lateral gene transfer event. Although a
Nitrobacter
strain was isolated, members of the nitrite-oxidizing genus
Nitrobacter
were not detectable in the activated sludge by in situ hybridization. Therefore, we used the rRNA approach to investigate the abundance of other well-known nitrite-oxidizing bacterial genera. Three different methods were used for DNA extraction from the activated sludge. For each DNA preparation, almost full-length genes encoding small-subunit rRNA were separately amplified and used to generate three 16S rDNA libraries. By comparative sequence analysis, 2 of 60 randomly selected clones could be assigned to the nitrite-oxidizing bacteria of the genus
Nitrospira
. Based on these clone sequences, a specific 16S rRNA-targeted probe was developed. FISH of the activated sludge with this probe demonstrated that
Nitrospira
-like bacteria were present in significant numbers (9% of the total bacterial counts) and frequently occurred in coaggregated microcolonies with
N. mobilis
.
Estimation of the State of the Bacterial Cell Wall by Fluorescent In Situ Hybridization
Authors: Bidnenko Elena , Mercier Carine , Tremblay Josselyne , Tailliez Patrick , Kulakauskas Saulius 
Published: December 2019

ABSTRACT
Fluorescent in situ hybridization (FISH) is now a widely used method for identification of bacteria at the single-cell level. With gram-positive bacteria, the thick peptidoglycan layer of a cell wall presents a barrier for entry of horseradish peroxidase (HRP)-labeled probes. Therefore, such probes do not give any signal in FISH unless cells are first treated with enzymes which hydrolyze the peptidoglycan. We explored this feature of FISH to detect cells which have undergone permeabilization due to expression of autolytic enzymes. Our results indicate that FISH performed with HRP-labeled probes provides a sensitive method to estimate the states of cell walls of individual gram-positive bacteria.
Screening of a Fosmid Library of Marine Environmental Genomic DNA Fragments Reveals Four Clones Related to Members of the Order
Planctomycetales
Authors: Vergin Kevin L. , Urbach Ena , Stein Jeffery L. , DeLong Edward F. , Lanoil Brian D. , Giovannoni Stephen J. 
Published: December 2019

ABSTRACT

A fosmid library with inserts containing approximately 40 kb of marine bacterial DNA (J. L. Stein, T. L. Marsh, K. Y. Wu, H. Shizuya, and E. F. DeLong, J. Bacteriol. 178:591–599, 1996) yielded four clones with 16S rRNA genes from the order
Planctomycetales
. Three of the clones belong to the
Pirellula
group and one clone belongs to the
Planctomyces
group, based on phylogenetic and signature nucleotide analyses of full-length 16S rRNA genes. Sequence analysis of the ends of the genes revealed a consistent mismatch in a widely used bacterium-specific 16S rRNA PCR amplification priming site (27F), which has also been reported in some thermophiles and spirochetes.
In Vitro and In Vivo Characterization of a Small-Colony Variant of the Primary Form of
Photorhabdus luminescens
MD (
Enterobacteriaceae
)
Authors: Hu Kaiji , Webster John M. 
Published: December 2019

ABSTRACT

A small-colony variant (Vsm) of the primary form (Vp) of
Photorhabdus luminescens
MD from in vitro and in vivo cultures is described. Unlike the primary form, Vp, the Vsm variant is not the preferred diet of its nematode symbiont, a
Heterorhabditis
sp., does not support development and reproduction of the nematode, and is less pathogenic than Vp to
Galleria mellonella
larvae. Vsm cells were carried by 25% of infective juveniles, but they comprised a very low percentage (∼0.4%) of the total cells carried by the juvenile. In vitro subculture and in vivo injection into the larvae with either Vp or Vsm always produced a mixture of both Vp and Vsm. In nematode-bacterium-infected
G. mellonella
larvae, the Vp population in the hemocoel was high (4 × 10
9
to 5 × 10
9
CFU/g of wet insect tissue) at 24 h after infection, decreased about 10-fold by 48 h, and then regained a high level at day 5 before decreasing at day 7 and then remaining relatively constant through day 15 postinfection. The Vsm population, under the same conditions as those of Vp, increased gradually to a high level (9 × 10
8
CFU/g of wet insect tissue) at day 5 postinfection and then declined gradually through day 15.
Comparative Study of Pressure-Induced Germination of
Bacillus subtilis
Spores at Low and High Pressures
Authors: Wuytack Elke Y. , Boven Steven , Michiels Chris W. 
Published: December 2019

ABSTRACT

We have studied pressure-induced germination of
Bacillus subtilis
spores at moderate (100 MPa) and high (500 to 600 MPa) pressures. Although we found comparable germination efficiencies under both conditions by using heat sensitivity as a criterion for germination, the sensitivity of pressure-germinated spores to some other agents was found to depend on the pressure used. Spores germinated at 100 MPa were more sensitive to pressure (>200 MPa), UV light, and hydrogen peroxide than were those germinated at 600 MPa. Since small, acid-soluble proteins (SASPs) and dipicolinic acid (DPA) are known to be involved in spore resistance to UV light and hydrogen peroxide, we studied the fate of these compounds during pressure germination. DPA was released upon both low- and high-pressure germination, but SASP degradation, which normally accompanies nutrient-induced germination, occurred upon low-pressure germination but not upon high-pressure germination. These results adequately explain the UV and hydrogen peroxide resistance of spores germinated at 600 MPa. The resistance to pressure inactivation of 600-MPa-germinated spores could also, at least partly, be attributed to α/β-type SASPs, since mutants deficient in α/β-type SASPs were more sensitive to inactivation at 600 MPa. Further, germination at 100 MPa resulted in rapid ATP generation, as is the case in nutrient-induced germination, but no ATP was formed during germination at 600 MPa. These results suggest that spore germination can be initiated by low- and high-pressure treatments but is arrested at an early stage in the latter case. The implications for the use of high pressure as a preservation treatment are discussed.
Confirmation of the Human-Pathogenic Microsporidia
Enterocytozoon bieneusi
,
Encephalitozoon intestinalis
, and
Vittaforma corneae
in Water
Authors: Dowd Scot E. , Gerba Charles P. , Pepper Ian L. 
Published: December 2019

ABSTRACT

Microsporidia, as a group, cause a wide range of infections, though two species of microsporidia in particular,
Enterocytozoon bieneusi
and
Encephalitozoon intestinalis
, are associated with gastrointestinal disease in humans. To date, the mode of transmission and environmental occurrence of microsporidia have not been elucidated due to lack of sensitive and specific screening methods. The present study was undertaken with recently developed methods to screen several significant water sources. Water concentrates were subjected to community DNA extraction followed by microsporidium-specific PCR amplification, PCR sequencing, and database homology comparison. A total of 14 water concentrates were screened; 7 of these contained human-pathogenic microsporidia. The presence of
Encephalitozoon intestinalis
was confirmed in tertiary sewage effluent, surface water, and groundwater; the presence of
Enterocytozoon bieneusi
was confirmed in surface water; and the presence of
Vittaforma corneae
was confirmed in tertiary effluent. Thus, this study represents the first confirmation, to the species level, of human-pathogenic microsporidia in water, indicating that these human-pathogenic microsporidia may be waterborne pathogens.
Assessment of Reductive Acetogenesis with Indigenous Ruminal Bacterium Populations and
Acetitomaculum ruminis
Authors: le Van Tricia D. , Robinson Joseph A. , Ralph John , Greening Richard C. , Smolenski Walter J. , Leedle Jane A. Z. , Schaefer Daniel M. 
Published: December 2019

ABSTRACT

The objective of this study was to evaluate the role of reductive acetogenesis as an alternative H
2
disposal mechanism in the rumen. H
2
/CO
2
-supported acetogenic ruminal bacteria were enumerated by using a selective inhibitor of methanogenesis, 2-bromoethanesulfonic acid (BES). Acetogenic bacteria ranged in density from 2.5 × 10
5
cells/ml in beef cows fed a high-forage diet to 75 cells/ml in finishing steers fed a high-grain diet. Negligible endogenous acetogenic activity was demonstrated in incubations containing ruminal contents, NaH
13
CO
3
, and 100% H
2
gas phase since [U-
13
C]acetate, as measured by mass spectroscopy, did not accumulate. Enhancement of acetogenesis was observed in these incubations when methanogenesis was inhibited by BES and/or by the addition of an axenic culture of the rumen acetogen
Acetitomaculum ruminis
190A4 (10
7
CFU/ml). To assess the relative importance of population density and/or H
2
concentration for reductive acetogenesis in ruminal contents, incubations as described above were performed under a 100% N
2
gas phase. Both selective inhibition of methanogenesis and
A. ruminis
190A4 fortification (>10
5
CFU/ml) were necessary for the detection of reductive acetogenesis under H
2
-limiting conditions. Under these conditions, H
2
accumulated to 4,800 ppm. In contrast, H
2
accumulated to 400 ppm in incubations with active methanogenesis (without BES). These H
2
concentrations correlated well with the pure culture H
2
threshold concentrations determined for
A. ruminis
190A4 (3,830 ppm) and the ruminal methanogen 10-16B (126 ppm). The data demonstrate that ruminal methanogenic bacteria limited reductive acetogenesis by lowering the H
2
partial pressure below the level necessary for H
2
utilization by
A. ruminis
190A4.
Sources of
Listeria monocytogenes
Contamination in a Cold-Smoked Rainbow Trout Processing Plant Detected by Pulsed-Field Gel Electrophoresis Typing
Authors: Autio Tiina , Hielm Sebastian , Miettinen Maria , Sjöberg Anna-Maija , Aarnisalo Kaarina , Björkroth Johanna , Mattila-Sandholm Tiina , Korkeala Hannu 
Published: December 2019

ABSTRACT

Sites of
Listeria monocytogenes
contamination in a cold-smoked rainbow trout (
Oncorhynchus mykiss
) processing plant were detected by sampling the production line, environment, and fish at different production stages. Two lots were monitored. The frequency of raw fish samples containing
L. monocytogenes
was low. During processing, the frequency of fish contaminated with
L. monocytogenes
clearly rose after brining, and the most contaminated sites of the processing plant were the brining and postbrining areas. A total of 303 isolates from the raw fish, product, and the environment were characterized by pulsed-field gel electrophoresis (PFGE). PFGE yielded nine pulsotypes, which formed four clusters. The predominating
L. monocytogenes
pulsotypes of the final product were associated with brining and slicing, whereas contaminants of raw fish were not detected in the final product. Air-mediated contamination in the plant could not be proved. In accordance with these results, an
L. monocytogenes
eradication program was planned. The use of hot steam, hot air, and hot water seemed to be useful in eliminating
L. monocytogenes
. None of the control samples taken in the 5 months after the eradication program was implemented contained
L. monocytogenes
.
Morphological and Biochemical Properties of a
Sphaerotilus
sp. Isolated From Paper Mill Slimes
Authors: Pellegrin Véronique , Juretschko Stefan , Wagner Michael , Cottenceau Gilles 
Published: December 2019

ABSTRACT

Four strains of filamentous bacteria were isolated from slimes collected in different paper mill factories. Morphological and physiological characterization of the isolates indicated an affiliation with the genus
Sphaerotilus
. However, while the physiological properties of the isolates were almost identical, pronounced physiological differences between the isolates and
Sphaerotilus natans
DSM 6575
T
, DSM 565, and DSM 566 with respect to their ability to metabolize complex polysaccharides, sugars, polyalcohols, or organic acids as carbon sources were detected. In contrast to the analyzed culture collection strains of
S. natans
, all paper mill isolates were able to grow at elevated temperatures of up to 40°C. Comparative sequence analysis of nearly complete 16S ribosomal DNA (rDNA) sequences from the four new isolates demonstrated that the retrieved sequences were highly similar to each other (99.6 to 99.8% similarity) and to previously published partial 16S rDNA sequences of
S. natans
DSM 6575
T
and ATCC 15291. Polyphasic characterization of the isolated
Sphaerotilus
strains revealed interesting adaptations of the strains to the environmental paper mill conditions with regard to temperature tolerance and utilization of cellulose and starch.
Bacterial Resistance to Ultrasonic Waves under Pressure at Nonlethal (Manosonication) and Lethal (Manothermosonication) Temperatures
Authors: Pagán R. , Mañas P. , Raso J. , Condón S. 
Published: December 2019

ABSTRACT

The decimal reduction times of
Streptococcus faecium
,
Listeria monocytogenes
,
Salmonella enteritidis
, and
Aeromonas hydrophila
corresponding to heat treatment at 62°C were 7.1, 0.34, 0.024, and 0.0096 min, and those corresponding to manosonication treatment (40°C, 200 kPa, 117 μm) were 4.0, 1.5, 0.86, and 0.90 min, respectively. The manosonication decimal reduction times of the four species investigated decreased sixfold when the amplitude was increased from 62 to 150 μm and fivefold when the relative pressure was raised from 0 to 400 kPa. In
L. monocytogenes
,
S. enteritidis
, and
A. hydrophila
, the lethal effect of manothermosonication was the result of the addition of the lethal effects of heat and manosonication, whereas in
S. faecium
it was a synergistic effect.
Chlorine Inactivation of
Sphingomonas
Cells Attached to Goethite Particles in Drinking Water
Authors: Gauthier Vincent , Redercher Stéphane , Block Jean-Claude 
Published: December 2019

ABSTRACT
Bacteria in drinking water, attached or not attached to goethite particles, were disinfected with chlorine. No additional protection was provided to the bacteria by their attachment to particles, and the limited efficiency of inactivation by chlorine was attributed to the presence of bacterial aggregates in both types of suspension.
In Situ, Real-Time Catabolic Gene Expression: Extraction and Characterization of Naphthalene Dioxygenase mRNA Transcripts from Groundwater
Authors: Wilson Mark S. , Bakermans Corien , Madsen Eugene L. 
Published: December 2019

ABSTRACT

We developed procedures for isolating and characterizing in situ-transcribed mRNA from groundwater microorganisms catabolizing naphthalene at a coal tar waste-contaminated site. Groundwater was pumped through 0.22-μm-pore-size filters, which were then frozen in dry ice-ethanol. RNA was extracted from the frozen filters by boiling sodium dodecyl sulfate lysis and acidic phenol-chloroform extraction. Transcript characterization was performed with a series of PCR primers designed to amplify
nahAc
homologs. Several primer pairs were found to amplify
nahAc
homologs representing the entire diversity of the naphthalene-degrading genes. The environmental RNA extract was reverse transcribed, and the resultant mixture of cDNAs was amplified by PCR. A digoxigenin-labeled probe mixture was produced by PCR amplification of groundwater cDNA. This probe mixture hybridized under stringent conditions with the corresponding PCR products from naphthalene-degrading bacteria carrying a variety of
nahAc
homologs, indicating that diverse dioxygenase transcripts had been retrieved from groundwater. Diluted and undiluted cDNA preparations were independently amplified, and 28 of the resulting PCR products were cloned and sequenced. Sequence comparisons revealed two major groups related to the dioxygenase genes
ndoB
and
dntAc
, previously cloned from
Pseudomonas putida
NCIB 9816-4 and
Burkholderia
sp. strain DNT, respectively. A distinctive subgroup of sequences was found only in experiments performed with the undiluted cDNA preparation. To our knowledge, these results are the first to directly document in situ transcription of genes encoding naphthalene catabolism at a contaminated site by indigenous microorganisms. The retrieved sequences represent greater diversity than has been detected at the study site by culture-based approaches.
Ferric Iron Reduction by Bacteria Associated with the Roots of Freshwater and Marine Macrophytes
Authors: King G. M. , Garey Meredith A. 
Published: December 2019

ABSTRACT

In vitro assays of washed, excised roots revealed maximum potential ferric iron reduction rates of >100 μmol g (dry weight)
−1
day
−1
for three freshwater macrophytes and rates between 15 and 83 μmol (dry weight)
−1
day
−1
for two marine species. The rates varied with root morphology but not consistently (fine root activity exceeded smooth root activity in some but not all cases). Sodium molybdate added at final concentrations of 0.2 to 20 mM did not inhibit iron reduction by roots of marine macrophytes (
Spartina alterniflora
and
Zostera marina
). Roots of a freshwater macrophyte,
Sparganium eurycarpum
, that were incubated with an analog of humic acid precursors, anthroquinone disulfate (AQDS), reduced freshly precipitated iron oxyhydroxide contained in dialysis bags that excluded solutes with molecular weights of >1,000; no reduction occurred in the absence of AQDS. Bacterial enrichment cultures and isolates from freshwater and marine roots used a variety of carbon and energy sources (e.g., acetate, ethanol, succinate, toluene, and yeast extract) and ferric oxyhydroxide, ferric citrate, uranate, and AQDS as terminal electron acceptors. The temperature optima for a freshwater isolate and a marine isolate were equivalent (approximately 32°C). However, iron reduction by the freshwater isolate decreased with increasing salinity, while reduction by the marine isolate displayed a relatively broad optimum salinity between 20 and 35 ppt. Our results suggest that by participating in an active iron cycle and perhaps by reducing humic acids, iron reducers in the rhizoplane of aquatic macrophytes limit organic availability to other heterotrophs (including methanogens) in the rhizosphere and bulk sediments.
Identification of Residues in Domain III of
Bacillus thuringiensis
Cry1Ac Toxin That Affect Binding and Toxicity
Authors: Lee Mi Kyong , You Taek H. , Gould Fred L. , Dean Donald H. 
Published: December 2019

ABSTRACT

Alanine substitution mutations in the Cry1Ac domain III region, from amino acid residues 503 to 525, were constructed to study the functional role of domain III in the toxicity and receptor binding of the protein to
Lymantria dispar
,
Manduca sexta
, and
Heliothis virescens
. Five sets of alanine block mutants were generated at the residues
503
SS
504
,
506
NNI
508
,
509
QNR
511
,
522
ST
523
, and
524
ST
525
. Single alanine substitutions were made at the residues
509
Q,
510
N,
511
R, and
513
Y. All mutant proteins produced stable toxic fragments as judged by trypsin digestion, midgut enzyme digestion, and circular dichroism spectrum analysis. The mutations,
503
SS
504
-AA,
506
NNI
508
-AAA,
522
ST
523
-AA,
524
ST
525
-AA, and
510
N-A affected neither the protein’s toxicity nor its binding to brush border membrane vesicles (BBMV) prepared from these insects. Toward
L. dispar
and
M. sexta
, the
509
QNR
511
-AAA,
509
Q-A,
511
R-A, and
513
Y-A mutant toxins showed 4- to 10-fold reductions in binding affinities to BBMV, with 2- to 3-fold reductions in toxicity. Toward
H. virescens
, the
509
QNR
511
-AAA,
509
Q-A,
511
R-A, and
513
Y-mutant toxins showed 8- to 22-fold reductions in binding affinities, but only
509
QNR
511
-AAA and
511
R-A mutant toxins reduced toxicity by approximately three to four times. In the present study, greater loss in binding affinity relative to toxicity has been observed. These data suggest that the residues
509
Q,
511
R, and
513
Y in domain III might be only involved in initial binding to the receptor and that the initial binding step becomes rate limiting only when it is reduced more than fivefold.