bluuebunny/crossref_metadata_2025 huggingface dataset:
The Gut of the Soil MicroarthropodFolsomia candida(Collembola) Is a Frequently Changeable but Selective Habitat and a Vector for Microorganisms
Authors: Thimm Torsten , Hoffmann Andrea , Borkott Heinz , Charles Munch Jean , Tebbe Christoph C.
Published: December 2019

Interaction potentials between soil microarthropods and microorganisms were investigated withFolsomia candida(Insecta, Collembola) in microcosm laboratory experiments. Microscopic analysis revealed that the volumes of the simple, rod-shaped guts of adult specimens varied with their feeding activity, from 0.7 to 11.2 nl. A dense layer of bacterial cells, associated with the peritrophic membrane, was detected in the midgut by scanning electron microscopy. Depending on the molting stage, which occurred at intervals of approximately 4 days, numbers of heterotrophic, aerobic gut bacteria changed from 4.9 × 102to 2.3 × 106CFU per specimen. A total of 11 different taxonomic bacterial groups and the filamentous fungusAcremonium charticolawere isolated from the guts of fiveF. candidaspecimens. The most abundant isolate was related toErwinia amylovora(96.2% DNA sequence similarity to its 16S rRNA gene).F. candidapreferred to feed onPseudomonas putidaand three indigenous gut isolates rather than eight different type culture strains. When luciferase reporter gene-tagged bacterial strains were pulse fed toF. candida, gut isolates were continuously shed for 8 days to several weeks butEscherichia coliHB101 was shed for only 1 day. Ratios of ingested to released bacterial cells demonstrated that populations of nonindigenous gut bacteria likeSinorhizobium melilotiL33 andE. coliHB101 were reduced by more than 4 orders of magnitude but that the population of gut isolateAlcaligenes faecalisHR4 was reduced only 500-fold. This work demonstrates thatF. candidarepresents a frequently changeable but selective habitat for bacteria in terrestrial environments and that microarthropods have to be considered factors that modify soil microbial communities.
In Situ Detection of High Levels of Horizontal Plasmid Transfer in Marine Bacterial Communities
Authors: Dahlberg Cecilia , Bergström Maria , Hermansson Malte
Published: December 2019

Gene transfer of the conjugative plasmid pBF1 from
Pseudomonas putida
to indigenous bacteria in seawater was investigated with a detection system for gene transfer based on the green fluorescent protein (GFP) (C. Dahlberg et al., Mol. Biol. Evol. 15:385–390, 1998). pBF1 was tagged with the
gfp
gene controlled by a
lac
promoter which is down regulated in the donor cell by a chromosomal repressor (
lacI
q
). The plasmid donor cells (
Pseudomonas putida
KT2442) subsequently do not express
gfp
. Transfer to recipient strains lacking the repressor results in expression of
gfp
. The transconjugant can subsequently be detected by epifluorescence microscopy on a single-cell level. By using this method, transfer of pBF1::
gfp
and expression of the
gfp
gene were first shown to occur during nutrient-limiting conditions to several defined recipient bacteria in artificial seawater. Second, we measured transfer of pBF1 from
P. putida
to the marine bacterial community directly in seawater samples, on a single-cell level, without limiting the detection of gene transfer to the culturable fraction of bacteria. Plasmid transfer was detected on surfaces and in bulk seawater. Seawater bacteria with different morphologies were shown to receive the plasmid. Gene transfer frequencies of 2.3 × 10
−6
to 2.2 × 10
−4
transconjugants per recipient were recorded after 3 days of incubation.
Association of Marine Archaea with the Digestive Tracts of Two Marine Fish Species
Authors: van der Maarel Marc J. E. C. , Artz Rebekka R. E. , Haanstra René , Forney Larry J.
Published: December 2019

Recent studies have shown that archaea which were always thought to live under strict anoxic or extreme environmental conditions are also present in cold, oxygenated seawater, soils, the digestive tract of a holothurian deep-sea-deposit feeder, and a marine sponge. In this study, we show, by using PCR-mediated screening in other marine eukaryotes, that marine archaea are also present in the digestive tracts of flounder and grey mullet, two fish species common in the North Sea, in fecal samples of flounder, and in suspended particulate matter of the North Sea water column. No marine archaea could be detected in the digestive tracts of mussels or the fecal pellets of a copepod species. The archaeal 16S ribosomal DNA clone libraries of feces of flounder and the contents of the digestive tracts of grey mullet and flounder were dominated by group II marine archaea. The marine archaeal clones derived from flounder and grey mullet digestive tracts and feces formed a distinct cluster within the group II marine archaea, with 76.7 to 89.8% similarity to previously described group II clones. Fingerprinting of the archaeal community of flounder digestive tract contents and feces by terminal restriction fragment length polymorphism of archaeal 16S rRNA genes after restriction with
Hha
I showed a dominant fragment at 249 bp, which is likely to be derived from group II marine archaea. Clones of marine archaea that were closely related to the fish-associated marine archaea clones were obtained from suspended particulate matter of the water column at two stations in the North Sea. Terminal restriction fragment length polymorphism fingerprinting of the archaeal community present in suspended particulate matter showed the same fragment pattern as was found for the archaeal community of the flounder digestive tract contents and feces. These data demonstrate that marine archaea are present in the digestive tracts and feces of very common marine fish. It is possible that the marine archaea associated with the digestive tracts of marine fish are liberated into the water column through the feces and subsequently contribute to the marine archaeal community of suspended particulate matter.
Influence of
Lactobacillus
spp. from an Inoculant and of
Weissella
and
Leuconostoc
spp. from Forage Crops on Silage Fermentation
Authors: Cai Yimin , Benno Yoshimi , Ogawa Masuhiro , Ohmomo Sadahiro , Kumai Sumio , Nakase Takashi
Published: December 2019

Lactobacillus
spp. from an inoculant and
Weissella
and
Leuconostoc
spp. from forage crops were characterized, and their influence on silage fermentation was studied. Forty-two lactic acid-producing cocci were obtained from forage crops and grasses. All isolates were gram-positive, catalase-negative cocci that produced gas from glucose, and produced more than 90% of their lactate in the
d
-isomer form. These isolates were divided into groups A and B by sugar fermentation patterns. Two representative strains from the two groups, FG 5 and FG 13, were assigned to the species
Weissella paramesenteroides
and
Leuconostoc pseudomesenteroides
, respectively, on the basis of DNA-DNA relatedness. Strains FG 5, FG 13, and SL 1 (
Lactobacillus casei
), isolated from a commercial inoculant, were used as additives to alfalfa and Italian ryegrass silage preparations. Lactic acid bacterium counts were higher in all additive-treated silages than in the control silage at an early stage of ensiling. During silage fermentation, inoculation with SL 1 more effectively inhibited the growth of aerobic bacteria and clostridia than inoculation with strain FG 5 or FG 13. SL 1-treated silages stored well. However, the control and FG 5- and FG 13-treated silages had a significantly (
P
< 0.05) higher pH and butyric acid and ammonia nitrogen contents and significantly (
P
< 0.05) lower lactate content than SL 1-treated silage. Compared with the control silage, SL 1 treatments reduced the proportion of
d
-(−)-lactic acid, gas production, and dry matter loss in two kinds of silage, but the FG 5 and FG 13 treatments gave similar values in alfalfa silages and higher values (
P
< 0.05) in Italian ryegrass silage. The results confirmed that heterofermentative strains of
W. paramesenteroides
FG 5 and
L. pseudomesenteroides
FG 13 did not improve silage quality and may cause some fermentation loss.
Purification and Characterization of a High-Molecular-Weight Insecticidal Protein Complex Produced by the Entomopathogenic Bacterium
Photorhabdus luminescens
Authors: Bowen David J. , Ensign Jerald C.
Published: December 2019

Photorhabdus luminescens
is a gram-negative enteric bacterium that is found in association with entomopathogenic nematodes of the family
Heterorhabditidae
. The nematodes infect a variety of soil-dwelling insects. Upon entering an insect host, the nematode releases
P. luminescens
cells from its intestinal tract, and the bacteria quickly establish a lethal septicemia. When grown in peptone broth, in the absence of the nematodes, the bacteria produce a protein toxin complex that is lethal when fed to, or injected into the hemolymph of,
Manduca sexta
larvae and several other insect species. The toxin purified as a protein complex which has an estimated molecular weight of 1,000,000 and contains no protease, phospholipase, or hemolytic activity and only a trace of lipase activity. The purified toxin possesses insecticidal activity whether injected or given orally. Analyses of the denatured complex by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed it to be composed of several protein subunits ranging in size from 30 to 200 kDa. The complex was further separated by native gel electrophoresis into three components, two of which retained insecticidal activity. The purified native toxin complex was found to be active in nanogram concentrations against insects representing four orders of the class
Insecta
.
The Requirement of RpoN (Sigma Factor ς
54
) in Denitrification by
Pseudomonas stutzeri
Is Indirect and Restricted to the Reduction of Nitrite and Nitric Oxide
Authors: Härtig Elisabeth , Zumft Walter G.
Published: December 2019

The
rpoN
region of
Pseudomonas stutzeri
was cloned, and an
rpoN
null mutant was constructed. RpoN was not essential for denitrification in this bacterium but affected the expression levels and enzymatic activities of cytochrome
cd
1
nitrite reductase and nitric oxide reductase, whereas those of respiratory nitrate reductase and nitrous oxide reductase were comparable to wild-type levels. Since the transcription of the structural genes
nirS
and
norCB
, coding for nitrite reductase and the nitric oxide reductase complex, respectively, proceeded unabated, our data indicate a posttranslational process for the two key enzymes of denitrification depending on RpoN.
Humic Acids as Electron Acceptors for Anaerobic Microbial Oxidation of Vinyl Chloride and Dichloroethene
Authors: Bradley Paul M. , Chapelle Francis H. , Lovley Derek R.
Published: December 2019

Anaerobic oxidation of [1,2-
14
C]vinyl chloride and [1,2-
14
C]dichloroethene to
14
CO
2
under humic acid-reducing conditions was demonstrated. The results indicate that waterborne contaminants can be oxidized by using humic acid compounds as electron acceptors and suggest that natural aquatic systems have a much larger capacity for contaminant oxidation than previously thought.
Transformation of the Ionic X-Ray Contrast Agent Diatrizoate and Related Triiodinated Benzoates by
Trametes versicolor
Authors: Rode Ulrike , Müller Rudolf
Published: December 2019

Iodinated X-ray contrast agents are considered to be nondegradable by microorganisms. The decomposition of the ionic X-ray contrast agents Diatrizoate (3,5-di(acetamido)-2,4,6-triiodobenzoic acid) and Iodipamide (3,3′-adipoyl-diimino-di(2,4,6-triiodobenzoic acid) and related triiodinated benzoates (Acetrizoate [3-acetylamino-2,4,6-triiodobenzoic acid] and Aminotrizoate [3-amino-2,4,6-triiodobenzoic acid]) by
Trametes versicolor
has been investigated. The fungus was able to transform all tested triiodinated benzoates cometabolically. During transformation of these compounds, iodide was released, but deiodination was not complete.
T. versicolor
liberated traces of
14
CO
2
from uniformly ring-
14
C-labeled Diatrizoate (3,5-di(acetamido)-2,4,6-triiodobenzoate). Various extracellular metabolites were detected during transformation of the different substances. In the transformation of Diatrizoate, the three main metabolites were identified as 3,5-di(acetamido)-2,6-diiodobenzoic acid, 3,5-di(acetamido)-2,4-diiodobenzoic acid, and 3,5-di(acetamido)-2-iodobenzoic acid, suggesting reductive deiodinations in steps as initial transformation steps.
Reverse Transcriptase PCR Detection of Astrovirus, Hepatitis A Virus, and Poliovirus in Experimentally Contaminated Mussels: Comparison of Several Extraction and Concentration Methods
Authors: Traore Ousmane , Arnal Charlotte , Mignotte Berengere , Maul Armand , Laveran Henri , Billaudel Sylviane , Schwartzbrod Louis
Published: December 2019

Four methods of extraction and three methods of concentration of three enteric viruses from mussels were comparatively evaluated by reverse transcriptase PCR (RT-PCR). Shellfish were experimentally contaminated by immersion in seawater seeded with astrovirus, hepatitis A virus, or poliovirus. Sixty-gram samples of mussel tissues were processed by using borate buffer, glycine solution, saline beef, and saline beef-Freon extraction methods. The viruses were concentrated by precipitation with polyethylene glycol 6000 (PEG 6000) or PEG 8000 or by organic flocculation. RT-PCR was performed with RNA extracts from crude shellfish extracts and concentrates with and without Sephadex LH20 filtration. The glycine solution and borate buffer extraction methods resulted in significantly more RT-PCR-positive samples than the saline beef extraction method. We assessed the efficiency of 20 combinations of extraction and concentration methods. The borate buffer-organic flocculation, borate buffer-PEG 6000, and glycine solution-PEG 6000 combinations gave RT-PCR-positive results for all 27 samples analyzed for the three viruses. Detoxification of the samples by Sephadex LH20 filtration significantly decreased the efficiency of RT-PCR virus detection.
Variations of Bacterial Populations in Human Feces Measured by Fluorescent In Situ Hybridization with Group-Specific 16S rRNA-Targeted Oligonucleotide Probes
Authors: Franks Alison H. , Harmsen Hermie J. M. , Raangs Gerwin C. , Jansen Gijsbert J. , Schut Frits , Welling Gjalt W.
Published: December 2019

Six 16S rRNA-targeted oligonucleotide probes were designed, validated, and used to quantify predominant groups of anaerobic bacteria in human fecal samples. A set of two probes was specific for species of the
Bacteroides fragilis
group and the species
Bacteroides distasonis
. Two others were designed to detect species of the
Clostridium histolyticum
and the
Clostridium lituseburense
groups. Another probe was designed for the genera
Streptococcus
and
Lactococcus
, and the final probe was designed for the species of the
Clostridium coccoides-Eubacterium rectale
group. The temperature of dissociation of each of the probes was determined. The specificities of the probes for a collection of target and reference organisms were tested by dot blot hybridization and fluorescent in situ hybridization (FISH). The new probes were used in initial FISH experiments to enumerate human fecal bacteria. The combination of the two
Bacteroides
-specific probes detected a mean of 5.4 × 10
10
cells per g (dry weight) of feces; the
Clostridium coccoides-Eubacterium rectale
group-specific probe detected a mean of 7.2 × 10
10
cells per g (dry weight) of feces. The
Clostridium histolyticum
,
Clostridium lituseburense
, and
Streptococcus-Lactococcus
group-specific probes detected only numbers of cells ranging from 1 × 10
7
to 7 × 10
8
per g (dry weight) of feces. Three of the newly designed probes and three additional probes were used in further FISH experiments to study the fecal flora composition of nine volunteers over a period of 8 months. The combination of probes was able to detect at least two-thirds of the fecal flora. The normal biological variations within the fecal populations of the volunteers were determined and indicated that these variations should be considered when evaluating the effects of agents modulating the flora.
Genetic Characterization and Physiological Role of Endopeptidase O from
Lactobacillus helveticus
CNRZ32
Authors: Chen Yo-Shen , Steele James L.
Published: December 2019

A previously identified insert expressing an endopeptidase from a
Lactobacillus helveticus
CNRZ32 genomic library was characterized. Nucleotide sequence analysis revealed an open reading frame of 1,941 bp encoding a putative protein of 71.2 kDa which contained a zinc-protease motif. Protein homology searches revealed that this enzyme has 40% similarity with endopeptidase O (PepO) from
Lactococcus lactis
P8-2-47. Northern hybridization revealed that
pepO
is monocistronic and is expressed throughout the growth phase. CNRZ32 derivatives lacking PepO activity were constructed via gene replacement. Enzyme assays revealed that the PepO mutant had significantly reduced endopeptidase activity when compared to CNRZ32 with two of the three substrates examined. Growth studies indicated that PepO has no detectable effect on growth rate or acid production by
Lactobacillus helveticus
CNRZ32 in amino acid defined or skim milk medium.
Genetic Analysis of
Comamonas acidovorans
Polyhydroxyalkanoate Synthase and Factors Affecting the Incorporation of 4-Hydroxybutyrate Monomer
Authors: Sudesh Kumar , Fukui Toshiaki , Doi Yoshiharu
Published: December 2019

The polyhydroxyalkanoate (PHA) synthase gene of
Comamonas acidovorans
DS-17 (

phaC
Ca

) was cloned by using the synthase gene of
Alcaligenes eutrophus
as a heterologous hybridization probe. Complete sequencing of a 4.0-kbp
Sma
I-
Hin
dIII (SH40) subfragment revealed the presence of a 1,893-bp PHA synthase coding region which was followed by a 1,182-bp β-ketothiolase gene (

phaA
Ca

). Both the translated products of these genes showed significant identity, 51.1 and 74.2%, respectively, to the primary structures of the products of the corresponding genes in
A. eutrophus
. The arrangement of PHA biosynthesis genes in
C. acidovorans
was also similar to that in
A. eutrophus
except that the third gene,
phaB
, coding for acetoacetyl-coenzyme A reductase, was not found in the region downstream of

phaA
Ca

. The cloned fragment complemented a PHA-negative mutant of
A. eutrophus
, PHB
−
4, resulting in poly-3-hydroxybutyrate accumulation of up to 73% of the dry cell weight when fructose was the carbon source. The heterologous expression enabled the incorporation of 4-hydroxybutyrate (4HB) and 3-hydroxyvalerate monomers. The PHA synthase of
C. acidovorans
does not appear to show any preference for 4-hydroxybutyryl-coenzyme A as a substrate. This leads to the suggestion that in
C. acidovorans
, it is the metabolic pathway, and not the specificity of the organism’s PHA synthase, that drives the incorporation of 4HB monomers, resulting in the efficient accumulation of PHA with a high 4HB content.
New Nitrogen-Fixing Microorganisms Detected in Oligotrophic Oceans by Amplification of Nitrogenase (
nifH
) Genes
Authors: Zehr Jonathan P. , Mellon Mark T. , Zani Sabino
Published: December 2019

Oligotrophic oceanic waters of the central ocean gyres typically have extremely low dissolved fixed inorganic nitrogen concentrations, but few nitrogen-fixing microorganisms from the oceanic environment have been cultivated. Nitrogenase gene (
nifH
) sequences amplified directly from oceanic waters showed that the open ocean contains more diverse diazotrophic microbial populations and more diverse habitats for nitrogen fixers than previously observed by classical microbiological techniques. Nitrogenase genes derived from unicellular and filamentous cyanobacteria, as well as from the α and γ subdivisions of the class
Proteobacteria
, were found in both the Atlantic and Pacific oceans.
nifH
sequences that cluster phylogenetically with sequences from sulfate reducers or clostridia were found associated with planktonic crustaceans. Nitrogenase sequence types obtained from invertebrates represented phylotypes distinct from the phylotypes detected in the picoplankton size fraction. The results indicate that there are in the oceanic environment several distinct potentially nitrogen-fixing microbial assemblages that include representatives of diverse phylotypes.
Induction of Acid Resistance of
Salmonella typhimurium
by Exposure to Short-Chain Fatty Acids
Authors: Kwon Y. M. , Ricke S. C.
Published: December 2019

Exposure to short-chain fatty acids (SCFA) is one of the stress conditions
Salmonella typhimurium
encounters during its life cycle, because SCFA have been widely used as food preservatives and SCFA are also present at high concentrations in the gastrointestinal tracts of host animals. The effects of SCFA on the acid resistance of the organism were examined in an attempt to understand the potential role of SCFA in the pathogenesis of
S. typhimurium
. The percent survival of
S. typhimurium
at pH 3.0 was determined after exposure to SCFA for 1 h at pH 7.0. The percent acid survival, which varied depending on the SCFA species and the concentration used, was 42 after exposure to 100 mM propionate at pH 7.0 under aerobic incubation conditions, while less than 1% could survive without exposure. The SCFA-induced acid resistance was markedly enhanced by anaerobiosis (64%), lowering pH conditions (138% at pH 5.0), or increasing incubation time (165% with 4 h) during exposure to propionic acid. When protein synthesis during exposure to propionate was blocked by chloramphenicol, the percent acid survival was less than 1, indicating that the protein synthesis induced by exposure to propionate is required for the induction of the acid resistance. The percent acid survival determined with the isogenic mutant strains defective in acid tolerance response revealed that AtrB protein is necessary for the full induction of acid resistance by exposure to propionate, while unexpectedly, inactivation of PhoP significantly increased acid resistance over that of the wild type (
P
< 0.05). The results suggest that the virulence of
S. typhimurium
may be enhanced by increasing acid resistance upon exposure to SCFA during its life cycle and further enhanced by anaerobiosis, low pH, and prolonged exposure time.
Discrimination of Psychrotrophic and Mesophilic Strains of the
Bacillus cereus
Group by PCR Targeting of Major Cold Shock Protein Genes
Authors: Francis Kevin P. , Mayr Ralf , von Stetten Felix , Stewart Gordon S. A. B. , Scherer Siegfried
Published: December 2019

Detection of psychrotrophic strains (those able to grow at or below 7°C) of the
Bacillus cereus
group (
Bacillus cereus
,
Bacillus thuringiensis
, and
Bacillus mycoides
) in food products is at present extremely slow with conventional microbiology. This is due to an inability to discriminate these cold-adapted strains from their mesophilic counterparts (those able to grow only above 7°C) by means other than growth at low temperature, which takes 5 to 10 days for detection. Here we report the development of a single PCR assay that, using major cold shock protein-specific primers and appropriate annealing temperatures, is capable of both rapidly identifying bacteria of the
B. cereus
group and discriminating between psychrotrophic and mesophilic strains. It is intended that this development help to more accurately predict the shelf life of refrigerated pasteurized food and dairy products and to reduce the incidence of food poisoning by psychrotrophic strains of the
B. cereus
group.
Purification and Properties of Two Thermostable Alkaline Xylanases from an Alkaliphilic
Bacillus
sp
Authors: Gessesse Amare
Published: December 2019

Two xylanases, designated XylA and XylB, were purified from the culture supernatant of the alkaliphilic
Bacillus
sp. strain AR-009. The molecular masses of the two enzymes were estimated to be 23 kDa (XylA) and 48 kDa (XylB) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimum pHs for activity were 9 for XylA and 9 to 10 for XylB. The temperature optima for the activity of XylA were 60°C at pH 9 and 70°C at pH 8. XylB was optimally active at 75°C at pH 9 and 70°C at pH 8. Both enzymes were stable in a broad pH range and showed good stability when incubated at 60 and 65°C in pH 8 and 9 buffers.
Expression of
Phanerochaete chrysosporium
Genes Encoding Lignin Peroxidases, Manganese Peroxidases, and Glyoxal Oxidase in Wood
Authors: Janse Bernard J. H. , Gaskell Jill , Akhtar Masood , Cullen Daniel
Published: December 2019

Expression of
Phanerochaete chrysosporium
genes encoding ligninolytic enzymes was assessed in wood. Poly(A) RNA was extracted from colonized wood chips by magnetic capture, and specific transcripts were quantified by competitive reverse transcriptase PCR. mRNA levels varied substantially among lignin peroxidase genes, and transcript patterns were dramatically different from those in previous studies with defined media.
Degradation of 3-Chlorobenzoate under Low-Oxygen Conditions in Pure and Mixed Cultures of the Anoxygenic PhotoheterotrophRhodopseudomonas palustrisDCP3 and an AerobicAlcaligenesSpecies
Authors: Krooneman Janneke , van den Akker Sytske , Pedro Gomes Teresa M. , Forney Larry J. , Gottschal Jan C.
Published: December 2019

The presence or absence of molecular oxygen has been shown to play a crucial role in the degradability of haloaromatic compounds. In the present study, it was shown that anaerobic phototrophic 3-chlorobenzoate (3CBA) metabolism byRhodopseudomonas palustrisDCP3 is oxygen tolerant up to a concentration of 3 μM O2. Simultaneous oxidation of an additional carbon source permitted light-dependent anaerobic 3CBA degradation at oxygen input levels which, in the absence of such an additional compound, would result in inhibition of light-dependent dehalogenation. Experiments under the same experimental conditions with strain DCP3 in coculture with an aerobic 3CBA-utilizing heterotroph,Alcaligenessp. strain L6, revealed that light-dependent dehalogenation of 3CBA did not occur. Under both oxygen limitation (O2< 0.1 μM) and low oxygen concentrations (3 μM O2), all the 3CBA was metabolized by the aerobic heterotroph. These data suggest that biodegradation of (halo)aromatics by photoheterotrophic bacteria such asR. palustrisDCP3 may be restricted to anoxic photic environments.
Ergosterol Content in Various Fungal Species and Biocontaminated Building Materials
Authors: Pasanen Anna-Liisa , Yli-Pietilä Kati , Pasanen Pertti , Kalliokoski Pentti , Tarhanen Juhani
Published: December 2019

This paper reports the ergosterol content for microbial cultures of six filamentous fungi, three yeast species, and one actinomycete and the ergosterol levels in 40 samples of building materials (wood chip, gypsum board, and glass wool) contaminated by microorganisms. The samples were hydrolyzed in alkaline methanol, and sterols were silylated and analyzed by gas chromatography-mass spectrometry. The average ergosterol content varied widely among the fungal species over the range of 2.6 to 42 μg/ml of dry mass or 0.00011 to 17 pg/spore or cell. Ergosterol could not be detected in the actinomycete culture. The results for both the fungal cultures and building material samples supported the idea that the ergosterol content reflects the concentration of filamentous fungi but it underestimates the occurrence of yeast cells. The ergosterol content in building material samples ranged from 0.017 to 68 μg/g of dry mass of material. A good agreement between the ergosterol concentration and viable fungal concentrations was detected in the wood chip (
r
> 0.66,
P
≤ 0.009) and gypsum board samples (
r
> 0.48,
P
≤ 0.059), whereas no relationship between these factors was observed in the glass wool samples. For the pooled data of the building materials, the ergosterol content correlated significantly with the viable fungal levels (
r
> 0.63,
P
< 0.0001). In conclusion, the ergosterol concentration could be a suitable marker for estimation of fungal concentrations in contaminated building materials with certain reservations, including the underestimation of yeast concentrations.
Production of Biogenic Mn Oxides by
Leptothrix discophora
SS-1 in a Chemically Defined Growth Medium and Evaluation of Their Pb Adsorption Characteristics
Authors: Nelson Yarrow M. , Lion Leonard W. , Ghiorse William C. , Shuler Michael L.
Published: December 2019

Biogenic Mn oxides were produced by the bacterium
Leptothrix discophora
SS-1 (= ATCC 3182) in a chemically defined mineral salts medium, and the Pb binding and specific surface area of these oxides were characterized. Growth of SS-1 in the defined medium with pyruvate as a carbon and energy source required the addition of vitamin B
12
. Complete oxidation of Mn(II) within 60 h required the addition of ≥0.1 μM FeSO
4
. Pb adsorption isotherms were determined for the biogenic Mn oxides (and associated cells with their extracellular polymer) and compared to the Pb adsorption isotherms of cells and exopolymer alone, as well as to abiotic Mn oxides. The Pb adsorption to cells and exopolymer with biogenic Mn oxides (0.8 mmol of Mn per g) at pH 6.0 and 25°C was 2 orders of magnitude greater than the Pb adsorption to cells and exopolymer alone (on a dry weight basis). The Pb adsorption to the biogenic Mn oxide was two to five times greater than the Pb adsorption to a chemically precipitated abiotic Mn oxide and several orders of magnitude greater than the Pb adsorption to two commercially available crystalline MnO
2
minerals. The N
2
Brunauer-Emmet-Teller specific surface areas of the biogenic Mn oxide and fresh Mn oxide precipitate (224 and 58 m
2
/g, respectively) were significantly greater than those of the commercial Mn oxide minerals (0.048 and 4.7 m
2
/g). The Pb adsorption capacity of the biogenic Mn oxide also exceeded that of a chemically precipitated colloidal hydrous Fe oxide under similar solution conditions. These results show that amorphous biogenic Mn oxides similar to those produced by SS-1 may play a significant role in the control of trace metal phase distribution in aquatic systems.
Cloning and Characterization of the Polyhydroxybutyrate Depolymerase Gene of
Pseudomonas stutzeri
and Analysis of the Function of Substrate-Binding Domains
Authors: Ohura Takeshi , Kasuya Ken-Ichi , Doi Yoshiharu
Published: December 2019

The extracellular polyhydroxybutyrate (PHB) depolymerase gene (

phaZ
Pst

) of
Pseudomonas stutzeri
was cloned and sequenced.

phaZ
Pst

was composed of 1,728 bp encoding a protein of 576 amino acids. Analyses of the N-terminal amino acid sequence and the matrix-assisted laser desorption/ionization–time-of-flight (MALDI-TOF) mass spectrum of the purified enzyme showed that the mature enzyme consisted of 538 amino acids with a deduced molecular mass of 57,506 Da. Analysis of the deduced amino acid sequence of the protein revealed a domain structure containing a catalytic domain, putative linker region, and two putative substrate-binding domains (SBDI and SBDII). The putative linker region was similar to the repeating units of the cadherin-like domain of chitinase A from
Vibrio harveyi
and chitinase B from
Clostridium paraputrificum
. The binding characteristics of SBDs to poly([
R
]-3-hydroxybutyrate) [P(3HB)] and chitin granules were characterized by using fusion proteins of SBDs with glutathione S -transferase (GST). These GST fusion proteins with SBDII and SBDI showed binding activity toward P(3HB) granules but did not bind on chitin granules. It has been suggested that the SBDs of the depolymerase interact specifically with the surface of P(3HB). In addition, a kinetic analysis for the enzymatic hydrolysis of 3-hydroxybutyrate oligomers of various sizes has suggested that the catalytic domain of the enzyme recognizes at least two monomeric units as substrates.
Hybridization Analysis of Chesapeake Bay Virioplankton
Authors: Wommack K. Eric , Ravel Jacques , Hill Russell T. , Colwell Rita R.
Published: December 2019

It has been hypothesized that, by specifically lysing numerically dominant host strains, the virioplankton may play a role in maintaining clonal diversity of heterotrophic bacteria and phytoplankton populations. If viruses selectively lyse only those host species that are numerically dominant, then the number of a specific virus within the virioplankton would be expected to change dramatically over time and space, in coordination with changes in abundance of the host. In this study, the abundances of specific viruses in Chesapeake Bay water samples were monitored, using nucleic acid probes and hybridization analysis. Total virioplankton in a water sample was separated by pulsed-field gel electrophoresis and hybridized with nucleic acid probes specific to either single viral strains or a group of viruses with similar genome sizes. The abundances of specific viruses were inferred from the intensity of the hybridization signal. By using this technique, a virus comprising 1/1,000 of the total virioplankton abundance (ca. 10
4
PFU/ml) could be detected. Titers of either a single virus species or a group of viruses changed over time, increasing to peak abundance and then declining to low or undetectable levels, and were geographically localized in the bay. Peak signal intensities, i.e., peak abundances of virus strains, were 10-fold greater than the low background level. Furthermore, virus species were found to be restricted to a particular depth, since probes specific to viruses from bottom water did not hybridize with virus genomes from surface water at the same geographical location. Overall, changes in abundances of specific viruses within the virioplankton were episodic, supporting the hypothesis that viral infection influences, if not controls, clonal diversity within heterotrophic bacteria and phytoplankton communities.
Polycyclic Aromatic Hydrocarbon Degradation by a New Marine Bacterium,
Neptunomonas naphthovorans
gen. nov., sp. nov
Authors: Hedlund Brian P. , Geiselbrecht Allison D. , Bair Timothy J. , Staley James T.
Published: December 2019

Two strains of bacteria were isolated from creosote-contaminated Puget Sound sediment based on their ability to utilize naphthalene as a sole carbon and energy source. When incubated with a polycyclic aromatic hydrocarbon (PAH) compound in artificial seawater, each strain also degraded 2-methylnaphthalene and 1-methylnaphthalene; in addition, one strain, NAG-2N-113, degraded 2,6-dimethylnaphthalene and phenanthrene. Acenaphthene was not degraded when it was used as a sole carbon source but was degraded by both strains when it was incubated with a mixture of seven other PAHs. Degenerate primers and the PCR were used to isolate a portion of a naphthalene dioxygenase iron-sulfur protein (ISP) gene from each of the strains. A phylogenetic analysis of PAH dioxygenase ISP deduced amino acid sequences showed that the genes isolated in this study were distantly related to the genes encoding naphthalene dioxygenases of
Pseudomonas
and
Burkholderia
strains. Despite the differences in PAH degradation phenotype between the new strains, the dioxygenase ISP deduced amino acid fragments of these organisms were 97.6% identical. 16S ribosomal DNA-based phylogenetic analysis placed these bacteria in the gamma-3 subgroup of the
Proteobacteria
, most closely related to members of the genus
Oceanospirillum
. However, morphologic, physiologic, and genotypic differences between the new strains and the oceanospirilla justify the creation of a novel genus and species,
Neptunomonas naphthovorans
. The type strain of
N. naphthovorans
is strain NAG-2N-126.
Pleiotropic Effects of Adaptation to a Single Carbon Source for Growth on Alternative Substrates
Authors: Velicer Gregory J.
Published: December 2019

It is frequently assumed that populations of genetically modified microorganisms will perform their intended function and then disappear from the environment due to inherent fitness disadvantages resulting from their genetic alteration. However, modified organisms used in bioremediation can be expected to adapt evolutionarily to growth on the anthropogenic substrate that they are intended to degrade. If such adaptation results in improved competitiveness for alternative, naturally occurring substrates, then this will increase the likelihood that the modified organisms will persist in the environment. In this study, bacteria capable of degrading the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) were used to test the effects of evolutionary adaptation to one substrate on fitness during growth on an alternative substrate. Twenty lineages of bacteria were allowed to evolve under abundant resource conditions on either 2,4-D or succinate as their sole carbon source. The competitiveness of each evolved line was then measured relative to that of its ancestor for growth on both substrates. Only three derived lines showed a clear drop in fitness on the alternative substrate after demonstrable adaptation to their selective substrate, while five derived lines showed significant simultaneous increases in fitness on both their selective and alternative substrates. These data demonstrate that adaptation to an anthropogenic substrate can pleiotropically increase competitiveness for an alternative natural substrate and therefore increase the likelihood that a genetically modified organism will persist in the environment.
Influence of Osmolarity and the Presence of an Osmoprotectant on
Lactococcus lactis
Growth and Bacteriocin Production
Authors: Uguen Patricia , Hamelin Jack , Le Pennec Jean-Paul , Blanco Carlos
Published: December 2019

Growth inhibition of
Lactococcus lactis
provoked by increasing osmolarity is reversed when glycine betaine (GB) or its analogs are added to a defined medium. Lacticin 481 production increased sharply with growth medium osmolarity in the absence of osmoprotectant but remained unaffected when GB was supplied in media of increasing osmolarity.
2-Bromoethanesulfonate, Sulfate, Molybdate, and Ethanesulfonate Inhibit Anaerobic Dechlorination of Polychlorobiphenyls by Pasteurized Microorganisms
Authors: Ye Dingyi , Quensen John F. , Tiedje James M. , Boyd Stephen A.
Published: December 2019

Dechlorination of Aroclor 1242 by pasteurized microorganisms was inhibited by 2-bromoethanesulfonate (BES), sulfate, molybdate, and ethanesulfonate. Consumption of these anions and production of sulfide from BES were detected. The inhibition could not be relieved by hydrogen. Taken together these results suggest that pattern M dechlorination is mediated by spore-forming sulfidogenic bacteria. These results also suggest that BES may inhibit anaerobic dechlorination by nonmethanogens by more than one mechanism.
Highly Sensitive Protein Translation Assay for Trichothecene Toxicity in Airborne Particulates: Comparison with Cytotoxicity Assays
Authors: Yike Iwona , Allan Terry , Sorenson William G. , Dearborn Dorr G.
Published: December 2019

Screening assays for environmental mycotoxins in bulk samples currently use cytotoxicity in cell cultures, but their application to air particulate samples often lacks sensitivity and specificity for fungal spores. An assay based on inhibition of protein synthesis using translation of firefly luciferase in a rabbit reticulocyte system has been developed for the detection of trichothecene mycotoxins found in the spores of toxigenic fungi. Ethanol extracts of air particulates trapped on polycarbonate filters are ultrafiltered and applied at several dilutions to a translation reaction mixture. The activity of translated luciferase is measured directly in a luminometer, eliminating the need for radioisotopes and time-consuming sample processing. Parallel standard curves using a commercially available trichothecene provide for expression of the results in T-2 toxin equivalents per cubic meter of air. The assay can be completed in 2 h and is readily applicable to multiple samples. Comparison to the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cytotoxicity assay indicates a 400-fold increase in sensitivity of trichothecene detection in addition to a much higher specificity for these toxins. Initial field testing indicates a strong correlation between the measured level of toxicity and the presence of toxigenic fungi detected with microbiological methods. In conclusion, this luciferase translation assay offers a rapid and highly sensitive and specific method for quantitative detection of trichothecene mycotoxin activity in air particulate samples.
The Outer Membrane of Gram-Negative Bacteria Inhibits Antibacterial Activity of Brochocin-C
Authors: Gao Yan , van Belkum Marco J. , Stiles Michael E.
Published: December 2019

Brochocin-C is a two-peptide bacteriocin produced by
Brochothrix campestris
ATCC 43754 that has a broad activity spectrum comparable to that of nisin. Brochocin-C has an inhibitory effect on EDTA-treated gram-negative bacteria,
Salmonella enterica
serovar Typhimurium lipopolysaccharide mutants, and spheroplasts of Typhimurium strains LT2 and SL3600. Brochocin-C treatment of cells and spheroplasts of strains of LT2 and SL3600 resulted in hydrolysis of ATP. The outer membrane of gram-negative bacteria protects the cytoplasmic membrane from the action of brochocin-C. It appears that brochocin-C is similar to nisin and possibly does not require a membrane receptor for its function; however, the difference in effect of the two bacteriocins on intracellular ATP indicates that they cause different pore sizes in the cytoplasmic membrane.
Possible Role of Xanthobaccins Produced by
Stenotrophomonas
sp. Strain SB-K88 in Suppression of Sugar Beet Damping-Off Disease
Authors: Nakayama Takato , Homma Yoshihisa , Hashidoko Yasuyuki , Mizutani Junya , Tahara Satoshi
Published: December 2019

Three antifungal compounds, designated xanthobaccins A, B, and C, were isolated from the culture fluid of
Stenotrophomonas
sp. strain SB-K88, a rhizobacterium of sugar beet that suppresses damping-off disease. Production of xanthobaccin A in culture media was compared with the disease suppression activities of strain SB-K88 and less suppressive strains that were obtained by subculturing. Strain SB-K88 was applied to sugar beet seeds, and production of xanthobaccin A in the rhizosphere of seedlings was confirmed by using a test tube culture system under hydroponic culture conditions; 3 μg of xanthobaccin A was detected in the rhizosphere on a per-plant basis. Direct application of purified xanthobaccin A to seeds suppressed damping-off disease in soil naturally infested by
Pythium
spp. We suggest that xanthobaccin A produced by strain SB-K88 plays a key role in suppression of sugar beet damping-off disease.
Two Cellobiohydrolase-Encoding Genes from
Aspergillus niger
Require
d
-Xylose and the Xylanolytic Transcriptional Activator XlnR for Their Expression
Authors: Gielkens Marco M. C. , Dekkers Ester , Visser Jaap , de Graaff Leo H.
Published: December 2019

Two cellobiohydrolase-encoding genes,
cbhA
and
cbhB
, have been isolated from the filamentous fungus
Aspergillus niger
. The deduced amino acid sequence shows that CbhB has a modular structure consisting of a fungus-type cellulose-binding domain (CBD) and a catalytic domain separated by a Pro/Ser/Thr-rich linker peptide. CbhA consists only of a catalytic domain and lacks a CBD and linker peptide. Both proteins are homologous to fungal cellobiohydrolases in family 7 of the glycosyl hydrolases. Northern blot analysis showed that the transcription of the
cbhA
and
cbhB
genes is induced by
d
-xylose but not by sophorose and, in addition, requires the xylanolytic transcriptional activator XlnR.
Use of PCR-Based Methods for Rapid Differentiation of
Lactobacillus delbrueckii
subsp.
bulgaricus
and
L. delbrueckii
subsp.
lactis
Authors: Torriani Sandra , Zapparoli Giacomo , Dellaglio Franco
Published: December 2019

Two PCR-based methods, specific PCR and randomly amplified polymorphic DNA PCR (RAPD-PCR), were used for rapid and reliable differentiation of
Lactobacillus delbrueckii
subsp.
bulgaricus
and
L. delbrueckii
subsp.
lactis
. PCR with a single combination of primers which targeted the proline iminopeptidase (
pepIP
) gene of
L. delbrueckii
subsp.
bulgaricus
allowed amplification of genomic fragments specific for the two subspecies when either DNA from a single colony or cells extracted from dairy products were used. A numerical analysis of the RAPD-PCR patterns obtained with primer M13 gave results that were consistent with the results of specific PCR for all strains except
L. delbrueckii
subsp.
delbrueckii
LMG 6412
T
, which clustered with
L. delbrueckii
subsp.
lactis
strains. In addition, RAPD-PCR performed with primer 1254 provided highly polymorphic profiles and thus was superior for distinguishing individual
L. delbrueckii
strains.
High-Level Production of Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate) by Fed-Batch Culture of Recombinant
Escherichia coli
Authors: Choi Jong-il , Lee Sang Yup
Published: December 2019

Fermentation strategies for production of high concentrations of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] with different 3-hydroxyvalerate (3HV) fractions by recombinant
Escherichia coli
harboring the
Alcaligenes latus
polyhydroxyalkanoate biosynthesis genes were developed. Fed-batch cultures of recombinant
E. coli
with the pH-stat feeding strategy facilitated production of high concentrations and high contents of P(3HB-co-3HV) in a chemically defined medium. When a feeding solution was added in order to increase the glucose and propionic acid concentrations to 20 g/liter and 20 mM, respectively, after each feeding, a cell dry weight of 120.3 g/liter and a relatively low P(3HB-co-3HV) content, 42.5 wt%, were obtained. Accumulation of a high residual concentration of propionic acid in the medium was the reason for the low P(3HB-co-3HV) content. An acetic acid induction strategy was used to stimulate the uptake and utilization of propionic acid. When a fed-batch culture and this strategy were used, we obtained a cell concentration, a P(3HB-co-3HV) concentration, a P(3HB-co-3HV) content, and a 3HV fraction of 141.9 g/liter, 88.1 g/liter, 62.1 wt%, and 15.3 mol%, respectively. When an improved nutrient feeding strategy, acetic acid induction, and oleic acid supplementation were used, we obtained a cell concentration, a P(3HB-co-3HV) concentration, a P(3HB-co-3HV) content, and a 3HV fraction of 203.1 g/liter, 158.8 g/liter, 78.2 wt%, and 10.6 mol%, respectively; this resulted in a high level of productivity, 2.88 g of P(3HB-co-3HV)/liter-h.
Purification and Characterization of Monovalent Cation-Activated Levodione Reductase from
Corynebacterium aquaticum
M-13
Authors: Wada Masaru , Yoshizumi Ayumi , Nakamori Shigeru , Shimizu Sakayu
Published: December 2019

(6
R
)-2,2,6-Trimethyl-1,4-cyclohexanedione (levodione) reductase was isolated from a cell extract of the soil isolate
Corynebacterium aquaticum
M-13. This enzyme catalyzed regio- and stereoselective reduction of levodione to (4
R
,6
R
)-4-hydroxy-2,2,6-trimethylcyclohexanone (actinol). The relative molecular mass of the enzyme was estimated to be 142,000 Da by high-performance gel permeation chromatography and 36,000 Da by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme required NAD
+
or NADH as a cofactor, and it catalyzed reversible oxidoreduction between actinol and levodione. The enzyme was highly activated by monovalent cations, such as K
+
, Na
+
, and NH
4
+
. The NH
2
-terminal and partial amino acid sequences of the enzyme showed that it belongs to the short-chain alcohol dehydrogenase/reductase family. This is the first report of levodione reductase.