bluuebunny/crossref_metadata_2025 huggingface dataset:
Contribution of Methanotrophic and Nitrifying Bacteria to CH
4
and NH
4
+
Oxidation in the Rhizosphere of Rice Plants as Determined by New Methods of Discrimination
Authors: Bodelier Paul L. E. , Frenzel Peter
Published: December 2019

Methanotrophic and nitrifying bacteria are both able to oxidize CH
4
as well as NH
4
+
. To date it is not possible to estimate the relative contribution of methanotrophs to nitrification and that of nitrifiers to CH
4
oxidation and thus to assess their roles in N and C cycling in soils and sediments. This study presents new options for discrimination between the activities of methanotrophs and nitrifiers, based on the competitive inhibitor CH
3
F and on recovery after inhibition with C
2
H
2
. By using rice plant soil as a model system, it was possible to selectively inactivate methanotrophs in soil slurries at a CH
4
/CH
3
F/NH
4
+
molar ratio of 0.1:1:18. This ratio of CH
3
F to NH
4
+
did not affect ammonia oxidation, but methane oxidation was inhibited completely. By using the same model system, it could be shown that after 24 h of exposure to C
2
H
2
(1,000 parts per million volume), methanotrophs recovered within 24 h while nitrifiers stayed inactive for at least 3 days. This gave an “assay window” of 48 h when only methanotrophs were active. Applying both assays to model microcosms planted with rice plants demonstrated a major contribution of methanotrophs to nitrification in the rhizosphere, while the contribution of nitrifiers to CH
4
oxidation was insignificant.
Molecular Characterization of a Phage-Encoded Resistance System in
Lactococcus lactis
Authors: McGrath Stephen , Seegers Jos F. M. L. , Fitzgerald Gerald F. , van Sinderen Douwe
Published: December 2019

A specific fragment of the genome of Tuc2009, a temperate lactococcal bacteriophage, was shown to contain several open reading frames, whose deduced protein products exhibited similarities to proteins known to be involved in DNA replication and modification. In this way, a putative single-stranded binding protein, replisome organizer protein, topoisomerase I, and a methylase were identified. When the genetic information coding for the putative replisome organizer protein of Tuc2009, Rep
2009
, was supplied on a high-copy-number plasmid vector, it was shown to confer a phage-encoded resistance (Per) phenotype on its lactococcal host UC509.9. The presence of this recombinant plasmid was shown to cause a marked reduction in Tuc2009 DNA replication, suggesting that the observed phage resistance was due to titration of a factor, or factors, required for Tuc2009 DNA replication. Further experiments delineated the phage resistance-conferring region to a 160-bp fragment rich in direct repeats. Gel retardation experiments, which indicated a protein-DNA interaction between this 160-bp fragment and the Rep
2009
protein, were performed. UC509.9 strains harboring plasmids with randomly mutated versions of this fragment were shown to display a variable phage resistance phenotype, depending on the position of the mutations.
Role of
Bacillus thuringiensis
Toxin Domains in Toxicity and Receptor Binding in the Diamondback Moth
Authors: Ballester V. , Granero F. , de Maagd R. A. , Bosch D. , Ménsua J. L. , Ferré J.
Published: December 2019

The toxic fragment of
Bacillus thuringiensis
crystal proteins consists of three distinct structural domains. There is evidence that domain I is involved in pore formation and that domain II is involved in receptor binding and specificity. It has been found that, in some cases, domain III is also important in determining specificity. Furthermore, involvement of domain III in binding has also been reported recently. To investigate the role of toxin domains in the diamondback moth (
Plutella xylostella
), we used hybrid toxins with domain III substitutions among Cry1C, Cry1E, and Cry1Ab. Neither Cry1E nor G27 (a hybrid with domains I and II from Cry1E and domain III from Cry1C) was toxic, whereas Cry1C and F26 (the reciprocal hybrid) were equally toxic. H04 (a hybrid with domains I and II from Cry1Ab and domain III from Cry1C) showed toxicity that was of a similar level as that of Cry1Ab and significantly higher than that of Cry1C. Binding assays with
125
I-Cry1C showed that Cry1C and F26 competed for the same binding sites on midgut membrane vesicles, whereas Cry1E, G27, and H04 did not bind to these sites. Our results show that, in contrast to findings in other insects for the toxins and hybrids used here, toxin specificity as well as specificity of binding to membrane vesicles in the diamondback moth is mediated by domain II (and/or I) and not by domain III.
Interactions between Carbon and Nitrogen Metabolism in
Fibrobacter succinogenes
S85: a
1
H and
13
C Nuclear Magnetic Resonance and Enzymatic Study
Authors: Matheron Christelle , Delort Anne-Marie , Gaudet Genevieve , Liptaj Tibor , Forano Evelyne
Published: December 2019

The effect of the presence of ammonia on [1-
13
C]glucose metabolism in the rumen fibrolytic bacterium
Fibrobacter succinogenes
S85 was studied by
13
C and
1
H nuclear magnetic resonance (NMR). Ammonia halved the level of glycogen storage and increased the rate of glucose conversion into acetate and succinate 2.2-fold and 1.4-fold, respectively, reducing the succinate-to-acetate ratio. The
13
C enrichment of succinate and acetate was precisely quantified by
13
C-filtered spin-echo difference
1
H-NMR spectroscopy. The presence of ammonia did not modify the
13
C enrichment of succinate C-2 (without ammonia, 20.8%, and with ammonia, 21.6%), indicating that the isotopic dilution of metabolites due to utilization of endogenous glycogen was not affected. In contrast, the presence of ammonia markedly decreased the
13
C enrichment of acetate C-2 (from 40 to 31%), reflecting enhanced reversal of the succinate synthesis pathway. The reversal of glycolysis was unaffected by the presence of ammonia as shown by
13
C-NMR analysis. Study of cell extracts showed that the main pathways of ammonia assimilation in
F. succinogenes
were glutamate dehydrogenase and alanine dehydrogenase. Glutamine synthetase activity was not detected. Glutamate dehydrogenase was active with both NAD and NADP as cofactors and was not repressed under ammonia limitation in the culture. Glutamate-pyruvate and glutamate-oxaloacetate transaminase activities were evidenced by spectrophotometry and
1
H NMR. When cells were incubated in vivo with [1-
13
C]glucose, only
13
C-labeled aspartate, glutamate, alanine, and valine were detected. Their labelings were consistent with the proposed amino acid synthesis pathway and with the reversal of the succinate synthesis pathway.
Adhesion of
Acinetobacter venetianus
to Diesel Fuel Droplets Studied with In Situ Electrochemical and Molecular Probes
Authors: Baldi Franco , Ivos̆ević Nadica , Minacci Andrea , Pepi Milva , Fani Renato , Svetlic̆ić Vesna , Z̆utić Vera
Published: December 2019

The adhesion of a recently described species,
Acinetobacter venetianus
VE-C3 (F. Di Cello, M. Pepi, F. Baldi, and R. Fani, Res. Microbiol. 148:237–249, 1997), to diesel fuel (a mixture of C
12
to C
28
n
-alkanes) and
n
-hexadecane was studied and compared to that of
Acinetobacter
sp. strain RAG-1, which is known to excrete the emulsifying lipopolysaccharide, emulsan. Oxygen consumption rates, biomass, cell hydrophobicity, electrophoretic mobility, and zeta potential were measured for the two strains. The dropping-mercury electrode (DME) was used as an in situ adhesion sensor. In seawater, RAG-1 was hydrophobic, with an electrophoretic mobility (μ) of −0.38 × 10
−8
m
2
V
−1
s
−1
and zeta potential (ζ) of −4.9 mV, while VE-C3 was hydrophilic, with μ of −0.81 × 10
−8
m
2
V
−1
s
−1
and ζ of −10.5 mV. The microbial adhesion to hydrocarbon (MATH) test showed that RAG-1 was always hydrophobic whereas the hydrophilic VE-C3 strain became hydrophobic only after exposure to
n
-alkanes. Adhesion of VE-C3 cells to diesel fuel was partly due to the production of capsular polysaccharides (CPS), which were stained with the lectin concanavalin A (ConA) conjugated to fluorescein isothiocyanate and observed in situ by confocal microscopy. The emulsan from RAG-1, which was negative to ConA, was stained with Nile Red fluorochrome instead. Confocal microscope observations at different times showed that VE-C3 underwent two types of adhesion: (i) cell-to-cell interactions, preceding the cell adhesion to the
n
-alkane, and (ii) incorporation of nanodroplets of
n
-alkane into the hydrophilic CPS to form a more hydrophobic polysaccharide–
n
-alkane matrix surrounding the cell wall. The incorporation of
n
-alkanes as nanodroplets into the CPS of VE-C3 cells might ensure the partitioning of the bulk apolar phase between the aqueous medium and the outer cell membrane and thus sustain a continuous growth rate over a prolonged period.
Detection of
Salmonella
spp. and
Listeria monocytogenes
in Suspended Organic Waste by Nucleic Acid Extraction and PCR
Authors: Burtscher Carola , Fall Papa A. , Wilderer Peter A. , Wuertz Stefan
Published: December 2019

A nucleic acid-based method for the detection of the bacterial pathogens
Salmonella
spp. and
Listeria monocytogenes
in biological waste was developed. The detection limits were less than 10 cells per ml of biological waste. The method does not include a phenol extraction step and can be easily performed in 1 to 2 days.
Whey Fermentation by
Anaerobiospirillum succiniciproducens
for Production of a Succinate-Based Animal Feed Additive
Authors: Samuelov Nissim S. , Datta Rathin , Jain Mahendra K. , Zeikus J. Gregory
Published: December 2019

Anaerobic fermentation processes for the production of a succinate-rich animal feed supplement from raw whey were investigated with batch, continuous, and variable-volume fed-batch cultures with
Anaerobiospirillum succiniciproducens
. The highest succinate yield, 90%, was obtained in a variable-volume fed-batch process in comparison to 80% yield in a batch cultivation mode. In continuous culture, succinate productivity was 3 g/liter/h, and the yield was 60%. Under conditions of excess CO
2
, more than 90% of the whey-lactose was consumed, with an end product ratio of 4 succinate to 1 acetate. Under conditions of limited CO
2
, lactose was only partially consumed and lactate was the major end product, with lower levels of ethanol, succinate, and acetate. When the succinic acid in this fermentation product was added to rumen fluid, it was completely consumed by a mixed rumen population and was 90% decarboxylated to propionate on a molar basis. The whey fermentation product formed under excess CO
2
, which contained mainly organic acids and cells, could potentially be used as an animal feed supplement.
Expression of the
Escherichia coli pntA
and
pntB
Genes, Encoding Nicotinamide Nucleotide Transhydrogenase, in
Saccharomyces cerevisiae
and Its Effect on Product Formation during Anaerobic Glucose Fermentation
Authors: Anderlund Mikael , Nissen Torben L. , Nielsen Jens , Villadsen John , Rydström Jan , Hahn-Hägerdal Bärbel , Kielland-Brandt Morten C.
Published: December 2019

We studied the physiological effect of the interconversion between the NAD(H) and NADP(H) coenzyme systems in recombinant
Saccharomyces cerevisiae
expressing the membrane-bound transhydrogenase from
Escherichia coli
. Our objective was to determine if the membrane-bound transhydrogenase could work in reoxidation of NADH to NAD
+
in
S. cerevisiae
and thereby reduce glycerol formation during anaerobic fermentation. Membranes isolated from the recombinant strains exhibited reduction of 3-acetylpyridine-NAD
+
by NADPH and by NADH in the presence of NADP
+
, which demonstrated that an active enzyme was present. Unlike the situation in
E. coli
, however, most of the transhydrogenase activity was not present in the yeast plasma membrane; rather, the enzyme appeared to remain localized in the membrane of the endoplasmic reticulum. During anaerobic glucose fermentation we observed an increase in the formation of 2-oxoglutarate, glycerol, and acetic acid in a strain expressing a high level of transhydrogenase, which indicated that increased NADPH consumption and NADH production occurred. The intracellular concentrations of NADH, NAD
+
, NADPH, and NADP
+
were measured in cells expressing transhydrogenase. The reduction of the NADPH pool indicated that the transhydrogenase transferred reducing equivalents from NADPH to NAD
+
.
Overexpression of the
Saccharomyces cerevisiae
Mannosylphosphodolichol Synthase-Encoding Gene in
Trichoderma reesei
Results in an Increased Level of Protein Secretion and Abnormal Cell Ultrastructure
Authors: Kruszewska Joanna S. , Butterweck Arno H. , Kurza˛tkowski Wiesław , Migdalski Andrzej , Kubicek Christian P. , Palamarczyk Graz˙yna
Published: December 2019

Production of extracellular proteins plays an important role in the physiology of
Trichoderma reesei
and has potential industrial application. To improve the efficiency of protein secretion, we overexpressed in
T. reesei
the
DPM1
gene of
Saccharomyces cerevisiae
, encoding mannosylphosphodolichol (MPD) synthase, under homologous, constitutively acting expression signals. Four stable transformants, each with different copy numbers of tandemly integrated
DPM1
, exhibited roughly double the activity of MPD synthase in the respective endoplasmic reticulum membrane fraction. On a dry-weight basis, they secreted up to sevenfold-higher concentrations of extracellular proteins during growth on lactose, a carbon source promoting formation of cellulases. Northern blot analysis showed that the relative level of the transcript of
cbh1
, which encodes the major cellulase (cellobiohydrolase I [CBH I]), did not increase in the transformants. On the other hand, the amount of secreted CBH I and, in all but one of the transformants, intracellular CBH I was elevated. Our results suggest that posttranscriptional processes are responsible for the increase in CBH I production. The carbohydrate contents of the extracellular proteins were comparable in the wild type and in the transformants, and no hyperglycosylation was detected. Electron microscopy of the
DPM1
-amplified strains revealed amorphous structure of the cell wall and over three times as many mitochondria as in the control. Our data indicate that molecular manipulation of glycan biosynthesis in
Trichoderma
can result in improved protein secretion.
Hyperproduction of Tryptophan by
Corynebacterium glutamicum
with the Modified Pentose Phosphate Pathway
Authors: Ikeda Masato , Katsumata Ryoichi
Published: December 2019

A classically derived tryptophan-producing
Corynebacterium glutamicum
strain was recently significantly improved both by plasmid-mediated amplification of the genes for the rate-limiting enzymes in the terminal pathways and by construction of a plasmid stabilization system so that it produced more tryptophan. This engineered strain, KY9218 carrying pKW9901, produced 50 g of tryptophan per liter from sucrose after 80 h in fed-batch cultivation without antibiotic pressure. Analysis of carbon balances showed that at the late stage of the fermentation, tryptophan yield decreased with a concomitant increase in CO
2
yield, suggesting a transition in the distribution of carbon flow from aromatic biosynthesis toward the tricarboxylic acid cycle via glycolysis. To circumvent this transition by increasing the supply of erythrose 4-phosphate, a direct precursor of aromatic biosynthesis, the transketolase gene of
C. glutamicum
was coamplified in the engineered strain by using low- and high-copy-number plasmids which were compatible with the resident plasmid pKW9901. The presence of the gene in low copy numbers contributed to improvement of tryptophan yield, especially at the late stage, and led to accumulation of more tryptophan (57 g/liter) than did its absence, while high-copy-number amplification of the gene resulted in a tryptophan production level even lower than that resulting from the absence of the gene due to reduced growth and sugar consumption. In order to assemble all the cloned genes onto a low-copy-number plasmid, the high-copy-number origin of pKW9901 was replaced with the low-copy-number one, generating low-copy-number plasmid pSW9911, and the transketolase gene was inserted to yield pIK9960. The pSW9911-carrying producer showed almost the same fermentation profiles as the pKW9901 carrier in fed-batch cultivation without antibiotic pressure. Under the same culture conditions, however, the pIK9960 carrier achieved a final tryptophan titer of 58 g/liter, which represented a 15% enhancement over the titers achieved by the pKW9901 and pSW9911 carriers.
Characterization of a
Pseudomonas putida
Allylic Alcohol Dehydrogenase Induced by Growth on 2-Methyl-3-Buten-2-ol
Authors: Malone Vincent F. , Chastain Amy J. , Ohlsson John T. , Poneleit Loelle S. , Nemecek-Marshall Michele , Fall Ray
Published: December 2019

We have been working to develop an enzymatic assay for the alcohol 2-methyl-3-buten-2-ol (232-MB), which is produced and emitted by certain pines. To this end we have isolated the soil bacterium
Pseudomonas putida
MB-1, which uses 232-MB as a sole carbon source. Strain MB-1 contains inducible 3-methyl-2-buten-1-ol (321-MB) and 3-methyl-2-buten-1-al dehydrogenases, suggesting that 232-MB is metabolized by isomerization to 321-MB followed by oxidation. 321-MB dehydrogenase was purified to near-homogeneity and found to be a tetramer (151 kDa) with a subunit mass of 37,700 Da. It catalyzes NAD
+
-dependent, reversible oxidation of 321-MB to 3-methyl-2-buten-1-al. The optimum pH for the oxidation reaction was 10.0, while that for the reduction reaction was 5.4. 321-MB dehydrogenase oxidized a wide variety of aliphatic and aromatic alcohols but exhibited the highest catalytic specificity with allylic or benzylic substrates, including 321-MB, 3-chloro-2-buten-1-ol, and 3-aminobenzyl alcohol. The N-terminal sequence of the enzyme contained a region of 64% identity with the TOL plasmid-encoded benzyl alcohol dehydrogenase of
P. putida
. The latter enzyme and the chromosomally encoded benzyl alcohol dehydrogenase of
Acinetobacter calcoaceticus
were also found to catalyze 321-MB oxidation. These findings suggest that 321-MB dehydrogenase and other bacterial benzyl alcohol dehydrogenases are broad-specificity allylic and benzylic alcohol dehydrogenases that, in conjunction with a 232-MB isomerase, might be useful in an enzyme-linked assay for 232-MB.
Optimization of Simultaneous Chemical and Biological Mineralization of Perchloroethylene
Authors: Büyüksönmez Fati˙h , Hess Thomas F. , Crawford Ronald L. , Paszczynski Andrzej , Watts Richard J.
Published: December 2019

Optimization of the simultaneous chemical and biological mineralization of perchloroethylene (PCE) by modified Fenton’s reagent and
Xanthobacter flavus
was investigated by using a central composite rotatable experimental design. Concentrations of PCE, hydrogen peroxide, and ferrous iron and the microbial cell number were set as variables. Percent mineralization of PCE to CO
2
was investigated as a response. A second-order, quadratic response surface model was generated and fit the data adequately, with a correlation coefficient of 0.72. Analysis of the results showed that the PCE concentration had no significant effect within the tested boundaries of the model, while the other variables, hydrogen peroxide and iron concentrations and cell number, were significant at α = 0.05 for the mineralization of PCE. The
14
C radiotracer studies showed that the simultaneous chemical and biological reactions increased the extent of mineralization of PCE by more than 10% over stand-alone Fenton reactions.
Delineation of Key Amino Acid Side Chains and Peptide Domains for Antimicrobial Properties of Divercin V41, a Pediocin-Like Bacteriocin Secreted by
Carnobacterium divergens
V41
Authors: Bhugaloo-Vial Parwin , Douliez Jean-Paul , Mollé Daniel , Dousset Xavier , Boyaval Patrick , Marion Didier
Published: December 2019

Divercin V41 (DV41) is a class IIa bacteriocin produced by
Carnobacterium divergens
V41. This antilisterial peptide is homologous to pediocin PA-1 and contains two disulfide bonds. To establish the structure-activity relationships of this specific family of bacteriocin, chemical modifications and enzymatic hydrolysis were performed on DV41. Alteration of the net charge of this cationic bacteriocin by succinylation and acetylation revealed that, in a certain range, the electrostatic interactions were surprisingly not necessary for the activity of DV41. Cleavage of DV41 by endoproteinase Asp-N released two fragments N1[1–17] and N2[18–43] corresponding to the conserved hydrophilic N-terminal and the variable hydrophobic C-terminal sequences, respectively. Inhibitory assays showed that only the C-terminal fragment was active, and after trypsin cleavage at Lys42 or disulfide reduction it lost its inhibitory activity. These results suggested that both hydrophobicity and folding imposed by the Cys25-Cys43 disulfide bond were essential for antilisterial activity of the C-terminal hydrophobic peptide. Chemical oxidation of tryptophan residues by N-bromosuccinimide demonstrated that these residues were crucial for inhibitory activity since modification of any one of them rendered DV41 inactive. On the contrary, only the modification of all the three tyrosine residues caused a total loss of antilisterial activity. These latter results strengthened previous results suggesting that the N-terminal domain containing the YGNGV consensus sequence was not involved in the binding of DV41 to a potential specific receptor on listerial cells.
Evaluation of Bottlenecks in the Late Stages of Protein Secretion in
Bacillus subtilis
Authors: Bolhuis Albert , Tjalsma Harold , Smith Hilde E. , de Jong Anne , Meima Rob , Venema Gerard , Bron Sierd , van Dijl Jan Maarten
Published: December 2019

Despite a high capacity for secretion of homologous proteins, the secretion of heterologous proteins by
Bacillus subtilis
is frequently inefficient. In the present studies, we have investigated and compared bottlenecks in the secretion of four heterologous proteins:
Bacillus lichenifomis
α-amylase (AmyL),
Escherichia coli
TEM β-lactamase (Bla), human pancreatic α-amylase (HPA), and a lysozyme-specific single-chain antibody. The same expression and secretion signals were used for all four of these proteins. Notably, all identified bottlenecks relate to late stages in secretion, following translocation of the preproteins across the cytoplasmic membrane. These bottlenecks include processing by signal peptidase, passage through the cell wall, and degradation in the wall and growth medium. Strikingly, all translocated HPA was misfolded, its stability depending on the formation of disulfide bonds. This suggests that the disulfide bond oxidoreductases of
B. subtilis
cannot form the disulfide bonds in HPA correctly. As the secretion bottlenecks differed for each heterologous protein tested, it is anticipated that the efficient secretion of particular groups of heterologous proteins with the same secretion bottlenecks will require the engineering of specifically optimized host strains.
Isolation and Characterization of a Sulfate-Reducing Bacterium That Anaerobically Degrades Alkanes
Authors: So Chi Ming , Young L. Y.
Published: December 2019

An alkane-degrading, sulfate-reducing bacterial strain, AK-01, was isolated from an estuarine sediment with a history of chronic petroleum contamination. The bacterium is a short, nonmotile, non-spore-forming, gram-negative rod. It is mesophilic and grows optimally at pH 6.9 to 7.0 and at an NaCl concentration of 1%. Formate, fatty acids (C
4
to C
16
) and hydrogen were readily utilized as electron donors. Sulfate, sulfite, and thiosulfate were used as electron acceptors, but sulfur, nitrite, and nitrate were not. Phenotypic characterization and phylogenetic analysis based on 16S rRNA gene sequence indicate that AK-01 is most closely related to the genera
Desulfosarcina
,
Desulfonema
, and
Desulfococcus
in the delta subdivision of the class
Proteobacteria
. It is phenotypically and phylogenetically different from strains Hxd3 and TD3, two previously reported isolates of alkane-degrading, sulfate-reducing bacteria. The alkanes tested to support growth of AK-01 had chain lengths of C
13
to C
18
. 1-Alkenes (C
15
and C
16
) and 1-alkanols (C
15
and C
16
) also supported growth. The doubling time for growth on hexadecane was 3 days, about four times longer than that for growth on hexadecanoate. Mineralization of hexadecane was indicated by the recovery of
14
CO
2
from cultures grown on [1-
14
C]hexadecane. Degradation of hexadecane was dependent on sulfate reduction. The stoichiometric ratio (as moles of sulfate reduced per mole of hexadecane degraded) was 10.6, which is very close to the theoretical ratio of 12.25, assuming a complete oxidation to CO
2
. Anaerobic alkane degradation by sulfate reducers may be a more widespread phenomenon than was previously thought.
Composition of Toluene-Degrading Microbial Communities from Soil at Different Concentrations of Toluene
Authors: Hubert Casey , Shen Yin , Voordouw Gerrit
Published: December 2019

Toluene-degrading bacteria were isolated from hydrocarbon-contaminated soil by incubating liquid enrichment cultures and agar plate cultures in desiccators in which the vapor pressure of toluene was controlled by dilution with vacuum pump oil. Incubation in desiccators equilibrated with either 100, 10, or 1% (wt/wt) toluene in vacuum pump oil and testing for genomic cross-hybridization resulted in four genomically distinct strains (standards) capable of growth on toluene (strains Cstd1, Cstd2, Cstd5, and Cstd7). The optimal toluene concentrations for growth of these standards on plating media differed considerably. Cstd1 grew best in an atmosphere equilibrated with 0.1% (wt/wt) toluene, but Cstd5 failed to grow in this atmosphere. Conversely, Cstd5 grew well in the presence of 10% (wt/wt) toluene, which inhibited growth of Cstd1. 16S ribosomal DNA sequencing and cross-hybridization analysis indicated that both Cstd1 and Cstd5 are members of the genus
Pseudomonas
. An analysis of the microbial communities in soil samples that were incubated with 10% (wt/wt) toluene with reverse sample genome probing indicated that
Pseudomonas
strain Cstd5 was the dominant community member. However, incubation of soil samples with 0.1% (wt/wt) toluene resulted in a community that was dominated by
Pseudomonas
strain Q7, a toluene degrader that has been described previously (Y. Shen, L. G. Stehmeier, and G. Voordouw, Appl. Environ. Microbiol. 64:637–645, 1998). Q7 was not able to grow by itself in an atmosphere equilibrated with 0.1% (wt/wt) toluene but grew efficiently in coculture with Cstd1, suggesting that toluene or metabolic derivatives of toluene were transferred from Cstd1 to Q7.
Nisin Production by a Mixed-Culture System Consisting of
Lactococcus lactis
and
Kluyveromyces marxianus
Authors: Shimizu Hiroshi , Mizuguchi Taiji , Tanaka Eiji , Shioya Suteaki
Published: December 2019

To control the pH during antimicrobial peptide (nisin) production by a lactic acid bacterium,
Lactococcus lactis
subsp.
lactis
(ATCC11454), a novel method involving neither addition of alkali nor a separation system such as a ceramic membrane filter and electrodialyzer was developed. A mixed culture of
L. lactis
and
Kluyveromyces marxianus
, which was isolated from kefir grains, was utilized in the developed system. The interaction between lactate production by
L. lactis
and its assimilation by
K. marxianus
was used to control the pH. To utilize the interaction of these microorganisms to maintain high-level production of nisin, the kinetics of growth of, and production of lactate, acetate, and nisin by,
L. lactis
were investigated. The kinetics of growth of and lactic acid consumption by
K. marxianus
were also investigated. Because the pH of the medium could be controlled by the lactate consumption of
K. marxianus
and the specific lactate consumption rate of
K. marxianus
could be controlled by changing the dissolved oxygen (DO) concentration, a cascade pH controller coupled with DO control was developed. As a result, the pH was kept constant because the lactate level was kept low and nisin accumulated in the medium to a high level compared with that attained using other pH control strategies, such as with processes lacking pH control and those in which pH is controlled by addition of alkali.
Nitrogen, Carbon, and Sulfur Metabolism in Natural
Thioploca
Samples
Authors: Otte Sandra , Kuenen J. Gijs , Nielsen Lars P. , Paerl Hans W. , Zopfi Jakob , Schulz Heide N. , Teske Andreas , Strotmann Bettina , Gallardo Victor A. , Jørgensen Bo B.
Published: December 2019

Filamentous sulfur bacteria of the genus
Thioploca
occur as dense mats on the continental shelf off the coast of Chile and Peru. Since little is known about their nitrogen, sulfur, and carbon metabolism, this study was undertaken to investigate their (eco)physiology.
Thioploca
is able to store internally high concentrations of sulfur globules and nitrate. It has been previously hypothesized that these large vacuolated bacteria can oxidize sulfide by reducing their internally stored nitrate. We examined this nitrate reduction by incubation experiments of washed
Thioploca
sheaths with trichomes in combination with
15
N compounds and mass spectrometry and found that these
Thioploca
samples produce ammonium at a rate of 1 nmol min
−1
mg of protein
−1
. Controls showed no significant activity. Sulfate was shown to be the end product of sulfide oxidation and was observed at a rate of 2 to 3 nmol min
−1
mg of protein
−1
. The ammonium and sulfate production rates were not influenced by the addition of sulfide, suggesting that sulfide is first oxidized to elemental sulfur, and in a second independent step elemental sulfur is oxidized to sulfate. The average sulfide oxidation rate measured was 5 nmol min
−1
mg of protein
−1
and could be increased to 10.7 nmol min
−1
mg of protein
−1
after the trichomes were starved for 45 h. Incorporation of
14
CO
2
was at a rate of 0.4 to 0.8 nmol min
−1
mg of protein
−1
, which is half the rate calculated from sulfide oxidation. [2-
14
C]acetate incorporation was 0.4 nmol min
−1
mg of protein
−1
, which is equal to the CO
2
fixation rate, and no
14
CO
2
production was detected. These results suggest that
Thioploca
species are facultative chemolithoautotrophs capable of mixotrophic growth. Microautoradiography confirmed that
Thioploca
cells assimilated the majority of the radiocarbon from [2-
14
C]acetate, with only a minor contribution by epibiontic bacteria present in the samples.
Development of Bacterial Contamination during Production of Yeast Extracts
Authors: Barrette Julie , Champagne Claude P. , Goulet Jacques
Published: December 2019

Baker’s yeast suspensions having bacterial populations of 106and 108CFU/ml were subjected to autolysis processes designed to obtain yeast extracts (YE). The bacterial contaminants added to the yeast cell suspensions were produced with spent broths obtained from a commercial yeast production plant and contained 59% cocci (Leuconostoc,Aerococcus,Lactococcus) as well as 41% bacilli (Bacillus). Autolyses were conducted at four different pH levels (4.0, 5.5, 7.0, and 8.5) and with two autolysis-promoting agents (ethyl acetate and chitosan). Processing parameters were more important than the initial bacterial population in the development of contaminating bacteria during manufacture of YE. Drops in the viable bacterial population after a 24-h autolysis were observed when pH was adjusted to 4.0 or when ethyl acetate was added. A significant interaction was found between the effects of pH and autolysis promoters on the bacterial population in YE, indicating that the activity of ethyl acetate, as opposed to that of chitosan, was not influenced by pH.
Low-Molecular-Weight Sulfonates, a Major Substrate for Sulfate Reducers in Marine Microbial Mats
Authors: Visscher Pieter T. , Gritzer Rachel F. , Leadbetter Edward R.
Published: December 2019

Several low-molecular-weight sulfonates were added to microbial mat slurries to investigate their effects on sulfate reduction. Instantaneous production of sulfide occurred after taurine and cysteate were added to all of the microbial mats tested. The rates of production in the presence of taurine and cysteate were 35 and 24 μM HS−h−1in a stromatolite mat, 38 and 36 μM HS−h−1in a salt pond mat, and 27 and 18 μM HS−h−1in a salt marsh mat, respectively. The traditionally used substrates lactate and acetate stimulated the rate of sulfide production 3 to 10 times more than taurine and cysteate stimulated the rate of sulfide production in all mats, but when ethanol, glycolate, and glutamate were added to stromatolite mat slurries, the resulting increases were similar to the increases observed with taurine and cysteate. Isethionate, sulfosuccinate, and sulfobenzoate were tested only with the stromatolite mat slurry, and these compounds had much smaller effects on sulfide production. Addition of molybdate resulted in a greater inhibitory effect on acetate and lactate utilization than on sulfonate use, suggesting that different metabolic pathways were involved. In all of the mats tested taurine and cysteate were present in the pore water at nanomolar to micromolar concentrations. An enrichment culture from the stromatolite mat was obtained on cysteate in a medium lacking sulfate and incubated anaerobically. The rate of cysteate consumption by this enrichment culture was 1.6 pmol cell−1h−1. Compared to the results of slurry studies, this rate suggests that organisms with properties similar to the properties of this enrichment culture are a major constituent of the sulfidogenic population. In addition, taurine was consumed at some of highest dilutions obtained from most-probable-number enrichment cultures obtained from stromatolite samples. Based on our comparison of the sulfide production rates found in various mats, low-molecular-weight sulfonates are important sources of C and S in these ecosystems.
Nanomolar Levels of Dimethylsulfoniopropionate, Dimethylsulfonioacetate, and Glycine Betaine Are Sufficient To Confer Osmoprotection to
Escherichia coli
Authors: Cosquer Anne , Pichereau Vianney , Pocard Jean-Alain , Minet Jacques , Cormier Michel , Bernard Théophile
Published: December 2019

We combined the use of low inoculation titers (300 ± 100 CFU/ml) and enumeration of culturable cells to measure the osmoprotective potentialities of dimethylsulfoniopropionate (DMSP), dimethylsulfonioacetate (DMSA), and glycine betaine (GB) for salt-stressed cultures of
Escherichia coli
. Dilute bacterial cultures were grown with osmoprotectant concentrations that encompassed the nanomolar levels of GB and DMSP found in nature and the millimolar levels of osmoprotectants used in standard laboratory osmoprotection bioassays. Nanomolar concentrations of DMSA, DMSP, and GB were sufficient to enhance the salinity tolerance of
E. coli
cells expressing only the ProU high-affinity general osmoporter. In contrast, nanomolar levels of osmoprotectants were ineffective with a mutant strain (GM50) that expressed only the low-affinity ProP osmoporter. Transport studies showed that DMSA and DMSP, like GB, were taken up via both ProU and ProP. Moreover, ProU displayed higher affinities for the three osmoprotectants than ProP displayed, and ProP, like ProU, displayed much higher affinities for GB and DMSA than for DMSP. Interestingly, ProP did not operate at substrate concentrations of 200 nM or less, whereas ProU operated at concentrations ranging from 1 nM to millimolar levels. Consequently,
proU
+
strains of
E. coli
, but not the
proP
+
strain GM50, could also scavenge nanomolar levels of GB, DMSA, and DMSP from oligotrophic seawater. The physiological and ecological implications of these observations are discussed.
Kinetics, Stereospecificity, and Expression of the Malolactic Enzyme
Authors: Arthurs Ceri E. , Lloyd David
Published: December 2019

Mass spectrometric measurement of carbon dioxide production was used to study malolactic fermentation (MLF) in
Lactobacillus collinoides
isolated from cider. The kinetics and stereospecificity of the malolactic enzyme (MLE) were studied, and the stoichiometry of the reaction sequence was investigated. The optimum pH for activity of the MLE was 4.9. MLF was more rapid (in both intact cells and cell extracts) when
l
-malic acid was used than when
d
-malic acid or the racemic mixture was added. The enzyme was found to be constitutively present in
L. collinoides
. Addition of
l
-malic acid (37 mM) to the growth medium resulted in increased MLE activity; addition of the
d
isomer alone or the racemic mixture resulted in lower activities. Addition of the main sugars in apple juice (fructose, sucrose, and glucose) to the growth medium in the presence of malic acid repressed production of MLE to similar extents in all three cases; in the absence of malic acid, instead of inhibiting MLF, addition of sugars to the growth medium somewhat increased the residual MLE activity.
Isolation, Characterization, and Avenacin Sensitivity of a Diverse Collection of Cereal-Root-Colonizing Fungi
Authors: Carter Jon P. , Spink John , Cannon Paul F. , Daniels Michael J. , Osbourn Anne E.
Published: December 2019

A total of 161 fungal isolates were obtained from the surface-sterilized roots of field-grown oat and wheat plants in order to investigate the nature of the root-colonizing fungi supported by these two cereals. Fungi were initially grouped according to their colony morphologies and then were further characterized by ribosomal DNA sequence analysis. The collection contained a wide range of ascomycetes and also some basidiomycete fungi. The fungi were subsequently assessed for their abilities to tolerate and degrade the antifungal oat root saponin, avenacin A-1. Nearly all the fungi obtained from oat roots were avenacin A-1 resistant, while both avenacin-sensitive and avenacin-resistant fungi were isolated from the roots of the non-saponin-producing cereal, wheat. The majority of the avenacin-resistant fungi were able to degrade avenacin A-1. These experiments suggest that avenacin A-1 is likely to influence the development of fungal communities within (and possibly also around) oat roots.
Optical Characteristics of the Phototroph
Thiocapsa roseopersicina
and Implications for Real-Time Monitoring of the Bacteriochlorophyll Concentration
Authors: Gitelson A. , Stark R. , Dor I. , Michelson O. , Yacobi Y. Z.
Published: December 2019

Optical characteristics of a
Thiocapsa roseopersicina
culture and environmental samples containing
T. roseopersicina
were investigated in the spectral range of 400 to 1,100 nm (absorption coefficient, diffuse attenuation coefficient, and reflectance). Specific absorption coefficients of
T. roseopersicina
at wavelengths of 480, 520, 550, 580, 805, 860, and 880 nm were determined. It is suggested that the optical properties of
T. roseopersicina
in the near-infrared range of 800 to 930 nm, confirmed in this study, may be used for development of remote sensing techniques for real-time monitoring of
T. roseopersicina
and other bacteriochlorophyll
a
-containing microbes.
Use of Antibiotic Resistance Analysis To Identify Nonpoint Sources of Fecal Pollution
Authors: Wiggins B. A. , Andrews R. W. , Conway R. A. , Corr C. L. , Dobratz E. J. , Dougherty D. P. , Eppard J. R. , Knupp S. R. , Limjoco M. C. , Mettenburg J. M. , Rinehardt J. M. , Sonsino J. , Torrijos R. L. , Zimmerman M. E.
Published: December 2019

A study was conducted to determine the reliability and repeatability of antibiotic resistance analysis as a method of identifying the sources of fecal pollution in surface water and groundwater. Four large sets of isolates of fecal streptococci (from 2,635 to 5,990 isolates per set) were obtained from 236 samples of human sewage and septage, cattle and poultry feces, and pristine waters. The patterns of resistance of the isolates to each of four concentrations of up to nine antibiotics were analyzed by discriminant analysis. When isolates were classified individually, the average rate of correct classification (ARCC) into four possible types (human, cattle, poultry, and wild) ranged from 64 to 78%. When the resistance patterns of all isolates from each sample were averaged and the resulting sample-level resistance patterns were classified, the ARCCs were much higher (96 to 100%). These data confirm that there are measurable and consistent differences in the antibiotic resistance patterns of fecal streptococci isolated from various sources of fecal pollution and that antibiotic resistance analysis can be used to classify and identify these sources.
Chloramphenicol Inhibition of Denitrifying Enzyme Activity in Two Agricultural Soils
Authors: Murray Robert E. , Knowles Roger
Published: December 2019

Chloramphenicol, at concentrations greater than 0.1 g/liter (0.3 mM), inhibited the denitrifying enzyme activity (DEA) of slurries of humisol and sandy loam soils by disrupting the activity of existing nitrate reductase enzymes. When the concentration of chloramphenicol was increased from 0.1 to 2.0 g/liter (6.0 mM), the rate of nitrite production from nitrate decreased by 25 to 46%. The rate of NO production from nitrate decreased by 20 to 39%, and the rate of N
2
O production from nitrate, in the presence of acetylene (DEA), decreased by 21 to 61%. The predicted values of DEA at 0 g of chloramphenicol/liter computed from linear regressions of DEA versus chloramphenicol concentration were 18 to 43% lower than DEA measurements made in the absence of chloramphenicol and within a few per cent of DEA rates measured in the presence of 0.1 g of chloramphenicol/liter. We conclude that DEA assays should be carried out with a single (0.1-g/liter) chloramphenicol concentration. Chloramphenicol at concentrations greater than 0.1 g/liter inhibits the activity of existing denitrifying enzymes and should not be used in DEA assays.
Depth Penetration and Detection of pH Gradients in Biofilms by Two-Photon Excitation Microscopy
Authors: Vroom Jurrien M. , De Grauw Kees J. , Gerritsen Hans C. , Bradshaw David J. , Marsh Philip D. , Watson G. Keith , Birmingham John J. , Allison Clive
Published: December 2019

Deep microbial biofilms are a major problem in many industrial, environmental, and medical settings. Novel approaches are needed to understand the structure and metabolism of these biofilms. Two-photon excitation microscopy (TPE) and conventional confocal laser scanning microscopy (CLSM) were compared quantitatively for the ability to visualize bacteria within deep in vitro biofilms. pH gradients within these biofilms were determined by fluorescence lifetime imaging, together with TPE. A constant-depth film fermentor (CDFF) was inoculated for 8 h at 50 ml · h
−1
with a defined mixed culture of 10 species of bacteria grown in continuous culture. Biofilms of fixed depths were developed in the CDFF for 10 or 11 days. The microbial compositions of the biofilms were determined by using viable counts on selective and nonselective agar media; diverse mixed-culture biofilms developed, including aerobic, facultative, and anaerobic species. TPE was able to record images four times deeper than CLSM. Importantly, in contrast to CLSM images, TPE images recorded deep within the biofilm showed no loss of contrast. The pH within the biofilms was measured directly by means of fluorescence lifetime imaging; the fluorescence decay of carboxyfluorescein was correlated with biofilm pH and was used to construct a calibration curve. pH gradients were detectable, in both the lateral and axial directions, in steady-state biofilms. When biofilms were overlaid with 14 mM sucrose for 1 h, distinct pH gradients developed. Microcolonies with pH values of below pH 3.0 were visible, in some cases adjacent to areas with a much higher pH (>5.0). TPE allowed resolution of images at significantly greater depths (as deep as 140 μm) than were possible with CLSM. Fluorescence lifetime imaging allowed the in situ, real-time imaging of pH and the detection of sharp gradients of pH within microbial biofilms.
Use of Single-Strand Conformation Polymorphism Analysis To Examine the Variability of the
rpoS
Sequence in Environmental Isolates of Salmonellae
Authors: Jordan Suzanne J. , Dodd Christine E. R. , Stewart Gordon S. A. B.
Published: December 2019

The natural environment places its resident microflora under stress, which may often result in adaptation by the microflora in order to increase the probability of survival. One such mechanism that has been postulated involves
rpoS
, which encodes a sigma factor that is known to enhance survival upon exposure to stress. The present work aimed to examine the genetic variability of
rpoS
in a selection of
Salmonella enterica
subspecies environmental isolates with an automated single-strand conformation polymorphism analysis technique. The results indicated that sequence variation does occur and that these changes are mainly located in two areas: at the center and near the end of the coding region. The variability was generally at the single-base level, although one strain (
S. arizonae
) did demonstrate significant differences in nucleotide sequence.
Distribution and Evolution of the Xylanase Genes
xynA
and
xynB
and Their Homologues in Strains of
Butyrivibrio fibrisolvens
Authors: Dalrymple Brian P. , Swadling Yolande , Layton Ingrid , Gobius Kari S. , Xue Gang-Ping
Published: December 2019

The ruminal bacterium
Butyrivibrio fibrisolvens
is being engineered by the introduction of heterologous xylanase genes in an attempt to improve the utilization of plant material in ruminants. However, relatively little is known about the diversity and distribution of the native xylanase genes in strains of
B. fibrisolvens
. In order to identify the most appropriate hosts for such modifications, the xylanase genotypes of 28 strains from the three 16S ribosomal DNA (rDNA) subgroups of
Butyrivibrio fibrisolvens
have been investigated. Only 4 of the 20 strains from 16S rDNA group 2 contained homologues of the strain Bu49
xynA
gene. However, these four
xynA
-containing strains, and two other group 2 strains, contained members of a second xylanase gene family clearly related to
xynA
(subfamily I). Homologues of
xynB
, a second previously described xylanase gene from
B. fibrisolvens
, were identified only in three of the seven group 1 strains and not in the group 2 and 3 strains. However, six of the group 1 strains contained one or more members of the two subfamilies of homologues of
xynA
. The distribution of genes and the nucleotide sequence relationships between the members of the two
xynA
subfamilies are consistent with the progenitor of all strains of
B. fibrisolvens
having contained a
xynA
subfamily I gene. Since many xylanolytic strains of
B. fibrisolvens
did not contain members of either of the
xynA
subfamilies or of the
xynB
family, at least one additional xylanase gene family remains to be identified in
B. fibrisolvens
.