bluuebunny/crossref_metadata_2025_split huggingface dataset:
Functional Complementation of Nontoxic Mutant Binary Toxins of
Bacillus sphaericus
1593M Generated by Site-Directed Mutagenesis
Authors: Shanmugavelu M. , Rajamohan F. , Kathirvel M. , Elangovan G. , Dean D. H. , Jayaraman Kunthala 
Published: December 2019

ABSTRACT

Alanine residues were substituted by site-directed mutagenesis at selected sites of the N- and C-terminal regions of the binary toxin (51- and 42-kDa peptides) of
B. sphaericus
1593M, and the mutant toxins were cloned and expressed in
Escherichia coli
. Bioassays with mosquito larvae, using binary toxins derived from individual mutants, showed that the substitution of alanine at some sites in both the 51-kDa and the 42-kDa peptides resulted in a total loss of activity. Surprisingly, after mixing two nontoxic derivatives of the same peptide, i.e., one mutated at the N-terminal end and the other mutated at the C-terminal end of either the 51-kDa or the 42-kDa peptide, the toxicity was restored. This result indicates that the altered binary toxins can functionally complement each other by forming oligomers.
The H
+
-ATPase in the Plasma Membrane of
Saccharomyces cerevisiae
Is Activated during Growth Latency in Octanoic Acid-Supplemented Medium Accompanying the Decrease in Intracellular pH and Cell Viability
Authors: Viegas Cristina A. , Almeida Paulo F. , Cavaco Miguel , Sá-Correia Isabel 
Published: December 2019

ABSTRACT

Saccharomyces cerevisiae
plasma membrane H
+
-ATPase activity was stimulated during octanoic acid-induced latency, reaching maximal values at the early stages of exponential growth. The time-dependent pattern of ATPase activation correlated with the decrease of cytosolic pH (pH
i
). The cell population used as inoculum exhibited a significant heterogeneity of pH
i
, and the fall of pH
i
correlated with the loss of cell viability as determined by plate counts. When exponential growth started, only a fraction of the initial population was still viable, consistent with the role of the physiology and number of viable cells in the inoculum in the duration of latency under acid stress.
Exopolysaccharide II Production Is Regulated by Salt in the Halotolerant Strain
Rhizobium meliloti
EFB1
Authors: Lloret Javier , Wulff Brande B. H. , Rubio Jose M. , Downie J. Allan , Bonilla Ildefonso , Rivilla Rafael 
Published: December 2019

ABSTRACT

The halotolerant strain
Rhizobium meliloti
EFB1 modifies the production of extracellular polysaccharides in response to salt. EFB1 colonies grown in the presence of 0.3 M NaCl show a decrease in mucoidy, and in salt-supplemented liquid medium this organism produces 40% less exopolysaccharides. We isolated transposon-induced mutant that, when grown in the absence of salt, had a colony morphology (nonmucoid) similar to the colony morphology of the wild type grown in the presence of salt. Calcofluor fluorescence, proton nuclear magnetic resonance spectroscopy, and genetic analysis of the mutant indicated that galactoglucan, which is not produced under normal conditions by other
R. meliloti
strains, is produced by strain EFB1 and that production of this compound decreases when the organism is grown in the presence of salt. The mutant was found to be affected in a genetic region highly homologous to genes for galactoglucan production in
R. meliloti
Rm2011 (
expE
genes). However, sequence divergence occurs in a putative
expE
promoter region. A transcriptional fusion of the promoter with
lacZ
demonstrated that, unlike
R. meliloti
Rm2011, galactoglucan is produced constitutively by EFB1 and that its expression is reduced 10-fold during exponential growth in the presence of salt.
Uptake of Choline and Its Conversion to Glycine Betaine by Bacteria in Estuarine Waters
Authors: Kiene Ronald P. 
Published: December 2019

ABSTRACT

The uptake and degradation of nanomolar levels of [
methyl
-
14
C]choline in estuarine water samples and in seawater filtrate cultures composed mainly of natural free-living bacteria was studied. Uptake of [
14
C]choline exhibited Michaelis-Menten kinetics, with

K
t

+

S
n

values of 1.7 to 2.9 nM in filtrate cultures and 1.7 to 4.1 nM in estuarine-water samples.
V
max
values ranged from 0.5 to 3.3 nM · h
−1
. The uptake system for choline in natural microbial assemblages therefore displays very high affinity and appears able to scavenge this compound at the concentrations expected in seawater. Uptake of choline was inhibited by some natural structural analogs and
p
-chloromercuribenzoate, indicating that the transporter may be multifunctional and may involve a thiol binding site. When 11 nM [
14
C]choline was added to water samples, a significant fraction (>50%) of the methyl carbon was respired to CO
2
in incubations lasting 10 to 53 h. Cells taking up [
14
C]choline produced [
14
C]glycine betaine ([
14
C]GBT), and up to 80% of the radioactivity retained by cells was in the form of GBT, a well-known osmolyte. Alteration of the salinity in filtrate cultures affected the relative proportion of [
14
C]choline degraded or converted to [
14
C]GBT, without substantially affecting the total metabolism of choline. Increasing the salinity from 14 to 25 or 35 ppt caused more [
14
C]GBT to be produced from choline but less
14
CO
2
to be produced than in the controls. Lowering the salinity to 7 ppt decreased [
14
C]GBT production and increased
14
CO
2
production slightly. Intracellular accumulations of [
14
C]GBT in the salt-stressed cultures were osmotically significant (34 mM). Choline may be used as an energy substrate by estuarine bacteria and may also serve as a precursor of the osmoprotectant GBT, particularly as bacteria are mixed into higher-salinity waters.
Influence of Structural Properties and Kinetic Constraints on
Bacillus cereus
Growth
Authors: Stecchini Mara Lucia , Del Torre Manuela , Sarais Ileana , Saro Onorio , Messina Mariella , Maltini Enrico 
Published: December 2019

ABSTRACT

The influence of structural properties and kinetic constraints on the behavior of
Bacillus cereus
was investigated on agar media. Dimensional criteria were used to study the growth in bacterial colonies. The architecture of the agar gel as modified by the agar content was found to influence the colony size, and smaller colonies were observed on media containing 50 to 70 g of agar liter
−1
. Except at low nutrient levels, colonies responded to nutrient gradients by decreasing in size the farther away they were from the nutrient source, and the decrease in colony size was influenced by the agar content. The diffusivities of glucose and a protein (insulin-like growth factor) were not affected by the gel architecture, suggesting that other factors, such as mechanical factors, could influence microbial growth in the agar systems used. Increasing the viscosity of the liquid phase of the agar media by adding polyvinylpyrrolidone resulted in a reduction in colony size. When the agar concentration was increased, the colony areas were not influenced by the viscosity of the system.
Methanol Promotes Atmospheric Methane Oxidation by Methanotrophic Cultures and Soils
Authors: Benstead J. , King G. M. , Williams H. G. 
Published: December 2019

ABSTRACT

Two methanotrophic bacteria,
Methylobacter albus
BG8 and
Methylosinus trichosporium
OB3b, oxidized atmospheric methane during batch growth on methanol. Methane consumption was rapidly and substantially diminished (95% over 9 days) when washed cell suspensions were incubated without methanol in the presence of atmospheric methane (1.7 ppm). Methanotrophic activity was stimulated after methanol (10 mM) but not methane (1,000 ppm) addition.
M. albus
BG8 grown in continuous culture for 80 days with methanol retained the ability to oxidize atmospheric methane and oxidized methane in a chemostat air supply. Methane oxidation during growth on methanol was not affected by methane deprivation. Differences in the kinetics of methane uptake (apparent

K
m

and
V
max
) were observed between batch- and chemostat-grown cultures. The
V
max
and apparent

K
m

values (means ± standard errors) for methanol-limited chemostat cultures were 133 ± 46 nmol of methane 10
8
cells
−1
h
−1
and 916 ± 235 ppm of methane (1.2 μM), respectively. These values were significantly lower than those determined with batch-grown cultures (
V
max
of 648 ± 195 nmol of methane 10
8
cells
−1
h
−1
and apparent

K
m

of 5,025 ± 1,234 ppm of methane [6.3 μM]). Methane consumption by soils was stimulated by the addition of methanol. These results suggest that methanol or other nonmethane substrates may promote atmospheric methane oxidation in situ.
Characterization of Root-Associated Methanotrophs from Three Freshwater Macrophytes:
Pontederia cordata
,
Sparganium eurycarpum
, and
Sagittaria latifolia
Authors: Calhoun A. , King G. M. 
Published: December 2019

ABSTRACT

Root-associated methanotrophic bacteria were enriched from three common aquatic macrophytes:
Pontederia cordata
,
Sparganium eurycarpum
, and
Sagittaria latifolia
. At least seven distinct taxa belonging to groups I and II were identified and presumptively assigned to the genera
Methylosinus
,
Methylocystis
,
Methylomonas
, and
Methylococcus
. Four of these strains appeared to be novel on the basis of partial 16S ribosomal DNA sequence analysis. The root-methanotroph association did not appear to be highly specific, since multiple methanotrophs were isolated from each of the three plant species. Group II methanotrophs were isolated most frequently; though less common, group I isolates accounted for three of the seven distinct methanotrophs. Apparent

K
m

values for methane uptake by representative cultures ranged from 3 to >17 μM; for five of the eight cultures examined, apparent

K
m

values agreed well with apparent

K
m

estimates for plant roots, suggesting that these strains may be representative of those active in situ.
Isolation of Copper Biochelates from
Methylosinus trichosporium
OB3b and Soluble Methane Monooxygenase Mutants
Authors: Téllez Carlos M. , Gaus Kristen P. , Graham David W. , Arnold Robert G. , Guzman Roberto Z. 
Published: December 2019

ABSTRACT

Methylosinus trichosporium
OB3b produces an extracellular copper-binding ligand (CBL) with high affinity for copper. Wild-type cells and mutants that express soluble methane monooxygenase (sMMO) in the presence and absence of copper (sMMO
c
) were used to obtain cell exudates that were separated and analyzed by size exclusion high-performance liquid chromatography. A single chromatographic peak, when present, contained most of the aqueous-phase Cu(II) present in the culture medium. In mutant cultures that were unable to acquire copper, extracellular CBL accumulated to high levels both in the presence and in the absence of copper. Conversely, in wild-type cultures containing 5 μM Cu(II), extracellular CBL was maintained at a low, steady level during exponential growth, after which the external ligand was rapidly consumed. When Cu(II) was omitted from the growth medium, the wild-type organism produced the CBL at a rate that was proportional to cell density. After copper was added to this previously Cu-deprived culture, the CBL and copper concentrations in the medium decreased at approximately the same rate. Apparently, the extracellular CBL was produced throughout the period of cell growth, in the presence and absence of Cu(II), by both the mutant and wild-type cultures and was reinternalized or otherwise utilized by the wild-type cultures when it was bound to copper. CBL produced by the mutant strain facilitated copper uptake by wild-type cells, indicating that the extracellular CBLs produced by the mutant and wild-type organisms are functionally indistinguishable. CBL from the wild-type strain did not promote copper uptake by the mutant. The molecular weight of the CBL was estimated to be 500, and its association constant with copper was 1.4 × 10
16
M
−1
. CBL exhibited a preference for copper, even in the presence of 20-fold higher concentrations of nickel. External complexation may play a role in normal copper acquisition by
M. trichosporium
OB3b. The sMMO
c
phenotype is probably related to the mutant’s inability to take up CBL-complexed copper, not to a defective CBL structure.
A Rapid Method for Screening Large Numbers of Environmental Microorganisms for Antiviral Activity
Authors: Maullu Carlo , Lampis Giorgio , Deidda Delia , Petruzzelli Sabrina , Pompei Raffaello 
Published: December 2019

ABSTRACT
A new method for screening microbial colonies endowed with antiviral activity is described. It is based on close contact between microbial agar cultures and agar-covered virus-infected-cell monolayers and allows the screening of large numbers of colonies in just a few months.
Production of Pediocin PA-1 by
Lactococcus lactis
Using the Lactococcin A Secretory Apparatus
Authors: Horn Nikki , Martínez María I. , Martínez José M. , Hernández Pablo E. , Gasson Michael J. , Rodríguez Juan M. , Dodd Helen M. 
Published: December 2019

ABSTRACT

The class II bacteriocins pediocin PA-1, from
Pediococcus acidilactici
, and lactococcin A, from
Lactococcus lactis
subsp.
lactis
bv. diacetylactis WM4 have a number of features in common. They are produced as precursor peptides containing similar amino-terminal leader sequences with a conserved processing site (Gly-Gly at positions −1 and −2). Translocation of both bacteriocins occurs via a dedicated secretory system. Because of the strong antilisterial activity of pediocin PA-1, its production by lactic acid bacteria strains adapted to dairy environments would considerably extend its application in the dairy industry. In this study, the lactococcin A secretory system was adapted for the expression and secretion of pediocin PA-1. A vector containing an in-frame fusion of sequences encoding the
lcnA
promoter, the lactococcin A leader, and the mature pediocin PA-1, was introduced into
L. lactis
IL1403. This strain is resistant to pediocin PA-1 and encodes a lactococcin translocation apparatus. The resulting
L. lactis
strains secreted a bacteriocin with an antimicrobial activity of approximately 25% of that displayed by the parental pediocin-producing
P. acidilactici
347. A noncompetitive indirect enzyme-linked immunosorbent assay with pediocin PA-1-specific antibodies and amino-terminal amino acid sequencing confirmed that pediocin PA-1 was being produced by the heterologous host.
The Citrate Transport System of
Lactococcus lactis
subsp.
lactis
biovar diacetylactis Is Induced by Acid Stress
Authors: García-Quintáns Nieves , Magni Christian , de Mendoza Diego , López Paloma 
Published: December 2019

ABSTRACT

Citrate transport in
Lactococcus lactis
subsp.
lactis
biovar diacetylactis is catalyzed by citrate permease P (CitP), which is encoded by the plasmidic
citP
gene. We have shown previously that
citP
is included in the
citQRP
operon, which is mainly transcribed from the P1 promoter in
L. lactis
subsp.
lactis
biovar diacetylactis. Furthermore, transcription of
citQRP
and citrate transport are not induced by the presence of citrate in the growth medium. In this work, we analyzed the influence of the extracellular pH on the expression of
citP
. The citrate transport system is induced by natural acidification of the medium during cell growth and by a shift to media buffered at acidic pHs. This inducible response to acid stress takes place at the transcriptional level and seems to be due to increased utilization of the P1 promoter. Increased transcription correlates with increased synthesis of CitP and results in higher citrate transport activity catalyzed by the cells. Finally, this acid stress response seems to provide
L. lactis
subsp.
lactis
biovar diacetylactis with a selective advantage resulting from cometabolism of glucose and citrate at low pHs.
Phylogeny of the Main Bacterial 16S rRNA Sequences in Drentse A Grassland Soils (The Netherlands)
Authors: Felske Andreas , Wolterink Arthur , Van Lis Robert , Akkermans Antoon D. L. 
Published: December 2019

ABSTRACT

The main bacteria in peaty, acid grassland soils in the Netherlands were investigated by ribosome isolation, temperature gradient gel electrophoresis, hybridization, cloning, and sequencing. Instead of using only 16S rDNA to determine the sequences present, we focused on rRNA to classify and quantify the most active bacteria. After direct ribosome isolation from soil, a partial amplicon of bacterial 16S rRNA was generated by reverse transcription-PCR. The sequence-specific separation by temperature gradient gel electrophoresis yielded soil-specific fingerprints, which were compared to signals from a clone library of genes coding for 16S rRNA. Cloned 16S rDNA sequences matching with intense bands in the fingerprint were sequenced. The relationships of the sequences to those of cultured organisms of known phylogeny were determined. Most of the amplicons originated from organisms closely related to
Bacillus
species. Such sequences were also detected by direct dot blot hybridization on soil rRNA: a probe specific for
Firmicutes
with low G+C content counted for about 50% of all bacterial rRNA. The bacterial activity in Drentse A grassland soil could be estimated by direct dot blot hybridization and sequencing of clones; it was found that about 65% of all the bacterial ribosomes originated from
Firmicutes
. The most active bacteria apparently were
Bacillus
species, from which about half of the sequences derived. Other sequences similar to those of gram-positive bacteria were only remotely related to known
Firmicutes
with a high G+C content. Other sequences were related to
Proteobacteria
, mainly the alpha subclass.
Quinone Profiling of Bacterial Communities in Natural and Synthetic Sewage Activated Sludge for Enhanced Phosphate Removal
Authors: Hiraishi Akira , Ueda Yoko , Ishihara Junko 
Published: December 2019

ABSTRACT

Respiratory quinones were used as biomarkers to study bacterial community structures in activated sludge reactors used for enhanced biological phosphate removal (EBPR). We compared the quinone profiles of EBPR sludges and standard sludges, of natural sewage and synthetic sewage, and of plant scale and laboratory scale systems. Ubiquinone (Q) and menaquinone (MK) components were detected in all sludges tested at molar MK/Q ratios of 0.455 to 0.981. The differences in MK/Q ratios were much larger when we compared different wastewater sludges (i.e., raw sewage and synthetic sewage) than when we compared sludges from the EBPR and standard processes or plant scale and laboratory scale systems. In all sludges tested a Q with eight isoprene units (Q-8) was the most abundant quinone. In the MK fraction, either tetrahydrogenated MK-8 or MK-7 was the predominant type, and there was also a significant proportion of MK-6 to MK-8 in most cases. A numerical cluster analysis of the profiles showed that the sludges tested fell into two major clusters; one included all raw sewage sludges, and the other consisted of all synthetic sewage sludges, independent of the operational mode and scale of the reactors and the phosphate accumulation. These data suggested that Q-8-containing species belonging to the class
Proteobacteria
(i.e., species belonging to the beta subclass) were the major constituents of the bacterial populations in the EBPR sludge, as well as in standard activated sludge. Members of the class
Actinobacteria
(gram-positive bacteria with high DNA G+C contents) were the second most abundant group in both types of sludge. The bacterial community structures in activated sludge processes may be affected more by the nature of the influent wastewater than by the introduction of an anaerobic stage into the process or by the scale of the reactors.
Effects of Bacterial Host and Dichloromethane Dehalogenase on the Competitiveness of Methylotrophic Bacteria Growing with Dichloromethane
Authors: Gisi Daniel , Willi Laurent , Traber Hubert , Leisinger Thomas , Vuilleumier Stéphane 
Published: December 2019

ABSTRACT

Methylobacterium
sp. strain DM4 and
Methylophilus
sp. strain DM11 can grow with dichloromethane (DCM) as the sole source of carbon and energy by virtue of homologous glutathione-dependent DCM dehalogenases with markedly different kinetic properties (the
k
cat
values of the enzymes of these strains are 0.6 and 3.3 s
−1
, respectively, and the

K
m

values are 9 and 59 μM, respectively). These strains, as well as transconjugant bacteria expressing the DCM dehalogenase gene (
dcmA
) from DM11 or DM4 on a broad-host-range plasmid in the background of
dcmA
mutant DM4-2cr, were investigated by growing them under growth-limiting conditions and in the presence of an excess of DCM. The maximal growth rates and maximal levels of dehalogenase for chemostat-adapted bacteria were higher than the maximal growth rates and maximal levels of dehalogenase for batch-grown bacteria. The substrate saturation constant of strain DM4 was much lower than the

K
m

of its associated dehalogenase, suggesting that this strain is adapted to scavenge low concentrations of DCM. Strains and transconjugants expressing the DCM dehalogenase from strain DM11, on the other hand, had higher growth rates than bacteria expressing the homologous dehalogenase from strain DM4. Competition experiments performed with pairs of DCM-degrading strains revealed that a strain expressing the dehalogenase from DM4 had a selective advantage in continuous culture under substrate-limiting conditions, while strains expressing the DM11 dehalogenase were superior in batch culture when there was an excess of substrate. Only DCM-degrading bacteria with a
dcmA
gene similar to that from strain DM4, however, were obtained in batch enrichment cultures prepared with activated sludge from sewage treatment plants.
Production of the Carotenoids Lycopene, β-Carotene, and Astaxanthin in the Food Yeast
Candida utilis
Authors: Miura Yutaka , Kondo Keiji , Saito Toshiko , Shimada Hiroshi , Fraser Paul D. , Misawa Norihiko 
Published: December 2019

ABSTRACT

The food-grade yeast
Candida utilis
has been engineered to confer a novel biosynthetic pathway for the production of carotenoids such as lycopene, β-carotene, and astaxanthin. The exogenous carotenoid biosynthesis genes were derived from the epiphytic bacterium
Erwinia uredovora
and the marine bacterium
Agrobacterium aurantiacum
. The carotenoid biosynthesis genes were individually modified based on the codon usage of the
C. utilis
glyceraldehyde 3-phosphate dehydrogenase gene and expressed in
C. utilis
under the control of the constitutive promoters and terminators derived from
C. utilis
. The resultant yeast strains accumulated lycopene, β-carotene, and astaxanthin in the cells at 1.1, 0.4, and 0.4 mg per g (dry weight) of cells, respectively. This was considered to be a result of the carbon flow into ergosterol biosynthesis being partially redirected to the nonendogenous pathway for carotenoid production.
Specificity of Milk Peptide Utilization by
Lactococcus lactis
Authors: Juillard Vincent , Guillot Alain , Le Bars Dominique , Gripon Jean-Claude 
Published: December 2019

ABSTRACT

To study the substrate specificity of the oligopeptide transport system of
Lactococcus lactis
for its natural substrates, the growth of
L. lactis
MG1363 was studied in a chemically defined medium containing milk peptides or a tryptic digest of α
s2
-casein as the source of amino acids. Peptides were separated into acidic, neutral, and basic pools by solid-phase extraction or by cation-exchange liquid chromatography. Their ability to sustain growth and the time course of their utilization demonstrated the preferential use of hydrophobic basic peptides with molecular masses ranging between 600 and 1,100 Da by
L. lactis
MG1363 and the inability to use large, acidic peptides. These peptide utilization preferences reflect the substrate specificity of the oligopeptide transport system of the strain, since no significant cell lysis was inferred. Considering the free amino acid content of milk and these findings on peptide utilization, it was demonstrated that the cessation of growth of
L. lactis
MG1363 in milk was due to deprivation of leucine and methionine.