bluuebunny/crossref_metadata_2025 huggingface dataset:
Engineering of a Cold-Adapted Protease by Sequential Random Mutagenesis and a Screening System
Authors: Taguchi Seiichi , Ozaki Akiyoshi , Momose Haruo
Published: December 2019

A cold-adapted protease subtilisin was successfully isolated by evolutionary engineering based on sequential in vitro random mutagenesis and an improved method of screening (H. Kano, S. Taguchi, and H. Momose, Appl. Microbiol. Biotechnol. 47:46–51, 1997). The mutant subtilisin, termed m-63, exhibited a catalytic efficiency (expressed as the
k
cat
/

K
m

value) 100% higher than that of the wild type at 10°C when
N
-succinyl-
l
-Ala-
l
-Ala-
l
-Pro-
l
-Phe-
p
-nitroanilide was used as a synthetic substrate. This cold adaptation was achieved with three mutations, Val to Ile at position 72 (V72I), Ala to Thr at position 92 (A92T), and Gly to Asp at position 131 (G131D), and it was found that an increase in substrate affinity (i.e., a decreased

K
m

value) was mostly responsible for the increased activity. Analysis of kinetic parameters revealed that the V72I mutation contributed negatively to the activity but that the other two mutations, A92T and G131D, overcame the negative contribution to confer the 100% increase in activity. Besides suppression of the activity-negative mutation (V72I) by A92T and G131D, suppression of structural stability was observed in measurements of activity retention at 60°C and circular dichroism spectra at 10°C.
Immunomagnetic Capture PCR for Rapid Concentration and Detection of Hepatitis A Virus from Environmental Samples
Authors: Jothikumar N. , Cliver Dean O. , Mariam Tadesse W.
Published: December 2019

We studied the concentration of hepatitis A virus (HAV) from environmental samples by membrane filter-based urea-arginine phosphate buffer and its detection by using immunomagnetic capture (IC) reverse transcription (RT)-PCR (IC PCR). Magnetic beads coated with anti-HAV rabbit antibodies were used for enrichment and concentration of HAV from environmental samples. IC PCR is sensitive enough to detect as few as 0.04 PFU of cell culture-adapted HAV in inoculated water and sewage samples. IC PCR is specific and does not yield positive reactions with poliovirus 1, HAV RNA, or selected bacteriophages. IC concentrates viruses suspended in small volumes to microliter volumes that can be used directly in RT-PCR. IC concentration of viruses from sewage samples without concentration of inhibitory substances is important for successful RT-PCR detection. In a field trial, 2 of 18 raw sewage samples tested by IC PCR were positive for HAV.
The Use of Fluorogenic Substrates To Measure Fungal Presence and Activity in Soil
Authors: Miller Morten , Palojärvi Ansa , Rangger Andrea , Reeslev Morten , Kjøller Annelise
Published: December 2019

Our objective was to determine if 4-methylumbelliferyl-labelled enzyme substrates could be used to detect and quantify specific components of chitinase and cellulase activities as specific indicators of the presence and activity of fungal biomass. The fluorogenic substrates 4-methylumbelliferyl (MUF)
N
-acetyl-β-
d
-glucosaminide and MUF β-
d
-lactoside were used for the detection and quantification of β-
N
-acetylglucosaminidase (EC
3.2.1.30
) (NAGase) and endo 1,4-β-glucanase (EC
3.2.1.4
)/cellobiohydrolase (EC
3.2.1.91
) (CELase), respectively. Culture screenings on solid media showed a widespread ability to produce NAGase among a taxonomically diverse selection of fungi on media with and without added chitin. NAGase activity was expressed only in a limited number of bacteria and on media supplemented with chitin. The CELase activity was observed only in a limited number of fungi and bacteria. Bacterial CELase activity was expressed on agar media containing a cellulose-derived substrate. In soil samples, NAGase activity was significantly correlated with estimates of fungal biomass, based on the content of two fungus-specific indicator molecules, 18:2ω6 phospholipid fatty acid (PLFA) and ergosterol. CELase activity was significantly correlated with the PLFA-based estimate of fungal biomass in the soil, but no correlation was found with ergosterol-based estimates of fungal biomass.
Size-Selective Predation on Groundwater Bacteria by Nanoflagellates in an Organic-Contaminated Aquifer
Authors: Kinner N. E. , Harvey R. W. , Blakeslee K. , Novarino G. , Meeker L. D.
Published: December 2019

Time series incubations were conducted to provide estimates for the size selectivities and rates of protistan grazing that may be occurring in a sandy, contaminated aquifer. The experiments involved four size classes of fluorescently labeled groundwater bacteria (FLB) and 2- to 3-μm-long nanoflagellates, primarily
Spumella guttula
(Ehrenberg) Kent, that were isolated from contaminated aquifer sediments (Cape Cod, Mass.). The greatest uptake and clearance rates (0.77 bacteria · flagellate
−1
· h
−1
and 1.4 nl · flagellate
−1
· h
−1
, respectively) were observed for 0.8- to 1.5-μm-long FLB (0.21-μm
3
average cell volume), which represent the fastest growing bacteria within the pore fluids of the contaminated aquifer sediments. The 19:1 to 67:1 volume ratios of nanoflagellate predators to preferred bacterial prey were in the lower end of the range commonly reported for other aquatic habitats. The grazing data suggest that the aquifer nanoflagellates can consume as much as 12 to 74% of the unattached bacterial community in 1 day and are likely to have a substantive effect upon bacterial degradation of organic groundwater contaminants.
Reverse Transcriptase (RT) Inhibition of PCR at Low Concentrations of Template and Its Implications for Quantitative RT-PCR
Authors: Chandler Darrell P. , Wagnon Christina A. , Bolton Harvey
Published: December 2019

Numerous instances of reverse transcriptase (RT) inhibition of the PCR were observed while developing nonquantitative uncoupled RT-PCR techniques for detecting nitrogenase and ammonia monooxygenase gene expression in situ. The inhibitory effect of RT on the PCR was removed with increasing template concentrations beyond 10
5
to 10
6
copies. Including T4 gene 32 protein during the reverse transcription phase of the RT-PCR reaction increased the RT-PCR product yield by as much as 483%; if gene 32 protein was introduced after reverse transcription but prior to the PCR phase, no improvement in product yield was observed. Addition of 1 μg of exogenous calf thymus DNA or yeast tRNA did little to relieve RT inhibition of the PCR on both genomic DNA and mRNA templates. These results suggest that RT inhibition of the PCR is mediated through direct interaction with the specific primer-template combination (DNA and RNA) and point to specific assay modifications for estimating the extent of RT inhibition and counteracting some of the inhibitory effect. Furthermore, the working hypothesis of RT inhibition below a 10
5
to 10
6
copy threshold has important implications for quantitative RT-PCR studies. In particular, competitive, quantitative RT-PCR systems will consistently underestimate the actual RNA concentration. Hence, enumerations of RNA templates below 10
5
to 10
6
copies will be relative to an internal standard and will not be an absolute measure of RNA abundance in situ.
In Situ Gene Expression in Mixed-Culture Biofilms: Evidence of Metabolic Interactions between Community Members
Authors: Møller Søren , Sternberg Claus , Andersen Jens Bo , Christensen Bjarke Bak , Ramos Juan Luis , Givskov Michael , Molin Søren
Published: December 2019

Microbial communities growing in laboratory-based flow chambers were investigated in order to study compartmentalization of specific gene expression. Among the community members studied, the focus was in particular on
Pseudomonas putida
and a strain of an
Acinetobacter
sp., and the genes studied are involved in the biodegradation of toluene and related aromatic compounds. The upper-pathway promoter (
Pu
) and the
meta
-pathway promoter (
Pm
) from the TOL plasmid were fused independently to the gene coding for the green fluorescent protein (GFP), and expression from these promoters was studied in
P. putida
, which was a dominant community member. Biofilms were cultured in flow chambers, which in combination with scanning confocal laser microscopy allowed direct monitoring of promoter activity with single-cell spatial resolution. Expression from the
Pu
promoter was homogeneously induced by benzyl alcohol in both community and pure-culture biofilms, while the
Pm
promoter was induced in the mixed community but not in a pure-culture biofilm. By sequentially adding community members, induction of
Pm
was shown to be a consequence of direct metabolic interactions between an
Acinetobacter
species and
P. putida
. Furthermore, in fixed biofilm samples organism identity was determined and gene expression was visualized at the same time by combining GFP expression with in situ hybridization with fluorescence-labeled 16S rRNA targeting probes. This combination of techniques is a powerful approach for investigating structure-function relationships in microbial communities.
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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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.
Spatial and Temporal Deposition of Adhesive Extracellular Polysaccharide Capsule and Fimbriae by
Hyphomonas
Strain MHS-3
Authors: Quintero Ernesto J. , Busch Kathryn , Weiner Ronald M.
Published: December 2019

Hyphomonas
strain MHS-3, a member of a genus of primary colonizers of surfaces immersed in marine water, synthesizes two structures that mediate adhesion to solid substrata, namely, capsular exopolysaccharide and fimbriae. Specific stains, gold-labelled lectins, and monoclonal antibodies, along with transmission electron microscopy of synchronized populations, revealed that both structures are polarly and temporally expressed. The timed synthesis and placement of the fimbriae and capsule correlated with the timing and locus of MHS-3 adhesion.
Biodegradation of Metal-EDTA Complexes by an Enriched Microbial Population
Authors: Thomas Russell A. P. , Lawlor Kirsten , Bailey Mark , Macaskie Lynne E.
Published: December 2019

A mixed culture utilizing EDTA as the sole carbon source was isolated from a mixed inoculum of water from the River Mersey (United Kingdom) and sludge from an industrial effluent treatment plant. Fourteen component organisms were isolated from the culture, including representatives of the genera
Methylobacterium
,
Variovorax
,
Enterobacter
,
Aureobacterium
, and
Bacillus
. The mixed culture biodegraded metal-EDTA complexes slowly; the biodegradability was in the order Fe>Cu>Co>Ni>Cd. By incorporation of inorganic phosphate into the medium as a precipitant ligand, heavy metals were removed in parallel to EDTA degradation. The mixed culture also utilized a number of possible EDTA degradation intermediates as carbon sources.
Bacterial Oxidation of Mercury Metal Vapor, Hg(0)
Authors: Smith Tracy , Pitts Keith , McGarvey Jeffery A. , Summers Anne O.
Published: December 2019

We used metalloregulated luciferase reporter fusions and spectroscopic quantification of soluble Hg(II) to determine that the hydroperoxidase-catalase, KatG, of
Escherichia coli
can oxidize monatomic elemental mercury vapor, Hg(0), to the water-soluble, ionic form, Hg(II). A strain with a mutation in
katG
and a strain overproducing KatG were used to demonstrate that the amount of Hg(II) formed is proportional to the catalase activity. Hg(0) oxidation was much decreased in stationary-phase cells of a strain lacking KatG, suggesting that the monofunctional hydroperoxidase KatE is less effective at this reaction. Unexpectedly, Hg(0) oxidation also occurred in a strain lacking both KatE and KatG, suggesting that activities other than hydroperoxidases may carry out this reaction. Two typical soil bacteria,
Bacillus
and
Streptomyces
, also oxidize Hg(0) to Hg(II). These observations establish for the first time that bacteria can contribute, as do mammals and plants, to the oxidative phase of the global Hg cycle.
Temperature and Sporulation of Aquatic Hyphomycetes
Authors: Chauvet Eric , Suberkropp Keller
Published: December 2019

Temperature appears to be an important factor affecting the occurrence and distribution of aquatic hyphomycetes, the dominant leaf litter-decomposing fungi in streams. We compared conidium production by eight species of aquatic hyphomycetes grown on yellow poplar leaves in stream-simulating microcosms at three temperatures (15, 20, and 25°C). The greatest conidium production occurred at 15°C for one species, 20°C for two species, and 25°C for two species. Two species produced similar numbers of conidia at 20 and 25°C, and one species produced similar numbers of conidia at all three temperatures. Linear growth rates were determined on malt extract agar. Six species had the same pattern of temperature responses for growth on malt extract agar as for sporulation on leaves, as shown by the positive correlations between the two parameters at the three temperatures. The species examined also exhibited differences in number of conidia produced from a similar amount of leaf material at a given temperature. These differences appeared to be due primarily to differences in individual conidium mass (determined by weighing conidia produced from cultures), as shown by the relationship of the type
Y = k/X
(
r
2
= 0.96), where
Y
is the number of conidia produced,
X
is the individual conidium mass in milligrams, and
k
is a constant empirically determined to be 2.11. This finding supports the hypothesis that aquatic hyphomycetes allocate similar amounts of their resources to reproduction but vary with respect how these resources are partitioned into reproductive units (conidia).
Enterohemorrhagic
Escherichia coli
O157:H7 Present in Radish Sprouts
Authors: Itoh Yoshinori , Sugita-Konishi Yoshiko , Kasuga Fumiko , Iwaki Masaaki , Hara-Kudo Yukiko , Saito Noriko , Noguchi Yoko , Konuma Hirotaka , Kumagai Susumu
Published: December 2019

Using cultivation, immunofluorescence microscopy, and scanning electron microscopy, we demonstrated the presence of viable enterohemorrhagic
Escherichia coli
O157:H7 not only on the outer surfaces but also in the inner tissues and stomata of cotyledons of radish sprouts grown from seeds experimentally contaminated with the bacterium. HgCl
2
treatment of the outer surface of the hypocotyl did not kill the contaminating bacteria, which emphasized the importance of either using seeds free from
E. coli
O157:H7 in the production of radish sprouts or heating the sprouts before they are eaten.
Development of a Direct In Situ PCR Method for Detection of Specific Bacteria in Natural Environments
Authors: Tani Katsuji , Kurokawa Ken , Nasu Masao
Published: December 2019

We applied HNPP (2-hydroxy-3-naphthoic acid-2′-phenylanilide phosphate) to direct in situ PCR for the routine detection of specific bacterial cells at the single-cell level. PCR was performed on glass slides with digoxigenin-labeled dUTP. The digoxigenin-labeled PCR products were detected with alkaline phosphatase-labeled antidigoxigenin antibody and HNPP which was combined with Fast Red TR. A bright red fluorescent signal was produced from conversion to HNP (dephosphorylated form) by alkaline phosphatase. We used the ECOL DNA primer set for amplification of ribosomal DNA of
Escherichia coli
to identify cells specifically at the single-cell level in a bacterial mixture. High-contrast images were obtained under an epifluorescence microscope with in situ PCR. By image analysis,
E. coli
cells in polluted river water also were detected.
An Origin of Transfer (
oriT
) on the Conjugative Element pRS01 from
Lactococcus lactis
subsp.
lactis
ML3
Authors: Mills David A. , Phister Trevor G. , Dunny Gary M. , McKay Larry L.
Published: December 2019

Previous analysis of the Tra1 region of the conjugative element pRS01 from
Lactococcus lactis
subsp.
lactis
ML3 suggested that an origin of transfer (
oriT
) was present. Deletion derivatives of this cloned Tra1 region were assayed for mobilization in the presence of the wild-type pRS01 element in
trans
. The pRS01
oriT
was localized to a 446-nucleotide segment in the intergenic region between open reading frames
ltrD
and
ltrE
. Sequence analysis of this region revealed a cluster of direct and inverted repeat structures characteristic of
oriT
regions associated with other conjugative systems.
Laser Microsurgery Permits Fungal Plasma Membrane Single-Ion-Channel Resolution at the Hyphal Tip
Authors: Véry Anne-Aliénor , Davies Julia M.
Published: December 2019

A method for formation of high-electrical-resistance seals on the
Neurospora crassa
plasma membrane, allowing resolution of single-ion-channel activity by patch clamp electrophysiology, is reported. Laser microsurgery permits access to the hyphal apex without enzymatic cell wall digestion and loss of morphological polarity. Cell wall reformation is delayed by brefeldin. This method can allow full characterization of apical plasma membrane channels, which are implicated in tip growth.
Characterization of Laccases and Peroxidases from Wood-Rotting Fungi (FamilyCoprinaceae)
Authors: Heinzkill Marion , Bech Lisbeth , Halkier Torben , Schneider Palle , Anke Timm
Published: December 2019

Panaeolus sphinctrinus,Panaeolus papilionaceus, andCoprinus friesiiare described as producers of ligninolytic enzymes.P. papilionaceusandP. sphinctrinusboth produced a laccase. In addition,P. sphinctrinusproduced a manganese peroxidase.C. friesiisecreted a laccase and two peroxidases similar to the peroxidase ofCoprinus cinereus. The purified laccases and peroxidases were characterized by broad substrate specificities, significant enzyme activities at alkaline pH values, and remarkably high pH optima. The two peroxidases ofC. friesiiremained active at pH 7.0 and 60°C for up to 60 min of incubation. The peroxidases were inhibited by sodium azide and ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), whereas the laccases were inhibited by sodium azide andN,N-diethyldithiocarbamic acid. As determined by native polyacrylamide gel electrophoresis and isoelectric focusing, all three fungi produced laccase isoenzymes.
Overproduction of
l
-Cysteine and
l
-Cystine by
Escherichia coli
Strains with a Genetically Altered Serine Acetyltransferase
Authors: Nakamori Shigeru , Kobayashi Shin-ichiro , Kobayashi Chitose , Takagi Hiroshi
Published: December 2019

Organisms that overproduced
l
-cysteine and
l
-cystine from glucose were constructed by using
Escherichia coli
K-12 strains.
cysE
genes coding for altered serine acetyltransferase, which was genetically desensitized to feedback inhibition by
l
-cysteine, were constructed by replacing the methionine residue at position 256 of the serine acetyltransferase protein with 19 other amino acid residues or the termination codon to truncate the carboxy terminus from amino acid residues 256 to 273 through site-directed mutagenesis by using PCR. A cysteine auxotroph, strain JM39, was transformed with plasmids having these altered
cysE
genes. The serine acetyltransferase activities of most of the transformants, which were selected based on restored cysteine requirements and ampicillin resistance, were less sensitive than the serine acetyltransferase activity of the wild type to feedback inhibition by
l
-cysteine. At the same time, these transformants produced approximately 200 mg of
l
-cysteine plus
l
-cystine per liter, whereas these amino acids were not detected in the recombinant strain carrying the wild-type serine acetyltransferase gene. However, the production of
l
-cysteine and
l
-cystine by the transformants was very unstable, presumably due to a cysteine-degrading enzyme of the host, such as cysteine desulfhydrase. Therefore, mutants that did not utilize cysteine were derived from host strain JM39 by mutagenesis with
N
-methyl-
N
′-nitro-
N
-nitrosoguanidine. When a newly derived host was transformed with plasmids having the altered
cysE
genes, we found that the production of
l
-cysteine plus
l
-cystine was markedly increased compared to production in JM39.
High Levels of Endemicity of 3-Chlorobenzoate-Degrading Soil Bacteria
Authors: Fulthorpe R. R. , Rhodes A. N. , Tiedje J. M.
Published: December 2019

Soils samples were obtained from pristine ecosystems in six regions on five continents. Two of the regions were boreal forests, and the other four were Mediterranean ecosystems. Twenty-four soil samples from each of four or five sites in each of the regions were enriched by using 3-chlorobenzoate (3CBA), and 3CBA mineralizers were isolated from most samples. These isolates were analyzed for the ability to mineralize 3CBA, and genotypes were determined with repetitive extragenic palindromic PCR genomic fingerprints and restriction digests of the 16S rRNA genes (amplified ribosomal DNA restriction analysis [ARDRA]). We found that our collection of 150 stable 3CBA-mineralizing isolates included 48 genotypes and 44 ARDRA types, which formed seven distinct clusters. The majority (91%) of the genotypes were unique to the sites from which they were isolated, and each genotype was found only in the region from which it was isolated. A total of 43 of the 44 ARDRA types were found in only one region. A few genotypes were repeatedly found in one region but not in any other continental region, suggesting that they are regionally endemic. A correlation between bacterial genotype and vegetative community was found for the South African samples. These results suggest that the ability to mineralize 3CBA is distributed among very diverse genotypes and that the genotypes are not globally dispersed.
Heterobinary Adhesins Based on the
Escherichia coli
FimH Fimbrial Protein
Authors: Schembri Mark A. , Klemm Per
Published: December 2019

The FimH adhesin of
Escherichia coli
type 1 fimbriae confers the ability to bind to
d
-mannosides by virtue of a receptor-binding domain located in its N-terminal region. This protein was engineered into a heterobifunctional adhesin by introducing a secondary binding site in the C-terminal region. The insertion of histidine clusters into this site resulted in coordination of various metal ions by recombinant cells expressing chimeric FimH proteins. In addition, libraries consisting of random peptide sequences inserted into the FimH display system and screened by a “panning” technique were used to identify specific sequences conferring the ability to adhere to Ni
2+
and Cu
2+
. Recombinant cells expressing heterobifunctional FimH adhesins could adhere simultaneously to both metals and saccharides. Finally, combining the metal-binding modifications with alterations in the natural receptor-binding region demonstrated the ability to independently modulate the binding of FimH to two ligands simultaneously.
A Rapid PCR-Based DNA Test for Enterotoxic
Bacillus cereus
Authors: Mäntynen Vesa , Lindström Kristina
Published: December 2019

The occurrence of DNA sequences encoding the hemolysin HblA complex and
Bacillus cereus
enterotoxin BceT, which have recently been confirmed as enterotoxins, was studied in
Bacillus
spp. To amplify these DNA sequences, PCR primer systems for the B component of
hblA
and for
bceT
DNA sequences were developed. The results from the amplification of
hblA
sequences correlated well with results obtained with the
B. cereus
enterotoxin (diarrheal type) test kit (RPLA kit), but not with the results of the
Bacillus
diarrheal enterotoxin visual immunoassay (BDE kit). Except for two thermophilic strains, all strains that were positive in PCR amplification assays with the
hblA
primers were also positive when tested with the RPLA kit. The
hblA
DNA sequence was found in 33 strains, and these strains were closely related according to 16S rDNA-RFLP analysis, except
B. pasteurii
. In PCR amplifications with the
bceT
primers only the model strain gave a positive signal. It is concluded that screening of the hemolysin HblA complex by the PCR method allows faster detection of enterotoxin production than does testing with the RPLA enterotoxin kit.