bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation ofaflRand Its Product, AflR, Associated with Aflatoxin Biosynthesis
Authors: Liu Biing-Hui , Chu Fun Sun
Published: December 2019

We studied the role of the regulatory geneaflRand its product, AflR, in the biosynthesis of aflatoxin inAspergillus. Western blot and enzyme-linked immunosorbent assay analyses revealed that aflatoxin B1accumulation was directly related to AflR expression and was regulated by various environmental and nutritional conditions, including temperature, air supply, carbon source, nitrogen source, and zinc availability. Expression of an aflatoxin biosynthetic pathway structural gene,omtA, was regulated by the presence of AflR. Induction patterns foraflRmRNA and AflR were correlated with that foromtAmRNA in an aflatoxin-producing strain ofAspergillus parasiticus. Analysis of non-aflatoxin-producing strains ofA. flavus,A. sojae, andA. oryzaegrown in medium suitable for aflatoxin B1production showed that bothaflRmRNA and AflR production were present; however,omtAmRNA production was not detected in any of these examined strains. AflR in theA. oryzaestrain was regulated by carbon source and temperature in a manner similar to that seen withA. parasiticus.
Development of PCR Primer Systems for Amplification of Nitrite Reductase Genes (nirKandnirS) To Detect Denitrifying Bacteria in Environmental Samples
Authors: Braker Gesche , Fesefeldt Andreas , Witzel Karl-Paul
Published: December 2019

A system was developed for the detection of denitrifying bacteria by the amplification of specific nitrite reductase gene fragments with PCR. Primer sequences were found for the amplification of fragments from both nitrite reductase genes (nirKandnirS) after comparative sequence analysis. Whenever amplification was tried with these primers, the knownnirtype of denitrifying laboratory cultures could be confirmed. Likewise, the method allowed a determination of thenirtype of five laboratory strains. ThenirKgene could be amplified fromBlastobacter denitrificans,Alcaligenes xylosoxidans, andAlcaligenessp. (DSM 30128); thenirSgene was amplified fromAlcaligenes eutrophusDSM 530 and from the denitrifying isolate IFAM 3698. For each of the two genes, at least one primer combination amplified successfully for all of the test strains. Specific amplification products were not obtained with nondenitrifying bacteria or with strains of the othernirtype. The specificity of the amplified products was confirmed by subsequent sequencing. These results suggest the suitability of the method for the qualitative detection of denitrifying bacteria in environmental samples. This was shown by applying one generally amplifying primer combination for eachnirgene developed in this study to total DNA preparations from aquatic habitats.
Temperature Gradient Gel Electrophoresis Analysis of 16S rRNA from Human Fecal Samples Reveals Stable and Host-Specific Communities of Active Bacteria
Authors: Zoetendal Erwin G. , Akkermans Antoon D. L. , De Vos Willem M.
Published: December 2019

The diversity of the predominant bacteria in the human gastrointestinal tract was studied by using 16S rRNA-based approaches. PCR amplicons of the V6 to V8 regions of fecal 16S rRNA and ribosomal DNA (rDNA) were analyzed by temperature gradient gel electrophoresis (TGGE). TGGE of fecal 16S rDNA amplicons from 16 individuals showed different profiles, with some bands in common. Fecal samples from two individuals were monitored over time and showed remarkably stable profiles over a period of at least 6 months. TGGE profiles derived from 16S rRNA and rDNA amplicons showed similar banding patterns. However, the intensities of bands with similar mobilities differed in some cases, indicating a different contribution to the total active fraction of the prominent fecal bacteria. Most 16S rRNA amplicons in the TGGE pattern of one subject were identified by cloning and sequence analysis. Forty-five of the 78 clones matched 15 bands, and 33 clones did not match any visible band in the TGGE pattern. Nested PCR of amplified 16S rDNA indicated preferential amplification of a sequence corresponding to 12 of the 33 nonmatching clones with similar mobilities in TGGE. The sequences matching 15 bands in the TGGE pattern showed 91.5 to 98.7% homology to sequences derived from different
Clostridium
clusters. Most of these were related to strains derived from the human intestine. The results indicate that the combination of cloning and TGGE analysis of 16S rDNA amplicons is a reliable approach to monitoring different microbial communities in feces.
The Introduction into
Bacillus sphaericus
of the
Bacillus thuringiensis
subsp.
medellin cyt1Ab1
Gene Results in Higher Susceptibility of Resistant Mosquito Larva Populations to
B. sphaericus
Authors: Thiéry I. , Hamon S. , Delécluse A. , Orduz S.
Published: December 2019

The fragment containing the gene encoding the cytolytic Cyt1Ab1 protein from
Bacillus thuringiensis
subsp.
medellin
and its flanking sequences (I. Thiery, A. Delécluse, M. C. Tamayo, and S. Orduz, Appl. Environ. Microbiol. 63:468–473, 1997) was introduced into
Bacillus sphaericus
toxic strains 2362, 2297, and Iab872 by electroporation with the shuttle vector pMK3. Only small amounts of the protein were produced in recombinant strains 2362 and Iab872. The protein was detected in these strains only by Western blotting and immunodetection with antibody raised against Cyt1Ab1 protein. Large amounts of Cyt1Ab1 protein were produced in
B. sphaericus
recombinant strain 2297, and there was an additional crystal, other than that of the binary toxin, within the exosporium. The production of the Cyt1Ab1 protein in addition to the binary toxin did not increase the larvicidal activity of the
B. sphaericus
recombinant strain against susceptible mosquito populations of
Culex pipiens
or
Aedes aegypti
. However, it partially restored (10 to 20 times) susceptibility of the resistant mosquito populations of
C. pipiens
(SPHAE) and
Culex quinquefasciatus
(GeoR) to the binary toxin. The Cyt1Ab1 protein produced in recombinant
B. thuringiensis
SPL407(p
cyt1Ab1
) was synthesized in two types of crystal—one round and with various dense areas, surrounded by an envelope, and the other a regular cuboid crystal, very similar to that found in the
B. sphaericus
recombinant strain.
Construction of a Range of Derivatives of the Biological Control Strain
Agrobacterium rhizogenes
K84: a Study of Factors Involved in Biological Control of Crown Gall Disease
Authors: McClure Nicholas C. , Ahmadi Ali-Reza , Clare Bruce G.
Published: December 2019

The biological control strain
Agrobacterium rhizogenes
K84 is an effective agent in the control of
Agrobacterium
pathogens, the causative agents of crown gall disease. A number of factors are thought to play a role in the control process, including production of the specific agrocins 84 and 434, which differ in the spectra of pathogenic strains that they inhibit in vitro. A range of derivatives of strain K84 has been developed with every combination of the three resident plasmids, pAgK84, pAgK434, and pAtK84b, including a plasmid-free strain. These derivatives produced either both, one, or neither of the characterized agrocins 84 and 434 and were isolated by plasmid curing, conjugation, and Tn
5
transposon mutagenesis. The ability of the derivative strains to inhibit gall formation on almond roots was compared to that of the wild-type K84 parent. Treatment with the plasmid-free derivative did not result in a significant level of control of an
A. rhizogenes
pathogen based on numbers or dry weight of galls formed on injured almond roots. The presence of plasmid pAgK84, pAgK434, or pAtK84b significantly enhanced the biological control efficacy of K84 derivatives, and the highest level of control was observed with strains harboring two or more plasmids. The results observed with strains deficient in agrocin 434 production suggest that this product may play an important role in the biological control of
A. rhizogenes
pathogens. The involvement of plasmid pAgK84b in biological control has not previously been reported. This study supports the conclusion that multiple factors are involved in the success of strain K84 as a biological control agent.
A New Operation for Producing Disease-Suppressive Compost from Grass Clippings
Authors: Nakasaki Kiyohiko , Hiraoka Sachiko , Nagata Hiroyuki
Published: December 2019

This study evaluated the use of grass clippings discharged from golf courses as the raw material for production of a suppressive compost to control
Rhizoctonia
large-patch disease in mascarene grass.
Bacillus subtilis
N4, a mesophilic bacterium with suppressive effects on the pathogenic fungus
Rhizoctonia solani
AG2-2, was used as an inoculum in a procedure developed with the aim of controlling composting temperatures and inoculation timing. The population density of mesophilic bacteria in the raw material was reduced to around 5 log
10
CFU/g (dry weight) of composting material in the self-heating reaction at the initial stage of composting by maintaining a temperature of 80°C for 1 day. The inoculum was applied immediately, and the composting material was maintained at 40°C for 3 days. This served both to highly concentrate the suppressive bacterium and to achieve sporulation. The temperature was then raised to 60°C and maintained, enabling hygienic, high-speed composting while maintaining the population density of the suppressive bacterium as high as 8 log
10
CFU/g (dry weight) in the compost. The suppressiveness of compost made in this way was confirmed in a turf grass disease prevention assay.
Identification, Characterization, and In Situ Detection of a Fruit-Body-Specific Hydrophobin of
Pleurotus ostreatus
Authors: Peñas María M. , Ásgeirsdóttir Sigridur A. , Lasa Iñigo , Culiañez-Macià Francisco A. , Pisabarro Antonio G. , Wessels Joseph G. H. , Ramírez Lucía
Published: December 2019

Hydrophobins are small (length, about 100 ± 25 amino acids), cysteine-rich, hydrophobic proteins that are present in large amounts in fungal cell walls, where they form part of the outermost layer (rodlet layer); sometimes, they can also be secreted into the medium. Different hydrophobins are associated with different developmental stages of a fungus, and their biological functions include protection of the hyphae against desiccation and attack by either bacterial or fungal parasites, hyphal adherence, and the lowering of surface tension of the culture medium to permit aerial growth of the hyphae. We identified and isolated a hydrophobin (fruit body hydrophobin 1 [Fbh1]) present in fruit bodies but absent in both monokaryotic and dikaryotic mycelia of the edible mushroom
Pleurotus ostreatus
. In order to study the temporal and spatial expression of the
fbh1
gene, we determined the N-terminal amino acid sequence of Fbh1. We also synthesized and cloned the double-stranded cDNA corresponding to the full-length mRNA of Fbh1 to use it as a probe in both Northern blot and in situ hybridization experiments. Fbh1 mRNA is detectable in specific parts of the fruit body, and it is absent in other developmental stages.
A Method for DNA Extraction from the Desert Cyanobacterium
Chroococcidiopsis
and Its Application to Identification of
ftsZ
Authors: Billi Daniela , Grilli Caiola Maria , Paolozzi Luciano , Ghelardini Patrizia
Published: December 2019

A method was developed for extraction of DNA from
Chroococcidiopsis
that overcomes obstacles posed by bacterial contamination and the presence of a thick envelope surrounding the cyanobacterial cells. The method is based on the resistance of
Chroococcidiopsis
to lysozyme and consists of a lysozyme treatment followed by osmotic shock that reduces the bacterial contamination by 3 orders of magnitude. Then DNase treatment is performed to eliminate DNA from the bacterial lysate. Lysis of
Chroococcidiopsis
cells is achieved by grinding with glass beads in the presence of hot phenol. Extracted DNA is further purified by cesium-chloride density gradient ultracentrifugation. This method permitted the first molecular approach to the study of
Chroococcidiopsis
, and a 570-bp fragment of the gene
ftsZ
was cloned and sequenced.
Variable Cross-Resistance to Cry11B from
Bacillus thuringiensis
subsp.
jegathesan
in
Culex quinquefasciatus
(Diptera: Culicidae) Resistant to Single or Multiple Toxins of
Bacillus thuringienisis
subsp.
israelensis
Authors: Wirth Margaret C. , Delécluse Armelle , Federici Brian A. , Walton William E.
Published: December 2019

A novel mosquitocidal bacterium,
Bacillus thuringiensis
subsp.
jegathesan
, and one of its toxins, Cry11B, in a recombinant
B. thuringiensis
strain were evaluated for cross-resistance with strains of the mosquito
Culex quinquefasciatus
that are resistant to single and multiple toxins of
Bacillus thuringiensis
subsp.
israelensis
. The levels of cross-resistance (resistance ratios [RR]) at concentrations which caused 95% mortality (LC
95
) between
B. thuringiensis
subsp.
jegathesan
and the different
B. thuringiensis
subsp.
israelensis
-resistant mosquito strains were low, ranging from 2.3 to 5.1. However, the levels of cross-resistance to Cry11B were much higher and were directly related to the complexity of the
B. thuringiensis
subsp.
israelensis
Cry toxin mixtures used to select the resistant mosquito strains. The LC
95
RR obtained with the mosquito strains were as follows: 53.1 against
Cq
4D, which was resistant to Cry11A; 80.7 against
Cq
4AB, which was resistant to Cry4A plus Cry4B; and 347 against
Cq
4ABD, which was resistant to Cry4A plus Cry4B plus Cry11A. Combining Cyt1A with Cry11B at a 1:3 ratio had little effect on suppressing Cry11A resistance in
Cq
4D but resulted in synergism factors of 4.8 and 11.2 against strains
Cq
4AB and
Cq
4ABD, respectively; this procedure eliminated cross-resistance in the former mosquito strain and reduced it markedly in the latter strain. The high levels of activity of
B. thuringiensis
subsp.
jegathesan
and
B. thuringiensis
subsp.
israelensis
, both of which contain a complex mixture of Cry and Cyt proteins, against Cry4- and Cry11-resistant mosquitoes suggest that novel bacterial strains with multiple Cry and Cyt proteins may be useful in managing resistance to bacterial insecticides in mosquito populations.
An Automated Fluorescent PCR Method for Detection of Shiga Toxin-Producing
Escherichia coli
in Foods
Authors: Chen Shu , Xu Renlin , Yee Arlene , Wu Kai Yuan , Wang Chang-Ning , Read Susan , De Grandis Stephanie A.
Published: December 2019

An automated fluorescence-based PCR system (a model AG-9600 AmpliSensor analyzer) was investigated to determine whether it could detect Shiga toxin-producing
Escherichia coli
(STEC). The AmpliSensor PCR assay involves amplification-mediated disruption of a fluorogenic DNA signal duplex (AmpliSensor) that is homologous to conserved target sequences in a 323-bp amplified fragment of Shiga toxin genes
stx
1
,
stx
2
, and
stx
e
. Using the Amplisensor assay, we detected 113 strains of STEC belonging to 50 different serotypes, while 18 strains of non-Shiga-toxin-producing
E. coli
and 68 strains of other bacteria were not detected. The detection limits of the assay were less than 1 to 5 CFU per PCR mixture when pure cultures of five reference strains were used and 3 CFU per 25 g of food when spiked ground beef samples that were preenriched overnight were used. The performance of the assay was also evaluated by using 53 naturally contaminated meat samples and 48 raw milk samples. Thirty-two STEC-positive samples that were confirmed to be positive by the culture assay were found to be positive when the AmpliSensor assay was used. Nine samples that were found to be positive when the PCR assay was used were culture negative. The system described here is an automated PCR-based system that can be used for detection of all serotypes of STEC in food or clinical samples.
Effect of Specific Growth Rate on Fermentative Capacity of Baker’s Yeast
Authors: Van Hoek Pim , Van Dijken Johannes P. , Pronk Jack T.
Published: December 2019

The specific growth rate is a key control parameter in the industrial production of baker’s yeast. Nevertheless, quantitative data describing its effect on fermentative capacity are not available from the literature. In this study, the effect of the specific growth rate on the physiology and fermentative capacity of an industrial
Saccharomyces cerevisiae
strain in aerobic, glucose-limited chemostat cultures was investigated. At specific growth rates (dilution rates,
D
) below 0.28 h
−1
, glucose metabolism was fully respiratory. Above this dilution rate, respirofermentative metabolism set in, with ethanol production rates of up to 14 mmol of ethanol · g of biomass
−1
· h
−1
at
D
= 0.40 h
−1
. A substantial fermentative capacity (assayed offline as ethanol production rate under anaerobic conditions) was found in cultures in which no ethanol was detectable (
D
< 0.28 h
−1
). This fermentative capacity increased with increasing dilution rates, from 10.0 mmol of ethanol · g of dry yeast biomass
−1
· h
−1
at
D
= 0.025 h
−1
to 20.5 mmol of ethanol · g of dry yeast biomass
−1
· h
−1
at
D
= 0.28 h
−1
. At even higher dilution rates, the fermentative capacity showed only a small further increase, up to 22.0 mmol of ethanol · g of dry yeast biomass
−1
· h
−1
at
D
= 0.40 h
−1
. The activities of all glycolytic enzymes, pyruvate decarboxylase, and alcohol dehydrogenase were determined in cell extracts. Only the in vitro activities of pyruvate decarboxylase and phosphofructokinase showed a clear positive correlation with fermentative capacity. These enzymes are interesting targets for overexpression in attempts to improve the fermentative capacity of aerobic cultures grown at low specific growth rates.
Seasonal Community and Population Dynamics of Pelagic Bacteria and Archaea in a High Mountain Lake
Authors: Pernthaler Jakob , Glöckner Frank-Oliver , Unterholzner Stefanie , Alfreider Albin , Psenner Roland , Amann Rudolf
Published: December 2019

The seasonal variations in community structure and cell morphology of pelagic procaryotes from a high mountain lake (Gossenköllesee, Austria) were studied by in situ hybridization with rRNA-targeted fluorescently labeled oligonucleotide probes (FISH) and image-analyzed microscopy. Compositional changes and biomass fluctuations within the assemblage were observed both in summer and beneath the winter ice cover and are discussed in the context of physicochemical and biotic parameters. Proteobacteria of the beta subclass (beta-proteobacteria) formed a dominant fraction of the bacterioplankton (annual mean, 24% of the total counts), whereas alpha-proteobacteria were of similar relative importance only during spring (mean, 11%). Bacteria of the
Cytophaga-Flavobacterium
cluster, although less abundant, constituted the largest fraction of the filamentous morphotypes during most of the year, thus contributing significantly to the total microbial biomass. Successive peaks of threadlike and rod-shaped archaea were observed during autumn thermal mixing and the period of ice cover formation, respectively. A set of oligonucleotide probes targeted to single phylotypes was constructed from 16S rRNA-encoding gene clone sequences. Three distinct populations of uncultivated microbes, affiliated with the alpha- and beta-proteobacteria, were subsequently monitored by FISH. About one-quarter of all of the beta-proteobacteria (range, 6 to 53%) could be assigned to only two phylotypes. The bacterial populations studied were annually recurrent, seasonally variable, and vertically stratified, except during the periods of lake overturn. Their variability clearly exceeded the fluctuations of the total microbial assemblage, suggesting that the apparent stability of total bacterioplankton abundances may mask highly dynamic community fluctuations.
Metal Accumulation and Vanadium-Induced Multidrug Resistance by Environmental Isolates of
Escherichia hermannii
and
Enterobacter cloacae
Authors: Hernández Alicia , Mellado Rafael P. , Martínez José L.
Published: December 2019

Contaminated soils from an oil refinery were screened for the presence of microorganisms capable of accumulating either nickel, vanadium, or both metals. Three strains of bacteria that belonged to the family
Enterobacteriaceae
were selected. Two of them were
Escherichia hermannii
strains, and outer membrane profile (OMP) analysis showed that they were similar to a strain of clinical origin; the other one was an
Enterobacter cloacae
strain that differed from clinical isolates. The selected bacteria accumulated both nickel and vanadium. Growth in the presence of vanadium induced multidrug resistance phenotypes in
E. hermannii
and
E. cloacae
. Incubation with this metal changed the OMP profile of
E. hermannii
but did not produce variations in the expression of the major OMPs of
E. cloacae.
Molecular Detection, Isolation, and Physiological Characterization of Functionally Dominant Phenol-Degrading Bacteria in Activated Sludge
Authors: Watanabe Kazuya , Teramoto Maki , Futamata Hiroyuki , Harayama Shigeaki
Published: December 2019

DNA was isolated from phenol-digesting activated sludge, and partial fragments of the 16S ribosomal DNA (rDNA) and the gene encoding the largest subunit of multicomponent phenol hydroxylase (LmPH) were amplified by PCR. An analysis of the amplified fragments by temperature gradient gel electrophoresis (TGGE) demonstrated that two major 16S rDNA bands (bands R2 and R3) and two major LmPH gene bands (bands P2 and P3) appeared after the activated sludge became acclimated to phenol. The nucleotide sequences of these major bands were determined. In parallel, bacteria were isolated from the activated sludge by direct plating or by plating after enrichment either in batch cultures or in a chemostat culture. The bacteria isolated were classified into 27 distinct groups by a repetitive extragenic palindromic sequence PCR analysis. The partial nucleotide sequences of 16S rDNAs and LmPH genes of members of these 27 groups were then determined. A comparison of these nucleotide sequences with the sequences of the major TGGE bands indicated that the major bacterial populations, R2 and R3, possessed major LmPH genes P2 and P3, respectively. The dominant populations could be isolated either by direct plating or by chemostat culture enrichment but not by batch culture enrichment. One of the dominant strains (R3) which contained a novel type of LmPH (P3), was closely related to
Valivorax paradoxus
, and the result of a kinetic analysis of its phenol-oxygenating activity suggested that this strain was the principal phenol digester in the activated sludge.
Analysis of the Influence of Environmental Parameters on
Clostridium botulinum
Time-to-Toxicity by Using Three Modeling Approaches
Authors: Schaffner Donald W. , Ross William H. , Montville Thomas J.
Published: December 2019

This study used the technique of waiting time modeling to analyze the combined effects of temperature, pH, carbohydrate, protein, and lipid on the time-to-toxicity of
Clostridium botulinum
56A. Waiting time models can be used whenever the time to the occurrence of some event is the variable of interest. In the case of the time-to-toxicity data, the variable is the time from the beginning of an experiment until a tube is identified as positive. The statistical analysis used the SAS procedure LIFEREG and included determination of the form of the response surface, identification of the error distribution, and simplification of the response surface. We found that increasing the macromolecule concentration decreased the probability of toxin formation. The probability of toxin formation also decreased at lower temperatures and at pHs further from the optimum. The waiting time modeling approach to developing models for botulinal toxin formation compared favorably with other approaches but had one specific advantage. Waiting time models have the inherent advantage that safety concerns regarding predictions are automatically quantified in the analysis by formally identifying a distribution of times-to-toxicity. The use of this time-to-toxicity distribution permits a customizable margin of safety (e.g., one in a million) not possible with other approaches.
Gene Cloning, DNA Sequencing, and Expression of Thermostable β-Mannanase from
Bacillus stearothermophilus
Authors: Ethier Nathalie , Talbot Guylaine , Sygusch Jurgen
Published: December 2019

A DNA genomic library constructed from
Bacillus stearothermophilus
, a gram-positive, facultative thermophilic aerobe that secretes a thermostable β-mannanase, was screened for mannan hydrolytic activity. Recombinant β-mannanase activity was detected on the basis of the clearing of halos around
Escherichia coli
colonies grown on a dye-labelled substrate, Remazol brilliant blue-locust bean gum. The nucleotide sequence of the mannanase gene,
manF
, corresponded to an open reading frame of 2,085 bp that codes for a 32-amino-acid signal peptide and a mature protein with a molecular mass of 76,089 Da. From sequence analysis, ManF belongs to glycosyl hydrolase family 5 and exhibits higher similarity to eukaryotic than to bacterial mannanases. The
manF
coding sequence was subcloned into the pH6EX3 expression plasmid and expressed in
E. coli
as a recombinant fusion protein containing a hexahistidine N-terminal sequence. The fusion protein has thermostability similar to the native enzyme and was purified by Ni
2+
affinity chromatography. The values for the kinetic parameters
V
max
and

K
m

were 384 U/mg and 2.4 mg/ml, respectively, for the recombinant mannanase and were comparable to those of the native enzyme.
Kinetic Bias in Estimates of Coastal Picoplankton Community Structure Obtained by Measurements of Small-Subunit rRNA Gene PCR Amplicon Length Heterogeneity
Authors: Suzuki Marcelino , Rappé Michael S. , Giovannoni Stephen J.
Published: December 2019

Marine bacterioplankton diversity was examined by quantifying natural length variation in the 5′ domain of small-subunit (SSU) rRNA genes (rDNA) amplified by PCR from a DNA sample from the Oregon coast. This new technique, length heterogeneity analysis by PCR (LH-PCR), determines the relative proportions of amplicons originating from different organisms by measuring the fluorescence emission of a labeled primer used in the amplification reaction. Relationships between the sizes of amplicons and gene phylogeny were predicted by an analysis of 366 SSU rDNA sequences from cultivated marine bacteria and from bacterial genes cloned directly from environmental samples. LH-PCR was used to compare the distribution of bacterioplankton SSU rDNAs from a coastal water sample with that of an SSU rDNA clone library prepared from the same sample and also to examine the distribution of genes in the PCR products from which the clone library was prepared. The analysis revealed that the relative frequencies of genes amplified from natural communities are highly reproducible for replicate sets of PCRs but that a bias possibly caused by the reannealing kinetics of product molecules can skew gene frequencies when PCR product concentrations exceed threshold values.
Spectrum of the Reductive Dehalogenation Activity of
Desulfitobacterium frappieri
PCP-1
Authors: Dennie D. , Gladu I. , Lépine F. , Villemur R. , Bisaillon J.-G. , Beaudet R.
Published: December 2019

Desulfitobacterium frappieri
PCP-1 was induced for
ortho
- and
para
-dechlorinating activities by different chlorophenols. Dehalogenation rates ranging from 25 to 1,158 nmol/min/mg of cell protein were observed according to the chlorophenol tested and the position of the chlorine removed.
D. frappieri
shows a broad substrate specificity; in addition to tetrachloroethylene and pentachloropyridine, strain PCP-1 can dehalogenate at
ortho
,
meta
, and
para
positions a large variety of aromatic molecules with substituted hydroxyl or amino groups. Reactions of O demethylation and reduction of nitro to amino substituents on aromatic molecules were also observed.
Effects of Starvation on Physiological Activity and Chlorine Disinfection Resistance in
Escherichia coli
O157:H7
Authors: Lisle John T. , Broadaway Susan C. , Prescott Annette M. , Pyle Barry H. , Fricker Colin , McFeters Gordon A.
Published: December 2019

Escherichia coli
O157:H7 can persist for days to weeks in microcosms simulating natural conditions. In this study, we used a suite of fluorescent, in situ stains and probes to assess the influence of starvation on physiological activity based on membrane potential (rhodamine 123 assay), membrane integrity (LIVE/DEAD
Bac
Light kit), respiratory activity (5-cyano-2,3-di-4-tolyl-tetrazolium chloride assay), intracellular esterase activity (ScanRDI assay), and 16S rRNA content. Growth-dependent assays were also used to assess substrate responsiveness (direct viable count [DVC] assay), ATP activity (MicroStar assay), and culturability (R2A agar assay). In addition, resistance to chlorine disinfection was assessed. After 14 days of starvation, the DVC values decreased, while the values in all other assays remained relatively constant and equivalent to each other. Chlorine resistance progressively increased through the starvation period. After 29 days of starvation, there was no significant difference in chlorine resistance between control cultures that had not been exposed to the disinfectant and cultures that had been exposed. This study demonstrates that
E. coli
O157:H7 adapts to starvation conditions by developing a chlorine resistance phenotype.
AbiQ, an Abortive Infection Mechanism fromLactococcus lactis
Authors: Emond Eric , Dion Eric , Walker Shirley A. , Vedamuthu Ebenezer R. , Kondo Jeffery K. , Moineau Sylvain
Published: December 2019

Lactococcus lactisW-37 is highly resistant to phage infection. The cryptic plasmids from this strain were coelectroporated, along with the shuttle vector pSA3, into the plasmid-free hostL. lactisLM0230. In addition to pSA3, erythromycin- and phage-resistant isolates carried pSRQ900, an 11-kb plasmid fromL. lactisW-37. This plasmid made the host bacteria highly resistant (efficiency of plaquing <10−8) to c2- and 936-like phages. pSRQ900 did not confer any resistance to phages of the P335 species. Adsorption, cell survival, and endonucleolytic activity assays showed that pSRQ900 encodes an abortive infection mechanism. The phage resistance mechanism is limited to a 2.2-kbEcoRV/BclI fragment. Sequence analysis of this fragment revealed a complete open reading frame (abiQ), which encodes a putative protein of 183 amino acids. A frameshift mutation withinabiQcompletely abolished the resistant phenotype. The predicted peptide has a high content of positively charged residues (pI = 10.5) and is, in all likelihood, a cytosolic protein. AbiQ has no homology to known or deduced proteins in the databases. DNA replication assays showed that phage c21 (c2-like) and phage p2 (936-like) can still replicate in cells harboring AbiQ. However, phage DNA accumulated in its concatenated form in the infected AbiQ+cells, whereas the AbiQ−cells contained processed (mature) phage DNA in addition to the concatenated form. The production of the major capsid protein of phage c21 was not hindered in the cells harboring AbiQ.
Genetic Characterization and Heterologous Expression of Brochocin-C, an Antibotulinal, Two-Peptide Bacteriocin Produced by
Brochothrix campestris
ATCC 43754
Authors: McCormick John K. , Poon Alison , Sailer Miloslav , Gao Yan , Roy Ken L. , McMullen Lynn M. , Vederas John C. , Stiles Michael E. , Van Belkum Marco J.
Published: December 2019

Brochocin-C, produced by
Brochothrix campestris
ATCC 43754, is active against many strains of the closely related meat spoilage organism
Brochothrix thermosphacta
and a wide range of other gram-positive bacteria, including spores of
Clostridium botulinum
. Purification of the active compound and genetic characterization of brochocin-C revealed that it is a chromosomally encoded, two-peptide nonlantibiotic bacteriocin. Both peptides of brochocin-C are ribosomally synthesized as prepeptides that are typical of class II bacteriocins. They are cleaved following Gly-Gly cleavage sites to yield the mature peptides, BrcA and BrcB, containing 59 and 43 amino acids, respectively. Fusion of the nucleotides encoding the signal peptide of the bacteriocin divergicin A in front of the structural genes for either BrcA or BrcB allowed independent expression of each component by the general protein secretion pathway. This revealed the two-component nature of brochocin-C and the necessity for both peptides for activity. A 53-amino-acid peptide encoded downstream of
brcB
functions as the immunity protein (BrcI) for brochocin-C. In addition, the cloned chromosomal fragment revealed open reading frames downstream of
brcI
, designated
brcT
and
brcD
, that encode proteins with homology to ATP-binding cassette translocator and accessory proteins, respectively, involved in the secretion of Gly-Gly-type bacteriocins.
A Holistic Approach for Determining the Entomopathogenic Potential of
Bacillus thuringiensis
Strains
Authors: Masson Luke , Erlandson Martin , Puzstai-Carey Marianne , Brousseau Roland , Juárez-Pérez Victor , Frutos Roger
Published: December 2019

The
cry
gene content of
Bacillus thuringiensis
subsp.
aizawai
HD-133 was analyzed by a combination of high-pressure liquid chromatography (HPLC) and exclusive PCR. A total of six
cry
genes were detected in genomic DNA purified from HD-133, four from the
cry1
family (
cry1Aa
,
cry1Ab
,
cry1C
, and
cry1D
) as well as a gene each from the
cry2
(
cry2B
) and the
cry1I
families. To directly determine which genes were expressed and crystallized in the purified parasporal inclusions, solubilized and trypsinized HD-133 crystals were subjected to chromatographic separation by HPLC. Only three proteins, Cry1Ab, Cry1C, and Cry1D, were found, in a 60/37/3 ratio. Dot blot analysis of total mRNA purified from HD-133 showed that both the
cry2B
and
cry1I
genes, but not the
cry1Aa
gene, were transcribed. Cloning and sequencing of the
cry1Aa
gene revealed an inserted DNA sequence within the
cry
coding sequence, resulting in a disrupted reading frame. Taken together, our results show that combining crystal protein analysis with a genetic approach is a highly complementary and powerful way to assess the potential of
B. thuringiensis
isolates for new insecticidal genes and specificities. Furthermore, based on the number of cryptic genes found in HD-133, the total
cry
gene content of
B. thuringiensis
strains may be higher than previously thought.
Procaryotic Expression of Single-Chain Variable-Fragment (scFv) Antibodies: Secretion in L-Form Cells of
Proteus mirabilis
Leads to Active Product and Overcomes the Limitations of Periplasmic Expression in
Escherichia coli
Authors: Rippmann Jörg F. , Klein Michaela , Hoischen Christian , Brocks Bodo , Rettig Wolfgang J. , Gumpert Johannes , Pfizenmaier Klaus , Mattes Ralf , Moosmayer Dieter
Published: December 2019

Recently it has been demonstrated that L-form cells of
Proteus mirabilis
(L VI), which lack a periplasmic compartment, can be efficiently used in the production and secretion of heterologous proteins. In search of novel expression systems for recombinant antibodies, we compared levels of single-chain variable-fragment (scFv) production in
Escherichia coli
JM109 and
P. mirabilis
L VI, which express four distinct scFvs of potential clinical interest that show differences in levels of expression and in their tendencies to form aggregates upon periplasmic expression. Production of all analyzed scFvs in
E. coli
was limited by the severe toxic effect of the heterologous product as indicated by inhibition of culture growth and the formation of insoluble aggregates in the periplasmic space, limiting the yield of active product. In contrast, the L-form cells exhibited nearly unlimited growth under the tested production conditions for all scFvs examined. Moreover, expression experiments with
P. mirabilis
L VI led to scFv concentrations in the range of 40 to 200 mg per liter of culture medium (corresponding to volume yields 33- to 160-fold higher than those with
E. coli
JM109), depending on the expressed antibody. In a translocation inhibition experiment the secretion of the scFv constructs was shown to be an active transport coupled to the signal cleavage. We suppose that this direct release of the newly synthesized product into a large volume of the growth medium favors folding into the native active structure. The limited aggregation of scFv observed in the
P. mirabilis
L VI supernatant (occurring in a first-order-kinetics manner) was found to be due to intrinsic features of the scFv and not related to the expression process of the host cells. The
P. mirabilis
L VI supernatant was found to be advantageous for scFv purification. A two-step chromatography procedure led to homogeneous scFv with high antigen binding activity as revealed from binding experiments with eukaryotic cells.
Characterization of Cell Lysis in
Pseudomonas putida
Induced upon Expression of Heterologous Killing Genes
Authors: Ronchel M. Carmen , Molina Lázaro , Witte Angela , Lutbiz Werner , Molin Søren , Ramos Juan L. , Ramos Cayo
Published: December 2019

Active biological containment systems are based on the controlled expression of killing genes. These systems are of interest for the
Pseudomonadaceae
because of the potential applications of these microbes as bioremediation agents and biopesticides. The physiological effects that lead to cell death upon the induction of expression of two different heterologous killing genes in nonpathogenic
Pseudomonas putida
KT2440 derivatives have been analyzed.
P. putida
CMC4 and CMC12 carry in their chromosomes a fusion of the P
A1-04/03
promoter to the
Escherichia coli gef
gene and the φX174 lysis gene
E
, respectively. Expression of the killing genes is controlled by the LacI protein, whose expression is initiated from the XylS-dependent Pm promoter. Under induced conditions, killing of
P. putida
CMC12 cells mediated by φX174 lysis protein E was faster than that observed for
P. putida
CMC4, for which the Gef protein was the killing agent. In both cases, cell death occurred as a result of impaired respiration, altered membrane permeability, and the release of some cytoplasmic contents to the extracellular medium.
Detection and Isolation of Novel Rhizopine-Catabolizing Bacteria from the Environment
Authors: Gardener Brian B. McSpadden , de Bruijn Frans J.
Published: December 2019

Microbial rhizopine-catabolizing (Moc) activity was detected in serial dilutions of soil and rhizosphere washes. The activity observed generally ranged between 10
6
and 10
7
catabolic units per g, and the numbers of nonspecific culture-forming units were found to be approximately 10 times higher. A diverse set of 37 isolates was obtained by enrichment on
scyllo
-inosamine-containing media. However, none of the bacteria that were isolated were found to contain DNA sequences homologous to the known
mocA
,
mocB
, and
mocC
genes of
Sinorhizobium meliloti
L5-30. Twenty-one of the isolates could utilize an SI preparation as the sole carbon and nitrogen source for growth. Partial sequencing of 16S ribosomal DNAs (rDNAs) amplified from these strains indicated that five distinct bacterial genera (
Arthrobacter
,
Sinorhizobium
,
Pseudomonas
,
Aeromonas
, and
Alcaligenes
) were represented in this set. Only 6 of these 21 isolates could catabolize 3-
O
-methyl-
scyllo
-inosamine under standard assay conditions. Two of these, strains D1 and R3, were found to have 16S rDNA sequences very similar to those of
Sinorhizobium meliloti
. However, these strains are not symbiotically effective on
Medicago sativa
, and DNA sequences homologous to the
nodB
and
nodC
genes were not detected in strains D1 and R3 by Southern hybridization analysis.
Flow Cytometric Analysis of the In Situ Accessibility of
Escherichia coli
16S rRNA for Fluorescently Labeled Oligonucleotide Probes
Authors: Fuchs Bernhard Maximilian , Wallner Günter , Beisker Wolfgang , Schwippl Ines , Ludwig Wolfgang , Amann Rudolf
Published: December 2019

In situ identification of whole fixed bacterial cells by hybridization with fluorescently labeled, rRNA-targeted oligonucleotide probes is often limited by low signal intensities. In addition to an impermeability of the cell periphery and a low cellular rRNA content, the three-dimensional structure of the ribosome may hinder the access of oligonucleotides to their target sites. Until now, a systematic study on the accessibility of 16S rRNA target sites had not been done. Here, we report fluorescence intensities obtained with more than 200 oligonucleotide probes (mostly 18-mers) used with whole fixed cells of
Escherichia coli
DSM 30083
T
. Two overlapping sets of adjacent oligonucleotides, 171 in total, were designed to cover the full length of the 16S rRNA. The two sets are shifted by 5 to 13 nucleotides. The probes were labeled with carboxyfluorescein, and signal intensities of hybridized cells were quantified by flow cytometry. Care was taken that the signal intensity of cells was dependent solely on the in situ accessibility of probe target sites. The brightest signal resulted from probe Eco1482, complementary to positions 1482 to 1499. With this probe, the fluorescence was 1.7 times brighter than that of the standard bacterial probe EUB338 and 44 times brighter than that of the worst probe, Eco468. The distribution of probe-conferred cell fluorescence in six arbitrarily set brightness classes (classes I to VI; 100 to 81%, 80 to 61%, 60 to 41%, 40 to 21%, 20 to 6%, and 5 to 0% of the brightness with Eco1482, respectively) was as follows: I, 4%; II, 14%; III, 21%; IV, 29%, V, 19%; and VI, 13%. A more detailed analysis of helices 6, 18, and 23 with additional probes demonstrated that a shift of the target region by only a few bases could result in a decline of cell fluorescence from >80 to <10%. Considering the high evolutionary conservation of 16S rRNA, the in situ accessibility map of
E. coli
should facilitate a more rational selection of probe target sites for other species as well.
Phylogenetic Affiliation of Soil Bacteria That Degrade Aliphatic Polyesters Available Commercially as Biodegradable Plastics
Authors: Suyama Tetsushi , Tokiwa Yutaka , Ouichanpagdee Pornpimol , Kanagawa Takahiro , Kamagata Yoichi
Published: December 2019

Thirty-nine morphologically different soil bacteria capable of degrading poly(β-hydroxyalkanoate), poly(ɛ-caprolactone), poly(hexamethylene carbonate), or poly(tetramethylene succinate) were isolated. Their phylogenetic positions were determined by 16S ribosomal DNA sequencing, and all of them fell into the classes
Firmicutes
and
Proteobacteria
. Determinations of substrate utilization revealed characteristic patterns of substrate specificities.
Effects of Bacterial Antibiotic Production on Rhizosphere Microbial Communities from a Culture-Independent Perspective
Authors: Robleto Eduardo A. , Borneman James , Triplett Eric W.
Published: December 2019

The effects of antibiotic production on rhizosphere microbial communities of field-grown
Phaseolus vulgaris
were assessed by using ribosomal intergenic spacer analysis. Inoculum strains of
Rhizobium etli
CE3 differing only in trifolitoxin production were used. Trifolitoxin production dramatically reduced the diversity of trifolitoxin-sensitive members of the α subdivision of the class
Proteobacteria
with little apparent effect on most microbes.
Effects of Halides on Plasmid-Mediated Silver Resistance in
Escherichia coli
Authors: Gupta Amit , Maynes Maria , Silver Simon
Published: December 2019

Silver resistance of sensitive
Escherichia coli
J53 and resistance plasmid-containing J53(pMG101) was affected by halides in the growth medium. The effects of halides on Ag
+
resistance were measured with AgNO
3
and silver sulfadiazine, both on agar and in liquid. Low concentrations of chloride made the differences in MICs between sensitive and resistant strains larger. High concentrations of halides increased the sensitivities of both strains to Ag
+
.
Influence of Temperature and Pressure on the Lethality of Ultrasound
Authors: Raso J. , Pagán R. , Condón S. , Sala F. J.
Published: December 2019

A specially designed resistometer was constructed, and the lethal effect on
Yersinia enterocolitica
of ultrasonic waves (UW) at different static pressures (manosonication [MS]) and of combined heat-UW under pressure treatments (manothermosonication [MTS]) was investigated. During MS treatments at 30°C and 200 kPa, the increase in the amplitude of UW of 20 kHz from 21 to 150 μm exponentially decreased decimal reduction time values (
D
MS
) from 4 to 0.37 min. When pressure was increased from 0 to 600 kPa at a constant amplitude (150 μm) and temperature (30°C),
D
MS
values decreased from 1.52 to 0.20 min. The magnitude of this decrease in
D
MS
declined progressively as pressure was increased. The influence of pressure on
D
MS
values was greater with increased amplitude of UW. Pressure alone of as much as 600 kPa did not influence the heat resistance of
Y. enterocolitica
(
D
60
= 0.094;
z
= 5.65). At temperatures of as much as 58°C, the lethality of UW under pressure was greater than that of heat treatment alone at the same temperature. At higher temperatures, this difference disappeared. Heat and UW under pressure seemed to act independently. The lethality of MTS treatments appeared to result from the added effects of UW under pressure and the lethal effect of heat. The individual contributions of heat and of UW under pressure to the total lethal effect of MTS depended on temperature. The inactivating effect of UW was not due to titanium particles eroded from the sonication horn. The addition to the MS media of cysteamine did not increase the resistance of
Y. enterocolitica
to MS treatment. MS treatment caused cell disruption.
A Cold-Adapted Lipase of an Alaskan Psychrotroph,
Pseudomonas
sp. Strain B11-1: Gene Cloning and Enzyme Purification and Characterization
Authors: Choo Dong-Won , Kurihara Tatsuo , Suzuki Takeshi , Soda Kenji , Esaki Nobuyoshi
Published: December 2019

A psychrotrophic bacterium producing a cold-adapted lipase upon growth at low temperatures was isolated from Alaskan soil and identified as a
Pseudomonas
strain. The lipase gene (
lipP
) was cloned from the strain and sequenced. The amino acid sequence deduced from the nucleotide sequence of the gene (924 bp) corresponded to a protein of 308 amino acid residues with a molecular weight of 33,714. LipP also has consensus motifs conserved in other cold-adapted lipases, i.e., Lipase 2 from Antarctic
Moraxella
TA144 (G. Feller, M. Thiry, J. L. Arpigny, and C. Gerday, DNA Cell Biol. 10:381–388, 1991) and the mammalian hormone-sensitive lipase (D. Langin, H. Laurell, L. S. Holst, P. Belfrage, and C. Holm, Proc. Natl. Acad. Sci. USA 90:4897–4901, 1993): a pentapeptide, GDSAG, containing the putative active-site serine and an HG dipeptide. LipP was purified from an extract of recombinant
Escherichia coli
C600 cells harboring a plasmid coding for the
lipP
gene. The enzyme showed a 1,3-positional specificity toward triolein.
p
-Nitrophenyl esters of fatty acids with short to medium chains (C
4
and C
6
) served as good substrates. The enzyme was stable between pH 6 and 9, and the optimal pH for the enzymatic hydrolysis of tributyrin was around 8. The activation energies for the hydrolysis of
p
-nitrophenyl butyrate and
p
-nitrophenyl laurate were determined to be 11.2 and 7.7 kcal/mol, respectively, in the temperature range 5 to 35°C. The enzyme was unstable at temperatures higher than 45°C. The

K
m

of the enzyme for
p
-nitrophenyl butyrate increased with increases in the assay temperature. The enzyme was strongly inhibited by Zn
2+
, Cu
2+
, Fe
3+
, and Hg
2+
but was not affected by phenylmethylsulfonyl fluoride and bis-nitrophenyl phosphate. Various water-miscible organic solvents, such as methanol and dimethyl sulfoxide, at concentrations of 0 to 30% (vol/vol) activated the enzyme.