bluuebunny/crossref_metadata_2025 huggingface dataset:
Microcosm Enrichment of Biphenyl-Degrading Microbial Communities from Soils and Sediments
Authors: Wagner-Döbler I. , Bennasar A. , Vancanneyt M. , Strömpl C. , Brümmer I. , Eichner C. , Grammel I. , Moore E. R. B.
Published: December 2019

A microcosm enrichment approach was employed to isolate bacteria which are representative of long-term biphenyl-adapted microbial communities. Growth of microorganisms was stimulated by incubating soil and sediment samples from polluted and nonpolluted sites with biphenyl crystals. After 6 months, stable population densities between 8 × 10
9
and 2 × 10
11
CFU/ml were established in the microcosms, and a large percentage of the organisms were able to grow on biphenyl-containing minimal medium plates. A total of 177 biphenyl-degrading strains were subsequently isolated and characterized by their ability to grow on biphenyl in liquid culture and to accumulate a yellow
meta
cleavage product when they were sprayed with dihydroxybiphenyl. Isolates were identified by using a polyphasic approach, including fatty acid methyl ester (FAME) analysis, 16S rRNA gene sequence comparison, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell proteins, and genomic fingerprinting based on sequence variability in the 16S-23S ribosomal DNA intergenic spacer region. In all of the microcosms, isolates identified as
Rhodococcus opacus
dominated the cultivable microbial community, comprising a cluster of 137 isolates with very similar FAME profiles (Euclidean distances, <10) and identical 16S rRNA gene sequences. The
R. opacus
isolates from the different microcosms studied could not be distinguished from each other by any of the fingerprint methods used. In addition, three other FAME clusters were found in one or two of the microcosms analyzed; these clusters could be assigned to
Alcaligenes
sp.,
Terrabacter
sp., and
Bacillus thuringiensis
on the basis of their FAME profiles and/or comparisons of the 16S rRNA gene sequences of representatives. Thus, the microcosm enrichments were strongly dominated by gram-positive bacteria, especially the species
R. opacus
, independent of the pollution history of the original sample.
R. opacus
, therefore, is a promising candidate for development of effective long-term inocula for polychlorinated biphenyl bioremediation.
A Novel Insecticidal Toxin from
Photorhabdus luminescens
, Toxin Complex a (Tca), and Its Histopathological Effects on the Midgut of
Manduca sexta
Authors: Blackburn Michael , Golubeva Elena , Bowen David , Ffrench-Constant Richard H.
Published: December 2019

Photorhabdus luminescens
is a bacterium which is mutualistic with entomophagous nematodes and which secretes high-molecular-weight toxin complexes following its release into the insect hemocoel upon nematode invasion. Thus, unlike other protein toxins from
Bacillus thuringiensis
(δ-endotoxins and Vip’s),
P. luminescens
toxin (Pht) normally acts from within the insect hemocoel. Unexpectedly, therefore, the toxin complex has both oral and injectable activities against a wide range of insects. We have recently fractionated the protein toxin and shown it to consist of several native complexes, the most abundant of which we have termed Toxin complex a (Tca). This complex is highly active against the lepidopteran
Manduca sexta
. In view of the difference in the normal mode of delivery of
P. luminescens
toxin and the apparent communality in the histopathological effects of other gut-active toxins from
B. thuringiensis
, as well as cholesterol oxidase, we were interested in investigating the effects of purified Tca protein on larvae of
M. sexta
. Here we report that the histopathology of the
M. sexta
midgut is similar to that for other novel midgut-active toxins. Following oral ingestion of Tca by
M. sexta
, we observed an acceleration in the blebbing of the midgut epithelium into the gut lumen and eventual lysis of the epithelium. The midgut shows a similar histopathology following injection of Tca into the insect hemocoel. These results not only show that Tca is a highly active oral insecticide but also confirm the similar histopathologies of a range of very different gut-active toxins, despite presumed differences in modes of action and/or delivery. The implications for the mode of action of Tca are discussed.
Heat Shock Response in the Thermophilic Enteric Yeast
Arxiozyma telluris
Authors: Deegenaars Michelle L. , Watson Kenneth
Published: December 2019

Heat stress tolerance was examined in the thermophilic enteric yeast
Arxiozyma telluris
. Heat shock acquisition of thermotolerance and synthesis of heat shock proteins hsp 104, hsp 90, hsp 70, and hsp 60 were induced by a mild heat shock at temperatures from 35 to 40°C for 30 min. The results demonstrate that a yeast which occupies a specialized ecological niche exhibits a typical heat shock response.
Unstable Expression and Thermal Instability of a Species-Specific Cell Surface Epitope Associated with a 66-Kilodalton Antigen Recognized by Monoclonal Antibody EM-7G1 within Serotypes of
Listeria monocytogenes
Grown in Nonselective and Selective Broths
Authors: Nannapaneni Ramakrishna , Story Robert , Bhunia Arun K. , Johnson Michael G.
Published: December 2019

Conditions that resulted in unstable expression and heat instability of a cell surface epitope associated with a 66-kDa antigen in
Listeria monocytogenes
serotypes were identified with the probe monoclonal antibody (MAb) EM-7G1 in an enzyme-linked immunosorbent assay. This epitope appeared to be absent in three serotypes (serotypes 3b, 4a, and 4c), which did not react with MAb EM-7G1 irrespective of the enrichment broth tested. The remaining 10 serotypes were detected by MAb EM-7G1 only when cells were grown in nonselective brain heart infusion broth (BHI) or selective
Listeria
enrichment broth (LEB). When cells were grown in
Listeria
repair broth (LRB), only 6 of the 13 serotypes were detected by MAb EM-7G1, and recognition of serogroup 4 was completely lost. None of the 13 serotypes was detected by MAb EM-7G1 when cells were grown in two other commonly used
Listeria
-selective media, UVM1 broth and Fraser broth (FRB), indicating that possible loss of epitope expression occurred under these conditions. MAb EM-7G1 maintained species specificity without cross-reacting with live or heat-killed cells of six other
Listeria
spp. (
Listeria ivanovii
,
Listeria innocua
,
Listeria seeligeri
,
Listeria welshimeri
,
Listeria grayi
, and
Listeria murrayi
) irrespective of the enrichment conditions tested. Due to heat instability of the cell surface epitope when it was exposed to 80 or 100°C for 20 min, MAb EM-7G1 is suitable for detection of live cells of
L. monocytogenes
in BHI or LEB but not in LRB, UVM1, or FRB enrichment medium.
Localization and Solubilization of the Iron(III) Reductase of
Geobacter sulfurreducens
Authors: Gaspard Sarra , Vazquez Francisco , Holliger Christof
Published: December 2019

The iron(III) reductase activity of
Geobacter sulfurreducens
was determined with the electron donor NADH and the artificial electron donor horse heart cytochrome
c
. The highest reduction rates were obtained with Fe(III) complexed by nitrilotriacetic acid as an electron acceptor. Fractionation experiments indicated that no iron(III) reductase activity was present in the cytoplasm, that approximately one-third was found in the periplasmic fraction, and that two-thirds were associated with the membrane fraction. Sucrose gradient separation of the outer and cytoplasmic membranes showed that about 80% of the iron(III) reductase was present in the outer membrane. The iron(III) reductase could be solubilized from the membrane fraction with 0.5 M KCl showing that the iron(III) reductase was weakly bound to the membranes. In addition, solubilization of the iron(III) reductase from whole cells with 0.5 M KCl, without disruption of cells, indicated that the iron(III) reductase is a peripheral protein on the outside of the outer membrane. Redox difference spectra of KCl extracts showed the presence of
c
-type cytochromes which could be oxidized by ferrihydrite. Only one activity band was observed in native polyacrylamide gels stained for the iron(III) reductase activity. Excision of the active band from a preparative gel followed by extraction of the proteins and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of high-molecular-mass, cytochrome-containing proteins in this iron(III) reductase activity band. From these experimental data it can be hypothesized that the iron(III) reductase of
G. sulfurreducens
is a peripheral outer membrane protein that might contain a
c
-type cytochrome.
Role of the Regulatory Gene
areA
of
Aspergillus oryzae
in Nitrogen Metabolism
Authors: Christensen Tove , Hynes Michael J. , Davis Meryl A.
Published: December 2019

The
areA
gene of
Aspergillus oryzae
was cloned by cross-hybridization with the
Aspergillus nidulans areA
gene and was found to encode an 866-amino-acid protein that is very similar to other fungal nitrogen regulatory proteins. The
A. oryzae areA
gene can complement
A. nidulans areA
loss-of-function mutations. Functional analyses indicated that the N-terminal region of the
A. oryzae
AreA protein was dispensable for function and revealed a probable acidic activation domain in the protein. C-terminal truncation of the protein resulted in derepression of several nitrogen-controlled activities in
A. nidulans
, while deletions extending into the conserved GATA type zinc finger region abolished the activator function. The
A. oryzae areA
gene was inactivated by replacement with the
A. oryzae pyrG
gene. Strains containing the resulting
areA
deletion grew as well as the wild-type strain on glutamine but were unable to grow vigorously on other nitrogen sources, including ammonium. While
A. oryzae
exhibited reduced growth on 10 mM ammonium, the results of growth tests indicated that
areA
mutants of both
A. oryzae
and
A. nidulans
were affected in utilization of low concentrations of ammonium. The levels of the major nitrogen assimilatory enzymes, NADP-linked glutamate dehydrogenase (EC
1.4.1.4
) and glutamine synthetase (EC
6.3.1.2
), were determined. In both
A. oryzae
and
A. nidulans areA
mutants, the NADP-glutamate dehydrogenase levels were reduced, whereas the glutamine synthetase levels were not affected. These results suggest that the AreA protein may play an important role in the regulation of nitrogen assimilation in addition to its previously established regulatory role in nitrogen catabolism.
Nucleic Acid (DNA, RNA) Quantification and RNA/DNA Ratio Determination in Marine Sediments: Comparison of Spectrophotometric, Fluorometric, and HighPerformance Liquid Chromatography Methods and Estimation of Detrital DNA
Authors: Dell’Anno A. , Fabiano M. , Duineveld G. C. A. , Kok A. , Danovaro R.
Published: December 2019

In this study, we compared three methods for extraction and quantification of RNA and DNA from marine sediments: (i) a spectrophotometric method using the diphenylamine assay; (ii) a fluorometric method utilizing selective fluorochromes (thiazole orange for total nucleic acids and Hoechst 33258 for DNA); and (iii) a high-pressure liquid chromatography (HPLC) method which uses a specific column to separate RNA and DNA and UV absorption of the nucleic acids for quantification. Sediment samples were collected in the oligotrophic Cretan Sea (eastern Mediterranean, from 40 to 1,540 m in depth) and compared to the distribution and composition of the benthic microbial assemblages (i.e., bacteria and microprotozoa). DNA concentrations measured spectrophotometrically and by HPLC were not significantly different, while fluorometric yields were significantly lower. Such differences appear mainly due to fact that the stain-DNA complex is strongly dependent on the DNA composition and structure. RNA concentrations determined by the three methods displayed some differences; fluorometric and spectrophotometric methods obtain RNA concentration by difference and therefore may be biased by DNA estimates. By contrast, the HPLC method provides independent assessments of RNA and DNA concentrations. We tentatively estimated the contribution of the detrital DNA to the total DNA pools in two ways. The two calculations provided quite similar results indicating that the majority of the DNA pool in the deep-sea sediments was detrital. Microbial RNA generally accounted for almost the entire sedimentary RNA pools below 100-m depth. RNA concentrations were found to decrease along the Cretan shelf and slope. The RNA/DNA ratio calculated by using fluorometric DNA concentrations was significantly correlated with values of sediment community oxygen consumption only below 100-m depth (dominated by the microbial biomass). These data suggest that the RNA/DNA ratio, based on fluorometric estimates of DNA, can be used as an indicator of benthic metabolic activity, but only when metazoan contribution to the microbial DNA is negligible.
Substrate Specificity of and Product Formation by Muconate Cycloisomerases: an Analysis of Wild-Type Enzymes and Engineered Variants
Authors: Vollmer Martin Dominik , Hoier Helga , Hecht Hans-Jürgen , Schell Ursula , Gröning Janosch , Goldman Adrian , Schlömann Michael
Published: December 2019

Muconate cycloisomerases play a crucial role in the bacterial degradation of aromatic compounds by converting
cis,cis
-muconate, the product of catechol ring cleavage, to (4
S
)-muconolactone. Chloromuconate cycloisomerases catalyze both the corresponding reaction and a dehalogenation reaction in the transformation of chloroaromatic compounds. This study reports the first thorough examination of the substrate specificity of the muconate cycloisomerases from
Pseudomonas putida
PRS2000 and
Acinetobacter
“
calcoaceticus
” ADP1. We show that they transform, in addition to
cis,cis
-muconate, 3-fluoro-, 2-methyl-, and 3-methyl-
cis,cis
-muconate with high specificity constants but not 2-fluoro-, 2-chloro-, 3-chloro-, or 2,4-dichloro-
cis,cis
-muconate. Based on known three-dimensional structures, variants of
P. putida
muconate cycloisomerase were constructed by site-directed mutagenesis to contain amino acids found in equivalent positions in chloromuconate cycloisomerases. Some of the variants had significantly increased specificity constants for 3-chloro- or 2,4-dichloromuconate (e.g., A271S and I54V showed 27- and 22-fold increases, respectively, for the former substrate). These kinetic improvements were not accompanied by a change from protoanemonin to
cis,cis
-dienelactone as the product of 3-chloro-
cis,cis
-muconate conversion. The rate of 2-chloro-
cis,cis
-muconate turnover was not significantly improved, nor was this compound dehalogenated to any significant extent. However, the direction of 2-chloro-
cis,cis
-muconate cycloisomerization could be influenced by amino acid exchange. While the wild-type enzyme discriminated only slightly between the two possible cycloisomerization directions, some of the enzyme variants showed a strong preference for either (+)-2-chloro- or (+)-5-chloromuconolactone formation. These results show that the different catalytic characteristics of muconate and chloromuconate cycloisomerases are due to a number of features that can be changed independently of each other.
Purification and Characterization of
l
-Methionine γ-Lyase from
Brevibacterium linens
BL2
Authors: Dias Benjamin , Weimer Bart
Published: December 2019

l
-Methionine γ-lyase (EC
4.4.1.11
) was purified to homogeneity from
Brevibacterium linens
BL2, a coryneform bacterium which has been used successfully as an adjunct bacterium to improve the flavor of Cheddar cheese. The enzyme catalyzes the α,γ elimination of methionine to produce methanethiol, α-ketobutyrate, and ammonia. It is a pyridoxal phosphate-dependent enzyme, with a native molecular mass of approximately 170 kDa, consisting of four identical subunits of 43 kDa each. The purified enzyme had optimum activity at pH 7.5 and was stable at pHs ranging from 6.0 to 8.0 for 24 h. The pure enzyme had its highest activity at 25°C but was active between 5 and 50°C. Activity was inhibited by carbonyl reagents, completely inactivated by
dl
-propargylglycine, and unaffected by metal-chelating agents. The pure enzyme had catalytic properties similar to those of
l
-methionine γ-lyase from
Pseudomonas putida
. Its

K
m

for the catalysis of methionine was 6.12 mM, and its maximum rate of catalysis was 7.0 μmol min
−1
mg
−1
. The enzyme was active under salt and pH conditions found in ripening Cheddar cheese but susceptible to degradation by intracellular proteases.
Growth Rate Regulation of rRNA Content of a Marine
Synechococcus
(Cyanobacterium) Strain
Authors: Binder Brian J. , Liu Ying Chun
Published: December 2019

The relationship between growth rate and rRNA content in a marine
Synechococcus
strain was examined. A combination of flow cytometry and whole-cell hybridization with fluorescently labeled 16S rRNA-targeted oligonucleotide probes was used to measure the rRNA content of
Synechococcus
strain WH8101 cells grown at a range of light-limited growth rates. The sensitivity of this approach was sufficient for the analysis of rRNA even in very slowly growing
Synechococcus
cells (μ = 0.15 day
−1
). The relationship between growth rate and cellular rRNA content comprised three phases: (i) at low growth rates (<∼0.7 day
−1
), rRNA cell
−1
remained approximately constant; (ii) at intermediate rates (∼0.7 − 1.6 day
−1
), rRNA cell
−1
increased proportionally with growth rate; and (iii) at the highest, light-saturated rates (>∼1.6 day
−1
), rRNA cell
−1
dropped abruptly. Total cellular RNA (as measured with the nucleic acid stain SYBR Green II) was well correlated with the probe-based measure of rRNA and varied in a similar manner with growth rate. Mean cell volume and rRNA concentration (amount of rRNA per cubic micrometer) were related to growth rate in a manner similar to rRNA cell
−1
, although the overall magnitude of change in both cases was reduced. These patterns are hypothesized to reflect an approximately linear increase in ribosome efficiency with increasing growth rate, which is consistent with the prevailing prokaryotic model at low growth rates. Taken together, these results support the notion that measurements of cellular rRNA content might be useful for estimating in situ growth rates in natural
Synechococcus
populations.
Direct Determination of Carbon and Nitrogen Contents of Natural Bacterial Assemblages in Marine Environments
Authors: Fukuda Rumi , Ogawa Hiroshi , Nagata Toshi , Koike Isao
Published: December 2019

In order to better estimate bacterial biomass in marine environments, we developed a novel technique for direct measurement of carbon and nitrogen contents of natural bacterial assemblages. Bacterial cells were separated from phytoplankton and detritus with glass fiber and membrane filters (pore size, 0.8 μm) and then concentrated by tangential flow filtration. The concentrate was used for the determination of amounts of organic carbon and nitrogen by a high-temperature catalytic oxidation method, and after it was stained with 4′,6-diamidino-2-phenylindole, cell abundance was determined by epifluorescence microscopy. We found that the average contents of carbon and nitrogen for oceanic bacterial assemblages were 12.4 ± 6.3 and 2.1 ± 1.1 fg cell
−1
(mean ± standard deviation;
n
= 6), respectively. Corresponding values for coastal bacterial assemblages were 30.2 ± 12.3 fg of C cell
−1
and 5.8 ± 1.5 fg of N cell
−1
(
n
= 5), significantly higher than those for oceanic bacteria (two-tailed Student’s
t
test;
P
< 0.03). There was no significant difference (
P
> 0.2) in the bacterial C:N ratio (atom atom
−1
) between oceanic (6.8 ± 1.2) and coastal (5.9 ± 1.1) assemblages. Our estimates support the previous proposition that bacteria contribute substantially to total biomass in marine environments, but they also suggest that the use of a single conversion factor for diverse marine environments can lead to large errors in assessing the role of bacteria in food webs and biogeochemical cycles. The use of a factor, 20 fg of C cell
−1
, which has been widely adopted in recent studies may result in the overestimation (by as much as 330%) of bacterial biomass in open oceans and in the underestimation (by as much as 40%) of bacterial biomass in coastal environments.
Identification and Activities In Situ of
Nitrosospira
and
Nitrospira
spp. as Dominant Populations in a Nitrifying Fluidized Bed Reactor
Authors: Schramm Andreas , de Beer Dirk , Wagner Michael , Amann Rudolf
Published: December 2019

Bacterial aggregates from a chemolithoautotrophic, nitrifying fluidized bed reactor were investigated with microsensors and rRNA-based molecular techniques. The microprofiles of O
2
, NH
4
+
, NO
2
−
, and NO
3
−
demonstrated the occurrence of complete nitrification in the outer 125 μm of the aggregates. The ammonia oxidizers were identified as members of the
Nitrosospira
group by fluorescence in situ hybridization (FISH). No ammonia- or nitrite-oxidizing bacteria of the genus
Nitrosomonas
or
Nitrobacter
, respectively, could be detected by FISH. To identify the nitrite oxidizers, a 16S ribosomal DNA clone library was constructed and screened by denaturing gradient gel electrophoresis and selected clones were sequenced. The organisms represented by these sequences formed two phylogenetically distinct clusters affiliated with the nitrite oxidizer
Nitrospira moscoviensis
. 16S rRNA-targeted oligonucleotide probes were designed for in situ detection of these organisms. FISH analysis showed that the dominant populations of
Nitrospira
spp. and
Nitrosospira
spp. formed separate, dense clusters which were in contact with each other and occurred throughout the aggregate. A second, smaller, morphologically and genetically different population of
Nitrospira
spp. was restricted to the outer nitrifying zones.
Microbial Desulfurization of a Crude Oil Middle-Distillate Fraction: Analysis of the Extent of Sulfur Removal and the Effect of Removal on Remaining Sulfur
Authors: Grossman M. J. , Lee M. K. , Prince R. C. , Garrett K. K. , George G. N. , Pickering I. J.
Published: December 2019

Rhodococcus
sp. strain ECRD-1 was evaluated for its ability to desulfurize a 232 to 343°C middle-distillate (diesel range) fraction of Oregon basin (OB) crude oil. OB oil was provided as the sole source of sulfur in batch cultures, and the extent of desulfurization and the chemical fate of the residual sulfur in the oil after treatment were determined. Gas chromatography (GC), flame ionization detection, and GC sulfur chemiluminesce detection analysis were used to qualitatively evaluate the effect of
Rhodococcus
sp. strain ECRD-1 treatment on the hydrocarbon and sulfur content of the oil, respectively. Total sulfur was determined by combustion of samples and measurement of released sulfur dioxide by infrared absorption. Up to 30% of the total sulfur in the middle distillate cut was removed, and compounds across the entire boiling range of the oil were affected. Sulfur K-edge X-ray absorption-edge spectroscopy was used to examine the chemical state of the sulfur remaining in the treated OB oil. Approximately equal amounts of thiophenic and sulfidic sulfur compounds were removed by ECRD-1 treatment, and over 50% of the sulfur remaining after treatment was in an oxidized form. The presence of partially oxidized sulfur compounds indicates that these compounds were en route to desulfurization. Overall, more than two-thirds of the sulfur had been removed or oxidized by the microbial treatment.
Phylogenetic Analysis of an Anaerobic, Trichlorobenzene-Transforming Microbial Consortium
Authors: von Wintzingerode Friedrich , Selent Burkhard , Hegemann Werner , Göbel Ulf B.
Published: December 2019

A culture-independent phylogenetic survey for an anaerobic trichlorobenzene-transforming microbial community was carried out. Small-subunit rRNA genes were PCR amplified from community DNA by using primers specific for
Bacteria
or
Euryarchaeota
and were subsequently cloned. Application of a new hybridization-based screening approach revealed 51 bacterial clone families, one of which was closely related to dechlorinating
Dehalobacter
species. Several clone sequences clustered to rDNA sequences obtained from a molecular study of an anaerobic aquifer contaminated with hydrocarbons and chlorinated solvents (Dojka et al., Appl. Env. Microbiol. 64:3869–3877, 1998).
Quantification of Hepatitis A Virus in Shellfish by Competitive Reverse Transcription-PCR with Coextraction of Standard RNA
Authors: Arnal Charlotte , Ferre-Aubineau Virginie , Mignotte Berangere , Imbert-Marcille Berthe Marie , Billaudel Sylviane
Published: December 2019

To quantify hepatitis A virus (HAV) in experimentally contaminated mussels, we developed an internal standard RNA with a 7-nucleotide deletion for competitive reverse transcription (RT)-PCR. Deposited directly into the sample, this standard was used both as extraction control and as quantification tool. After coextraction and competitive RT-PCR, standard and wild-type products were detected by differential hybridization with specific probes and a DNA enzyme immunoassay. The quantifiable range with this reproducible method was 10
4
to 10
7
copies of HAV/gram or 400 to 10
6
50% tissue culture infective doses/ml.
Expression, Regulation, and Mode of Action of the AbiG Abortive Infection System of
Lactococcus lactis
subsp.
cremoris
UC653
Authors: O’Connor Lisa , Tangney Mark , Fitzgerald Gerald F.
Published: December 2019

The abortive infection system AbiG is encoded by the lactococcal plasmid pCI750. The
abiG
locus (consisting of two genes,
abiGi
and
abiGii
) was examined by Northern blot analysis, revealing two transcripts of approximately 2.8 and 1.5 kb which were homologous to the two gene-specific probes. A transcriptional start site was mapped upstream of
abiGi
, and it appeared that the two genes were cotranscribed, resulting in the 2.8-kb transcript. The smaller transcript may be the result of independent transcription of
abiGii
within
abiGi
or of the presence of a weak terminator within
abiGii
. The locus was shown to be constitutively expressed. Evidence is presented for the possible existence of a second Abi mechanism on pCI750. Examination of phage sk1 RNA synthesis demonstrated that both the subcloned AbiG and, to a greater extent, pCI750 inhibited this process. pCI750 also severely inhibited synthesis of both early and late phage c2 transcripts, while the presence of the subclone resulted in a reduction in late transcript synthesis only.
Growth of
Listeria monocytogenes
and
Yersinia enterocolitica
on Cooked Modified-Atmosphere-Packaged Poultry in the Presence and Absence of a Naturally Occurring Microbiota
Authors: Barakat R. K. , Harris L. J.
Published: December 2019

Cooked poultry cuts were inoculated with five-strain composite mixtures of either
Listeria monocytogenes
or
Yersinia enterocolitica
(1,000 CFU/150-g piece), packaged in 44:56 CO
2
-N
2
, and stored at 3.5, 6.5, or 10°C for up to 5 weeks. Both
L. monocytogenes
and
Y. enterocolitica
grew under all test conditions. The presence of a naturally occurring microbiota did not influence the growth of either pathogen. Addition of lactate with the shelf life extender ALTA 2341 lengthened the lag phases of
L. monocytogenes
and
Y. enterocolitica
but did not prevent their growth.
Fate of Free DNA and Transformation of the Oral Bacterium
Streptococcus gordonii
DL1 by Plasmid DNA in Human Saliva
Authors: Mercer Derry K. , Scott Karen P. , Bruce-Johnson Wendy A. , Glover L. Anne , Flint Harry J.
Published: December 2019

Competitive PCR was used to monitor the survival of a 520-bp DNA target sequence from a recombinant plasmid, pVACMC1, after admixture of the plasmid with freshly sampled human saliva. The fraction of the target remaining amplifiable ranged from 40 to 65% after 10 min of exposure to saliva samples from five subjects and from 6 to 25% after 60 min of exposure. pVACMC1 plasmid DNA that had been exposed to degradation by fresh saliva was capable of transforming naturally competent
Streptococcus gordonii
DL1 to erythromycin resistance, although transforming activity decreased rapidly, with a half-life of approximately 50 s.
S. gordonii
DL1 transformants were obtained in the presence of filter-sterilized saliva and a 1-μg/ml final concentration of pVACMC1 DNA. Addition of filter-sterilized saliva instead of heat-inactivated horse serum to
S. gordonii
DL1 cells induced competence, although with slightly lower efficiency. These findings indicate that DNA released from bacteria or food sources within the mouth has the potential to transform naturally competent oral bacteria. However, further investigations are needed to establish whether transformation of oral bacteria can occur at significant frequencies in vivo.
A Mechanism of Resistance to Hydrogen Peroxide in
Vibrio rumoiensis
S-1
Authors: Ichise Nobutoshi , Morita Naoki , Hoshino Tamotsu , Kawasaki Kosei , Yumoto Isao , Okuyama Hidetoshi
Published: December 2019

A possible mechanism of resistance to hydrogen peroxide (H
2
O
2
) in
Vibrio rumoiensis
, isolated from the H
2
O
2
-rich drain pool of a fish processing plant, was examined. When
V. rumoiensis
cells were inoculated into medium containing either 5 mM or no H
2
O
2
, they grew in similar manners. A spontaneous mutant strain, S-4, derived from
V. rumoiensis
and lacking catalase activity did not grow at all in the presence of 5 mM H
2
O
2
. These results suggest that catalase is inevitably involved in the resistance and survival of
V. rumoiensis
in the presence of H
2
O
2
. Catalase activity was constitutively present in
V. rumoiensis
cells grown in the absence of H
2
O
2
, and its occurrence was dependent on the age of the cells, a characteristic which is observed for the HP II-type catalase of
Escherichia coli
. The presence of the HP II-type catalase in
V. rumoiensis
cells was evidenced by partial sequencing of the gene encoding the HP II-type catalase from this organism. A notable difference between
V. rumoiensis
and
E. coli
is that catalase is accumulated at very high levels (∼2% of the total soluble proteins) in
V. rumoiensis
, in contrast to the case for
E. coli
. When
V. rumoiensis
cells which had been exposed to 5 mM H
2
O
2
were centrifuged, most intracellular proteins, including catalase, were recovered in the medium. On the other hand, when
V. rumoiensis
cells were grown on plates containing various concentrations of H
2
O
2
, individual cells had a colony-forming ability inferior to those of
E. coli
,
Bacillus subtilis
, and
Vibrio parahaemolyticus
. Thus, it is suggested that when
V. rumoiensis
cells are exposed to high concentrations of H
2
O
2
, most cells will immediately be broken by H
2
O
2
. In addition, the cells which have had little or no damage will start to grow in a medium where almost all H
2
O
2
has been decomposed by the catalase released from broken cells.
Identification of
Bacillus
Spores by Matrix-Assisted Laser Desorption Ionization–Mass Spectrometry
Authors: Hathout Yetrib , Demirev Plamen A. , Ho Yen-Peng , Bundy Jonathan L. , Ryzhov Victor , Sapp Lisa , Stutler James , Jackman Joany , Fenselau Catherine
Published: December 2019

Unique patterns of biomarkers were reproducibly characterized by matrix-assisted laser desorption ionization (MALDI)–mass spectrometry and were used to distinguish
Bacillus
species members from one another. Discrimination at the strain level was demonstrated for
Bacillus cereus
spores. Lipophilic biomarkers were invariant in
Bacillus globigii
spores produced in three different media and in
B. globigii
spores stored for more than 30 years. The sensitivity was less than 5,000 cells deposited for analysis. Protein biomarkers were also characterized by MALDI analysis by using spores treated briefly with corona plasma discharge. Protein biomarkers were readily desorbed following this treatment. The effect of corona plasma discharge on the spores was examined.
Biodegradation of Pentachlorophenol in a Continuous Anaerobic Reactor Augmented with
Desulfitobacterium frappieri
PCP-1
Authors: Tartakovsky B. , Levesque M.-J. , Dumortier R. , Beaudet R. , Guiot S. R.
Published: December 2019

In this work, a strain of anaerobic pentachlorophenol (PCP) degrader,
Desulfitobacterium frappieri
PCP-1, was used to augment a mixed bacterial community of an anaerobic upflow sludge bed reactor degrading PCP. To estimate the efficiency of augmentation, the population of PCP-1 in the reactor was enumerated by a competitive PCR technique. The PCP-1 strain appeared to compete well with other microorganisms of the mixed bacterial community, with its population increasing from 10
6
to 10
10
cells/g of volatile suspended solids within a period of 70 days. Proliferation of strain PCP-1 allowed for a substantial increase of the volumetric PCP load from 5 to 80 mg/liter of reaction volume/day. A PCP removal efficiency of 99% and a dechlorination efficiency of not less than 90.5% were observed throughout the experiment, with 3-Cl-phenol and phenol being observable dechlorination intermediates.
Cold Shock Proteins and Low-Temperature Response of
Streptococcus thermophilus
CNRZ302
Authors: Wouters Jeroen A. , Rombouts Frank M. , de Vos Willem M. , Kuipers Oscar P. , Abee Tjakko
Published: December 2019

Low-temperature adaptation and cryoprotection were studied in the thermophilic lactic acid bacterium
Streptococcus thermophilus
CNRZ302.
S. thermophilus
actively adapts to freezing during a pretreatment at 20°C, resulting in an approximately 1,000-fold increased survival after four freeze-thaw cycles compared to mid-exponential-phase cells grown at an optimal temperature of 42°C. No adaptation is observed when cells are exposed to a temperature (10°C) below the minimal growth temperature of the strain (just below 15°C). By two-dimensional gel electrophoresis several 7-kDa cold-induced proteins were identified, which are the major induced proteins after a shift to 20°C. These cold shock proteins were maximally expressed at 20°C, while the induction level was low after cold shock to 10°C. To confirm the presence of
csp
genes in
S. thermophilus
, a PCR strategy was used which yielded products of different sizes. Sequence analysis revealed
csp
-like sequences that were up to 95% identical to those of
csp
genes of
S. thermophilus
ST1-1,
Streptococcus dysgalactiae
,
Streptococcus pyogenes
, and
Lactococcus lactis
. Northern blot analysis revealed a seven- to ninefold induction of
csp
mRNA after a temperature shift to 20°C, showing that this thermophilic bacterium indeed contains at least one cold-inducible
csp
gene and that its regulation takes place at the transcriptional level.
Registered Designation of Origin Areas of Fermented Food Products Defined by Microbial Phenotypes and Artificial Neural Networks
Authors: Lopes M. F. S. , Pereira C. I. , Rodrigues F. M. S. , Martins M. P. , Mimoso M. C. , Barros T. C. , Figueiredo Marques J. J. , Tenreiro R. P. , Almeida J. S. , Barreto Crespo M. T.
Published: December 2019

Cheese produced from raw ewes’ milk and
chouriço
, a Portuguese dry fermented sausage, are still produced in a traditional way in certain regions of Portugal by relying on colonization by microbial populations associated with the raw materials, equipment, and local environments. For the purpose of describing the product origins and types of these fermented foods, metabolic phenotypes can be used as descriptors of the product as well as to determine the presence of compounds with organoleptic value. The application of artificial neural networks to the metabolic profiles of bacterial isolates was assayed and allowed the separation of products from different regions. This method could then be used for the Registered Designation of Origin certification process of food products. Therefore, besides test panel results for these traditionally produced food products, another tool for validating products for the marketplace is available to the producers. The method can be improved for the detection of counterfeit products.
Natural Diversity of
Frankia
Strains in Actinorhizal Root Nodules from Promiscuous Hosts in the Family Myricaceae
Authors: Clawson Michael L. , Benson David R.
Published: December 2019

Actinorhizal plants invade nitrogen-poor soils because of their ability to form root nodule symbioses with N
2
-fixing actinomycetes known as
Frankia
.
Frankia
strains are difficult to isolate, so the diversity of strains inhabiting nodules in nature is not known. To address this problem, we have used the variability in bacterial 16S rRNA gene sequences amplified from root nodules as a means to estimate molecular diversity. Nodules were collected from 96 sites primarily in northeastern North America; each site contained one of three species of the family Myricaceae. Plants in this family are considered to be promiscuous hosts because several species are effectively nodulated by most isolated strains of
Frankia
in the greenhouse. We found that strain evenness varies greatly between the plant species so that estimating total strain richness of
Frankia
within myricaceous nodules with the sample size used was problematical. Nevertheless,
Myrica pensylvanica
, the common bayberry, was found to have sufficient diversity to serve as a reservoir host for
Frankia
strains that infect plants from other actinorhizal families.
Myrica gale
, sweet gale, yielded a few dominant sequences, indicating either symbiont specialization or niche selection of particular ecotypes. Strains in
Comptonia peregrina
nodules had an intermediate level of diversity and were all from a single major group of
Frankia
.
Characterization of a Tetrameric Inositol Monophosphatase from the Hyperthermophilic Bacterium
Thermotoga maritima
Authors: Chen Liangjing , Roberts Mary F.
Published: December 2019

Inositol monophosphatase (I-1-Pase) catalyzes the dephosphorylation step in the de novo biosynthetic pathway of inositol and is crucial for all inositol-dependent processes. An extremely heat-stable tetrameric form of I-1-Pase from the hyperthermophilic bacterium
Thermotoga maritima
was overexpressed in
Escherichia coli
. In addition to its different quaternary structure (all other known I-1-Pases are dimers), this enzyme displayed a 20-fold higher rate of hydrolysis of
d
-inositol 1-phosphate than of the
l
isomer. The homogeneous recombinant
T. maritima
I-1-Pase (containing 256 amino acids with a subunit molecular mass of 28 kDa) possessed an unusually high
V
max
(442 μmol min
−1
mg
−1
) that was much higher than the
V
max
of the same enzyme from another hyperthermophile,
Methanococcus jannaschii
. Although
T. maritima
is a eubacterium, its I-1-Pase is more similar to archaeal I-1-Pases than to the other known bacterial or mammalian I-1-Pases with respect to substrate specificity, Li
+
inhibition, inhibition by high Mg
2+
concentrations, metal ion activation, heat stability, and activation energy. Possible reasons for the observed kinetic differences are discussed based on an active site sequence alignment of the human and
T. maritima
I-1-Pases.
Protective Role of Catalase in
Pseudomonas aeruginosa
Biofilm Resistance to Hydrogen Peroxide
Authors: Elkins James G. , Hassett Daniel J. , Stewart Philip S. , Schweizer Herbert P. , McDermott Timothy R.
Published: December 2019

The role of the two known catalases in
Pseudomonas aeruginosa
in protecting planktonic and biofilm cells against hydrogen peroxide (H
2
O
2
) was investigated. Planktonic cultures and biofilms formed by the wild-type strain PAO1 and the
katA
and
katB
catalase mutants were compared for their susceptibility to H
2
O
2
. Over the course of 1 h, wild-type cell viability decreased steadily in planktonic cells exposed to a single dose of 50 mM H
2
O
2
, whereas biofilm cell viability remained at approximately 90% when cells were exposed to a flowing stream of 50 mM H
2
O
2
. The
katB
mutant, lacking the H
2
O
2
-inducible catalase KatB, was similar to the wild-type strain with respect to H
2
O
2
resistance. The
katA
mutant possessed undetectable catalase activity. Planktonic
katA
mutant cultures were hypersusceptible to a single dose of 50 mM H
2
O
2
, while biofilms displayed a 10-fold reduction in the number of culturable cells after a 1-h exposure to 50 mM H
2
O
2
. Catalase activity assays, activity stains in nondenaturing polyacrylamide gels, and
lacZ
reporter genes were used to characterize the oxidative stress responses of planktonic cultures and biofilms. Enzyme assays and catalase activity bands in nondenaturing polyacrylamide gels showed significant KatB catalase induction occurred in biofilms after a 20-min exposure to H
2
O
2
, suggesting that biofilms were capable of a rapid adaptive response to the oxidant. Reporter gene data obtained with a
katB
::
lacZ
transcriptional reporter strain confirmed
katB
induction and that the increase in total cellular catalase activity was attributable to KatB. Biofilms upregulated the reporter in the constant presence of 50 mM H
2
O
2
, while planktonic cells were overwhelmed by a single 50 mM dose and were unable to make detectable levels of β-galactosidase. The results of this study demonstrated the following: the constitutively expressed KatA catalase is important for resistance of planktonic and biofilm
P. aeruginosa
to H
2
O
2
, particularly at high H
2
O
2
concentrations; KatB is induced in both planktonic and biofilm cells in response to H
2
O
2
insult, but plays a relatively small role in biofilm resistance; and KatB is important to either planktonic cells or biofilm cells for acquired antioxidant resistance when initial levels of H
2
O
2
are sublethal.
Production of Chymotrypsin-Resistant
Bacillus thuringiensis
Cry2Aa1 δ-Endotoxin by Protein Engineering
Authors: Audtho Mongkon , Valaitis Algimantas P. , Alzate Oscar , Dean Donald H.
Published: December 2019

Cleavage of the Cry2Aa1 protoxin (molecular mass, 63 kDa) from
Bacillus thuringiensis
by midgut juice of gypsy moth (
Lymantria dispar
) larvae resulted in two major protein fragments: a 58-kDa fragment which was highly toxic to the insect and a 49-kDa fragment which was not toxic. In the midgut juice, the protoxin was processed into a 58-kDa toxin within 1 min, but after digestion for 1 h, the 58-kDa fragment was further cleaved within domain I, resulting in the protease-resistant 49-kDa fragment. Both the 58-kDa and nontoxic 49-kDa fragments were also found in vivo when
125
I-labeled toxin was fed to the insects. N-terminal sequencing revealed that the protease cleavage sites are at the C termini of Tyr49 and Leu144 for the active fragment and the smaller fragment, respectively. To prevent the production of the nontoxic fragment during midgut processing, five mutant proteins were constructed by replacing Leu144 of the toxin with Asp (L144D), Ala (L144A), Gly (L144G), His (L144H), or Val (L144V) by using a pair of complementary mutagenic oligonucleotides in PCR. All of the mutant proteins were highly resistant to the midgut proteases and chymotrypsin. Digestion of the mutant proteins by insect midgut extract and chymotrypsin produced only the active 58-kDa fragment, except that L144H was partially cleaved at residue 144.
Toxigenic Strains of
Bacillus licheniformis
Related to Food Poisoning
Authors: Salkinoja-Salonen M. S. , Vuorio R. , Andersson M. A. , Kämpfer P. , Andersson M. C. , Honkanen-Buzalski T. , Scoging A. C.
Published: December 2019

Toxin-producing isolates of
Bacillus licheniformis
were obtained from foods involved in food poisoning incidents, from raw milk, and from industrially produced baby food. The toxin detection method, based on the inhibition of boar spermatozoan motility, has been shown previously to be a sensitive assay for the emetic toxin of
Bacillus cereus
, cereulide. Cell extracts of the toxigenic
B. licheniformis
isolates inhibited sperm motility, damaged cell membrane integrity, depleted cellular ATP, and swelled the acrosome, but no mitochondrial damage was observed. The responsible agent from the
B. licheniformis
isolates was partially purified. It showed physicochemical properties similar to those of cereulide, despite having very different biological activity. The toxic agent was nonproteinaceous; soluble in 50 and 100% methanol; and insensitive to heat, protease, and acid or alkali and of a molecular mass smaller than 10,000 g mol
−1
. The toxic
B. licheniformis
isolates inhibited growth of
Corynebacterium renale
DSM 20688
T
, but not all inhibitory isolates were sperm toxic. The food poisoning-related isolates were beta-hemolytic, grew anaerobically and at 55°C but not at 10°C, and were nondistinguishable from the type strain of
B. licheniformis
, DSM 13
T
, by a broad spectrum of biochemical tests. Ribotyping revealed more diversity; the toxin producers were divided among four ribotypes when cut with
Pvu
II and among six when cut with
Eco
RI, but many of the ribotypes also contained nontoxigenic isolates. When ribotyped with
Pvu
II, most toxin-producing isolates shared bands at 2.8 ± 0.2, 4.9 ± 0.3, and 11.7 ± 0.5 or 13.1 ± 0.8 kb.
Diversity of Sulfate-Reducing Bacteria in Oxic and Anoxic Regions of a Microbial Mat Characterized by Comparative Analysis of Dissimilatory Sulfite Reductase Genes
Authors: Minz Dror , Flax Jodi L. , Green Stefan J. , Muyzer Gerard , Cohen Yehuda , Wagner Michael , Rittmann Bruce E. , Stahl David A.
Published: December 2019

Sequence analysis of genes encoding dissimilatory sulfite reductase (DSR) was used to identify sulfate-reducing bacteria in a hypersaline microbial mat and to evaluate their distribution in relation to levels of oxygen. The most highly diverse DSR sequences, most related to those of the
Desulfonema
-like organisms within the δ-proteobacteria, were recovered from oxic regions of the mat. This observation extends those of previous studies by us and others associating
Desulfonema
-like organisms with oxic habitats.
Aerobic Degradation of 1,1,1-Trichloroethane by
Mycobacterium
spp. Isolated from Soil
Authors: Yagi Osami , Hashimoto Akiko , Iwasaki Kazuhiro , Nakajima Mutsuyasu
Published: December 2019

Two strains of 1,1,1-trichloroethane (TCA)-degrading bacteria, TA5 and TA27, were isolated from soil and identified as
Mycobacterium
spp. Strains TA5 and TA27 could degrade 25 and 75 mg · liter of TCA
−1
cometabolically in the presence of ethane as a carbon source, respectively. The compound 2,2,2-trichloroethanol was produced as a metabolite of the degradation process.
Partial Purification and Characterization of
Bacillus thuringiensis
Cry1A Toxin Receptor A from
Heliothis virescens
and Cloning of the Corresponding cDNA
Authors: Oltean Daniela I. , Pullikuth Ashok K. , Lee Hyun-Ku , Gill Sarjeet S.
Published: December 2019

Although extensively studied, the mechanism of action of insecticidal
Bacillus thuringiensis
Cry toxins remains elusive and requires further elucidation. Toxin receptors in the brush border membrane demand particular attention as they presumably initiate the cascade of events leading to insect mortality after toxin activation. The 170-kDa Cry1Ac toxin-binding aminopeptidase from the tobacco budworm (
Heliothis virescens
) was partially purified, and its corresponding cDNA was cloned. The cDNA encodes a protein with a putative glycosyl phosphatidylinositol anchor and a polythreonine stretch clustered near the C terminus with predicted O-glycosylation. Partial purification of the 170-kDa aminopeptidase also resulted in isolation of a 130-kDa protein that was immunologically identical to the 170-kDa protein, and the two proteins had identical N termini. These proteins were glycosylated, as suggested by soybean agglutinin lectin blot results. Cry1Ac toxin affinity data for the two proteins indicated that the 130-kDa protein had a higher affinity than the 170-kDa protein. The data suggest that posttranslational modifications can have a significant effect on Cry1A toxin interactions with specific insect midgut proteins.