bluuebunny/crossref_metadata_2025 huggingface dataset:
PCR Detection of Cyclic Lipodepsinonapeptide-Producing
Pseudomonas syringae
pv. syringae and Similarity of Strains
Authors: Sorensen Kevin N. , Kim Kwang-Hee , Takemoto Jon Y.
Published: December 2019

Many strains of
Pseudomonas syringae
pv. syringae produce one of four classes of small cyclic lipodepsinonapeptides: syringomycins, syringostatins, syringotoxins, or pseudomycins. These metabolites are phytotoxic and growth inhibitory against a broad spectrum of fungi. Their production is dependent upon the expression of conserved biosynthesis and export genes
syrB
and
syrD
, respectively. PCR and oligonucleotide primers specific for a 752-bp fragment of
syrB
were used to identify cyclic lipodepsinonapeptide-producing strains of
P. syringae
pv. syringae. In contrast, PCR amplification with primers based on
syrD
did not always correlate with possession of the
syrD
gene, as indicated by Southern blot analysis, or with cyclic lipodepsinonapeptide production. Sequence comparisons of 400 nucleotides from the
syrB
PCR-amplified fragments showed 94% plot similarity among 27 strains. In a sequence phenogram, syringostatin and syringotoxin producers were grouped apart from syringomycin-producing strain B301D, with sequences that differed by eight and nine conserved base substitutions, respectively. PCR amplification of the 752-bp
syrB
fragment offers rapid and accurate detection of cyclic lipodepsinonapeptide-producing strains, and its sequence provides some predictive capabilities for identifying syringotoxin and syringostatin producers.
Nisin Resistance inListeria monocytogenesATCC 700302 Is a Complex Phenotype
Authors: Crandall Allison D. , Montville Thomas J.
Published: December 2019

Nisin resistance inListeria monocytogenesATCC 700302 is a complex phenotype involving alterations in both the cytoplasmic membrane and the cell wall and a requirement for divalent cations. In addition to a lower ratio of C15to C17fatty acids than in the wild-type strain (A. S. Mazzotta and T. J. Montville, J. Appl. Microbiol. 82:32–38, 1997), this nisin-resistant (Nisr) strain contained significantly more zwitterionic phosphatidylethanolamine and less anionic phosphatidylglycerol and cardiolipin. The extraction of cardiolipin was enhanced by a penicillin-lysozyme step to disrupt the cell wall. This study is the first to quantify the phosphatidylethanolamine component of theL. monocytogenescytoplasmic membrane. While these cytoplasmic membrane changes were induced by nisin, the Nisrstrain also showed altered sensitivities to cell wall-acting compounds, even when grown in the absence of nisin, suggesting a constitutive alteration in the strain’s cell wall. A model which integrates the roles of the cell membrane, cell wall, and divalent cations is presented. Finally, nisin resistance inL. monocytogenesATCC 700302 conferred cross-resistance to the class IIa bacteriocin pediocin PA-1 and the class IV leuconocin S.
Effects of Ammonium and Non-Ammonium Salt Additions on Methane Oxidation by
Methylosinus trichosporium
OB3b and Maine Forest Soils
Authors: King G. M. , Schnell S.
Published: December 2019

Additions of ammonium and non-ammonium salts inhibit atmospheric methane consumption by soil at salt concentrations that do not significantly affect the soil water potential. The response of soils to non-ammonium salts has previously raised questions about the mechanism of ammonium inhibition. Results presented here show that inhibition of methane consumption by non-ammonium salts can be explained in part by ion-exchange reactions: cations desorb ammonium, with the level of desorption varying as a function of both the cation and anion added; differential desorption results in differential inhibition levels. Differences in the extent of inhibition among ammonium salts can also be explained in part by the effects of anions on ammonium exchange. In contrast, only minimal effects of cations and anions are observed in liquid cultures of
Methylosinus trichosporium
OB3b. The comparable level of inhibition by equinormal concentrations of NH
4
Cl and (NH
4
)
2
SO
4
and the insensitivity of salt inhibition to increasing methane concentrations (from 10 to 100 ppm) are of particular interest, since both of these patterns are in contrast to results for soils. The greater inhibition of methane consumption for NH
4
Cl than (NH
4
)
2
SO
4
in soils can be attributed to increased ammonium adsorption by sulfate; increasing inhibition by non-ammonium salts with increasing methane concentrations can be attributed to desorbed ammonium and a physiological mechanism proposed previously for pure cultures.
Nitrospira
-Like Bacteria Associated with Nitrite Oxidation in Freshwater Aquaria
Authors: Hovanec Timothy A. , Taylor Lance T. , Blakis Andrew , Delong Edward F.
Published: December 2019

Oxidation of nitrite to nitrate in aquaria is typically attributed to bacteria belonging to the genus
Nitrobacter
which are members of the α subdivision of the class
Proteobacteria
. In order to identify bacteria responsible for nitrite oxidation in aquaria, clone libraries of rRNA genes were developed from biofilms of several freshwater aquaria. Analysis of the rDNA libraries, along with results from denaturing gradient gel electrophoresis (DGGE) on frequently sampled biofilms, indicated the presence of putative nitrite-oxidizing bacteria closely related to other members of the genus
Nitrospira
. Nucleic acid hybridization experiments with rRNA from biofilms of freshwater aquaria demonstrated that
Nitrospira
-like rRNA comprised nearly 5% of the rRNA extracted from the biofilms during the establishment of nitrification. Nitrite-oxidizing bacteria belonging to the α subdivision of the class
Proteobacteria
(e.g.,
Nitrobacter
spp.) were not detected in these samples. Aquaria which received a commercial preparation containing
Nitrobacter
species did not show evidence of
Nitrobacter
growth and development but did develop substantial populations of
Nitrospira
-like species. Time series analysis of rDNA phylotypes on aquaria biofilms by DGGE, combined with nitrite and nitrate analysis, showed a correspondence between the appearance of
Nitrospira
-like bacterial ribosomal DNA and the initiation of nitrite oxidation. In total, the data suggest that
Nitrobacter winogradskyi
and close relatives were not the dominant nitrite-oxidizing bacteria in freshwater aquaria. Instead, nitrite oxidation in freshwater aquaria appeared to be mediated by bacteria closely related to
Nitrospira moscoviensis
and
Nitrospira marina.
Choline Derivatives Involved in Osmotolerance of
Penicillium fellutanum
Authors: Park Yong-Il , Gander John E.
Published: December 2019

Penicillium fellutanum
is osmotolerant and xerotolerant when cultured in a low-phosphate medium containing 3 M NaCl. Glycerol and erythritol accumulated in cultures with NaCl concentrations up to 2 M; glycerol was the only detectable polyol in cultures containing 3 M NaCl. In cultures with 3 M NaCl, the intracellular levels of glycine betaine and choline-
O
-sulfate were 22- and 2.6-fold greater (70 and 46 mM), respectively, than those of cultures without added NaCl. The levels of glycine betaine and glycerol decreased in mycelia transferred from a medium containing 3 M NaCl into a fresh medium without added NaCl. NaCl at 3 M inhibited mycelial mass accumulation; this inhibition was partially corrected by supplementation of cultures with glycine betaine (2 mM) or choline-
O
-sulfate (10 mM). The presence of exogenous choline chloride (2 mM) in plate cultures protected the cells from stress from 3 M NaCl. The data suggest that glycine betaine and choline-
O
-sulfate are secondary osmoprotectants which are effective at the point that the cell is incapable of synthesizing more glycerol.
Production of Respirable Vesicles Containing Live
Legionella pneumophila
Cells by Two
Acanthamoeba
spp
Authors: Berk Sharon G. , Ting Rebecca S. , Turner Glenn W. , Ashburn Rebecca J.
Published: December 2019

Two
Acanthamoeba
species, fed at three temperatures, expelled vesicles containing living
Legionella pneumophila
cells. Vesicles ranged from 2.1 to 6.4 μm in diameter and theoretically could contain several hundred bacteria. Viable
L. pneumophila
cells were observed within vesicles which had been exposed to two cooling tower biocides for 24 h. Clusters of bacteria in vesicles were not dispersed by freeze-thawing and sonication. Such vesicles may be agents for the transmission of legionellosis associated with cooling towers, and the risk may be underestimated by plate count methods.
Phylogenetic Diversity of Ultraplankton Plastid Small-Subunit rRNA Genes Recovered in Environmental Nucleic Acid Samples from the Pacific and Atlantic Coasts of the United States
Authors: Rappé Michael S. , Suzuki Marcelino T. , Vergin Kevin L. , Giovannoni Stephen J.
Published: December 2019

The scope of marine phytoplankton diversity is uncertain in many respects because, like bacteria, these organisms sometimes lack defining morphological characteristics and can be a challenge to grow in culture. Here, we report the recovery of phylogenetically diverse plastid small-subunit (SSU) rRNA gene (rDNA) clones from natural plankton populations collected in the Pacific Ocean off the mouth of Yaquina Bay, Oreg. (OCS clones), and from the eastern continental shelf of the United States off Cape Hatteras, N.C. (OM clones). SSU rRNA gene clone libraries were prepared by amplifying rDNAs from nucleic acids isolated from plankton samples and cloning them into plasmid vectors. The PCR primers used for amplification reactions were designed to be specific for bacterial SSU rRNA genes; however, plastid genes have a common phylogenetic origin with bacteria and were common in both SSU rRNA gene clone libraries. A combination of restriction fragment length polymorphism analyses, nucleic acid sequencing, and taxon-specific oligonucleotide probe hybridizations revealed that 54 of the 116 OCS gene clones were of plastid origin. Collectively, clones from the OCS and OM libraries formed at least eight unique lineages within the plastid radiation, including gene lineages related to the classes
Bacillariophyceae
,
Cryptophyceae
,
Prymnesiophyceae
,
Chrysophyceae
, and
Prasinophyceae
; for a number of unique clones, no close phylogenetic neighbors could be identified with confidence. Only a group of two OCS rRNA gene clones showed close identity to the plastid SSU rRNA gene sequence of a cultured organism [
Emiliania huxleyi
(Lohmann) Hay and Mohler; 99.8% similar]. The remaining clones could not be identified to the genus or species level. Although cryptic species are not as prevalent among phytoplankton as they are among their bacterial counterparts, this genetic survey nonetheless uncovered significant new information about phytoplankton diversity.
Biodegradation of Phosphonomycin by
Rhizobium huakuii
PMY1
Authors: McGrath John W. , Hammerschmidt Friedrich , Quinn John P.
Published: December 2019

The biodegradation by
Rhizobium huakuii
PMY1 of up to 10 mM phosphonomycin as a carbon, energy, and phosphorus source with accompanying P
i
release is described. This biodegradation represents a further mechanism of resistance to this antibiotic and a novel, phosphate-deregulated route for organophosphonate metabolism by
Rhizobium
spp.
Differentiation of
Campylobacter
Isolates on the Basis of Sensitivity to Boiling in Water as Measured by PCR-Detectable DNA
Authors: Mohran Zaynab S. , Arthur Ray R. , Oyofo Buhari A. , Peruski Leonard F. , Wasfy Momtaz O. , Ismail Tharwat F. , Murphy James R.
Published: December 2019

Differential sensitivity for the release of PCR-detectable genomic DNA upon boiling in water is reported for 45
Campylobacter jejuni
and
Campylobacter coli
strains isolated in Egypt. All of the strains released PCR-detectable DNA when treated with proteinase K and sodium dodecyl sulfate. When DNA was extracted from these strains by boiling in water, nine (20%) of the strains were PCR negative or resistant to boiling, suggesting the presence of boiling-sensitive and boiling-resistant phenotypes.
Improvement of the Immunomagnetic Separation Method Selective for
Escherichia coli
O157 Strains
Authors: Tomoyasu Takahiro
Published: December 2019

Immunomagnetic separation is a useful enrichment method selective for
Escherichia coli
O157 cells against non-O157
E. coli
cells from a preenrichment culture. However,
E. coli
cells are adsorbed onto a solid surface nonspecifically. With the conventional immunomagnetic separation method, this nonspecific adsorption interfered with immunomagnetic separation. It was found that this interference could be reduced with a low-ionic-strength solution. When immunomagnetic separation was carried out with this solution, the proportion of
E. coli
O157 cells to non-O157
E. coli
cells increased from 9.6 to 31.4 times compared to the proportion obtained by the conventional immunomagnetic separation method. The effectiveness of this solution was successfully evaluated by the use of
E. coli
O157-spiked samples.
c
-Type Cytochromes and Manganese Oxidation in
Pseudomonas putida
MnB1
Authors: Caspi Ron , Tebo Bradley M. , Haygood M. G.
Published: December 2019

Pseudomonas putida
MnB1 is an isolate from an Mn oxide-encrusted pipeline that can oxidize Mn(II) to Mn oxides. We used transposon mutagenesis to construct mutants of strain MnB1 that are unable to oxidize manganese, and we characterized some of these mutants. The mutants were divided into three groups: mutants defective in the biogenesis of
c
-type cytochromes, mutants defective in genes that encode key enzymes of the tricarboxylic acid cycle, and mutants defective in the biosynthesis of tryptophan. The mutants in the first two groups were cytochrome
c
oxidase negative and did not contain
c
-type cytochromes. Mn(II) oxidation capability could be recovered in a
c
-type cytochrome biogenesis-defective mutant by complementation of the mutation.
The Transcriptional Activator XlnR Regulates Both Xylanolytic and Endoglucanase Gene Expression inAspergillus niger
Authors: van Peij Noël N. M. E. , Gielkens Marco M. C. , de Vries Ronald P. , Visser Jaap , de Graaff Leo H.
Published: December 2019

The expression of genes encoding enzymes involved in xylan degradation and two endoglucanases involved in cellulose degradation was studied at the mRNA level in the filamentous fungusAspergillus niger. A strain with a loss-of-function mutation in thexlnRgene encoding the transcriptional activator XlnR and a strain with multiple copies of this gene were investigated in order to define which genes are controlled by XlnR. The data presented in this paper show that the transcriptional activator XlnR regulates the transcription of thexlnB,xlnC, andxlnDgenes encoding the main xylanolytic enzymes (endoxylanases B and C and β-xylosidase, respectively). Also, the transcription of the genes encoding the accessory enzymes involved in xylan degradation, including α-glucuronidase A, acetylxylan esterase A, arabinoxylan arabinofuranohydrolase A, and feruloyl esterase A, was found to be controlled by XlnR. In addition, XlnR also activates transcription of two endoglucanase-encoding genes,eglAandeglB, indicating that transcriptional regulation by XlnR goes beyond the genes encoding xylanolytic enzymes and includes regulation of two endoglucanase-encoding genes.
Study of Toxin Production by Isolates of
Stachybotrys chartarum
and
Memnoniella echinata
Isolated during a Study of Pulmonary Hemosiderosis in Infants
Authors: Jarvis Bruce B. , Sorenson W. G. , Hintikka Eeva-Liisa , Nikulin Marjo , Zhou Yihong , Jiang Jian , Wang Shengun , Hinkley Simon , Etzel Ruth A. , Dearborn D.
Published: December 2019

A cluster of cases of pulmonary hemosiderosis among infants was reported in Cleveland, Ohio, during 1993 and 1994. These unusual cases appeared only in infants ranging in age from 1 to 8 months and were characterized by pulmonary hemorrhage, which caused the babies to cough up blood. A case-control study identified major home water damage (from plumbing leaks, roof leaks, or flooding) as a risk factor for development of pulmonary hemorrhage in these infants. Because of an interest in the possibility that trichothecene mycotoxins might be involved in this illness, a number of isolates of
Stachybotrys chartarum
were grown in the laboratory on rice, and extracts were prepared and analyzed both for cytotoxicity and for specific toxins. Two isolates of
Memnoniella echinata
, a fungus closely related to
S. chartarum
, were also included in these studies.
S. chartarum
isolates collected from the homes were shown to produce a number of highly toxic compounds, and the profiles of toxic compounds from
M. echinata
were similar; the most notable difference was the fact that the principal metabolites produced by
M. echinata
were griseofulvins.
Ellipsometric Measurement of Bacterial Films at Metal-Electrolyte Interfaces
Authors: Busalmen J. P. , de Sánchez S. R. , Schiffrin D. J.
Published: December 2019

Ellipsometric measurements were used to monitor the formation of a bacterial cell film on polarized metal surfaces (Al-brass and Ti). Under cathodic polarization bacterial attachment was measured from changes in the ellipsometric angles. These were fitted to an effective medium model for a nonabsorbing bacterial film with an effective refractive index (

n
f

) of 1.38 and a thickness (

d
f

) of 160 ± 10 nm. From the optical measurements a surface coverage of 17% was estimated, in agreement with direct microscopic observations. The influence of bacteria on the formation of oxide films was monitored by ellipsometry following the film growth in situ. A strong inhibition of metal oxide film formation was observed, which was assigned to the decrease in oxygen concentration due to the presence of bacteria. It is shown that the irreversible adhesion of bacteria to the surface can be monitored ellipsometrically. Electrophoretic mobility is proposed as one of the factors determining bacterial attachment. The high sensitivity of ellipsometry and its usefulness for the determination of growth of interfacial bacterial films is demonstrated.
Characteristics of Airborne Actinomycete Spores
Authors: Reponen T. A. , Gazenko S. V. , Grinshpun S. A. , Willeke K. , Cole E. C.
Published: December 2019

Airborne actinomycete spores, important contaminants in occupational and residential environments, were studied with respect to their (i) release into the air, (ii) aerodynamic and physical size while airborne, and (iii) survival after collection onto agar with an impactor. Three actinomycete species were selected for the tests to exemplify the three main spore types:
Streptomyces albus
for arthrospores,
Micromonospora halophytica
for aleuriospores, and
Thermoactinomyces vulgaris
for endospores. The results show that the incubation conditions (temperature, time, and nutrients) needed for the development of spores for their release into air are different from the conditions that are needed for colony growth only. Additional drying of
M. halophytica
and
T. vulgaris
cultures was needed before spores could be released from the culture. The aerodynamic sizes of the spores, measured with an aerodynamic particle sizer, ranged from 0.57 (
T. vulgaris
) to 1.28 μm (
M. halophytica
). The physical sizes of the spores, when measured with a microscope and an image analysis system, were found to be smaller than previously reported in the literature. The relative recovery of the spores on agar media ranged from 0.5 (
T. vulgaris
) to 35% (
S. albus
). The results indicate that the culturability of the collected airborne actinomycete spores varies widely and is affected by several variables, such as the species and the sampling flow rate. Therefore, alternatives to commonly used cultivation methods need to be developed for the enumeration of actinomycete spores.
Quantitative and Physiological Analyses of Chloride Dependence of Growth of
Halobacillus halophilus
Authors: Roeßler Markus , Müller Volker
Published: December 2019

A quantitative analysis of the Cl
−
dependence of growth of
Halobacillus halophilus
was performed. Optimal growth rates were obtained at Cl
−
concentrations of between 0.5 and 2.0 M, and the final yield was also strictly dependent on the Cl
−
concentration. Br
−
but not I
−
, SO
4
2−
, NO
2
−
, SO
2
−
, OCN
−
, SCN
−
, BO
2
−
, or BrO
3
−
could substitute for Cl
−
. To analyze the function of chloride, chloride concentration was determined. At low external Cl
−
(Cl
e
−
) concentrations, the growth rate was low and Cl
−
was excluded from the cytoplasm; increasing the Cl
e
−
concentration led to an increase in the growth rate and an energy-dependent uptake of Cl
−
, thus decreasing the Cl
e
−
/internal Cl
i
−
gradient from ≥10 at 0.1 M Cl
e
−
to a nearly constant value of 2 at Cl
e
−
concentrations which allowed optimal growth. Two membrane proteins with apparent molecular masses of 31 and 16 kDa which were identified to be specific for Cl
−
-grown cultures are possible candidates for a chloride uptake system.
Interlaboratory Comparison of Methods To Quantify Microsclerotia of
Verticillium dahliae
in Soil
Authors: Termorshuizen A. J. , Davis J. R. , Gort G. , Harris D. C. , Huisman O. C. , Lazarovits G. , Locke T. , Melero Vara J. M. , Mol L. , Paplomatas E. J. , Platt H. W. , Powelson M. , Rouse D. I. , Rowe R. C. , Tsror L.
Published: December 2019

In a comparison of different methods for estimating
Verticillium dahliae
in soil, 14 soil samples were analyzed in a blinded fashion by 13 research groups in seven countries, using their preferred methods. One group analyzed only four samples. Twelve soil samples were naturally infested, and two had known numbers of microsclerotia of
V. dahliae
added to them. In addition, a control was included to determine whether transport had an effect on the results. Results differed considerably among the research groups. There was a 118-fold difference between the groups with the lowest and highest mean estimates. Results of the other groups were evenly distributed between these extremes. In general, methods based on plating dry soil samples gave higher numbers of
V. dahliae
than did plating of an aqueous soil suspension. Recovery of
V. dahliae
from samples with added microsclerotia varied from 0 to 59%. Most of the variability within each analysis was at the petri dish level. The results indicate the necessity to check the performance of detection assays regularly by comparing recoveries with other laboratories, using a common set of soil samples. We conclude that wet plating assays are less accurate than dry plating assays.
Physiological Activity of
Campylobacter jejuni
Far below the Minimal Growth Temperature
Authors: Hazeleger Wilma C. , Wouters Jeroen A. , Rombouts Frank M. , Abee Tjakko
Published: December 2019

The behavior of
Campylobacter jejuni
at environmental temperatures was examined by determining the physiological activities of this human pathogen. The minimal growth temperatures were found to be 32 and 31°C for strains 104 and ATCC 33560, respectively. Both strains exhibited a sudden decrease in growth rate from the maximum to zero within a few degrees not only near the maximal growth temperature but also near the minimal growth temperature. This could be an indication that a temperature-dependent transition in the structure of a key enzyme(s) or regulatory compound(s) determines the minimal growth temperature. Oxygen consumption, catalase activity, ATP generation, and protein synthesis were observed at temperatures as low as 4°C, indicating that vital cellular processes were still functioning. PCR analysis showed that cold shock protein genes, which play a role in low-temperature adaptation in many bacteria, are not present in
C. jejuni
. The fact that chemotaxis and aerotaxis could be observed at all temperatures shows that the pathogen is able to move to favorable places at environmental temperatures, which may have significant implications for the survival of
C. jejuni
in the environment.
A New Genetic Locus in
Sinorhizobium meliloti
Is Involved in Stachydrine Utilization
Authors: Phillips Donald A. , Sande Eve S. , Vriezen J. A. C. , de Bruijn Frans J. , Le Rudulier Daniel , Joseph Cecillia M.
Published: December 2019

Stachydrine, a betaine released by germinating alfalfa seeds, functions as an inducer of nodulation genes, a catabolite, and an osmoprotectant in
Sinorhizobium meliloti
. Two stachydrine-inducible genes were found in
S. meliloti
1021 by mutation with a Tn
5-luxAB
promoter probe. Both mutant strains (S10 and S11) formed effective alfalfa root nodules, but neither grew on stachydrine as the sole carbon and nitrogen source. When grown in the absence or presence of salt stress, S10 and S11 took up [
14
C]stachydrine as well as wild-type cells did, but neither used stachydrine effectively as an osmoprotectant. In the absence of salt stress, both S10 and S11 took up less [
14
C]proline than wild-type cells did. S10 and S11 appeared to colonize alfalfa roots normally in single-strain tests, but when mixed with the wild-type strain, their rhizosphere counts were reduced more than 50% (
P
≤ 0.01) relative to the wild type. These results suggest that stachydrine catabolism contributes to root colonization. DNA sequence analysis identified the mutated locus in S11 as
putA
, and the
luxAB
fusion in that gene was induced by proline as well as stachydrine. DNA that restored the capacity of mutant S10 to catabolize stachydrine contained a new open reading frame,
stcD
. All data are consistent with the concept that
stcD
codes for an enzyme that produces proline by demethylation of
N
-methylproline, a degradation product of stachydrine.
Relatedness of Strains of
Xanthomonas fragariae
by Restriction Fragment Length Polymorphism, DNA-DNA Reassociation, and Fatty Acid Analyses
Authors: Roberts P. D. , Hodge N. C. , Bouzar H. , Jones J. B. , Stall R. E. , Berger R. D. , Chase A. R.
Published: December 2019

The levels of relatedness of strains of
Xanthomonas fragariae
collected over several years from locations in Canada and the United States were compared by determining fatty acid methyl ester profiles, restriction fragment length polymorphisms (RFLP) based on pulsed-field gel electrophoresis (PFGE) analysis, and DNA-DNA reassociation values. Based on qualitative and quantitative differences in fatty acid profiles, the strains were divided into nine groups and four groups by the MIDI “10% rule” and unweighted pair analysis, respectively. Restriction analysis of genomic DNA by PFGE with two endonucleases (
Xba
I and
Spe
I) revealed four distinct profiles. When a third endonuclease (
Vsp
I) was used, one group was divided into three subgroups. The profile of the American Type Culture Collection type strain differed from the profile of every other strain of
X. fragariae
. Considerable diversity was observed within
X. fragariae
, although the majority of the strains represented a clonal population. The four groups based on fatty acid profiles were similar to the four groups based on RFLP, but neither method related groups to the geographic origins of the strains. The DNA-DNA reassociation values were high for representative strains, providing evidence that all of the strains belong to the same species.
Enzymatic Conversion of Glucose to UDP-4-Keto-6-Deoxyglucose in
Streptomyces
spp
Authors: Liu Song Yu , Rosazza John P. N.
Published: December 2019

All of the 2,6-dideoxy sugars contained within the structure of chromomycin A
3
are derived from
d
-glucose. Enzyme assays were used to confirm the presence of hexokinase, phosphoglucomutase, UDPG pyrophosphorylase (UDPGP), and UDPG oxidoreductase (UDPGO), all of which are involved in the pathway of glucose activation and conversion into 2,6-dideoxyhexoses during chromomycin biosynthesis. Levels of the four enzymes in
Streptomyces
spp. cell extracts were correlated with the production of chromomycins. The pathway of sugar activation in
Streptomyces
spp. involves glucose 6-phosphorylation by hexokinase, isomerization to G-1-P catalyzed by phosphoglucomutase, synthesis of UDPG catalyzed by UDPGP, and formation of UDP-4-keto-6-deoxyglucose by UDPGO.
Purification and Substrate Specificities of Two α-l-Arabinofuranosidases fromAspergillus awamoriIFO 4033
Authors: Kaneko Satoshi , Arimoto Mitsue , Ohba Misako , Kobayashi Hideyuki , Ishii Tadashi , Kusakabe Isao
Published: December 2019

α-l-Arabinofuranosidases I and II were purified from the culture filtrate ofAspergillus awamoriIFO 4033 and had molecular weights of 81,000 and 62,000 and pIs of 3.3 and 3.6, respectively. Both enzymes had an optimum pH of 4.0 and an optimum temperature of 60°C and exhibited stability at pH values from 3 to 7 and at temperatures up to 60°C. The enzymes released arabinose fromp-nitrophenyl-α-l-arabinofuranoside,O-α-l-arabinofuranosyl-(1→3)-O-β-d-xylopyranosyl-(1→4)-d-xylopyranose, and arabinose-containing polysaccharides but not fromO-β-d-xylopyranosyl-(1→2)-O-α-l-arabinofuranosyl-(1→3)-O-β-d-xylopyranosyl-(1→4)-O-β-d-xylopyranosyl-(1→4)-d-xylopyranose. α-l-Arabinofuranosidase I also released arabinose fromO-β-d-xylopy-ranosyl-(1→4)-[O-α-l-arabinofuranosyl-(1→3)]-O-β-d-xylopyranosyl-(1→4)-d-xylopyranose. However, α-l-arabinofuranosidase II did not readily catalyze this hydrolysis reaction. α-l-Arabinofuranosidase I hydrolyzed all linkages that can occur between two α-l-arabinofuranosyl residues in the following order: (1→5) linkage > (1→3) linkage > (1→2) linkage. α-l-Arabinofuranosidase II hydrolyzed the linkages in the following order: (1→5) linkage > (1→2) linkage > (1→3) linkage. α-l-Arabinofuranosidase I preferentially hydrolyzed the (1→5) linkage of branched arabinotrisaccharide. On the other hand, α-l-arabinofuranosidase II preferentially hydrolyzed the (1→3) linkage in the same substrate. α-l-Arabinofuranosidase I released arabinose from the nonreducing terminus of arabinan, whereas α-l-arabinofuranosidase II preferentially hydrolyzed the arabinosyl side chain linkage of arabinan.
Comparison of a New
Thiomicrospira
Strain from the Mid-Atlantic Ridge with Known Hydrothermal Vent Isolates
Authors: Wirsen C. O. , Brinkhoff T. , Kuever J. , Muyzer G. , Molyneaux S. , Jannasch H. W.
Published: December 2019

A new autotrophic
Thiomicrospira
strain, MA-3, was isolated from the surface of a polymetal sulfide deposit collected at a Mid-Atlantic Ridge hydrothermal vent site. The DNA homology among three vent isolates,
Thiomicrospira crunogena
,
Thiomicrospira
sp. strain L-12, and
Thiomicrospira
sp. strain MA-3, was 99.3% or higher, grouping them as the same species,
T. crunogena
(type strain, ATCC 35932). The fact that
T. crunogena
and
Thiomicrospira
sp. strain L-12 were isolated from Pacific vent sites demonstrates a cosmopolitan distribution of this species.
Activities of
Bacillus thuringiensis
Insecticidal Crystal Proteins Cyt1Aa and Cyt2Aa against Three Species of Sheep Blowfly
Authors: Chilcott Chris N. , Wigley Peter J. , Broadwell Andrew H. , Park Danny J. , Ellar David J.
Published: December 2019

The toxicity of
Bacillus thuringiensis
Cyt1Aa protein to sheep blowfly larvae depends on its solubilization and proteolytic activation. Cyt1Aa crystals were not toxic. Full-length and trypsin-digested Cyt1Aa proteins were toxic to larvae of three species of sheep blowfly. Neither full-length nor trypsin-digested Cyt2A soluble crystal proteins were toxic.
Use of Phospholipid Fatty Acids and Carbon Source Utilization Patterns To Track Microbial Community Succession in Developing Compost
Authors: Carpenter-Boggs Lynne , Kennedy Ann C. , Reganold John P.
Published: December 2019

Carbon source utilization and phospholipid fatty acid analyses were used to track the rapidly changing microbial community in composting dairy waste. Microbial abilities to utilize common plant sugars increased during composting. Community phospholipid profiles changed significantly over time. Phospholipids suggested the presence of more thermophiles and fewer bacteria with continued compost development.
Evidence for Interspecies Gene Transfer in the Evolution of 2,4-Dichlorophenoxyacetic Acid Degraders
Authors: McGowan Catherine , Fulthorpe Roberta , Wright Alice , Tiedje J. M.
Published: December 2019

Small-subunit ribosomal DNA (SSU rDNA) from 20 phenotypically distinct strains of 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacteria was partially sequenced, yielding 18 unique strains belonging to members of the alpha, beta, and gamma subgroups of the class
Proteobacteria
. To understand the origin of 2,4-D degradation in this diverse collection, the first gene in the 2,4-D pathway,
tfdA
, was sequenced. The sequences fell into three unique classes found in various members of the beta and gamma subgroups of
Proteobacteria
. None of the α-Proteobacteria yielded
tfdA
PCR products. A comparison of the dendrogram of the
tfdA
genes with that of the SSU rDNA genes demonstrated incongruency in phylogenies, and hence 2,4-D degradation must have originated from gene transfer between species. Only those strains with
tfdA
sequences highly similar to the
tfdA
sequence of strain JMP134 (
tfdA
class I) transferred all the 2,4-D genes and conferred the 2,4-D degradation phenotype to a
Burkholderia cepacia
recipient.
Substrate Selectivity and Biochemical Properties of 4-Hydroxy-2-Keto-Pentanoic Acid Aldolase from
Escherichia coli
Authors: Pollard John R. , Rialland Delphine , Bugg Timothy D. H.
Published: December 2019

4-Hydroxy-2-keto-pentanoic acid aldolase from
Escherichia coli
was identified as a class I aldolase. The enzyme was found to be highly selective for the acetaldehyde acceptor but would accept α-ketobutyric acid or phenylpyruvic acid in place of the pyruvic acid carbonyl donor.
Oxidation of Morphine to 2,2′-Bimorphine by
Cylindrocarpon didymum
Authors: Stabler Peter J. , Bruce Neil C.
Published: December 2019

The oxidation of morphine by whole-cell suspensions and cell extracts of
Cylindrocarpon didymum
gave rise to the formation of 2,2′-bimorphine. The identity of 2,2′-bimorphine was confirmed by mass spectrometry and
1
H nuclear magnetic resonance spectroscopy.
C. didymum
also displayed activity with the morphine analogs hydromorphone, 6-acetylmorphine, and dihydromorphine, but not codeine or diamorphine, suggesting that a phenolic group at C-3 is essential for activity.
Heat Killing of
Bacillus subtilis
Spores in Water Is Not Due to Oxidative Damage
Authors: Setlow Barbara , Setlow Peter
Published: December 2019

The heat resistance of wild-type spores of
Bacillus subtilis
or spores (termed α
−
β
−
) lacking DNA protective α/β-type small, acid-soluble spore proteins was not altered by anaerobiosis or high concentrations of the free radical scavenging agents ethanethiol and ethanedithiol. Heat-killed wild-type and α
−
β
−
spores exhibited no increase in either protein carbonyl content or oxidized bases in DNA. These data strongly suggest that oxidative damage to spore macromolecules does not contribute significantly to spore killing by heat.
Bacteriophage Diversity in the North Sea
Authors: Wichels Antje , Biel Stefan S. , Gelderblom Hans R. , Brinkhoff Thorsten , Muyzer Gerard , Schütt Christian
Published: December 2019

In recent years interest in bacteriophages in aquatic environments has increased. Electron microscopy studies have revealed high numbers of phage particles (10
4
to 10
7
particles per ml) in the marine environment. However, the ecological role of these bacteriophages is still unknown, and the role of the phages in the control of bacterioplankton by lysis and the potential for gene transfer are disputed. Even the basic questions of the genetic relationships of the phages and the diversity of phage-host systems in aquatic environments have not been answered. We investigated the diversity of 22 phage-host systems after 85 phages were collected at one station near a German island, Helgoland, located in the North Sea. The relationships among the phages were determined by electron microscopy, DNA-DNA hybridization, and host range studies. On the basis of morphology, 11 phages were assigned to the virus family
Myoviridae
, 7 phages were assigned to the family
Siphoviridae
, and 4 phages were assigned to the family
Podoviridae
. DNA-DNA hybridization confirmed that there was no DNA homology between phages belonging to different families. We found that the 22 marine bacteriophages belonged to 13 different species. The host bacteria were differentiated by morphological and physiological tests and by 16S ribosomal DNA sequencing. All of the bacteria were gram negative, facultatively anaerobic, motile, and coccoid. The 16S rRNA sequences of the bacteria exhibited high levels of similarity (98 to 99%) with the sequences of organisms belonging to the genus
Pseudoalteromonas
, which belongs to the γ subdivision of the class
Proteobacteria
.
Association of EnterohemorrhagicEscherichia coliHemolysin with Serotypes of Shiga-Like-Toxin-ProducingEscherichia coliof Human and Bovine Origins
Authors: Gyles Carlton , Johnson Roger , Gao Anli , Ziebell Kim , Pierard Denis , Aleksic Stojanka , Boerlin Patrick
Published: December 2019

In this study we investigated whether the enterohemorrhagicEscherichia coli(EHEC) hemolysin geneehxAcould be used as an indicator of pathogenicity in Shiga-like-toxin-producingEscherichia coli(SLTEC) isolates. The isolates in a collection of 770 SLTEC strains of human and bovine origins were assigned to group 1 (230 human and 138 bovine SLTEC isolates belonging to serotypes frequently implicated in human disease), group 2 (85 human and 183 bovine isolates belonging to serotypes less frequently implicated in disease), and group 3 (134 bovine isolates belonging to serotypes not implicated in disease). PCR amplification was used to examine all of the SLTEC isolates for the presence ofehxAand the virulence-associated geneseae,slt-I, andslt-II. The percentages of human isolates in groups 1 and 2 that were positive forehxAwere 89 and 46%, respectively, and the percentages of bovine isolates in groups 1 to 3 that were positive forehxAwere 89, 51, and 52%, respectively. The percentages of human isolates in groups 1 and 2 that were positive foreaewere 92 and 27%, respectively, and the percentages of bovine isolates in groups 1 to 3 that were positive foreaewere 78, 15, and 19%, respectively. The frequencies of bothehxAandeaewere significantly higher for group 1 isolates than for group 2 isolates. The presence of theehxAgene was associated with serotype, as was the presence of theeaegene. Some serotypes, such as O117:H4, lacked botheaeandehxAand have been associated with severe disease, but only infrequently. Theslt-Igenes were more frequent in group 1 isolates than in group 2 isolates, and theslt-IIgenes were more frequent in group 2 isolates than in group 1 isolates. In a second experiment we determined the occurrence of theehxAandsltgenes inE. coliisolated from bovine feces. Fecal samples from 175 animals were streaked onto washed sheep erythrocyte agar plates. EightE. coli-like colonies representing all of the morphological types were transferred to MacConkey agar. A total of 1,080E. coliisolates were examined, and theehxAgene was detected in 12 independent strains, only 3 of which were positive forslt. We concluded that theehxAgene was less correlated with virulence than theeaegene was and that EHEC hemolysin alone has limited value for screening bovine feces for pathogenic SLTEC because of presence of theehxAgene in bovine isolates that are not SLTEC.
Prevalence of
Clostridium botulinum
in Finnish Trout Farms: Pulsed-Field Gel Electrophoresis Typing Reveals Extensive Genetic Diversity among Type E Isolates
Authors: Hielm Sebastian , Björkroth Johanna , Hyytiä Eija , Korkeala Hannu
Published: December 2019

The distribution of
Clostridium botulinum
serotypes A, B, E, and F in Finnish trout farms was examined. A total of 333 samples were tested with a neurotoxin-specific PCR assay.
C. botulinum
type E was found in 68% of the farm sediment samples, in 15% of the fish intestinal samples, and in 5% of the fish skin samples. No other serotypes were found. The spore counts determined by the most-probable-number method were considerably higher for the sediments than for the fish intestines and skin; the average values were 2,020, 166, and 310
C. botulinum
type E spores kg
−1
, respectively. The contamination rates in traditional freshwater ponds and marine net cages were high, but in concrete ponds equipped with sediment suction devices the contamination rates were significantly lower. Pulsed-field gel electrophoresis (PFGE) typing of 42 isolates obtained in this survey and 12 North American reference strains generated 28 pulsotypes upon visual inspection, suggesting that there was extensive genetic diversity and that the discriminatory power of PFGE typing in
C. botulinum
type E was high. A numerical analysis of
Sma
I-
Xma
I macrorestriction profiles confirmed these findings, as it divided the 54 isolates into 15 clusters at a similarity level of 76%. For this material, this level of similarity corresponded to a three-band difference in the macrorestriction profiles, which indicated that there is no genotypic proof of a close epidemiological relationship among the clusters.
Evolution of a Pathway for Chlorobenzene Metabolism Leads to Natural Attenuation in Contaminated Groundwater
Authors: van der Meer Jan Roelof , Werlen Christoph , Nishino Shirley F. , Spain Jim C.
Published: December 2019

Complete metabolism of chlorinated benzenes is not a feature that is generally found in aerobic bacteria but is thought to be due to a novel recombination of two separate gene clusters. Such a recombination could be responsible for adaptation of a natural microbial community in response to contamination with synthetic chemicals. This hypothesis was tested in a chlorobenzene (CB)-contaminated aquifer. CB-degrading bacteria from a contaminated site were characterized for a number of years by examining a combination of growth characteristics and DNA-DNA hybridization, PCR, and DNA sequence data. The genetic information obtained for the CB pathway of the predominant microorganism,
Ralstonia
sp. strain JS705, revealed a unique combination of (partially duplicated) genes for chlorocatechol degradation and genes for a benzene-toluene type of aromatic ring dioxygenase. The organism was detected in CB-polluted groundwater by hybridizing colonies cultivated on low-strength heterotrophic media with probes for the CB pathway. Southern hybridizations performed to determine the organization of the CB pathway genes and the 16S ribosomal DNA indicated that CB-degrading organisms isolated from different wells at the site were identical to JS705. Physiological characterization by the Biolog test system revealed some differences. The genes for the aromatic ring dioxygenase and dihydrodiol dehydrogenase of JS705 were detected in toluene and benzene degraders from the same site. Our results suggest that recent horizontal gene transfer and genetic recombination of existing genes between indigenous microorganisms were the mechanisms for evolution of the catabolic pathway. Evolution of the CB pathway seems to have created the capacity for natural attenuation of CB at the contaminated site.
PCR Detection ofCoxiella burnetiifrom Different Clinical Specimens, Especially Bovine Milk, on the Basis of DNA Preparation with a Silica Matrix
Authors: Lorenz Helga , Jäger Cornelie , Willems Hermann , Baljer Georg
Published: December 2019

For PCR detection ofCoxiella burnetiiin various clinical specimens we developed a sample preparation method in which silica binding of DNA was used. This method was found to be fast, easily performed with large numbers of samples, and equally sensitive for all of the specimens tested (livers, spleens, placentas, heart valves, milk, blood). The DNA preparation method described here can also be used as an initial step in any PCR-based examination of specimens. The procedure was tested with more than 600 milk samples, which were taken from 21 cows that were seropositive forC. burnetiiand reportedly had fertility problems (and therefore were suspected of shedding the agent through milk intermittently or continuously). Of the 21 cows tested, 6 were sheddingC. burnetiithrough milk. Altogether,C. burnetiiDNA was detected in 6% of the samples. There was no correlation between the shedding pattern and the serological results.
rRNA Stability in Heat-Killed and UV-Irradiated Enterotoxigenic
Staphylococcus aureus
and
Escherichia coli
O157:H7
Authors: McKillip John L. , Jaykus Lee-Ann , Drake Maryanne
Published: December 2019

Differentiation of viable cells from nonviable cells is of considerable importance in the development of methods to detect foodborne pathogens. To study the suitability of 16S rRNA as an indicator of cell viability in nucleic acid-based detection assays, we examined rRNA stability in two representative foodborne pathogens,
Escherichia coli
O157:H7 and enterotoxigenic
Staphylococcus aureus
, which were inactivated by extreme heat, moderate heat, and UV irradiation. Cell death under all conditions was confirmed by a failure to grow in brain heart infusion broth after incubation for 48 h at 37°C. rRNA stability was monitored by a Northern blot analysis, and detection was evaluated by using reverse transcription (RT)-PCR performed with two primer sets (which produced 325- and 1,400-bp amplicons). rRNA of neither pathogen was detected by Northern blot analysis and RT-PCR after cells were killed by autoclaving at 121°C for 15 min. In contrast, intact rRNA of both pathogens were detected by Northern blotting and could be amplified by RT-PCR up to 48 h after cells were killed by heat treatment at 80°C and UV irradiation at 254 nm. rRNA was a suitable target molecule for monitoring bacterial viability under extreme heat conditions, but the presence of rRNA was not correlated with viability following moderate heat inactivation or UV irradiation of cells.
Selection and Characterization of Microorganisms Utilizing Thaxtomin A, a Phytotoxin Produced by
Streptomyces scabies
Authors: Doumbou Cyr Lézin , Akimov Vladimir , Beaulieu Carole
Published: December 2019

Thaxtomin A is the main phytotoxin produced by
Streptomyces scabies
, a causal agent of potato scab. Thaxtomin A is a yellow compound composed of 4-nitroindol-3-yl-containing 2,5-dioxopiperazine. A collection of nonpathogenic streptomycetes isolated from potato tubers and microorganisms recovered from a thaxtomin A solution were examined for the ability to grow in the presence of thaxtomin A as a sole carbon or nitrogen source. Three bacterial isolates and two fungal isolates grew in thaxtomin A-containing media. Growth of these organisms resulted in decreases in the optical densities at 400 nm of culture supernatants and in 10% reductions in the thaxtomin A concentration. The fungal isolates were identified as a
Penicillium
sp. isolate and a
Trichoderma
sp. isolate. One bacterial isolate was associated with the species
Ralstonia pickettii
, and the two other bacterial isolates were identified as
Streptomyces
sp. strains. The sequences of the 16S rRNA genes were determined in order to compare thaxtomin A-utilizing actinomycetes to the pathogenic organism
S. scabies
and other
Streptomyces
species. The nucleotide sequences of the γ variable regions of the 16S ribosomal DNA of both thaxtomin A-utilizing actinomycetes were identical to the sequence of
Streptomyces mirabilis
ATCC 27447. When inoculated onto potato tubers, the three thaxtomin A-utilizing bacteria protected growing plants against common scab, but the fungal isolates did not have any protective effect.
Difluoromethane, a New and Improved Inhibitor of Methanotrophy
Authors: Miller Laurence G. , Sasson Caleb , Oremland Ronald S.
Published: December 2019

Difluoromethane (HFC-32; DFM) is compared to acetylene and methyl fluoride as an inhibitor of methanotrophy in cultures and soils. DFM was found to be a reversible inhibitor of CH
4
oxidation by
Methylococcus capsulatus
(Bath). Consumption of CH
4
in soil was blocked by additions of low levels of DFM (0.03 kPa), and this inhibition was reversed by DFM removal. Although a small quantity of DFM was consumed during these incubations, its remaining concentration was sufficiently elevated to sustain inhibition. Methanogenesis in anaerobic soil slurries, including acetoclastic methanogenesis, was unaffected by levels of DFM which inhibit methanotrophy. Low levels of DFM (0.03 kPa) also inhibited nitrification and N
2
O production by soils. DFM is proposed as an improved inhibitor of CH
4
oxidation over acetylene and/or methyl fluoride on the basis of its reversibility, its efficacy at low concentrations, its lack of inhibition of methanogenesis, and its low cost.